Professional Documents
Culture Documents
THE THE
SERIES SERIES
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Genetics,
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ABOUTABOUT ABOUT
THE
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Conifers represent
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represent 650 650
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genetic improvement
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genetic improvement of some of these species started about 60 years ago. started
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reviewing the the the
reviewing Editors
Editors Editors
genetics,
genetics, genetics, genomics
genomicsgenomics and
and breeding breeding
and of of
breeding conifers.
conifers. of conifers. Christophe Plomion • Jean Bousquet
Christophe
Christophe Plomion Plomion
• Jean • Jean Bousquet
Bousquet
ABOUT
ABOUT THE ABOUT THE
EDITORSEDITORS
THE EDITORS Chittaranjan Kole
Chittaranjan
Chittaranjan Kole Kole
Christophe
Christophe Christophe
PlomionPlomion Plomion
receivedreceived a Ph.D.
areceived
Ph.D. in Genetics
inaGenetics
Ph.D. in Geneticsand Plant
and Plant and Breeding
Plant
Breeding fromfrom from
Breeding
AgroCampus
AgroCampus AgroCampus Ouest,
Ouest, Rennes, Rennes,
Ouest, France. France.
Rennes,He He
France. is presently
He is presently
is presently deputy
deputy head head
deputyof the of theof the
head
Conifers
Conifers
Conifers
“Forest, Grassland
“Forest, Grassland
“Forest, Grassland and
and Fresh Fresh
and Water Water
Fresh Ecology”
Water Ecology”
Ecology” division
division ofdivision of
INRA. of INRA.HeINRA.also also
He He also
leadsleads research
leads
research in forest
research
in forest tree tree
in forest
genomicsgenomics
tree genomics
within within the “Biodiversity,
thewithin the “Biodiversity,
“Biodiversity, Genes Genes
and and and
Genes
Community”
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Community” researchresearch
INRA unit unit
research in
unit
in Bordeaux,Bordeaux,
in Bordeaux,
France.France. France.
Over Over
the Overthe last
last the 15
15 last 15
years,
years, heyears, he has published
he has published
has published 100
100 scientific scientific
100 scientific papers
papers inpapers in the
the fields fields
in the of molecular,
fields of molecular,
of molecular,
population
population populationand quantitative
and quantitative
and quantitative geneticsgenetics of forest
genetics
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of forest
trees.
Jean
Jean Bousquet Bousquet
Jean Bousquet is
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is professor and
and Canada Canada
and Research Research
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in Forest in
and and and
Forest
Environmental
Environmental
Environmental Genomics
GenomicsGenomics at Laval University
at Laval University
at Laval University in Quebec
in QuebecinCity. Quebec City.
OverCity.Over the past
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23 years, 23 heyears,he has
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publishedpublished
has 120 120
published scientific
120
scientific papers
scientific
papers inpapers in the
the in fields
fields of of
theof fields
phylogenetics,
phylogenetics,
phylogenetics, population
population population
geneticsgenetics and
genetics
and genomicsgenomics
and genomics of forest
of forest trees
of forest
trees and
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andtrees and their
symbionts.
symbionts. He isHe
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is co-director theofspruce
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genomics genomics genomics
projectproject ARBOREA.
project
ARBOREA. ARBOREA.
Christophe
Christophe Plomion
Christophe
Chittaranjan
Chittaranjan
Chittaranjan Kole
Jean Bousquet
JeanEditors
Jean Bousquet
N10379
Editors
Editors Kole
Bousquet
Science
Science Publishers
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Publishers Publishers Plomion
Plomion
Kole
Science
Science Science
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Publishers
9 7 891 75 87 189 570788817 501 789 878 109887 1 9 8
GENETICS, GENOMICS
AND BREEDING OF
CONIFERS
Genetics, Genomics and Breeding of Crop Plants
Series Editor
Chittaranjan Kole
Department of Genetics and Biochemistry
Clemson University
Clemson, SC
USA
Editors
Christophe Plomion
INRA
UMR BIOGECO
Cestas
France
Jean Bousquet
Centre d’étude de la forêt
Université Laval
Québec
Canada
Chittaranjan Kole
Department of Genetics and Biochemistry
Clemson University
Clemson, SC
USA
Science Publishers
Jersey, British Isles
Enfield, New Hampshire
CRC Press
Taylor & Francis Group
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© 2011 by Taylor & Francis Group, LLC
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Preface to the Series
Chittaranjan Kole
This page intentionally left blank
Preface to the Volume
Conifers are woody plants, the great majority being trees. They represent 650
species, some ranking as the largest, tallest, and longest living non-clonal
terrestrial organisms on Earth. They are of immense ecological importance,
dominating many terrestrial landscapes and representing the largest
terrestrial carbon sink. They are evolutionary distinct from angiosperm
trees on many accounts and with their extraordinary large genomes, they
provide a different view of plant genome biology and evolution. They are
also of great economic importance, as they are primarily used for timber
and paper production worldwide. Domestication of some of these species
was started about 60 years ago through traditional genetic improvement
programs. It has resulted in advances in overall growth, wood quality, pest
resistance and adaptation, but breeding still remains a slow process because
of long generation intervals typical of most conifers and because most traits
cannot be correctly evaluated at an early stage.
During the past 20 years, more and more sophisticated genomics tools
have been developed to describe the extreme plasticity and variability of
these species at different levels of integration (from genes up to phenotypes)
and are now being integrated into breeding to accelerate the domestication
process by a more precise exploitation of genetic diversity. Application of
genomic-based science is also playing an important role in understanding
the evolution, patterns of nucleotide variation and the molecular basis of
quantitative traits and adaptation. Altogether, this new knowledge is also
expected to help delineate more efficient gene conservation strategies.
This book will give the reader an in-depth review of the current state-
of-the-art of genetic and genomic research conducted in conifers. Each
chapter is the product of specialists in their field. Their goal was to report
on the latest trends and findings and at the same time, promote awareness
and make this knowledge accessible to the vast majority. Accordingly, the
chapters are well documented and illustrated. Their contribution is greatly
appreciated.
The book begins with an exhaustive description of the conifers in
terms of classification, geographical distribution, life history and ecology,
morphology and fossil history as well as phylogenetics (Chapter 1). It
is followed by a chapter devoted to their economic importance and the
xii Genetics, Genomics and Breeding of Conifers
8. Transcriptomics 323
John J. Mackay and Jeffrey F. D. Dean
9. Recent Advances in Proteomics and Metabolomics in 358
Gymnosperms
Rebecca Dauwe, Andrew Robinson and Shawn D. Mansfield
10. Toward the Conifer Genome Sequence 389
Michele Morgante and Emanuele De Paoli
11. Future Prospects 404
Jeffrey F.D. Dean
Index 439
Color Plate Section 449
List of Contributors
R. Alía
Department of Forest Ecology and Genetics, Center of Forest Research,
INIA, 28040 Madrid, Spain.
Email: alia@inia.es
B. Andersson
Skogforsk (Sävar), Box 3, S-918 21 Sävar, Sweden.
Email: bengt.andersson@skogforsk.se
Francesca Bagnoli
Plant Protection Institute, CNR, Via Madonna del Piano 10, 50019 Sesto
Fiorentino (FI), Italy.
Email: bagnoli@ipp.cnr.it
J.-C. Bastien
INRA—Centre de Recherche d’Orléans, 2163, Avenue de la Pomme de Pin,
CS 400001 ARDON, F-45075 Orléans Cedex 2, France.
Email: jean-charles.bastien@orleans.inra.fr
J. Beaulieu
Natural Resources Canada, P.O. Box 10380, Stn. Sainte-Foy, Québec, QC
G1V 4C7, Canada.
Email: jeanbeau@nrcan-rncan.gc.ca
Jean Bousquet
Centre d’étude de la forêt, Université Laval, Québec, Québec G1V 0A6,
Canada.
Email: jean.bousquet@sbf.ulaval.ca
R.D. Burdon
Scion (NZ Forest Research Institute Ltd.), Private Bag 3020, Rotorua 3010,
New Zealand.
Email: rowland.burdon@scionresearch.com
C. Burgarella
Department of Forest Ecology and Genetics, Center of Forest Research,
INIA, 28040 Madrid, Spain.
Email: concettaburgarella@hotmail.com
xvi Genetics, Genomics and Breeding of Conifers
Rebecca Dauwe
Department of Wood Science, Faculty of Forestry, 4030-2424 Main Mall,
Vancouver, BC, V6T 1Z4, Canada.
Email: rebecca.dauwe@u-picardie.fr
Jeffrey F.D. Dean
Warnell School of Forestry and Natural Resources, University of Georgia,
Athens, GA 30602, USA.
Email: jeffdean@uga.edu
Tel: +1-706-542-1710
Emanuele De Paoli
Istituto Agrario di San Michele all’Adige, Vie E. Mach 1, 38010 San Michele
all’Adige, Italy.
Email: emanuele.depaoli@iasma.it.
S. Dillon
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.
Email: shannon.dillon@csiro.au
W.S. Dvorak
North Carolina State University, Campus Box 8008, Raleigh, NC 27695-
8008, USA.
Email: w_dvorak@ncsu.edu
A.J. Eckert
Section of Evolution and Ecology and Center for Population Biology,
University of California at Davis, Davis, CA 95616, USA.
Email: ajeckert@ucdavis.edu
Bruno Fady
INRA, UR629, Ecologie des Forêts Méditerranéennes, Domaine Saint Paul,
Site Agroparc, 84914 Avignon, France.
Email: bruno.fady@avignon.inra.fr
Silvia Fineschi
Plant Protection Institute, CNR, Via Madonna del Piano 10, 50019 Sesto
Fiorentino (FI), Italy.
Email: fineschi@ipp.cnr.it
M.R. García-Gil
Umeå Plant Science Center, Swedish University of Agricultural Science, SE
901 83 Umeå, Sweden.
Email: m.rosario.garcia@genfys.slu.se
P.H. Garnier-Géré
INRA, UMR1202 Biodiversity Genes & Communities, 69 route d’Arcachon,
33612 Cestas Cedex, France.
Email: pauline@pierroton.inra.fr
List of Contributors xvii
David S. Gernandt
Departamento de Botánica, Instituto de Biología, Universidad Nacional
Autónoma de México, A.P. 70-233, México Distrito Federal 04510,
Mexico.
Email: dgernandt@ibiologia.unam.mx
S.C. González-Martínez
Department of Forest Ecology and Genetics, Center of Forest Research,
INIA, 28040 Madrid, Spain.
Email: santiago@inia.es
Tel: +34 913471499
D. Grivet
Department of Forest Ecology and Genetics, Center of Forest Research,
INIA, 28040 Madrid, Spain.
Email: dgrivet@inia.es
M. Heuertz
Department of Forest Ecology and Genetics, Center of Forest Research,
INIA, 28040 Madrid, Spain.
and
Université Libre de Bruxelles, Faculté des Sciences, Behavioural and
Evolutionary Ecology cp160/12, av. F.D. Roosevelt 50, 1050 Brussels,
Belgium.
Email: mheuertz@ulb.ac.be
Nathalie Isabel
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry
Centre, 1055 du P.E.P.S., P.O. Box 10380, Stn Sainte-Foy, Québec, Québec
G1V 4C7, Canada.
Email: nisabel@cfl.forestry.ca
Nurul Islam-Faridi
Forest Tree Molecular Cytogenetics, Southern Institute of Forest Genetics,
US Forest Service.
and
Dept. of Ecosystem Science & Management, Texas A&M University,
College Station, TX 77843, USA.
Email: nfaridi@tamu.edu
J.P. Jaramillo-Correa
Department of Forest Ecology and Genetics, Center of Forest Research,
INIA, 28040 Madrid, Spain; and
Department of Evolutionary Ecology, Ecology Institute, Universidad
Nacional Autónoma de México, Ciudad Universitaria, Tercer circuito
Exterior, Apartado Postal 70-275, México, D.F.
Email: jaramillo@miranda.ecologia.unam.mx
xviii Genetics, Genomics and Breeding of Conifers
J.N. King
British Columbia Forest Service, PO Box 9519 Stn Prov Govt, Victoria, B.C.
V8W 9C2, Canada.
Email: john.king@gov.bc.ca
T. Kondo
Forest Tree Breeding Centre, 3809-1 Ishi, Juo, Hitachi, Ibaraki 319-1301,
Japan.
Email: kontei@affrc.go.jp
J. Krakowski
British Columbia Ministry of Forests and Range, Box 335, Mesachie Lake,
B.C. V0R2N0, Canada.
Email: Jodie.Krakowski@gov.bc.ca
Konstantin V. Krutovsky
Department of Ecosystem Science and Management, Texas A&M University,
College Station, Texas 77843-2138, USA.
Email: k-krutovsky@tamu.edu
M. Lascoux
Program in Evolutionary Functional Genetics, Evolutionary Biology Centre,
Uppsala University, 75326 Uppsala, Sweden.
Email: Martin.Lascoux@ebc.uu.se
S.J. Lee
Forest Research, Northern Research Station, Roslin, Midlothian, EH25 9SY,
Scotland.
Email: steve.lee@forestry.gsi.gov.uk
Aaron Liston
Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon
State University, Corvallis, Oregon 97331, USA.
Email: listona@science.oregonstate.edu
John J. Mackay
Center for Forest Research, Laval University, Québec City, Québec, Canada,
G1V 0A6.
Email: John.mackay@sbf.ulaval.ca
Shawn D. Mansfield
Department of Wood Science, Faculty of Forestry, 4030-2424 Main Mall,
Vancouver, BC, V6T 1Z4, Canada.
Email: shawn.mansfield@ubc.ca
List of Contributors xix
S.E. McKeand
North Carolina State University, Campus Box 8002, Raleigh, NC 27695-8002,
USA.
Email: steve_mckeand@ncsu.edu
Michele Morgante
Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, Via
delle Scienze 208, 33100 Udine, Italy; and
Istituto di Genomica Applicata, Parco Scientifico e Tecnologico di Udine,
Via Linussio 51, 33100 Udine, Italy.
Email: michele.morgante@uniud.it
T.J. Mullin
BioSylve Forest Science NZ Limited, 45 Krokoro Road, Lower Hutt 5012,
New Zealand.
Email: tim.mullin@biosylve.com
D.B. Neale
Department of Plant Sciences, University of California at Davis, Davis, CA
95616, USA; and
Institute of Forest Genetics, Pacific Southwest Research Station, US
Department of Agriculture Forest Service, Placerville, CA 95667, USA.
Email: dbneale@ucdavis.edu
C. Dana Nelson
USDA Forest Service, Southern Research Station, Southern Institute of
Forest Genetics, 23332 Success Road, Saucier, MS 39574, USA.
Email: dananelson@fs.fed.us
Sylvie Oddou-Muratorio
INRA, UR629, Ecologie des Forêts Méditerranéennes, Domaine Saint Paul,
Site Agroparc, 84914 Avignon, France.
Email: sylvie.oddou@avignon.inra.fr
L. Pâques
INRA—Centre de Recherche d’Orléans, 2163 Avenue de la Pomme de Pin,
CS 400001 ARDON, F-45075 Orléans Cedex 2, France.
Email: luc.paques@orleans.inra.fr
Betty Pelgas
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry
Centre, 1055 du P.E.P.S., P.O. Box 10380, Stn Sainte-Foy, Québec, Québec
G1V 4C7, Canada.
Email: betty.pelgas@RNCan-NRCan.gc.ca
xx Genetics, Genomics and Breeding of Conifers
Andrea Piotti
Department of Environmental Sciences, University of Parma, Viale Usberti
11/A, 43100 Parma, Italy.
Email: andrea.piotti@nemo.unipr.it
A. Raffin
INRA (Pierroton), 69 route d’Arcachon, 33612 CESTAS Cedex, France.
Email: annie.raffin@pierroton.inra.fr
Kermit Ritland
Department of Forest Sciences, University of British Columbia, Vancouver,
British Columbia V6T 1Z4, Canada.
Email: kermit.ritland@ubc.ca
Andrew Robinson
Department of Wood Science, Faculty of Forestry, 4030-2424 Main Mall,
Vancouver, BC, V6T 1Z4, Canada.
Email: andrewrobinsonnz@gmail.com
J.H. Russell
British Columbia Ministry of Forests and Range, Box 335, Mesachie Lake,
B.C. V0R2N0, Canada.
Email: john.russell@gov.bc.ca
O. Savolainen
Department of Biology, University of Oulu, 90014 Oulu, Finland.
Email: outi.savolainen@oulu.fi
Federico Sebastiani
Plant Genetics Institute, CNR, Via Madonna del Piano 10, 50019 Sesto
Fiorentino (FI), Italy.
Email: federico.sebastiani@unifi.it
T. Skrøppa
Norwegian Forest and Landscape Institute, Høgskoleveien 8, 1432 Ås,
Norway.
Email: tore.skroppa@skogoglandskap.no
M. Stoehr
British Columbia Ministry of Forests, PO Box 9519, Stn Prov Govt, Victoria,
B.C. V8W 9C2, Canada.
Email: michael.stoehr@gov.bc.ca
John V. Syring
Department of Biology, Linfield College, 900 SE Baker St., McMinnville,
Oregon 97128, USA.
Email: jsyring@linfield.edu
List of Contributors xxi
Yoshihiko Tsumura
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687,
Japan.
Email: ytsumu@ffpri.affrc.go.jp
Giovanni G. Vendramin
Plant Genetics Institute, CNR, Via Madonna del Piano 10, 50019 Sesto
Fiorentino (FI), Italy.
Email: giovanni.vendramin@igv.cnr.it
Phillip L. Wilcox
Scion: New Zealand Forest Research Institute Ltd, Private Bag 3020, Rotorua
3046, New Zealand.
Email: phillip.wilcox@scionresearch.com
Ann Willyard
Biology Department, Hendrix College, 1600 Washington Ave, Conway,
Arkansas 72032, USA.
Email: willyard@hendrix.edu
A. Yanchuk
British Columbia Ministry of Forests, PO Box 9519, Stn Prov Govt, Victoria,
B.C. V8W 9C2, Canada.
Email: alvin.yanchuk@gov.bc.ca
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Abbreviations
µ Mutation rate
2-D Two-dimensional
2-DE Two-dimensional electrophoresis
ABC Approximate Bayesian computation
AFA Adaptive Force Acoustics
AFLP Amplified fragment length polymorphism
AGP Arabinogalactan protein
ANOVA Analysis of variance
AT Adenine-Thymine
ATRS Arabidopsis-type telomere repeat sequence
BAC Bacterial artificial chromosome
BHT Butylated hydroxytoluene
BIC Bayesian information criterion
BLUP Best Linear Unbiased Prediction
bp Base pairs
CAPS Cleaved amplified polymorphic sequence
CCA Canonical correlation analysis
CDA Canonical discriminate analysis
cDNA Complementary-DNA
CDS Complete coding sequences
Ch Chromosome
ChIP-Seq Chromatin ImmunoPrecipitation coupled with next-
generation sequencing
CID Carbon isotope discrimination
cM CentiMorgan
CMA Chromamycin A3
COLD-PCR CO-amplification at lower denaturation temperature-
PCR
COS Conserved orthologous set
cpDNA Chloroplast-DNA
cpSSR Chloroplast-SSR
DAPI 4’, 6-Diamidino -2-phenylindole
DArT Diversity array technology
DIGE Differential in gel electrophoresis
xxiv Genetics, Genomics and Breeding of Conifers
ABSTRACT
Conifers (Pinophyta) are woody trees or shrubs with simple leaves,
simple pollen cones, and compound or reduced ovulate cones. Despite
their dominance in many terrestrial landscapes, the 670 species of extant
conifers make up less than 0.3% of the species diversity of modern land
plants. The fossil record of conifers, which extends to the Carboniferous,
indicates that a much greater diversity is now extinct. Conifers occur
on six of the seven continents and include both widely distributed,
dominant species that form vast forests and narrow endemics. They rank
as the largest, tallest, and longest living non-clonal terrestrial organisms
on the Earth. Pinus is the largest extant genus with approximately 20
species distributed throughout the Northern Hemisphere. It is rivaled
in diversity in the Southern Hemisphere and the tropics by Podocarpus,
with approximately 105 species. Genetic diversity is often high in
conifers, promoted by large population size, outcrossing reproductive
systems, high mutation rates, and long distance dispersal of pollen and
sometimes seeds. Estimates of ages and mutation rates in the group are
expected to improve greatly as conceptual advances related to fossil
interpretation converge with the enormous quantities of new sequence
data being generated by genetic and phylogenetic studies of living
species. Contrasting patterns of organellar and nuclear inheritance
1
Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México,
A.P. 70-233, México Distrito Federal 04510, Mexico; e-mail: dgernandt@ibiologia.unam.mx
2
Biology Department, Hendrix College, 1600 Washington Ave, Conway, Arkansas 72032, USA;
e-mail: willyard@hendrix.edu
3
Department of Biology, Linfield College, 900 SE Baker St., McMinnville, Oregon 97128,
USA; e-mail: jsyring@linfield.edu
4
Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University,
Corvallis, Oregon 97331, USA; e-mail: listona@science.oregonstate.edu
*Correspondig author
2 Genetics, Genomics and Breeding of Conifers
make conifers an important system for studying pollen and seed flow,
hybridization, lineage sorting, and gene coalescence.
Keywords: conifers; ecology; fossils; molecular clock; phylogeny;
Pinophyta
Family # Gen- Representative # Species Representative Species Native Range Common Notes Genetic
era Genera or Names Resources
Subgenera
6
Genetics, Genomics and Breeding of Conifers
Family # Gen- Representative # Species Representative Species Native Range Common Notes Genetic
era Genera or Names Resources
Subgenera
8
Genetics, Genomics and Breeding of Conifers
Family # Gen- Representative # Species Representative Species Native Range Common Notes Genetic
era Genera or Names Resources
Subgenera
10
Family # Gen- Representative # Species Representative Species Native Range Common Notes Genetic
(e.g., Wollemia nobilis W.G. Jones, K.D. Hill & J.M. Allen). Many species
are rare and/or threatened with extinction (Farjon et al. 1999). In Pinus,
geographic ranges have been shown to decrease with increasing proximity
to the equator (Stevens and Enquist 1998), while species diversity increases
dramatically along this same gradient (Farjon et al. 1993).
Overall, the Northern Hemisphere contains about 70% of total conifer
diversity (Farjon 2001). Regions with high species diversity include
California, Mexico, the Chinese provinces of Sichuan and Yunnan, and
the eastern Himalayas, Japan, Taiwan, and New Caledonia (Farjon 2001).
Pinaceae comprises 11 genera and 238 species distributed throughout
Eurasia, North Africa, the Himalayas, and North and Central America.
Pinus, with approximately 120 species, is the largest genus. The only
member of Pinaceae that occurs naturally in the Southern Hemisphere
is Pinus merkusii Jungh. & de Vriese, with a distribution that crosses the
equator in Sumatra.
Podocarpaceae, with approximately 19 genera and 190 species, and
Araucariaceae, with three genera and approximately 42 species, are
distributed across the Southern Hemisphere and the tropics. Podocarpaceae
occurs in Africa, South America, Australia, South and East Asia, Indonesia,
and numerous other islands of the South Pacific. Other Podocarpaceae taxa
occur north of the equator in East Africa, Japan, China, Central America,
and Mexico. Podocarpus, with ca. 105 is the largest genus, and once better
studied, may eventually be shown to be more diverse than Pinus (Farjon
2001, 2003). Araucariaceae occurs in South America, South and East Asia,
Australia, and on islands throughout the South Pacific. The largest genera
are Agathis (ca. 23 species) and Araucaria (19 species).
Sciadopitys verticillata Siebold & Zucc., the sole representative of
Sciadopityaceae, is endemic to southern Japan. Taxaceae (6 genera and 24
species) occurs primarily in the Northern Hemisphere (North and Central
America, Eurasia, and the Himalayas), but Taxus sumatrana Miquel de
Laub. occurs south of the equator and the monotypic genus Austrotaxus
is endemic to New Caledonia. Taxus, with 10 species, is the largest genus.
Cupressaceae, with approximately 31 genera (approximately 18 monotypic)
and 165 species (Little 2006), occurs on every continent except Antarctica.
Juniperus, with ca. 67 species, is the largest genus.
chromosomes (Flory 1936; Page 1990), however this has not been examined
in a phylogenetic framework. Numbers can be conserved within genera, as
in Pinus (n = 12), or they can be highly variable, as in Dacrydium or Podocarpus
(Page 1990). Polyploidy has played a minor role in the evolution of conifers,
the only naturally occurring cases are tetraploid Fitzroya cupressoides and
hexaploid Sequoia sempervirens (Ahuja 2005).
Genetic diversity in conifers is generally high, promoted by large
population sizes, long life spans, outcrossing reproductive systems, high
mutation rates, and long distance dispersal of pollen, and sometimes
seeds (Hamrick et al. 1992; Ledig 1998). Hamrick et al. (1992) estimated
an average of 71.1% polymorphic loci and 16.9% expected heterozygosity
across representative gymnosperms heavily favoring conifers. Ledig (1998)
recognizes Pinus as one of the most variable of organisms with an average
of 70.4% polymorphic loci and typical expected heterozygosity of 13 to
16%. Quiroga and Premoli (2007) reported 57.0% polymorphic loci and
an expected heterozygosity of 14.8% in Podocarpus parlatorei Pilg., values
within the reported range for other conifers. Some conifers do have low
levels of genetic diversity. Most known examples are narrow endemics,
including Pinus torreyana Carrière (Provan et al. 1999), Picea chihuahuana
Martínez (Ledig et al. 1997), and Picea omorika (Pančić) Purk. (Ballian et al.
2006). In contrast, Pinus resinosa Aiton has low genetic diversity but a wide
geographic distribution in eastern North America (Walter and Epperson
2005). Due to their outcrossing reproductive system, the ability of pollen
to travel vast distances, and occasional long-distance seed dispersal, most
species of conifers show little among population differentiation (Ledig 1998).
Exceptions occur where drift is acting on small, fragmented populations
(Ledig et al. 1997; Ge et al. 1998; Ballian et al. 2006).
As a result of their life history traits, conifers will generally have large
effective population sizes (Ne), though variation by species is expected
according to individual history (Syring et al. 2007a, b). Across Pinus, Ne
estimates range from 1.7 × 104 in P. flexilis James to 1.2 × 105 in P. lambertiana
Dougl. (Syring et al. 2007b). Values for three species of Picea are on the same
order of magnitude as the higher Pinus estimates (1.2–1.5 × 105) (Bouillé
and Bousquet 2005). For comparison, reports from both inbreeding and
outcrossing angiosperm species are typically less than 1.0 × 104 (Schoen
and Brown 1991; Reusch et al. 2000). Large Ne promotes the retention of
allelic diversity and has implications for phylogenetic analyses (see below).
Because conifer species are less likely to form large, contiguous populations
in the Southern Hemisphere (Enright 1995), it is tempting to assume that
Ne will be larger for Northern Hemisphere species. However, geographic
range is known to be a poor predictor of Ne (Syring et al. 2007b). Future
estimates of Ne would prove informative.
18 Genetics, Genomics and Breeding of Conifers
2002). In Juniperus (Cupressaceae), the cone scales are fleshy and fused
into a bird dispersed “berry-like” structure. Multiple lineages of Pinus
(Pinaceae) have cones with relatively few scales and enlarged, functionally
wingless, bird-dispersed seeds. One of the two seeds per cone scale
often aborts, presumably allowing for the more extensive growth of the
surviving seed.
Wood. The exceptional size and height of many conifers with respect
to other living organisms is due in part to the strength of their wood
(secondary xylem), which is composed of thick walled vertical tracheids
with bordered pits and lacks vessels. In addition to conducting water and
nutrients, these cells provide much greater mechanical support than thin-
walled parenchyma cells (Greguss 1955). The type of pitting, together with
the arrangement of the horizontal rays, is diagnostic for conifer families.
The horizontal rays have also undergone specialization, from homogeneous,
thin walled parenchyma as seen in cycads, Ginkgo, and fossil conifer woods
similar to modern Araucariaceae and Podocarpaceae, to heterogeneous,
with variously pitted ray parenchyma and ray tracheids. Heterogeneous
rays are found in two separate lineages, Cupressaceae (Sequoia and
Metasequoia) and Pinaceae.
Pollen morphology and ovule orientation. The pollen grains of many
conifers have air bladders, or sacci. The presence of air bladders facilitates
pollen dispersal by wind, although their primary function is probably
to orient pollen grains on pollen drops exuded on the micropyle of the
ovulate cone, allowing germination towards the nucellar chamber (Doyle
1945; Tomlinson 1994). In many conifers, fertilization is facilitated by the
absorption of the pollen drop, which draws the pollen inside the nucellus.
Pollen drops appear to be functionally linked to ovule inversion and the
presence of pollen sacs (Tomlinson and Takaso 2002). Families with ovules
that are inverted during pollination (Pinaceae and Podocarpaceae) tend
to have saccate pollen, and families with erect ovules during pollination
(Araucariaceae, Sciadopityaceae, Taxaceae, and Cupressaceae) have
nonsaccate pollen.
codon positions of protein coding regions can be used to estimate silent, (also
called synonymous) relative substitution rates. With the addition of a fixed
calibration point and a generation time, these relative rates can be converted
to absolute rates. As with any use of a molecular clock, the assumption that
the selected calibration age matches the assigned divergence has a major
effect on the results. Dramatic lineage-specific (Gaut et al. 1996; Soltis et
al. 2002) and locus-specific rate differences have been reported among
plants (Senchina et al. 2003; Mower et al. 2007). Our knowledge of the true
range of rate variation for conifers (and for all plants) is just beginning to
unfold, but it seems prudent to examine the calibration assumptions if a
rate estimation falls far outside of reported ranges.
As an example of a useful molecular clock, two alternative calibration
points were examined in a combined analysis of cpDNA sequences and
morphological characters in Pinaceae (Gernandt et al. 2008). Pityostrobus
bernissartensis Alvin (Barremian/Aptian, ca. 123 Mya) was used to set a
minimum age for the divergence of Pinus from the Picea-Cathaya clade.
The oldest fossil record for an extant Pinaceae genus is Pseudolarix erensis
Krassilov (LePage and Basinger 1995) (Mongolia, Upper Oxfordian, ca.
155 Mya), but this lacks the anatomical details required for confident
determination. Regardless, using it to constrain the divergence of Pseudolarix
from its sister group results in age estimates that are only slightly older than
the Pityostrobus calibration.
The oldest representative of Pinus, P. belgica Alvin, has been placed
at strikingly different nodes in published studies. This fossil has been
associated with the divergence between Pinus and other modern Pinaceae
genera (Wang et al. 2000; García-Gil et al. 2003; Willyard et al. 2007) or at the
divergence of subgenera (Sokol and Williams 2005). Because Alvin (1960)
further ascribed P. belgica to subsection Pinus, its age has also been applied
to the divergence between representatives from the sections of subg. Pinus
(Krupkin et al. 1996; Dvornyk et al. 2002; Brown et al. 2004; Eckert and
Hall 2006). The association of the oldest Pinus fossil with a crown node has
been shown to result in unreasonable age and rate estimates (see below)
and a substantial distortion in biogeographical interpretations (Willyard et
al. 2007). Some alternative calibrations that have been published for Pinus
should also be approached with a generous dose of skepticism. For example,
an origin for the genus Pinus of 195 Mya (Kutil and Williams 2001) was
based on a presumed Jurassic origin (Miller 1977) that lacks explicit fossil
evidence. This calibration scenario was bolstered with the assumption that
both pine subgenera were already present by the Cretaceous. However,
reassignment of a fossil from the Magothy Formation in Delaware from
subgenus Strobus (Miller 1977) to subgenus Pinus (Miller and Malinky
1986) negates the support for a Jurassic origin for Pinus. Based on our
current understanding, the appearance of subg. Strobus dates to the Upper
The Conifers (Pinophyta) 23
1.3 Phylogenetics
1.3.1 Organellar Studies
Chloroplast DNA was the initial data source for comparative molecular
genetic studies of plants, and it has had an enormous impact on the fields
of phylogenetics and population genetics for almost 20 years. A pioneering
study of chloroplast-based phylogeny was conducted in Pinus (Strauss
and Doerksen 1990) and one of the first plant chloroplast genomes to be
completely sequenced was Pinus thunbergii Parl. (Wakasugi et al. 1994).
Another important early discovery was the absence of the ca. 25 Kbp
inverted repeat in Pinus (Strauss et al. 1988) and other conifers (Raubeson
and Jansen 1992), which typically have plastomes ca. 120,000–130,000 bp
in length.
In contrast to most other seed plants, the chloroplast is predominantly
paternally inherited in conifers (Mogensen 1996). However, small
percentages (9 of 361 progeny from a controlled cross) of maternal
chloroplast inheritance and heteroplasmy (3 of 80 open-pollinated seeds)
have been documented in Chamaecyparis obtusa Siebold & Zucc. (Shiraishi
et al. 2001). Early reports of limited maternal inheritance and heteroplasmy
in Pinus banksiana Lamb. × P. contorta hybrids (Dong et al. 1992) and in
Pinus radiata D. Don (Cato and Richardson 1996) were not as rigorously
documented, and require confirmation. The prevalence of this phenomenon
in other conifers remains to be evaluated.
The population dynamics of organellar DNA has important
consequences. The effective population size (Ne) of organelles is one-half
(in monoecious plants) to one-quarter (in dioecious plants) that of nuclear
genes (Birky et al. 1983), resulting in more rapid coalescence than nuclear
loci. Therefore, the retention of ancestral polymorphisms is predicted to
be less common at organellar loci. In practice, this means that geographic
partitioning of genetic diversity is often recovered, making plastid and
mitochondrial DNA very popular in phylogeographic studies of conifers
(reviewed in Petit et al. 2005). Note that most of these studies have only
considered single species in isolation, and that broader taxonomic sampling
may reveal alternative explanations for genetic differentiation. For example,
Liston et al. (2007) found that very strong cpDNA differentiation between
northern and southern populations of Pinus lambertiana was likely due to
introgression from Pinus albicaulis. The haploid nature of organellar genomes
makes them an effective tool for recognizing interspecific hybridization,
including both recent (Senjo et al. 1999; Liston et al. 2007; Wachowiak and
Prus-Glowacki 2008) and ancient (Wang and Szmidt 1994) events.
Reasons for the early popularity of chloroplast DNA include its haploid
nature, high copy number per cell, stable structure, rarity of recombination,
The Conifers (Pinophyta) 25
cpDNA regions (3,318 bp) (Willyard et al. 2007). In a more taxon-rich sample
across four loci, Syring et al. (2005) found divergence rates to be intermediate
to the nuclear ribosomal internal transcribed spacers (nrITS) and cpDNA,
with exons diverging 2.1-times faster than cpDNA, and introns diverging
1.3-times faster than nrITS. Both studies clearly illustrate the variability in the
divergence rates among nDNA. Willyard et al. (2007) documented a ca. 3.3-
fold difference (0.063 to 0.205 substitutions/sites) in silent substitution rates,
while Syring et al. (2005) found an eight-fold difference between the slowest
and fastest evolving regions. For gaining insight into the overall phylogenetic
pattern in Pinus, the range in divergence amongst loci has proven beneficial
in reconstructing a combination of both deep and shallow nodes (Syring et al.
2005, 2007b). The patterns of among-locus substitution rate variation reported
in Pinus are typical of the broader Pinaceae (Wang et al. 2000; Gros-Louis et
al. 2005) as well as the Cupressaceae (Kusumi et al. 2002).
Secondly, low-copy nDNA loci chosen from separate genetic linkage
groups provide multiple independent markers for use in phylogenetics, in
contrast to cpDNA markers which derive from a single linkage group. One of
the benefits of this independence is that these loci can be used for resolving
conflict between cpDNA and nrITS hypotheses. For example, conflicting
topologies in Pinus using cpDNA (Wang et al. 1999; Gernandt et al. 2005)
and nrDNA (Liston et al. 2003) have been partially resolved using multiple
low-copy markers (Syring et al. 2005). Multiple independent markers have
also revealed the complex phylogenetic history of Pinus chiapensis (Martínez)
Andresen (Syring et al. 2007a).
Thirdley, the exponential increase of expressed sequence tags (ESTs)
in GenBank (http: //www.ncbi.nlm.nih.gov/dbEST/dbEST summary.html) is
beginning to provide a nearly limitless supply of low-copy nuclear loci
for use in conifer phylogenetics. Nearly one million conifer EST markers
were submitted to GenBank by the end of 2009. However, coverage across
the conifers remains uneven (Table 1-1). Pinaceae has ca. 850,000, with ca.
470,000 markers from Picea (Picea glauca (Moench) Voss—ca. 300,000) and
ca. 365,000 markers from Pinus (Pinus taeda L.—ca. 330,000). Cupressaceae
and Taxaceae are poorly represented, and there are no EST entries for
Araucariaceae, Podocarpaceae, or Sciadopityaceae.
et al. 2004; Syring et al. 2005). The haploid genome size (1C) of conifers
ranges from 5.8–36.0 pg, which is roughly an order of magnitude higher
than the size of most angiosperm genomes (Ohri and Khoshoo 1986; Davies
et al. 1997; Grotkopp et al. 2004; Leitch et al. 2005). Large genome size acts
as a hindrance to the development and application of low-copy nuclear
markers for phylogenetic applications, as multigene families (Kinlaw and
Neale 1997) and repetitive and retrotransposon DNA (Friesen et al. 2001)
are abundant. Despite these obstacles, genomics and comparative mapping
efforts (Brown et al. 2001; Devey et al. 2004; Krutovsky et al. 2004; Pelgas et
al. 2006) reveal the scope of conserved synteny (linear gene order) among
species, and aid in the detection of orthology. Comparative analysis of
expressed sequence tag (EST) markers (Kirst et al. 2003; Ujino-Ihara et
al. 2005) has revealed the conservation of genetic structure across highly
divergent members of the seed plants, thereby providing aid in the choice
of loci to address the level of interest.
Arguably the most daunting obstacle to working with nDNA in conifers
is the slow coalescence (time to monophyly) of allele lineages within
species, a property demonstrated to impact both repeated gene families
(e.g., nrDNA, Gernandt et al. 2001; Campbell et al. 2005) and low copy
loci (Bouillé and Bousquet 2005; Syring et al. 2007b). Bouillé and Bousquet
(2005) demonstrated a striking case of non-coalescence across three low-
copy nuclear genes in three species of Picea. Allelic coalescence among
randomly selected alleles from these spruce species was estimated at 10–18
million years, values that overlapped with estimated divergence times
(13–20 Mya) for the species studied. Explicit tests in Pinus subsects. Strobus
and Cembroides demonstrated that allele lineages in 11 of 28 species tested
failed to coalesce at one low-copy locus (Syring et al. 2007b). For these 11
species, coalescent expectations indicate that reciprocal monophyly will be
more likely than paraphyly in 1.71 to 24.0 million years, and that complete
genome-wide coalescence in these species may require up to 76.3 million
years (Rosenberg 2003; Syring et al. 2007b).
The timing of speciation events and the historic effective population
size are critical factors impacting the rate of allelic coalescence. Both
factors interplay in determining whether species are genetically unique,
and whether gene trees can accurately trace phylogenetic history. Because
other conifer species share many life history traits with Picea and Pinus
(e.g., highly outcrossing, long-lived trees with large effective population
sizes), we should expect to encounter similar phylogenetic difficulties
in related genera and families (Bouillé and Bousquet 2005; Syring et al.
2007b). Issues of non-coalescence highlight the importance of considering
the magnitude of intraspecific diversity within the overall pattern of
phylogenetic divergence (Fig. 1-2). While allelic non-coalescence may be
highly problematic in the reconstruction of resolved phylogenies, it should
The Conifers (Pinophyta) 29
Figure 1-2 Example of allelic nonmonophyly. One of two most parsimonious trees derived
from phylogenetic analysis of the cesA locus (see Syring et al. 2007b). Bootstrap values from
1,000 replicates are shown near nodes. Bold arrows indicate two cases where alleles have
failed to coalesce, one in Pinus strobus (S) and the other in P. monticola (M). Note that one of
the purposes of this particular study was to determine the sister species to P. chiapensis. If only
a single sample had been sequenced per species then the choice of the individual could have
dramatically affected the conclusions.
Acknowledgements
We thank Matt Parks and Rich Cronn for comments on the manuscript.
Figure 1-1 was designed by Jesús Romero. Parts of the introduction,
classification and phylogeny, and morphology sections were translated and
modified from a chapter in “El árbol de la vida: sistemática y evolución de
los seres vivos” coauthored by David Gernandt and Alejandra Vázquez
Lobo and edited by Pablo Vargas Gómez and Rafael Zardoya for Editorial
Reverté. This work was supported by National Science Foundation grants
ATOL-0629508 to Sarah Mathews and DEB-0317103.
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The Conifers (Pinophyta) 39
ABSTRACT
This chapter reviews the historical context, economic importance,
objectives and achievements to-date for many of the more important
conifers undergoing domestication through genetic improvement
programs around the world. These provide examples of the context
in which genomic technologies will have an impact in forestry. Unlike
many other crop plants and livestock animals, forest trees have only
been exposed to a few cycles of breeding and selection, and most
retain very large amounts of genetic variation in natural populations.
These factors present both opportunities and hurdles in the effective
application of genomic technologies to existing operational breeding
programs.
Keywords: operational tree breeding, plantation programs, breeding,
selection, genetic testing, genomic technologies, molecular markers,
quatitative trait loci, Pinus, Picea, Pseudotsuga, Larix, Cryptomeria,
Chamaecyparis, Cupressus, Thuja, Cunninghamia, Sequoia
2.1 Introduction
Although conifers are generally regarded as undomesticated trees, genetic
improvement through breeding, selection and testing has had a significant
impact on the productivity and quality of plantations established in a
wide variety of species worldwide. Many conifers have been the target
of tree improvement efforts over the last 50 years, and many of these are
now well into their second, third or even fourth cycle of breeding. In the
context of these well-established programs, emerging genomic tools offer
the greatest potential for immediate impact and deployment of benefits to
production forests.
The purpose of this chapter is to describe the context in which genomics
can have an impact on current breeding and reforestation of conifers.
Descriptions are given for each species or group of species covering historical
perspectives, economic importance, breeding objectives, and achievements
to-date. In addition, some brief notes are given on the application of
genomics technologies, particularly with respect to their current use, or
lack thereof, in breeding and selection. While a wide range of species and
programs are discussed (see Table 2-1), the list is not exhaustive, although
we have attempted to capture some of the most important.
2.2 Pines—Pinus L.
The genus Pinus is the largest genus in the family Pinaceae and is widely
distributed throughout the Northern Hemisphere, with as many as 100
recognized species (Richardson 1998). Many of these are of great economic
importance for wood production and are the targets of intensive tree
improvement programs, some of the more important of which are discussed
here, organized as regional groups.
Family/genus
Species/group Country programs discussed
Pines (Pinus)
Northeastern North American Canada, United States
pines (Pinus strobus, P. resinosa, P.
banksiana, and P. rigida)
Lodgepole Pine (Pinus contorta) Canada, Sweden
Western White Pine (Pinus Canada, Unites States
monticola)
Southern Pines (Pinus taeda, P. United States, Brazil, Argentina, China,
elliottii, P. palustris, and P. echinata) South Africa, Swaziland, Zimbabwe,
Australia
Maritime pine (Pinus pinaster) France
Scots pine (Pinus sylvestris) Sweden, Finland, Germany, France,
Lithuania, Latvia, Poland, Spain
Radiata Pine (Pinus radiata) New Zealand, Chile, Australia, Spain,
South Africa
Spruces (Picea)
Black spruce (Picea mariana) Canada, United States
White spruce (Picea glauca) Canada, United States
Red spruce (Picea rubens) Canada
Sitka spruce (Picea sitchensis) Canada, Great Britain
Norway spruce (Picea abies) Norway, Sweden, Finland, Germany
Other Pinaceae
Douglas-fir (Pseudotsuga menziesii) Canada, United States, Germany,
Belgium, Fance, Italy, Spain, United
Kingdom, New Zealand, Argentina,
Chile
Larches in Europe (Larix spp.) France
Cypresses (Cupressaceae)
Sugi (Cryptomeria japonica) Japan
Other Cupressaceae including China, Japan, Korea, New Zealand,
the whitecedars (Chamaecyparis Greece, Italy, France, Canada, United
lawsoniana, C. nootkatensis, C. obtusa, States, Columbia, Mexico, El Salvador,
C. pisifera), the cypresses (Cupressus Guatemala, Honduras, Kenya, Rwanda,
lusitanica, C. macrocarpa, Uganda, Tanzania, South Africa
C, sempervirens), the arborvitae
(Thuja plicata), Chinese fir
(Cunninghamia lanceolata), Coastal
redwood (Sequoia sempervirens)
They were especially suitable for ship masts. By the end of the 19th
century, their extensive resources had been decimated, especially in eastern
Canada (Daoust and Beaulieu 2004), where they were extensively used in
shipbuilding for the British navy. The introduction of an exotic pathogen,
the white pine blister rust (Cronartium ribicola J.C. Fisher) in the early 20th
century decimated remaining eastern white pine stands and caused major
losses to advance regeneration. As a result, there are today only scattered
Economic Importance, Breeding Objectives and Achivements 43
Jack pine breeders aim at developing, for each breeding, zone varieties
that are improved for height growth and volume, cold hardiness and
reduced branching. Jack pine is known to develop undesirable branch and
form characteristics, especially on poor sites and when stand density is
not sufficiently high. Variation in branch and form traits has been shown
to be partially under genetic control and this can be exploited to improve
the species. Jack pine is also sensitive to pests such as scleroderris canker
and western gall rust (Endocronartium harknessii [J.P. Moore] Y. Hirat.) in the
western part of its natural range. However, studies have reported resistance
to various pests in the species (Yeatman and Teich 1969). More recently,
tree breeders have also focussed on wood traits in order to maintain fiber
attributes that give a competitive advantage to the industry using jack
pine.
Seeds were also collected in more than 100 natural stands in Quebec
and others were obtained from collaborators of neighboring provinces
and states to set up genecological tests including over 550 open-pollinated
progenies from over 225 seed sources. Early growth and phenological trait
assessments made it possible to study the genetic structure and patterns
of genetic variation in these white pine populations (Beaulieu et al. 1996;
Li et al. 1997a). Breeding values were also estimated for height, 12 years
after plantation; 14% gain was expected through selection of the best 50
progenies (Daoust and Beaulieu 2004). A new breeding population was set
up, grafting three plus-trees from each of the 50 selected families. Several
series of new progeny tests were also established in the 1990s and 2000s
including half- and full-sib families collected into the breeding orchards.
Recent efforts have focussed on the development of interspecific hybrids
resistant to blister rust. The most promising material developed in Ontario
for this purpose by Carl Heimburger and Louis Zsuffa was grafted and put
in a breeding orchard in Quebec to facilitate controlled crosses. Since the
early 2000s, over 130 control crosses have been made to create new hybrids.
Seedlings were inoculated with blister rust, and after two inoculation
phases, some of the hybrids appeared promising (G Daoust pers. comm.
2009). Somatic embryogenesis techniques are being used to propagate
them (Klimaszewska et al. 2001). For the short-term, these hybrid somatic
seedlings are being deployed to clonal trials, further testing resistance to
blister rust.
There are also some seed orchard facilities established in the Atlantic
region in Canada, by JD Irving Ltd in New Brunswick, the Department
of Natural Resources in Nova Scotia, the Department of Environment,
Energy and Forestry in Prince Edward Island. These seed orchards are now
producing the genetically improved seed required for their reforestation
programs.
Research on the genetics of eastern white pine in the eastern United
States began in the early 1950s with interspecific hybridization experiments
and early tests for resistance to the white pine weevil and the white pine
blister rust (Kriebel 2004). From the 1950s to the 1980s, extensive cooperative
tree improvement activities took place with range-wide and regional
provenance trials providing information on geographical variation in
adaptability and growth (Wright 1970; Kriebel 1982). Progeny testing was
also carried out which allowed estimating inheritance of growth traits
and the potential for genetic gain (Adams and Joly 1977; Kriebel 1978,
1983). Some of the progeny tests have been converted into seed orchards.
Results of hybridization experiments demonstrated that the two most
promising hybrids exhibiting desirable fiber attributes were P. strobus x
Pinus wallichiana A.B. Jackson and P. strobus x Pinus monticola Douglas ex
D. Don and their reciprocal crosses (Wright 1959; Kriebel 1972). Efforts to
Economic Importance, Breeding Objectives and Achivements 47
develop weevil and blister rust resistance in this species have not yet been
successful but continue with application of genetic engineering technologies
(Kriebel 2004). Ongoing genomics research at the CFS should lead to better
understanding of the interaction between the host and disease, and to the
development of efficient tools to select eastern white pine tolerant to blister
in the future. Eastern white pine is also highly sensitive to sulfur dioxide
and ozone, and genetic variation in tolerance to these air pollutants has
been investigated (Karnosky and Houston 1979).
to the southern part of the distribution in the United States. One unique
biological characteristic of “latifolia” pine particularly is the serotinous
“closed” seed cone, which is thought to have evolved as a regeneration
strategy in response to fire. Lodgepole pine’s basic adaptive strategy can be
described as a genetic “specialist” (Rehfeldt 1988) as it typically has strong
genetic clines, although these clines vary greatly across its natural range. All
varieties exhibit special ecological distributions or “niches”, which is not
surprising considering the large range of lodgepole pine. Below, we focus
on the most common of the three subspecies, var. latifolia.
This early effort was abandoned, but in 1983 a program based on the USDA
western regional Phase II programs of inoculation was initiated. Open-
pollinated seedlots were screened from 300 widely distributed candidate
parent trees from the coast and 300 from the interior regions of British
Columbia (Hunt 2004).
All three of these western white pine programs were influenced by that
initiated by Bingham, but were regionally adjusted. For example, in British
Columbia, where the rust severity is generally less than in the United States,
canker-free parents with intact lower branches were selected as candidate
trees. In the Inland Empire, stand infections could average more than 150
cankers per tree so while most selected candidates had fewer than three
cankers, canker-free trees were so rare that disease-free status could not
used as a criterion (McDonald et al. 2004).
lying areas have a tendency to be wet during the year. Improved silviculture
practices, like bedding, might make P. taeda more attractive as a species on
these sites in the future. The forestry plantation programs in Uruguay are
relatively new. Currently, 144,500 ha of loblolly pine have been established
with the prospect of much greater expansion (J Posse pers. comm. 2008).
The southern pines have been grown in South Africa since the 1890s
with expansion into other countries in southern and eastern Africa since
the late 1920s (Poynton 1977). In general, loblolly pine has always been
a secondary species to P. patula in most highland areas of the region. Its
main disadvantages are its relatively poor stem form (Poynton 1977), its
propensity to produce reaction wood at the base in some environments
(van der Sijde et al. 1985), and its adaptability to a limited number of sites
in the southern African environment. Recently, however, it has gained more
attention as an alternate to P. patula on some sites because of its resistance to
Pitch canker (Fusarium circinatum) and as sawmillers learn how to identify
and handle the reaction-wood problem.
Slash pine remains the most widely planted of the southern pines in
southern Africa, especially in South Africa, Swaziland and Zimbabwe, on the
drier and/or colder sites, for both pulpwood and sawtimber (Bridgwater et
al. 1997). In South Africa and Swaziland, 192,000 and 10,000 ha of slash pine
and 27,500 and 2,000 ha of loblolly pine have been established, respectively
(DWAF 2006). Lesser amounts occur in southern and eastern Africa as far
north as the equator (Bridgwater et al. 1997; Poynton 1977).
Loblolly pine and slash pine were introduced into Queensland,
Australia in 1917 and 1925, respectively (Bridgwater et al. 1997). Loblolly
pine was eventually found to be poorly adapted to the region but slash pine
did well on the excessively wet sites. On sites that are better drained, the
P. elliottii x P. caribaea var. hondurensis hybrid and pure Pinus caribaea are
now preferred with 62,120 and 50,345 ha established, respectively (I Last
pers. comm. 2008). The seeds for the pure species and hybrids all come
from advanced generation breeding orchards.
the southern pines in Latin America or in southern and eastern Africa, but
insect attacks are becoming more problematic in Southeast Asia (H Wang
pers. comm. 2008).
The southern pines, and in particular loblolly pine, have long been the
subject of marker studies, particularly for wood quality traits and disease
resistance. Most recently, the breeding programs for loblolly and slash
pine have entered a collaboration with the Conifer Translational Genomics
Network (CTGN), in the hopes of making genomics-assisted selection an
operational reality (http://www.pinegenome.org/ctgn/). Led by UC Davis, with
additional funding from the USDA and the US Forest Service, the CTGN
will genotype up 7,500 trees and analyze genetic variation at about 7,000
loci previously identified as single nucleotide polymorphisms (SNPs).
Phenotypic information will be associated with SNP variation, focussing
on stem volume, fusiform rust resistance, wood quality, and stem form,
with the goal to develop selection tools.
250,000 ha, located along rivers and the Atlantic coast, to a cultivated area
of one million ha. Such a progression was the result of the determination
of the local land owners and public authorities to stabilize coastal dunes,
drain 700,000 ha of marshes, and plant a new forest. Once the forest was
settled, the challenges of nature and of a changing economic background
had to be addressed repeatedly (Riou-Nivert 2002). Between 1939 and 1950,
fire destroyed 400,000 ha. In the 1950s, the resin market collapsed, due to
international competition and the emergence of oil by-products. Production
objectives were reoriented towards timber, supported by the progress in
silviculture and the breeding of improved varieties, marketed in the early
1980s. During the winter of 1985, an intense cold wave in southwestern
France destroyed 30,000 ha of Maritime pine plantations from Spanish
and Portugese provenances, which had been established during the major
reforestation effort following the fires of the 1940s. The genetic origin of
seed source stands is now systematically verified by a terpene test, and
seed harvest from non-local stands is forbidden. A hurricane in December
1999 felled more than 100,000 ha in Aquitaine: 28 million cubic meters
were levelled (http://agreste.agriculture.gouv.fr/). Once more, the Maritime
pine forest resource had to be reconstituted. Reforested areas increased,
reaching 23,000 ha/year, while 100% of plantations have been established
with seedlings from second-generation seed orchards (GPMF 2002).
Atlantic coast of Spain and Portugal and around the Mediterranean basin
(Spain, southeastern France, Italy, Tunisia, Algeria, and Morocco). Cold
resistance was identified as an important issue, especially when lowest
night temperatures in Aquitaine can reach –10°C or –15°C every few winters
(-20°C in February 1985). The local provenances were thus chosen to build
up a breeding population, despite their form defects: trunk flexuosity and
poor branching. A total of 380 plus-trees were selected phenotypically on
the coastal sand dunes of Aquitaine, based on height and diameter, and
visual scoring of stem form. This first phenotypic selection proved to be
efficient for improving growth and stem straightness, as shown by a progeny
test comparing plus-trees progeny with their non-selected neighbor-tree
progenies on two locations after 10 years old (Danjon 1995). In addition,
genetic variation among provenances and performance of crosses between
provenances were explored (Harfouche and Kremer 2000; Harfouche et al.
2000). Among all tested combinations, Landes x Corsica families proved
to be the best material for growth and form in Aquitaine conditions. A few
hundred clones from the Corsica provenance were selected in provenance
trials located in Aquitaine, based on growth, stem straightness, branch
quality, pyralis resistance (Dioryctria sylvestrella), and cold resistance. The
objective of this second population is to produce improved Landes x Corsica
varieties for better stem straightness and branch quality.
traits, the results showed a clear decrease (30 to 40%) in genetic variation
from the Aquitaine natural resource to the selected plus-tree founder
breeding population, and stabilization of variance from founder breeding
population to the next-generation breeding population. The pattern was
different for stem straightness, and difficult to interpret due to different
measurement methods over time. It was concluded that the recurrent
selection strategy based on one main population could sustain several
generations of breeding and selection, considering the level of additive
coefficients of variation for the selected traits and a status number of the
breeding population (population effective size, Lindgren et al. 1996) close
to 100.
For the next generation of the breeding population, the focus is on a
reduction of population census size and better management of pedigrees, to
optimize selection efficiency while producing regularly renewed varieties
with increasing genetic gains. Eight unrelated sublines were assembled
within the breeding population based on pedigrees and breeding values,
allowing the deployment of unrelated selections to clonal seed orchards.
Status number is used as an indicator of genetic diversity. Double-pair
mating designs are used to produce material for progeny tests and the base
of the next generation, while polycross testing is performed for parental
ranking. Trials are replicated on several contrasting sites, usually with
single-tree plots and a large number of replications per site.
Including new selection criteria is also a focus: studies on pests and
diseases resistance (Jactel et al. 1996; Kleinhentz et al. 1998; Burban et al.
1999; Lung-Escarmant and Guyon 2004), wood quality (Pot et al. 2002;
Bouffier et al. 2008c, 2009), and drought tolerance (Dubos et al. 2003; Dubos
and Plomion 2003; Nguyen-Queyrens and Boucher-Lannat 2003; Eveno et
al. 2008) are ongoing. Some new criteria have already been included in the
selection process: rust resistance (Melampsora pinitorca) is tested on future
seed orchard parents through a cut-shoot assessment (Desprez-Loustau
1990), wood density is evaluated at the family level in progeny trials with
an IML-Resi tool (Bouffier et al. 2008a), and branch quality is scored visually
in progeny tests (GPMF 2002).
Breeding forest tree varieties in the context of changing climate is another
challenge. Models predicting the evolution of climate in southwestern
France during the next decades show an elevation of air temperature and
a seasonal shift in precipitation distribution from spring and summer to
winter, which will likely result in decreased forest productivity (Loustau
et al. 2005). Although these are hypotheses, interest to improve drought
resistance has been increasing. Current varieties of Maritime pine in
Aquitaine were selected, tested and used in one breeding zone. Selection
is aimed at producing multipurpose varieties adapted to the different soil
types of Aquitaine, including dry, semi-humid and humid podzol soils. In
62 Genetics, Genomics and Breeding of Conifers
the near future, seed orchards could be rogued to favor clones that are better
adapted to the drier sites as and when their progeny tests are assessed in a
changing climate. As for future varieties, different strategies are considered:
locating progeny trials in more southern and drier sites as an anticipation of
future climate, infusing new diversity into the breeding population either
by selecting better adapted trees in the local provenance, using the national
network for Maritime pine natural genetic resources conservation, either
by selecting adapted inter-provenance combinations (Landes x Portugal
and Landes x Morocco progenies are already being tested), as well as
introducing new selection criteria for drought resistance, e.g., water-use
efficiency (Brendel et al. 2002), resistance to cavitation (Lopez et al. 2005),
and molecular markers for these traits.
with marker-assisted selection for complex traits such as wood quality and
drought resistance.
with Russia, Finland, and Sweden comprising the largest areas for timber
production. In addition, Scots pine has also been widely planted for timber
production beyond its natural distribution in western Europe, Eurasia, and
North America, and to a small extent even in Mexico and New Zealand
(Boratyńsky 1991).
In Sweden, commercial Scots pine forests occupy 12 million ha of
productive forest land (ca 50%), with a total stocking of about 1,100 million
m3, an annual cut of 30 million m3, and annual planting of 120 million
seedlings (ca 32% of total seedling production). According to the Swedish
Forest Agency (http://www.svo.se), the value of forest product exports in 2007
totalled 127,000 million SEK (ca US$18 billion), or 11 % of total exports and
4 % of GNP. More than 100,000 people are employed in the forestry sector
(2.2 % of all workers). Based on its share of total harvest volume, Scots pine
contributes roughly 30–40% to these figures.
In Finland, there are 13.6 million ha of Scots pine dominated forest,
representing 65% of the forest area and 50% of the standing volume (FFRI
2008). About 55,000 ha annually are artificially regenerated with pine, where
direct seeding is used on more than half of the area (requiring 20-times as
much seed as planting).
According to Russian Federal Agency of Forestry (A. Fedorkov, pers.
comm.), Scots pine in Russia covers 117 million ha (42 million in Europe and
75 million in Asia). It is the second most dominant species with a standing
inventory of 15,000 million m3, or 20% of total standing volume.
In other European countries, areas dominated with Scots pine are
considerably smaller, but still contribute significantly to total production
and are considered economically important.
wood products, and both chemical and mechanical pulps. The solid-wood
products cover both light structural and appearance-grade lumber, and the
latter can be quite high-value. While at present its long-fiber kraft pulp rates
as a commodity, it now tends to be a by-product rather than a primary one.
The mechanical pulps are now valued for magazine papers.
In New Zealand, radiata pine has since around 1925 been the mainstay
for replacing dwindling supplies of native timber species that are not readily
domesticated. It has since become the basis of major export industries,
with a total annual roundwood harvest of nearly 20 million m3, making the
country the 12th largest producer of coniferous roundwood and the third
largest exporter of coniferous logs. The contribution of the forestry sector,
including derived industries, which is around 95% based on radiata pine,
is estimated at around 3.5% of the GDP (over US$2.5 billion), and 10% of
the country’s total export receipts, based on 7% of the land area (MAF
2007). This has been despite a depressed state of the sector, due to a strong
New Zealand dollar, an historical focus of the corporates on producing
commodity products and on log exports, and some correction of over-
harvesting, factors that obscure the species’ full contribution to wealth. In
addition to producing wood, P. radiata makes important contributions to
soil conservation and provision of shelter, the shelter plantings containing
an additional timber resource.
In Chile, radiata pine is also the mainstay of a large forestry sector,
albeit less pre-eminent than in New Zealand. Annual roundwood harvest
is some 25 million m3 (INFOR 2007), the country being the ninth largest
producer of coniferous roundwood. Contributions to GDP of primary
production from the species are estimated at around US$ 2.3 billion, ca 2%
GDP (G Ortiz pers. comm.). The species also greatly dominates forestry
exports (ca. US$3.9 billion) (loc cit). The contribution has been helped by
very active government encouragement to reduce the economy’s extreme
exposure to the world market for copper. Many of the plantings have also
rehabilitated severely degraded land.
In Australia, radiata pine is also less pre-eminent in the forestry sector
than in New Zealand, being 73% of total softwood plantation area (Wu et
al. 2007), and it is oriented very much towards local markets. The species
probably contributes around 11 million m3 to the annual roundwood
harvest. In the solid-wood area, conifers comprise around three-quarters
of the sawtimber production (ABARE 2007), and are widely favored for
light construction, for which locally grown radiata pine is generally well
suited.
Economic Importance, Breeding Objectives and Achivements 71
and have been the focus of reforestation and breeding efforts. A few have
also become important far outside their natural range.
crown form and branch size, while maintaining a broad genetic base for
adaptability and pest resistance, especially to the white pine weevil (Pissodes
strobi [Peck]) in western Canada. More recently, emphasis has been put on
wood physical properties.
the late 1950s by the Canadian Forest Service (Morgenstern et al. 1981).
Reforestation of red spruce in Quebec has not been extensive, due to low
productivity when planted on open sites and its susceptibility to winter
drying and frost damage (Morgenstern et al. 1981; Beaulieu et al. 1989b), so
advanced-generation breeding had not been continued. On the other hand,
New Brunswick responded to an increased interest in planting red spruce
by reviving its program in 2004. Second-generation red spruce clonal seed
orchards had been established in 1999, and by 2007 they occupied 3.6 ha
(Tosh and Fullarton 2009).
As red spruce is highly susceptible to winter dessication and given the
relative paucity of breeding resources for this species, red spruce breeders
in the future may resort to genomic approaches to identify adaptive
polymorphisms and select trees that are more tolerant to winter drying
and frost damage. The current development of gene catalogs and SNP
directories for white spruce and black spruce should help accelerate the
application of MAS in red spruce.
Outside its natural range, Sitka spruce has played an important role
in plantation forestry, particularly in northern Europe (Hermann 1987). In
Great Britain, Sitka spruce is the most widely planted conifer, accounting
for nearly 700,000 ha of forest or 30% of the total forest estate (Forestry
Commission 2003). The species is well suited to areas of high rainfall and
lower quality agricultural soils that predominate in the north and west
of Britain. It is planted from Cornwall in the southwest England (latitude
51o N), through Wales and northwestern England, across northeastern
England and southern Scotland and up into the Scottish Highlands (latitude
58°N).
Although the species was originally described by Archibald Menzies
in 1792, it was not introduced into Britain until 1831 by David Douglas. By
the time the British Forestry Commission (the State Forestry Service) was
formed in 1919, experience from sample trees planted in arboreta and on
large estates had established the species as fast-growing, hardy in exposed
conditions and capable of growing on site types which at the time were
mainly planted with Norway spruce (Picea abies [L.] Karst.). The superior
growth of Sitka ultimately led to an increase in its popularity through the
1930s and beyond as the forest estate expanded under the then-government
policy of afforestation.
general conclusion was that material from around the QCI (54º N) was most
suitable for the bulk of Britain although in the milder areas of southwest
England and Wales, Washington sources (48º N) or even Oregon material
(45º N) were well adapted.
Plus-tree selection in Britain commenced during the early 1960s (Fletcher
and Faulkner 1972) and progressed through into the early 1980s. Over
1,800 candidate trees of predominately QCI origin were selected. Progeny
tests were established with open-pollinated seeds, with each candidate
evaluated in replicated trials established on an average of three sites, and
compared against standard controls of unimproved QCI and Washington
origin (Lee 2001). The trials were measured regularly for height and later
stem diameter, stem straightness and wood density. The best 340 plus
tress were identified, based on a multi-trait index combining 15-year stem
diameter, straightness and wood density, and these used as first-generation
breeding parents. In the second generation, the program expects to stratify
the breeding population into six sublines of equal mean genetic value, and
to apply positive assortative mating within sublines (Lee 2001).
Improved planting stock has been available from the Sitka spruce
breeding program since the early 1990s. Improved stock can be derived
either from seedlings raised from seed collected in progeny-tested clonal
seed orchards, or as rooted cuttings derived from stock plants originating
from controlled pollinations. The controlled pollination of selected seed
parents uses a polymix of 20 or so unrelated pollens, again from selected
trees. Predictions of genetic gain have been impressive, up to around 20%
for both stem diameter and stem straightness with minimal loss in wood
density. More recently, these half-sibling family mixtures used in the
production of stock plants and ultimately rooted cuttings, have given way
to full-sibling families (Lee 2006). Sawmill studies involving trees from some
of the earlier half-sib progeny tests have suggested end-of-rotation gains
for volume of around 25% relative to unimproved QCI material (Lee and
Matthews 2004), and an increase of high-end value sawlogs of up to 130%
(Mochan et al. 2008). Improved material is in high-demand and this is now
entirely satisfied from home-produced improved sources.
Despite having contrasting breeding objectives, groups in Canada
and Britain are collaborating to develop geneomic resources and identify
markers associated with a range of economically important traits, including
disease and insect resistance and wood density. In particular, the British
effort has invested in MAS, with an initial objective to identify a suite of
DNA-based markers, which could be used in the laboratory as surrogates
for direct field selection. Three large clonal trials were planted in 2004 on
climatically contrasting sites across Britain. Each trial contains the same
material; 1,500 clones from each of three full-sib families, along with the
usual QCI control (used also in the Canadian program). It is hoped that the
Economic Importance, Breeding Objectives and Achivements 85
orchard, the orchard thinned or a new orchard established with the best
progeny-tested parents.
In other countries, breeding programs were based on materials
selected from populations with high adaptive potential exhibited in
comparative provenance trials. The best individuals from families of the
best provenances were selected to produce seeds in orchards, or to create
a breeding population through controlled crosses. Some of these programs
also targeted mass production of rooted cuttings of tested clones (Birot 1982;
van de Sype and Roman-Amat 1989; Kleinschmit 1993). Of major concern
in the breeding strategies have been the breeding objectives; the sizes of
breeding and production populations required to maintain genetic diversity;
test design and efficiency; and identification of suitable regions where the
orchard seed should be recommended for use.
The principal breeding objectives in most programs are to improve
the value of production in future spruce stands and to mitigate risk under
variable environmental conditions. The selection criteria needed to achieve
these goals will vary among different breeding populations, based on the
varying regional conditions. Under the severe conditions in the northern
boreal forest, adaptation to the climatic conditions is crucial. Frost hardiness
in artificial freezing tests, the timing of flushing in spring and survival,
vitality and lack of injuries in field tests are therefore important target
traits. Spring frost events may also occur at more southern latitudes, and
selection for late bud flushing may also be important here. Selection for yield
is mostly based on height or diameter growth. Some programs aim to keep
stem and wood quality at the present level, while others also want to select
for improvement of quality traits. Another important target for breeding
has been resistance to root rot (Heterobasidion annosum), but research efforts
have not yet succeeded in developing reliable techniques for selection
of resistant materials. In the last decade, adaptation to changing climate
conditions has been an increasing concern. In Sweden, this objective has
been addressed by establishing a system of multiple breeding populations,
which are bred for adaptation to different combinations photoperiod and
temperature conditions, including combinations that lie outside of what is
normal under the present climatic conditions (Andersson 2002).
provenance collection, planted over more than 100 sites, has been the base
of a vast number of biosystematics studies and provided several European
institutes with genetic resources to start or diversify their breeding activity
(Kleinschmit and Bastien 1992).
In 1985, six European countries (Belgium, France, Germany, Italy,
Spain, and United Kingdom) agreed to collect a base population from a
broad genetic base of superior provenances in previous IUFRO tests to
provide accurate genetic parameter estimates for further breeding. This
base population, made of 1,000 open-pollinated progenies harvested at low
elevation from United States Pacific Northwest has been evaluated in field
tests, covering 270 ha in western Europe and straddling over 10 degrees of
latitude. Selection criteria were: adaptedness, expressed as survival, bud
flush and bud set (frost damage avoidance), stem quality, volume growth,
and wood quality (despite adverse genetic correlations with growth)
(Rozenberg et al. 2001).
In New Zealand, Douglas-fir improvement started with provenance
trials of large numbers of provenances in 1957 and 1959 from the United
States Pacific Northwest and northern California (Shelbourne et al. 2007).
Before the provenance trial results were known, a breeding population
was established based on plus-tree selections from 35–50 year-old stands,
probably from Washington provenances planted during the Depression in
Kaingaroa Forest in the Central North Island. Parents were selected and
grafted, and open-pollinated progeny tests established with little delay in
the early 1970s. However, early test results from the 1957 and 1959 tests at
age 13 years, showed superior growth of Californian and southern Oregon
provenances, causing the breeding program to stall for the following 14
years (Shelbourne et al. 2007).
In 1988, in the wake of high log prices, industry interest revived and
a new breeding program was started in New Zealand with 186 selections
(superline) composed largely of better coastal fogbelt provenances in the
1959 provenance trials and material of Fort Bragg origin. Plus-trees were
grafted in an archive and it was planned to progeny test these clones by
polycross and use pair crossing for forward selection. This strategy failed
to deliver sufficient seed or crosses, and has recently been revised to rely
on an open-pollinated testing strategy in the clonal archive for generation
turnover and breeding value estimation. It is intended that relatedness
among selections will be assessed by DNA pedigree analysis (Shelbourne
et al. 2007).
In Argentina, the growth potential of the land race is high. The principal
objectives of the breeding program initialized in 1998 by INTA Bariloche
were therefore: (1) to increase growth and improve form by selections
from the land race; (2) to supply improved seed from seed production
areas; (3) to broaden the genetic base from fast-growing Washington and
94 Genetics, Genomics and Breeding of Conifers
Oregon populations; (4) to assess genetic diversity of the land race; and (5)
to maintain adaptability
In Chile, the breeding program has objectives similar to those in
Argentina, nevertheless the propagation procedures are much more
developed; for example, rooting cutting propagation, management of donor
plants (hedges), and evaluation of flowering induction techniques.
this a number of selections are now being undertaken for wood stiffness,
diameter and stem straightness in both second-generation land-race stands
of Fort Bragg, Californian origin and in existing progeny trials (H Dungey
pers. comm.). Seedlots from the Fort Bragg land race have proven to be top
performers for volume growth in the 1959- and 1996-planted trials.
In South America, first-generation testing and breeding are underway
and early results have led to the establishment of seed orchards and seed
production areas in Argentina (Gallo et al. 2005).
Detailed marker association studies have been limited to cloned (rooted
cuttings) seedlings from a single full-sib family to identify QTLs for several
adaptive traits, such as spring bud flush and spring and fall frost hardiness
(Jermstad et al. 2001a, b). For spring flushing, there was congruence in QTL
presence and linkage group location from year-to-year, but not between
test sites, suggesting that different suites of genes are governing growth
initiation in different environments. Significant QTLs were also found for
spring and fall cold-hardiness, but their locations revealed that different
genes are responsible for the two cold-hardiness traits. In a follow-up
study, significant QTL x treatment interactions have been detected in the
same genetic background (Jermstad et al. 2003), indicating that QTLs as
tools for selection is still in a developmental state in Douglas-fir. In a more
recent study, again in the same family, Wheeler et al. (2005) showed that,
with a larger sample size, several QTLs for adaptive traits can be classified
as candidate genes.
As breeding zones are not (yet clearly) defined at national levels for
larch, breeding is usually for whole countries, except in the native range
and some areas unsuited for the species. Stable varieties across these large
areas are required and genotype-environment interaction is used as a
selection criterion.
For European larch, short-term, low-input breeding is used with the
final aim to release first-generation seed orchard varieties. For hybrid larch
as well, with very few exceptions, the strategy is generally restricted to first-
generation hybrids, identifying outstanding varieties combining favorable
parental traits. The French program for hybrid larch is an exception, where
for the last 20 years breeding has been strongly linked to research on sources
and prediction of interspecific heterosis, as well as conditions required to
benefit from F2-hybrids.
While abundant flowering and seed crops of European larch are
irregular, production of improved varieties from seed orchards works
rather well, especially on continental sites. In contrast, production of
first-generation hybrids, either by sexual or asexual means, has remained
problematic, which seriously impedes rapid expansion of hybrid larch
plantations. Over the last two decades, research work has focussed on the
improvement of propagation systems.
seed lots is highly variable from orchard to orchard and from year to year
(< 20% to > 60%) as first revealed by isozyme markers or more recently by
cytoplasmic DNA markers (Acheré et al. 2004). Poor climatic conditions
during pollination (frost damage, snow, alternating warm and cold days,
etc.) and differences in parental phenology are the main causes of these
failures.
select varieties with lower pollen production, and the other is to select
for low-allergenic pollen. The two major allergy proteins in the pollen of
sugi have been documented; Cry j 1 and Cry j 2. Efficiency of CO2 fixation
is a further breeding objective to address issues associated with global
warming.
2.5.2.3.2 Europe
Italian cypress is the Cupressaceae species with the most intensive tree
improvement program in Europe. It has both cultural and economic
importance. Since about 1927, the fungal pathogen Seiridium cardinale,
indigenous to California, has spread rapidly throughout the global range of
106 Genetics, Genomics and Breeding of Conifers
cypress and related species, including Europe, Asia, Africa, and Australasia,
causing widespread and increasing mortality. The main impact of Seiridium
fungi is death from stem canker, also termed cypress blight. Breeding
programs have thus targeted selection and breeding for disease resistance,
using clonal and sexual recombination to increase gains. To date, research
programs in Greece, Italy, and France have conducted the largest body of
work in breeding and improvement for canker resistance (Santini et al.
1997; Papageorgiou et al. 2005; P Raddi pers. comm.).
2.5.2.3.5 Africa
Introduced Cupressaceae, particularly Mexican cypress and Monterey
cypress, have been grown in plantation forests of tropical and subtropical
countries for decades. Monterey cypress was a preferred species in Kenya
for its greater yields, but due to its susceptibility to Seiridium canker disease,
has widely been replaced by Mexican cypress (Roux et al. 2005). Stem
taper, stem form, wood grain angle, stem branches, and susceptibility to
key diseases were considered in the selection of trees for the Kenyan tree
improvement program. The introduced cypress aphid (Cinara cupressi),
has been spreading throughout the region since 1986 severely damaging
stands, and is now the subject of selection and breeding for resistance, along
with cypress canker (Ciesla 1991; Mugasha et al. 1997). Beginning in the
late 1960s under individual corporate and government programs, South
Africa has reaped considerable economic benefits from a comprehensive tree
improvement program, including Mexican cypress. The predominance of
the private sector in forest management and research in tree improvement,
however, has limited the availability of information on these programs
(Denison 2001).
2.5.2.4.2 Europe
In Greece, dozens of canker-resistant clones of Cupressus lusitanica are
available for deployment, but base levels of resistance in testing populations
remain below 5% (Santini et al. 1997).
2.5.2.4.5 Africa
South Africa, Kenya, Rwanda, Uganda, Tanzania, and other countries
have established a network of Mexican cypress plantations for wood
production from selected material that have been assessed for variability
in growth and yield parameters. Most are from open-pollinated selections
but seed production areas are now widely used to produce seed that can
be transferred across cooperating countries on suitable sites, based on
provenance studies. In Kenya, the Kenya Tree Seed Centre is the central
repository and distribution center for forest seed and clone banks, and
also manages a network of seed orchards. Since the early 1960s, plus-trees,
provenance, and progeny testing have resulted in advanced-generation
gains of approximately 30% for Mexican cypress (Bernard 2001) over
unimproved yields.
Economic Importance, Breeding Objectives and Achivements 109
2.5.2.4.6 Summary
The combination of substantial additive variation for economic traits,
ease of grafting and cloning, precocious reproduction, and wide range of
ecological adaptations make the Cupressaceae an ideal taxonomic group
that has demonstrated many successes through tree improvement. Gains
can be substantial when selecting for one or several traits, with limited or
no trade-offs between growth and disease or insect resistance. Although
the wood is generally soft, rapid fiber production supports a diverse
range of forest products. The horticultural sector has long sought value in
the Cupressaceae, and the emerging non-timber forest products sector is
increasing its utilization for distilled oils, phytochemicals, bark, chips, and
green foliage. To date, prospects for marker-aided selection are limited,
given the lack of correlations identified between traits of interest and
molecular markers or QTLs for this taxon; however, short generations and
high gains from breeding programs indicate that phenotypic selection for
quantitative traits, supported by genetic and biochemical data is a viable
system for efficient improvement.
Acknowledgments
The authors are greatful to their colleagues who shared information freely
and in particular to Heidi Dungey, Richard Sniezko, James Turner, and Don
Zobel for their contributions.
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1
BioSylve Forest Science NZ Limited, 45 Korokoro Road, Lower Hutt 5012, NEW ZEALAND;
e-mail: tim.mullin@biosylve.com
2
Skogforsk (Sävar), Box 3, S-918 21 Sävar, Sweden; e-mail: bengt.andersson@skogforsk.se
3
INRA-Centre de Recherche d’Orléans, 2163, Avenue de la Pomme de Pin, CS 400001 ARDON,
F-45075 Orléans Cedex 2, FRANCE;
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e-mail: jean-charles.bastien@orleans.inra.fr
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4
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e-mail: jeanbeau@nrcan-rncan.gc.ca
5
Scion (NZ Forest Research Institute Ltd.), Private Bag 3020, Rotorua 3010, New Zealand;
e-mail: rowland.burdon@scionresearch.com
6
North Carolina State University, Campus Box 8008, Raleigh, NC 27695-8008, USA;
e-mail: w_dvorak@ncsu.edu
7
British Columbia Forest Service, PO Box 9519 Stn Prov Govt, Victoria, B.C. V8W 9C2, Canada;
e-mail: john.king@gov.bc.ca
8
Forest Tree Breeding Centre, 3809-1 Ishi, Juo, Hitachi, Ibaraki 319-1301, Japan;
e-mail: kontei@affrc.go.jp
9
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Canada;
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d
e-mail: john.russell@gov.bc.ca
10
Forest Research, Northern Research Station, Roslin, Midlothian, EH25 9SY, Scotland; e-mail:
steve.lee@forestry.gsi.gov.uk
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e-mail: steve_mckeand@ncsu.edu
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e-mail: annie.raffin@pierroton.inra.fr
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Norwegian Forest and Landscape Institute, Høgskoleveien 8, 1432 Ås, Norway;
e-mail: tore.skroppa@skogoglandskap.no
14
British Columbia Ministry of Forests, PO Box 9519 Stn Prov Govt, Victoria, B.C. V8W 9C2,
Canada;
e
e-mail: michael.stoehr@gov.bc.ca
f
e-mail: alvin.yanchuk@gov.bc.ca
3
Cytogenetics
M. Nurul Islam-Faridi1,* and C. Dana Nelson2
ABSTRACT
Fluorescent in situ hybridization (FISH) has become the most important
tool in molecular cytogenetics for positioning and characterizing DNA
sequences on chromosomes. Although numerous genetic linkage and
quantitative trait loci maps have been reported in conifer species,
little progress has been made on developing standard karyotypes
capable of identifying individual chromosomes. Standard karyotypes
based on a reference set of cytological landmarks will greatly facilitate
the integration of genetic and physical maps and their comparisons
between species. Such information has the potential to significantly
boost our knowledge of genome evolution and to guide marker-based
tree breeding and species conservation. To date 25 species of Pinus (the
most widely studied genus in the conifers), four of Picea, six of Larix and
one each of Abies and Pseudotsuga have been karyotyped using FISH.
A total of 19 loci (10 major and 9 intermediate to minor) of 18S rDNA
have been reported in Pinus taeda, and this is the highest number of 18S
rDNA loci observed in any plant species. Different patterns of 5S and
18S rDNA sites have also been reported among the Pinaceae genera,
with Pinus showing the most variation.
Keywords: Pinales, Pinaceae, Pinus, Fluorescent in situ hybridization,
Ribosomal DNA, Plant telomere repeat, Karyotype
3.1 Introduction
The conifers (Order Pinales = Coniferales, Division Pinophyta) consist
of at least five families and approximately 600 species (Whetton 2005;
1
US Forest Service, Southern Research Station, Southern Institute of Forest Genetics, Forest Tree
Molecular Cytogenetics Laboratory, Texas A & M University, College Station, Texas 77843, USA;
e-mail: nfaridi@tamu.edu
2
US Forest Service, Southern Research Station, Southern Institute of Forest Genetics, Harrison
Experimental Forest, Saucier, Mississippi 39574, USA.
*Corresponding author
Cytogenetics 129
a) b)
Figure 3-1 Fluorescent in situ hybridization images of ribosomal DNAs (18S-28S rDNA, 5S
rDNA) and telomere (ATRS) probes on Pinus echinata somatic metaphase chromosomes: a)
superimposed images of DAPI, Cy3 (red signals, 18S and 5S rDNA sites) and FITC (green
signals, ATRS sites) filters; b) super imposed images of DAPI and FITC filters.
Color image of this figure appears in the color plate section at the end of the book.
Figure 3-2 Diagrammatic representation of 18S and 5S rDNA loci in different Pinaceae genera.
All 18S and 5S rDNA patterns reported in the less extensively studied genera (i.e., Picea, Abies,
Pseudotsuga and Larix) are present in the more extensively studied genus Pinus (subgenera,
Pinus and Strobus).
Color image of this figure appears in the color plate section at the end of the book.
had low-copy centromeric ATRS sites, except that one Ch4 homolog had no
ATRS site. The other six chromosomes had intermediate ATRS signals in their
centromeric regions suggesting intermediate copy numbers (Islam-Faridi
et al. 2007). When carefully characterized (i.e., positioned and quantified),
ATRS has been found to be an excellent cytomolecular marker. Furthermore,
when ATRS is used in combination with other markers including the rDNA
gene families and DNA specific fluorochrome banding (discussed below),
informative karyotypes can be developed where individual chromosomes
are differentiated from each other and subdivided for more detailed analysis
(Fig. 3-3, for details see Islam-Faridi et al. 2007, also see http://www.srs.fs.usda.
gov/sifg/sifg/pinustaeda.html). In addition it has been suggested that telomere-
like DNA sequences located at interstitial and centromeric regions are likely
results of chromosomal rearrangements due to inversions, chromosome
translocations and fusions (Meyne et al. 1990; Biessmann and Mason 1994;
Fuchs et al. 1995; Schubert et al. 1995). Understanding these ATRS patterns
among and within genera will provide further information about the
divergence of the Pinaceae as well as about the remarkable conservation of
their basic karyotypes.
Figure 3-3 Fluorescent in situ hybridization image of Pinus taeda somatic metaphase
chromosomes probed with 18S-28S rDNA (red signals) and Arabidopsis-type telomere repeat
sequence (green signals). Numbers from 1 to 12 enumerate homologous chromosome pairs.
The ideogram of Pinus taeda in the right hand side box is based on 108 readings of each
measurement (see Islam-Faridi et al. 2007 for details).
Color image of this figure appears in the color plate section at the end of the book.
Cytogenetics 135
Figure 3-4 An inverted image of DAPI stained Pinus taeda chromosomes showing proximal
(i.e., centromeric) (triangles) and interstitial (arrows) DAPI (AT-rich) bands.
136 Genetics, Genomics and Breeding of Conifers
3.4 Conclusions
Well developed karyotypes that uniquely distinguish each chromosome
and chromosome arm and their graphical representation as ideograms
are critical tools in moving towards cytomolecular mapping for genome-
based applications. We and others have noted frequent instances where
the ideograms within genera or even within species differ for no identified
or apparent reason (Hizume et al. 2002a; Liu et al. 2003; Islam-Faridi et al.
2007), except where the differences appear to be due to variable chromosome
preparations and under sampling in data collection. To overcome this
limitation, it will be important for the conifer and especially Pinaceae
community to develop standardized, reference karyotypes for each major
taxa. Within Pinus the appropriate level of taxonomic discrimination at present
would seem to be at the subsection level. Standardization should include high-
quality chromosome preparations with minimal chromosome distortion and
absence of cell wall debris along with multiple cells measured from multiple
genotypes per taxa. Reference markers should include the major and minor
18S and 5S rDNA sites, major and intermediate ATRS bands (both centromeric
and interstitial), and chromosome specific probes as they are identified. The
later class of markers is now more readily within grasp as large-scale genome
projects are progressing in Pinus and Picea including the development and
analysis of bacterial artificial chromosome (BAC) libraries.
FISH has revolutionized plant cytogenetics and continues to yield new
insights into the genomes of Pinaceae genera and species. Implementing
FISH at a large-enough scale to cover the many Pinaceae taxa and to be
confident of probe locations and intensities is still a challenge. Continued
improvements in chromosome preparation techniques will aid in
developing the large-scale capabilities needed to meet the challenge along
with continued enhancements in microscopy technology and computerized
digital analyses. Implementing standardization methods across laboratories
and developing a standardized set of reference markers will facilitate the
development of well-referenced ideograms and cytomolecular maps. These
maps will be invaluable for whole genome mapping and sequencing.
Recent advances in flow sorting (e.g., Li et al. 2004; Šafář et al. 2004) and
laser capture microdissection (e.g., Zhou and Hu 2007) offer great promise
in providing Pinaceae cytogeneticists new tools for isolating and analyzing
individual chromosomes, chromosome arms or pieces of arms.
Acknowledgement
We thank colleagues Kostya Krutovsky, Tom Byram and George Hodnett
for valuable comments and suggestions regarding an earlier draft of this
manuscript and Michael Robinson and Robert Eaton for developing the
animated version of the Pinus taeda karyotype.
138 Genetics, Genomics and Breeding of Conifers
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studies of ribosomal genes and heterochromatin reveal conserved genome organization
among 11 Quercus species. Theor Appl Genet 99: 969–977.
4
Neutral Patterns of Genetic
Variation and Applications to
Conservation in Conifer Species
Francesca Bagnoli,1,a Bruno Fady,2,c Silvia Fineschi,1,b
Sylvie Oddou-Muratorio,2,d Andrea Piotti,3 Federico Sebastiani4,e
and Giovanni G. Vendramin4,f,*
ABSTRACT
This chapter describes how neutral genetic markers can be used for
the study of population and conservation genetics, phylogeography
and gene flow in conifers. It includes a comprehensive review of the
studies performed in these research fields. The chapter starts with a
review of the different kinds of neutral genetic markers most frequently
used in conifers in the recent literature. In the second part, it describes
how variation is organized within and among natural populations at
the three conifer genomes (chloroplast, mitochondrial and nuclear). In
the third part, it highlights how stochastic processes have shaped this
organization focusing on two large areas of investigation in population
genetics: phylogeography and gene flow. Finally, it demonstrates
1
Plant Protection Institute, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy;
a
e-mail: bagnoli@ipp.cnr.it
b
e-mail: fineschi@ipp.cnr.it
2
INRA, UR629, Ecologie des Forêts Méditerranéennes, Domaine Saint Paul, Site Agroparc,
84914 Avignon, France;
c
e-mail: bruno.fady @ avignon.inra.fr
d
e-mail: sylvie.oddou@avignon.inra.fr
3
Department of Environmental Sciences, University of Parma, Viale Usberti 11/A, 43100 Parma,
Italy; e-mail: andrea.piotti@nemo.unipr.it
4
Plant Genetics Institute, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy;
e
e-mail: federico.sebastiani@unifi.it
f
e-mail: giovanni.vendramin@igv.cnr.it
*Corresponding author
142 Genetics, Genomics and Breeding of Conifers
that neutral genetic markers and the information they generate are
fundamental for the conservation and management of genetic resources.
This chapter is addressed to plant molecular geneticists as well as plant
breeders in the public and private sectors.
Keywords: neutral markers; diversity; differentiation; phylogeography;
gene flow; conservation; conifers.
4.1 Introduction
Conifer forests are important reservoirs of biological diversity (at gene,
individual, and community level) as a consequence of their complex
history and environmental variation at local and regional scales. Conifers
are keystone organisms in European ecosystems; they directly support rich
plant and animal communities that rely on them and mediate nutrient and
water ecological cycles. Impacts of global change on forests are expected
to be acute, resulting in notable changes in species range, ecosystem
functioning and interactions among species. Because they are sessile but
long-lived, trees will either disappear, have to disperse to other places via
their seeds and pollen or be able to adapt in situ over a reduced number of
generations. To adapt, trees will rely more on standing genetic variation
and recombination than on new mutations (Aitken et al. 2008).
The genomics revolution of the last 10 years has improved our
understanding of the genetic make-up of living organisms. Together with
the achievements represented by complete genome sequences for an
increasing number of species, high throughput and parallel approaches are
available for the analysis of transcripts, proteins, insertional and chemically
induced mutants. All this information facilitates the understanding of the
function of genes in terms of their relationship to the phenotype. Despite
its great relevance, such an understanding could be of little value to
population and conservation genetics if it only elucidates the relationship
between genetic variant and a mutant phenotype but fails to elucidate the
relationship between genetic variation in gene sequences and phenotypic
variation in traits. The relationships between complex trait variation and
molecular diversity of genes can be studied based on a genomic approach.
However, the identification of genes responsible for trait variation is still a
difficult task, especially in long lived organisms such as forest trees. Work
in model plant species has started to unveil an ever-increasing number of
genes involved in the determination of traits of adaptive significance such
as phenology and abiotic stress tolerance/resistance. These progresses will
hopefully allow ecological and conservation genetics to analyze directly
variation in genes involved in adaptive processes rather than in neutral
markers as was traditionally done in the past.
Neutral genetic Variation in Conifers 143
Type of Substitution DNA Fragment DNA Fragment DNA Fragment DNA Fragment DNA DNA sequence
polymorphism of electrically length length length length sequence changes due to
charged non polymorphism due polymorphism polymorphism polymorphism changes due substitutions
synonymous to substitutions, due to due to due to to single point of nucleotides,
amino acids indels, inversions substitutions, substitutions, modifications substitutions indels, etc.
of nucleotides indels, indels, of number of of nucleotides
Frequency of Low High High High Medium to high Very high Low to
sites in the depending on very high
genome the genome depending on
the genome
146
Allozymes RFLP AFLP PCR-RFLP SSR SNP Sequencing
Cost Low Medium to high Low Medium to high Medium to high High Medium to
high
Neutral genetic Variation in Conifers 147
markers for several purposes, such as for population genetic studies and
construction of ultra high-density genetic maps. In most organisms studied
to date, SNPs are more prevalent in the non-coding than in the coding
regions of the genome (Soleimani et al. 2003).
As both SSR and SNP markers often require extensive and costly
molecular procedures for their identification and characterization, the
more versatile AFLP (amplified fragment length polymorphism, Vos et al.
1995) markers, for which no knowledge of the genes are required prior to
their use as population genetic markers, are in some cases used instead in
ecological studies (genome scan approach, Alvarez et al. 2009).
The choice of the most appropriate markers for a given study depends
on its objectives (Table 4-2) as well as many other factors, among which
species’ life history traits and availability of information and genome
origin (cpDNA, mtDNA and nuclear DNA) are the most important ones.
Having the possibility to choose among three types of genomes, either
bi-parentally (nuclear genome) or uni-parentally (organelles) inherited, is
a key feature in plants, unavailable in other eukaryotes. In angiosperms,
both mitochondria and chloroplasts are maternally inherited, although
exceptions are also known (Petit and Vendramin 2007). In gymnosperms,
however, mitochondria are generally maternally inherited (therefore
dispersed through seed), whereas chloroplasts are paternally inherited
(therefore dispersed through pollen and then through seeds), although
exceptions are known: for example, both chloroplasts and mitochondria
are paternally inherited in Cupressus, Araucaria, Podocarpus, Taxodium and
Metasequoia (Whittle and Johnston 2002).
Hybridization ++ +++ ++ ++ ++ + ++ ++ ++
Phylogeography – – ++ – – + + – +++
the only real difference among plastid genomes is related to the repeated
sequences (IR), the plastid genomes are classified as: a) Group I genomes,
which lack the large (20–25 kb) inverted repeat that characterizes most land
plants (certain legumes and conifers, see the pioneering paper of Strauss et
al. (1988) on the chloroplast genome structure of two conifers, Pseudotsuga
menziesii and Pinus radiata); b) Group II genomes, which contain inverted
repeats (almost all plants); c) Group III sort of oddball genomes, which have
tandem repeats (Euglena, a photosynthetic protist).
In the 1980s completely sequenced chloroplast genomes became
available, thus originating the development of consensus (if not universal)
primers of interest for intraspecific studies. The first conifer chloroplast
genomes completely sequenced were that of P. thunbergii (Wakasugi et
al. 1994) (119,707 bp) and P. koraiensis (116,866 bp); both genomes are
significantly smaller than those of most angiosperms (Steane 2005). Recently,
the improvements in second generation sequencing, made it possible to
assess genetic diversity at the genome scale and to sequence at a fraction
of the time and cost of traditional approaches (Duran et al. 2009). In this
way, the chloroplast genome of one spruce species (P. stichensis) and seven
pine species (P. contorta, P. lambertiana, P. gerardiana, P. krempfii, P. longaeva,
P. monophylla, and P. nelsonii) were sequenced by Cronn et al. (2008). The
genome sizes were very similar to the known genome sizes for P. thunbergii
and P. koraiensis.
The availability of full chloroplast genome sequences allowed designing
primers in conserved (generally coding) regions separated by more variable
regions (Petit and Vendramin 2007). The region amplified are either large
and may be used in combination with restriction enzymes (usually 4-bp
cutters) (Demesure et al. 1995; Dumolin-Lapègue et al. 1997) as is often the
case in angiosperms, or are very small (< 200 bp) but contain potentially
variable mononucleotide single strand repeats (cpSSRs) (Vendramin et al.
1996; Weising and Gardner 1999) as is often the case in conifers.
The occurrence of mononucleotide repeats within the chloroplast
genome of seed plants, bryophytes and algae was firstly documented by
Powell et al. (1995). Furthermore, Powell et al. (1995) also demonstrated that
simple mononucleotide repeats in the chloroplast genome of conifers exhibit
length variation, and that polymorphism within these regions may be used
to study both intra- and interspecific variability. Conifer cpSSR markers have
a high degree of transferability between species and primers designed in one
species can often be used in closely related species (Vendramin et al. 1996).
The haploid state and uni-parental transmission gives chloroplast genes and
genomes an effective population size approximately one-half of a nuclear
locus. This has the effect of making chloroplast genes more responsive
than nuclear genes to stochastic processes like drift and founder events, a
property that has been exploited for testing hypotheses of seed (and less
150 Genetics, Genomics and Breeding of Conifers
Because of their life history traits, conifers are thus able to maintain
large effective population sizes. A decrease in within population genetic
diversity is thus the result of random loss of alleles or haplotypes when
populations contract, for example as a result of habitat fragmentation (Young
et al. 1996). Species with large distribution areas and low within population
genetic diversity at nuclear or paternally inherited genes are thus rare.
When they occur, they indicate a dramatic demographic bottleneck effect
in the more or less recent past, as shown for Pinus pinea. This widespread
typically Mediterranean thermophilous conifer (Fady et al. 2004) has almost
no diversity at all chloroplast (Vendramin et al. 2008) and nuclear (Fallour
et al. 1997) loci investigated, most likely as a result of a major contraction
of its distribution area during one of the Quaternary glacial periods.
Biogeographic scale current distribution of within-population
genetic diversity is a powerful tool for understanding species history. For
example, within population genetic diversity of conifers is higher in the
Mediterranean than in temperate regions (Fady 2005). The Mediterranean
basin was a refugial zone for temperate and Mediterranean-type organisms
during the glacial cycles of the Quaternary (Hewitt 2000). There, species
distribution areas, and thus their effective population sizes, were much
smaller than their current ones, especially those of temperate species which
refugia were close to the southernmost extant of the ice cap that covered
most of northern Europe. These low diversity refugia were the front runners
of recolonization of Europe when climate warmed during the Holocene
circa 10,000 years ago. Mediterranean-type species on the contrary, were
not as strongly affected by recolonization and were able to maintain higher
effective population sizes during the glacial phases of the Quaternary,
particularly in the eastern Mediterranean (Fady and Conord 2010).
Lower effective population size can also explain why rare congeners
of widespread species (Gitzendanner and Soltis 2000 and see the example
of Picea omorika presented above) and marginal/rear edge populations of
species (Eckert et al. 2008a) tend to have lower levels of genetic diversity.
In self-compatible species (such as conifers), a decrease in population size
is not necessarily associated with an increase in consanguineous mating
(Leimu et al. 2006). However, in predominantly mixed-mating conifers,
which are mostly outcrossed in large and dense populations, marginality
is correlated with an increase in selfed reproductive events (Restoux et
al. 2008). Ultimately, a reduction in within population genetic diversity
will have consequences on individual fitness and population persistence
(Hughes et al. 2008). In conifers, pinpointing regions where demographic
bottlenecks shaped the populations’ genetic diversity and where diversity
is higher than average remain key issues in conservation planning and
genetic resource sampling (Fady and Conord 2010).
154 Genetics, Genomics and Breeding of Conifers
156
Species Distribution Markers
with more than one branch), which generally display higher haplotype
frequencies. From a geographical point of view, old alleles are expected to
distribute broadly, because they have had a long time to disperse, whereas
haplotypes with only one connection (singletons) are likely to be connected
to haplotypes of the same population because they evolved recently and
had no time to disperse (Freeland 2005).
Once the genealogical relationships among haplotypes have been
established, the next step is to identify the historical and geographical
factors that influenced the current distribution of haplotypes. In recent years
a growing number of methods and specific software have been developed
for these purposes: the most commonly used are listed in Table 4-4.
The most popular method in phylogeographic studies is the nested
clade phylogeographic analysis (NCPA), also known as nested clade
analysis (NCA) (Templeton et al. 1995). NCPA is able to distinguish
between recurrent gene flow and a variety of historical processes, such as
fragmentation, long distance colonization and range expansion (Pleines et
al. 2009). The method uses statistical parsimony to construct a statistically
supportable haplotype network as the one outlined above. Then, it tests
for an association between geography and haplotype distribution, and
works through an inference key to identify the processes that could have
produced the association. The oldest and the newest haplotypes are
located at the center and at the periphery of the network, respectively. As
a result, the nested arrangements correspond to evolutionary time, with
higher nested levels corresponding to earlier coalescent events (Freeland
2005). The following step is to overlap the clades with geography and to
calculate two measures of distance: the mean distance of clade members
from the geographical center of the clade (Dc) and the mean distance of
nested clade members from the geographical center of the nested clade
(Dn). The existence or not of a non-random association between genetic
lineages and geographical locations is verified by permutation tests and
if the hypothesis of no association (null hypothesis) can be excluded, an
a posteriori inference key is used to determine the most likely alternative
scenarios to explain the patterns that have been observed (Templeton 2004).
Hence, specific hypothesis about the geographical distribution of lineages
based on both organellar and nuclear sequence data can be tested using the
NCPA method, although this analysis is limited by sampling size, because
the network may be inaccurate if too few individuals or populations are
considered (Freeland 2005). During the last 10 years, the complex NCPA
analysis has been implemented in computer programs (TCS, Clement et
al. 2000; GeoDis, Posada et al. 2000) and more recently several approaches
have been developed to automate the procedure (Zhang et al. 2006; Panchall
2007).
160
Table 4-4 Some of the software used in phylogeographic studies.
several studies have been carried out on conifer DNA variation using the
organelle and the nuclear genomes. The main objectives of phylogeographic
studies are the identification of glacial refugia and the characterization
of migration and colonization dynamics. During the last decade the
literature on phylogeographic studies of European and American conifers
has considerably increased, whereas little is known about the role played
by climatic oscillations on plant species in Asia, Africa and the Southern
Hemisphere (Pleines et al. 2009). As for the first pioneer works on genetic
variation of tree populations, Abies, Picea and some Pinus species have been
the first object of phylogeographic studies. Phylogeographic studies on
plants demonstrated that this analytical approach may be used to address
unresolved issues concerning genetic exchange and differentiation within
and among conifer species. Very likely, the identification of glacial refugia
and the description of post-glacial colonization dynamics represent the most
innovative contribution that phylogeography has given to population studies.
The first phylogeographic results published on European forest tree species
revealed the important role played by southern refugia (Iberian, Italian and
Balkan). Later, when cryptic and more northern refugial areas were also
described, the complex history of vegetation during the climatic oscillation of
the Quaternary age was more clearly described and the accepted hypothesis
that temperate areas were exclusively colonized from southern refugia was
somewhat modified (Provan and Bennet 2008).
central and northern Europe which was done from northern refugia as for
Picea abies (see above).
Swiss stone pine, P. cembra, is a European species considered to be a
glacial relict. It occurs in two disjunct regions: the continental parts of the
Alps (central Alps), which is considered to be its core natural range, and
the Carpathian Mountains, where isolated populations exist. Höhn et al.
(2009) have investigated the post glacial history of this pine species. The
populations of P. cembra within the two parts of the species’ range share
many cpDNA haplotypes, suggesting a common gene pool conserved from
a previously large, continuous distribution range.
The phylogeographic studies of several Eurasian larch species
(L. decidua, L. sibirica, L. gmelinii, L. olgensis, L. kaempferi and L. sukaczewii)
helped to identify different glacial refugia and illustrated the post glacial
migration routes of the species (Semerikov and Lascoux 2003; Araki et al.
2008).
Moroccan populations of Cedrus atlantica (Atlas cedar) from the Rif,
Middle Atlas, and High Atlas mountains, were analyzed by Terrab et al.
(2006) and Cheddadi et al. (2009) using cpDNA markers. The populations
are separated by valleys and confront considerable barriers to gene flow and
poor geographic structure was revealed among the analyzed populations
(Terrab et al. 2006). The analysis of Moroccan and Turkish populations
recognized the existence of two C. libani taxa, one in Lebanon and one in
Turkey; moreover, Turkish populations probably emerged from several
refugia (Fady et al. 2008).
A recent study based on nuclear DNA was performed on cypress,
Cupressus sempervirens, in its distribution range (Bagnoli et al. 2009). This
species is supposed to have originated in the eastern Mediterranean area
and experienced a strong human impact during the last thousands of
years; as a consequence, the present distribution of the species around the
Mediterranean appears to be broader than it was originally. The authors
emphasized a different history of cypress compared to the current one based
entirely on human introduction of cypress in Italy, suggesting that probably
a mosaic of recently introduced trees and remnants of ancient, depauperate
populations exist today in central Mediterranean cypress range. It is further
suggested that, as already demonstrated for cork oak (Magri et al. 2007),
the timescale for understanding tree population dynamics, usually starting
from the end of the last glaciation, has to be repositioned to more ancient
times.
The range-wide population structure and phylogeography of Juniperus
thurifera L. revealed that the Strait of Gibraltar represented an efficient barrier
against gene flow between the Moroccan and European populations for a
very long time, and consequently support that the Moroccan populations
Neutral genetic Variation in Conifers 165
Time Spatial Type of gene flow Estimate Sampling Sampling Typical Principle of Methodological Software
scale scale inferred provided design * effort number of inference reference
polymorphic method
markers
required
Neσ²e
Historical
among Total gene flow 30 individuals/ Low Minimum The traditional Hardy and Spagedi
pop. (seed + pollen) population 3 SSR or 50 auto-correlation Vekemans, (Hardy and
with biparentally AFLP approach: 1999; Rousset Vekemans 2002),
/paternally regression of 1997
inherited differentiation Genepop
markers. Seed among (Rousset, 2008),
flow estimate population
with maternally (Fst/1- GeneAlex
inherited markers Fst) againt (Peakall and
(logarithm of) Smouse 2006)
168
Time Spatial Type of gene flow Estimate Sampling Sampling Typical Principle of Methodological Software
scale scale inferred provided design * effort number of inference reference
within Seed and pollen High The seedlings Burczyk et al., Nm+
pop. flow gradient on potential neighborhood 2006; Oddou- (Chybicki
phenotypic fathers within model or Muratorio and & Burczyk
variables a given Spatially Klein, 2008 unpublished)
affecting neighborhood explicit mating
male fertility • + phenotypic model
• de/dobs traits of
potential
fathers (size,
flowering…)
• selection • + all potential
gradient on parents
phenotypic within a given
variables neighbourhood
affecting (Np)
female/male
fertility
class (< 50 m). Sp. has the desirable characteristic of being comparable
among species or sites, allowing quantitative comparison among studies.
Species Method Plot area/ distance to the #mothers #seeds Selfing Migration Average Nep Reference
important, since the impact of the general shape of the pollen dispersal
kernel (leptokurtic vs. platykurtic kernels) and of the specific shape of its
tail (fat-tailed vs. thin-tailed kernels) on major processes in population
biology has been highlighted by various theoretical and experimental
studies. For instance, the pollen dispersal kernel allows one to gauge the
risk of contamination of seed crops by other fields (Bateman, 1947), while
the seed dispersal kernel affects strongly both the rate of colonization and
the diversity in newly-founded populations (e.g., Le Corre et al. 1997; Clark
1998; Nathan and Muller-Landau 2000).
Neighborhood models such as those proposed by Adams and Birkes
(1991), Adams (1992), Burczyk et al. (2002) and Oddou-Muratorio et al.
(2005) can be used to jointly estimate the pollen dispersal kernel and the
heterogeneity in fecundity among phenotypically or environmentally
defined groups of males. A great advantage of neighborhood models is
that they can decompose the inter-individual variance in male reproductive
success into a spatial component due to the positions of father-trees
relatively to mother-trees and to the pollen dispersal kernel, and into an
inter-individual variance of male fecundity (determining the effective
male reproductive density). These approaches have been used in a few
angiosperms and conifer species to investigate the shape and the range of
the pollen dispersal kernel and the variance of male fecundity due to a few
covariates individually measured on the putative fathers (Burczyk et al.
1996, 2002, 2004; Burczyk and Prat 1997; Bacles et al. 2005; Oddou-Muratorio
et al. 2005). For instance, in knobcone pine (Pinus atteanuata), Burczyk et
al. (1996) showed that distance and direction of individuals males from
mother trees and the size of males (tree height) played significant roles in
determining outcross mating patterns within a neighborhood.
A major drawback of paternity-based approaches is that they rely on
an exhaustive sampling of the males found in the vicinity of the sampled
females, requiring substantial sampling efforts as pollen can come from
males that are far from the sampled site (Smouse and Sork 2004). An
alternative strategy is the TWOGENER analysis of Smouse et al. (2001),
based on the differentiation among the inferred pollen pools of a sample
of females, spread across the landscape, and encapsulated in a synthetic
parameter Φft, that is analogous to FST, but which relates only to a single bout
of pollination. The virtue of this method is that, unlike paternity analysis,
it does not require exhaustive sampling of the adults of the population.
The global estimate of Φft, computed from the entire collection of sampled
mothers, is easily translated into an estimate of the mean pollination distance
and the effective number of pollinators (Smouse et al. 2001). As an extension
of TWOGENER, we can use the computation of pairwise Φft between the
pollen pools sampled by all pairs of sampled females to estimate multiple
parameters jointly, among them the adult density and the average distance
178 Genetics, Genomics and Breeding of Conifers
success was skewed, with six local adults that generated almost two thirds
(62.4%) of juveniles with local parents. They concluded that, although a few
local adults play an important role in the colonization process at treeline,
large levels of gene flow from outside were maintained, suggesting that the
potential advantages of local adults (such as local adaptation, proximity
to the colonization area, phenological synchrony) did not prevent a large
gamete immigration in such a harsh environment.
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5
Genetic Mapping in Conifers
Kermit Ritland,1,* Konstantin V. Krutovsky,2 Yoshihiko Tsumura,3
Betty Pelgas,4,a Nathalie Isabel4,b and Jean Bousquet5
ABSTRACT
This chapter summarizes the history and current status of genetic
mapping in conifers. We review the development of molecular markers,
methods to construct genetic maps, and the resulting conifer genetic
maps. Genetic maps are subdivided into (1) linkage maps of genetic
markers, (2) quantitative trait loci (QTL) maps, and (3) comparative
maps. Comparative maps involve alignment of marker genes and even
QTLs between species. Physical mapping is also briefly discussed.
Emphasis is placed up problems and approaches unique to conifers,
and the involvement of new genomics technologies.
Keywords: genetic markers, genetic mapping, quantitative trait loci
mapping, comparative mapping
5.1 Introduction
Genetic mapping is the ordering of specific genes or DNA fragments
(genetic markers) along a chromosome, based up observed frequencies of
recombination in pedigrees. It provides the approximate locations of these
1
Department of Forest Sciences, University of British Columbia, Vancouver, British Columbia
V6T 1Z4, Canada; e-mail: kermit.ritland@ubc.ca
2
Department of Ecosystem Science and Management, Texas A&M University, College Station,
Texas 77843-2138, USA; e-mail: k-krutovsky@tamu.edu
3
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan;
e-mail: ytsumu@ffpri.affrc.go.jp
4
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du
P.E.P.S., P.O. Box 10380, Stn Sainte-Foy, Québec, Québec G1V 4C7, Canada;
a
e-mail: betty.pelgas@RNCan-NRCan.gc.ca
b
e-mail: nisabel@cfl.forestry.ca
5
Canada Research Chair in Forest and Environmental Genomics, Centre d’étude de la forêt,
Université Laval, Québec, Québec G1V 0A6, Canada; e-mail: jean.bousquet@sbf.ulaval.ca
*Corresponding author
Genetic Mapping in Conifers 197
entities, which can serve as DNA “landmarks” for further studies (Ott
1999). Physical mapping, in contrast, uses various molecular techniques to
reassemble the actual DNA into contiguous stretches, such that numbers
of bases separating genes are approximately known, as in for example
the chloroplast genome (Tsumura et al. 1993) and the nuclear genome
(Amarasinghe and Carlson 1998). Quantitative trait loci (QTL) mapping
places the locations of putative genes underlying a quantitative trait
onto a genetic map (Lander and Botstein 1989). Conifers have enormous
genomes, on the order of tens of billions of nucleotides (Murray 1998). This
prohibits physical mapping, and suggests that marker/QTL mapping may
continue to dominate conifer genetics research (Neale et al. 1994; White
et al. 2007). In addition, the conserved nature of conifer evolution places
greater importance on comparing genetic and QTL maps (comparative
mapping) in conifers (Krutovsky et al. 2004) and transferring information
among these species.
Conifers provide unique opportunities but also problems for genetic
mapping. Most notably, the gametophyte allows direct observation of the
haploid product of maternal meiosis (Cairney and Pullman 2007). Secondly,
conifers are outbred, and issues in data analysis arise from the fact that
parents and grandparents are heterozygous for markers and QTL, requiring
more complex approaches for data analysis (Liu 1998). Thirdly, the large
genome size of conifers, a consequence of repeated DNA elements (Morse
et al. 2009), make protocols for marker screening more complex, and the
development of markers more difficult compared to most angiosperms
(Kinlaw and Neale 1997). Finally, the enormous evolutionary distance
between conifers and angiosperms, separated by 300 hundred million years
of evolution (Savard et al. 1994), makes gene identification and annotation in
conifers very difficult (Kirst et al. 2003; Ralph et al. 2008). Here, we review
the current state of conifer genome mapping, with reference to current
advances in genomics studies of conifers.
about 10% of SNPs identified in loblolly pine (Pinus taeda L., subgenus Pinus,
section Pinus, subsection Australes) amplified in white spruce (D Neale et al.
unpubl. data); these are currently being utilized in the Treenomix II project
for map synteny comparisons.
as of September 2010, contained 629,815 ESTs for Pinus and 542,939 ESTs
for Picea. Within Pinus, the numbers of ESTs are (in parenthesis) are: P. taeda
(328,756), P. contorta (40,483), P. banksiana (36,379), P. pinaster (34,044), and P.
radiata (7,538). Within Picea, the numbers are P. glauca (313,110), P. sitchensis
(186,637), P. engelmannii x P. glauca (28,174) and P. abies (14,224). Smaller
EST collections exist for other conifers including the family Cupressacea
(cedars), which has 72,146 ESTs deposited, mainly for Crytomeria japonica.
For in silico SNP development, a large number of ESTs are required, unless
the deposited ESTs are used to design primers to amplify a small panel of
individuals to find SNPs. For pure in silico marker development, a given
gene must have at least four overlapping ESTs, in which case a SNP will
be detected if two of four nucleotide sites differ in base composition (this
mostly rules out sequencing error).
Species Marker type Markers Linkage Map length, Expected cM per Reference
groups cM coverage, % marker
Abies nordmanniana AFLP, RAPD 556 19 1977 80 NA Hudson (2005)
Cunninghamia lanceolata AFLP 101/94 11 2283/2566 NA 23/27 Tong and Shi (2004)
Cryptomeria japonica RFLP, RAPD, Isozymes 91 13 887 NA 10 Mukai et al. (1995)
RAPD 84/119 14/21 1112/1756 40/62 13/15 Kuramoto et al. (2000)
AFLP, CAPS 91/132 19/23 1266/1992 50/80 16/18 Nikaido et al. (2000)
CAPS, Isozymes, SNP, 438 11 1372 96 3 Tani et al. (2003)
RAPD, RFLP, SSR
Larix decidua AFLP, ISSR, RAPD 117 17 1152 80 14 Arcade et al. (2000)
L. kaempferi AFLP, ISSR, RAPD 125 21 1206 81 14 Arcade et al. (2000)
Picea abies RAPD 165 17 3584 NA 22 Binelli and Bucci (1994)
RAPD 82 13 1385 NA 24 Skov & Wellendorf (1998)
AFLP, SSR 413 22 2198 77 9 Paglia et al. (1998)
AFLP, ESTP, rDNA, SSR 755 12 2035 NA 3 Acheré et al. (2004)
208
Species Marker type Markers Linkage Map length, Expected cM per Reference
Figure 5-1, based upon a meta-analysis of Table 5-1, shows how the
types of markers used in genetic maps have changed in the past 20 years.
In general, the numbers of maps have declined in the past five years.
From this graph, it is evident that RAPD and ISSR markers predominated
during 1995–2005, but their use has declined, as they cannot be transferred
among pedigrees to build additional maps. AFLPs had a big impact during
2000–2005; again, as they are anonymous markers, but their transferability
is limited. Isozymes (the hobbit in the corner) and RFLPs have had a
constant impact, but their numbers are still limited. SSRs and SNP/ESTP/
STS markers are obviously the markers of choice for future mapping. They
have seen increasing useage. These are all sequence based markers that can
be transferred among pedigrees and species.
18
16
Number of maps containing marker
1990-1994
1995-1999
14
2000-2004
2005-2009
12
10
0
AFLP RAPD/ISSR Isozyme RFLP SSR SNP/ESTP/STS
Marker type
Marker type
Figure 5-1 Trends in conifer maps over the past 20 years. I. Usage of the various classes of
genetic markers for conifer genetic mapping.
Figure 5-2, also based upon Table 5-1, shows how the number of markers
used in conifer genetic mapping has increased in the past 20 years. Figure
5-2a shows that the number of markers has obviously increased as expected,
but the variance of the number of markers has also increased. This does not
include plans from spruce and pine genome projects to radically increase
marker number to 5,000 and above. But total map length has remained almost
constant (Fig. 5.2b) as markers separated by 30 cM or less (ca. 100 markers
total) are sufficient to cover genome length. High density marker maps will
be of main use for assembling contigs from genome sequencing projects.
Genetic Mapping in Conifers 211
1400
800
600
400
200
1992 1994 1996 1998 2000 2002 2004 2006 2008 2010
4000
(b)
3000
Total map length
2000
1000
1992 1994 1996 1998 2000 2002 2004 2006 2008 2010
Year
Year
Figure 5-2 Trends in conifer maps over the past 20 years. II. (a) Numbers of markers linked
to maps, (b) Total map length explained by markers.
EST sequence data. Marker data were obtained for an additional 50 new
EST-SSR loci that did not segregate in either mapping population but which
were polymorphic in population surveys. One hundred and ninety four
mapped loci were given a functional GO assignment; 242 mapped loci were
assigned to a NCBI UniGene cluster. Unigene and GO assignments, along
with linkage data, aided in identifying duplicated and paralogous marker
loci on the map. This species may serve as a reference map in comparative
mapping with other pines and even other members of the Pinaceae family
such as spruce and Douglas-fir.
5.4.1.2 Spruce
Linkage mapping in spruce (Picea spp.) has been directed toward three species
of major economic importance: Picea abies, a European species, and P. glauca
and P. mariana, both primarily North American species. The first saturated
composite map for white spruce was reported by Gosselin et al. (2002), who
used 165 RAPD, SCAR and ESTP markers to join maps from two individuals.
They noted that co-dominant markers were needed to join the maps. In
Norway spruce, Acheré et al. (2004) developed the second map, involving
755 markers. Interestingly, 150 of these markers were tested for their pattern
of population differentiation differing from neutral expectations, and nine of
these markers were found to be “outliers”, or genes that showed excessive
population divergence, compared to the majority of markers, suggesting they
were linked to QTLs for adaptation (Acheré et al. 2005).
More recently, the Arborea project in Canada has constructed several
linkage maps involving both individual and composite maps for white
spruce and black spruce. A map for the black spruce × red spruce species
complex was constructed (Pelgas et al. 2005), and for white spruce alone
(Pelgas et al. 2006). Most notably, Pavy et al. (2008) assembled a white
spruce linkage map with markers assayed via the Illumina GoldenGate
SNP genotyping platform. The resulting composite map had 821 loci
including 461 AFLPs, 12 SSRs, 31 ESTPs and 317 gene SNPs, and map
coverage was > 98%. This map also positioned genes with SNPs involved in
among-population differentiation of eastern white spruce; 50 outlier SNPs
were identified (Namroud et al. 2008); these genes are putatively involved
in adaptive differentiation. An expanded white spruce composite map
containing 836 gene loci has recently been published (Pelgas et al. 2011).
The most recent white spruce gene composite map emerging from
the Arborea project integrates two pedigrees of 500 progeny and has an
increased resolution of 0.9 cM with 2,255 positioned loci including 455
AFLPs, 12 SSRs and 1,788 gene SNPs. The map covers 2,065.4 cM over 12
Genetic Mapping in Conifers 213
LGs. The average gene density is 1.16 cM. The current published spruce map
has 826 genes; the largest number of mapped genes in a conifer species.
5.4.1.3 Douglas-fir
In Pseudotsuga menziesii, the most recent marker development has focused
on ESTP and SNP markers (Krutovsky et al. 2004), which together with SSR
markers, have added to the RFLP and RAPD linkage maps (Jermstad et al.
1998). The most recently published genetic map of Douglas-fir consists of
376 markers, including 172 RFLP, 77 RAPD, 2 isozyme, 20 SSR, 4 sequence
tagged site (STS), and 101 expressed sequence tag (EST) markers (Krutovsky
et al. 2004). This map is organized into 22 LGs that have three or more
linked markers and spans 1,859 cM. Several hundred SNP markers were
developed recently (Eckert et al. 2009), and their mapping is under way.
When enough markers are mapped, the number of LGs should coalesce
into 13, corresponding to the 13 chromosome pairs in Douglas-fir. It would
be valuable to map additional ESTP, EST-SSR and SNP markers to create a
high-density map that can be used for QTL, candidate gene and physical
mapping to facilitate eventual complete Douglas-fir genome sequencing.
5.4.1.4 Sugi
Sugi (Cryptomeria japonica) has been planted widely throughout Japan over
an area of 4.5 million ha, accounting for 44% of all the Japanese artificial
forest. A second generation linkage map for Sugi was constructed by
integrating linkage data from two unrelated third-generation pedigrees.
The progeny segregation data of the first pedigree, which involved a cross
between half-sibs, were derived from cleaved amplified polymorphic
sequences (CAPS), SSRs, RFLPs, and SNPs (Tsumura et al. 1997; Iwata et
al. 2001). The data of the second pedigree, which involved a self-pollinated
individual, were derived from CAPS, isozyme markers, morphological
traits, RAPDs, and RFLPs. The co-dominant DNA markers such as CAPS,
RFLP and SNP were developed from ESTs and cDNA clones from several
kinds of cDNA libraries (Ujino-Ihara et al. 2000; Ujino-Ihara et al. 2005).
More than 95 % of the markers were gene-based markers.
Using JoinMap, linkage analyses were done for the first pedigree
assuming cross-pollination, and for the second pedigree assuming selfing.
Four hundred and thirty eight markers were assigned to 11 large LGs
(corresponding to the 11 chromosomes of C. japonica), 1 small LG, and
1 non-integrated LG from the second pedigree; their total length was
214 Genetics, Genomics and Breeding of Conifers
1,372.2 cM (Tani et al. 2003). On average, the consensus map showed one
marker every 3.0 cM. PCR-based co-dominant DNA marker such as CAPS,
microsatellite and SNP were distributed over all LGs and represented about
a half of mapped loci.
karyotype was presented recently for loblolly pine based on FISH and using
18S–28S rDNA, 5S rDNA, and an Arabidopsis-type telomere repeat sequence,
A-type TRS signals (Islam-Faridi et al. 2007). Statistically, only seven of
the 12 loblolly pine chromosomes could be distinguished by their relative
lengths. However, the position and relative strength of the rDNA and
telomeric sites made it possible to uniquely identify all of the chromosomes,
providing a reference karyotype for use in comparative genome analyses.
A dichotomous key was developed to aid in the identification of loblolly pine
chromosomes and their comparison to chromosomes of other Pinus spp.
A cytomolecular map was developed using the interstitial 18S–28S rDNA
and A-type TRS signals. A total of 54 bins were assigned, ranging from
three to five bins per chromosome. This is the first report of a chromosome-
anchored physical map for a conifer that includes a dichotomous key for
accurate and consistent identification of the loblolly pine chromosomes.
Recently, bacterial artificial chromosome (BAC) hybridization has been
developed as an alternative to rDNA hybridization, which allows very specific
identification of chromosomes, and such methods would be fruitful to apply
to conifers, particularly the Pinaceae, as chromosomal morphology is hardly
distinguishable among the dozen or so chromosomes. This method has been
used in many plant species (Zhang et al. 2004) but not in conifer.
The normal activity of physical mapping is to construct a library of
inserts, then to construct “tiling paths” to obtain an ordered set of clonal
inserts that span the entire genome. For coverage of a conifer genome
(5–10×), about two million BAC clones are needed, too large for practical
work. Nevertheless, BACs are useful for conifers, and there are currently
BAC libraries available for white spruce and loblolly pine. The spruce
library is unarrayed and about 5× coverage, while the loblolly pine library
is arrayed and about 8× coverage (Liu et al. 2009). Currently, both random
BACs and targeted BACs (BAC identified as having a gene of interest) are
being sequenced from both libraries (J MacKay et al. unpubl. data; DG
Peterson et al. unpubl. data; K Ritland et al. unpubl. data).
Figure 5-3 Comparison of homologous linkage groups between white spruce (Picea glauca)
and black spruce (species complex Picea mariana × P. rubens).
Color image of this figure appears in the color plate section at the end of the book.
gene order between the two genomes, consistent with the hypothesis of
conservative chromosomal evolution among even distantly related species
in the Pinaceae family. This study established a working framework that
the Pinaceae can be viewed as a single genetic system.
Homology of Pinaceae LGs was more recently extended to three spruce
species (Pelgas et al. 2006). Between spruce and loblolly pine, 26 of 29 anchor
markers were in synteny, identifying 11 homologous LGs. In this study,
orthology of anchor gene markers was checked by extensive resequencing
of single haploid megagametophytes in the various species. For the three
exceptions to synteny, sequencing of megagametophytes indicated at least
two cases of paralogy, while the third case remained dubious, implicating a
conserved gene family. Between spruce and Douglas-fir, synteny could be
assessed with 20 anchor markers, of which just one proved to be paralogous
after megagametophyte resequencing. Of the remaining markers, three were
not in synteny, including two markers on LG13 of Douglas-fir, confirming
that the supernumerary chromosome of Douglas-fir is the result of fission
(Krutovsky et al. 2004; Pelgas et al. 2005). The remaining marker, in synteny
between spruce and lodgepole pine, was translocated to a different LG in
Genetic Mapping in Conifers 219
for the plant kingdom, and suggests that the pine family (Pinaceae) can
be viewed as one genetic system, allowing genomic information to be
readily transferred, in contrast to angiosperm species with even one-tenth
evolutionary separation.
However, on these maps, there are three instances of apparent segmental
inversions, two between Douglas-fir and pine, and one between pine and
spruce. A case where a pair of markers is reversed is likely due to mistaken
orthology. However, between Douglas-fir and pine, four markers are
involved with an apparent rearrangement (involving orthologous markers
3–6 in pine, which are linked to Douglas-fir). To have four, instead of two,
markers involved in an apparent inversion provide much stronger evidence
of true orthology. This suggests that the genetic system is less homologous
in Douglas-fir, as indeed its time since evolutionary divergence is greater
than between pine and spruce, and that there are limits to the transfer of
genomic information between conifer taxa.
given individual is usually heterozygous for at least one locus over a small
(10 cM) genome interval.
Following this original work, Knott et al. (1997) analyzed the same data for
evidence of multiple QTL in the same linkage interval, finding discordant
results with Groover et al. (1994). Kaya et al. (1999) used the pedigree of
Groover et al. (1994), termed “qtl”, as well as second pedigree, “base”, used
previously by Devey et al. (1994b). Thirteen height and eight diameter QTLs
were detected, suggesting control by few genes of large effect. However,
a given QTL was rarely expressed in multiple years or multiple genetic
backgrounds.
A series of works then ensued with the “qtl” pedigree. Sewell et al.
(2000) used the “qtl” pedigree to infer physical traits of wood: wood specific
gravity (wsg), volume percentage of latewood (vol%) and microfibril angle
(mfa), in both earlywood and latewood. Nine unique QTLs were detected
for wood specific gravity, five for volume percentage of latewood, and
five for microfibril angle (mfa). Most QTL for specific gravity were specific
to either earlywood or latewood, whereas each mfa QTL occurred in both
earlywood and latewood. Sewell et al. (2002) found eight unique chemical
wood property QTLs, with differences among populations for QTL. Brown
et al. (2003) stressed that verification of QTL is necessary, comparing inferred
QTL among populations and within populations for different years. They
found that QTL expressed within pedigrees were more stable than QTL
expressed among pedigrees.
An unusual approach to QTL mapping, which takes advantage of the
conifer megagametophyte, was undertaken by Gwaze et al. (2003). As
megagametophytes are haploid, QTL haplotypes can be traced from the
offspring back to individual founders in outbred pedigrees by combining
founder-origin probabilities with fully informative flanking markers. A
large QTL accounting for 11.3 % of the phenotypic variance in the growth
rate was detected in a loblolly pine pedigree; the QTL haplotype was traced
from offspring to its founder, GP3.
quantity of delta 3-carene was associated with RAPD markers near the
major QTL. This was the first study of co-localization of QTL.
Markussen et al. (2003) found 10 QTLs for height or diameter and
40 QTLs for seven wood parameters in P. pinaster. They found that two
SSR markers linked to QTL also were linked in a QTL mapped for P. taeda
(Devey et al, 1999); such markers could be used for comparative QTL
studies. Using a second P. pinaster three-generation pedigree, Brendel et
al. (2002) found four QTLs for δ13C (the first time found in a tree) and two
QTLs for ring width, but they did not co-locate with the δ13C QTL. On the
same pedigree, Pot et al. (2006) detected 54 QTLs. QTL for different traits
in the same map position also showed genetic correlations as estimated by
traditional quantitative genetic analyses. Chagné et al. (2003) compared
QTL maps of Maritime pine and loblolly pine, using 32 common mapped
ESTP markers. The positions of two QTLs controlling wood density and
cell wall components were conserved between the two species. This was
the first ever comparison of QTL maps between conifer species.
5.6.3.6 Douglas-fir
A series of studies used a three-generation pedigree to examine various
classes of traits for QTL in Douglas-fir (Pseudotsuga menziesii). Jermstad
et al. (2001a) genotyped 192 progeny for 74 evenly distributed RFLP
markers found by Jermstad et al. (1998). Thirty three QTL for timing of
spring bud flush were found, and measurements for each of 3 years and
2 test sites showed that several QTLs influence the timing of bud flush
over multiple years within sites but not between sites, indicating major
QTL of consistent effect within sites but interactions with environment
between sites. Using the same material, Jermstad et al. (2001b) found 11
and 15 QTLs affecting fall and spring cold-hardiness, respectively. Three
different shoot tissues phenotyped for spring hardiness showed similar
QTL, while different tissues phenotyped for fall hardiness showed little
QTL similarity, supporting previous reports that spring tissues are more
synchronized than fall tissues.
226 Genetics, Genomics and Breeding of Conifers
Jermstad et al. (2003) again used the same pedigree and markers, but
for additional individuals totaling 460, to investigate QTL interactions of
many of the above traits with photoperiod, moisture stress, winter chilling,
and spring temperature. In the first investigation of QTL interaction
with environment, they found two QTL-by-treatment interactions for
growth initiation traits, and several QTL-by-treatment interactions for
growth cessation traits. Finally, Wheeler et al. (2005) evaluated QTL for
cold-hardiness via artificial freezing and various cold injury assessment
methods in two pedigrees of size 170 and 383. Six QTL were found in the
first pedigree, eight in the second, of which four were shared between the
pedigrees; 17 of 29 putative cold-hardiness candidate genes identified from
ESTs were located within the QTL intervals, thus identifying them as high
priority for association studies. These works with Douglas-fir demonstrate
a unique opportunity of working with trees: long-lived species allow
“immortal” pedigrees that can be repeatedly phenotyped for different traits
after genotyping.
Finally, QTL analyses are normally conducted in single pedigrees. In
contrast, Ukrainetz et al. (2008b) examined eight full-sib families, each of
size 40 progeny, for wood-related QTLs, using the software “QTL Express”
(Seaton et al. 2002). They found that wood fiber and density traits both
showed the lowest number of QTLs (3) with relatively small effects; wood
chemistry traits showed more QTLs (7), while ring density traits large
numbers of QTLs (78) and interesting patterns of temporal variation. Growth
traits gave just five QTLs but of major effect. These wood quality traits
are the widest suite of traits yet examined for QTL analysis in a conifer.
Moreover, examination of multiple families for QTL gives a population
perspective of the true extent of QTL variation.
5.6.3.9 Sugi
Yoshimaru et al. (1998) mapped QTLs for growth, flowering and rooting
ability in Sugi (Crypomeria. japonica). Growth is one of the most important
traits for timber-producing woody species and also for carbon dioxide
fixation to mitigate global warming. QTLs for juvenile growth, including
height and diameter of basal area, were mapped. Flowering is essential for
reproduction, but is not necessary for timber production. If the expression
of flowering could be controlled, it would be useful not only for breeding
but also for forestry and the environment. QTLs for male and female flowers
have been mapped at two locations each, respectively. The rooting ability of
this species is very important for clonal forestry in the southwestern part of
Japan, especially in Kyushu Island. QTLs for rooting ability were found but
there were not highly significant in the family used in the study (Yoshimaru
et al. 1998). Wood quality QTLs, specifically modulus of elasticity (an
important indicator of wood strength), have also been mapped in Sugi
(Kuramoto et al. 2000).
228 Genetics, Genomics and Breeding of Conifers
5.7 Prospects
In a seminal review, Remington and Purugganan (2003) stated that future
research in plants should expand the number of traits that are intensively
studied and make greater use of QTL mapping in wild plant taxa, especially
those undergoing adaptive radiations, while continuing to draw on insights
from model plants. Conifers are inherently non-domesticated (e.g., wild
plant taxa) and the resources provided by breeding programs and genome
projects will provide rich resources for testing of candidate gene-trait
associations in wild populations, genetic mapping in hybrid zones, and
microarray analyses of gene expression.
In conifers, comparative analyses of genetic maps will continue to be a
fertile ground for future studies. In sunflower species, a comparative study
showed that in the face of extensive hybridization and gene flow, species
integrity is maintained (Strasburg et al. 2009). There are many examples
of hybrid zones in conifer species, such as the hybridization between
Englemann spruce and white spruce in British Columbia. There have been
no such studies in conifers that compare patterns of genetic divergence
and diversity along chromosomal segments, which can reveal divergent
selection for speciation. In conifers, few studies involving “genome scans”
have been done (but see Namroud et al. 2008).
Another approach possible for conifers is to use “hitchhiking mapping”
to identify regions of recent selective sweeps, due to adaptive divergence.
This method starts from a genome scan using a randomly spaced set of
molecular markers followed by a fine-scale analysis in the flanking regions
of the candidate regions under selection. In fish, the hitchhiking approach
identified a selective sweep around candidate locus Stn90 (Makinen et
al. 2008). Fine scale genome maps will help identify candidate loci for
adaptation in conifers, particularly those involved with strong ecological
Genetic Mapping in Conifers 229
gradients, such as that found Sitka spruce from coastal California to coastal
Alaska (see Mimura and Aitken 2007).
Yet another new avenue for using QTL maps is “genetical genomics”,
which combines genetic mapping with gene expression analysis. It uses
variation of gene expression induced by segregation within mapping
populations to infer interactions among expressed genes or metabolites.
Gene networks, and even directed gene networks, can be inferred by the
joint analysis of marker genotypes and gene expression and metabolite levels
(Rockman 2008). In the Treenomix II project, two genetical genomic studies
are nearing completion. These involve a 22K member cDNA microarray,
hundreds of assayed metabolites, and scores for weevil resistance in both
white spruce pedigree (I Porth et al. unpubl. data; R Dauwe et al. unpubl.
data) and a Sitka spruce pedigree (S Verne et al. unpubl. data).
Recently a number of “next-generation” sequencing technologies have
been invented, which can sequence fragments of DNA at astoundingly
higher rates compared to Sanger sequencing. These include the Illumina/
Solexa, ABI/SOLiD, 454/Roche, Pacific Biosciences/SMRT and Helicos
(Morozova and Marra 2008). To date, these technologies have been
applied mostly in non-marker contexts, such as whole-genome sequencing
(Bentley et al. 2008), targeted resequencing (Gnirke et al. 2009), discovery
of transcription factor binding sites, transcript and non-coding RNA
expression profiling, and other functional genomic studies (Eveland et al.
2008). These technologies should greatly facilitate genotyping of mapping
populations for mapping through direct and parallel sequencing of multiple
individuals.
Finally, and last but not least, for the past several years, there has been
an initiative to sequence a conifer genome, starting with the seminal paper
of Neale et al. (1994). There are several initiatives such as the Pine Genome
Initiative (http://pinegenomeinitiative.org/) and the International Conifer
Genome Initiative (http://www.pinegenome.org). It is not clear what strategy
is the best, and current initiatives are exploring alternatives. Fine scale
genetic mapping will clearly enable the assembly of contigs based upon
shotgun sequencing (for example, in the monkeyflower genome project,
John Willis pers. comm.). A current goal of the Arborea project is to map
10,000 genes in white spruce (J Bousquet pers. comm.). Other workers in the
USA, Canada and Spain have embarked upon exploratory BAC sequencing
and gene enrichment of the repetitive genome to discover the structure of
conifer genomes, using “gene space” explorations developed such as for
maize (Liu et al. 2007). These approaches will interface with genetic mapping
to help assemble the first conifer genome.
230 Genetics, Genomics and Breeding of Conifers
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6
Patterns of Nucleotide Diversity
and Association Mapping
González-Martínez S.C.,1,a,* ,† Dillon S.,2,† Garnier-Géré P.H.,3,†
Krutovsky K.V.,4,† Alía R.,1,b Burgarella C.,1,c Eckert A.J.,5 García-
Gil M.R.,6 Grivet D.,1,d Heuertz M.,1,7 Jaramillo-Correa J.P.,1,8
Lascoux M.,9 Neale D.B.,10,11 Savolainen O.,12 Tsumura Y.13 and
Vendramin G.G.14
ABSTRACT
6.1 Introduction
Conifers are long-lived, sessile organisms that occupy extensive landscapes.
They are important forest components in many areas of the world, and
members of the pine family are especially abundant in cool to cold-
temperate and mountainous areas of the Northern Hemisphere.
Conifer forests are the key in terrestrial ecosystems and a major source
of biodiversity. They are also economically important, as they provide a
full suite of ecosystem services and resources for human use. Conifers are
important as a source of timber and other wood products, and are also
widely planted as ornamental trees and shrubs. The most important source
of softwood timber in the world is trees in the Pinaceae (pine family),
which are widely used for building and boat construction. Several species
of conifers are tapped or cut and steam-distilled for stem resins, which are
used as commercial sources of turpentine, tar oils, rosin, and pitch. Many
species of conifers are grown as ornamentals and a wide variety of cultivated
shrub forms have been selected for garden use. Recently, bark and leaves
of several species of yews have become important as the source of taxol
and related alkaloids, which disrupt the process of cell division and are
used in cancer therapy.
It should be stressed that despite the ancient use of forests by humans,
there is still abundant genetic variation present in natural populations of
conifers. Based on molecular marker studies, conifers display higher genetic
variation than other plant species (Hamrick and Godt 1996; Nybom and
Bartish 2000). Recent studies based on DNA-sequence data for several loci
(see below) also showed a considerable amount of genetic variation still
present in conifers, even in intensively-managed commercial species.
Conifers, however, differ in their life history traits from most other
species where extensive candidate gene studies are available. Most
importantly, they are long-lived and in many cases have large effective
population sizes, with highly efficient pollen flow between populations
(see Savolainen and Pyhäjärvi 2007). Despite the lack of self-incompatibility
system, most species produce predominantly outcrossed seed, as most selfed
embryos are eliminated by a system of embryonic lethals (Koelewijn et al.
1999). Extensive pollen flow also homogenizes allelic frequencies, such that,
for example, Swedish and Chinese populations of Scots pine (Pinus sylvestris
L.) are only little differentiated at the isozyme level (Wang et al. 1991). In
strong contrast, many species show steep clinal variation in adaptive traits
Patterns of Nucleotide Diversity and Association Mapping 241
Species Loci All Coding Non-codingb Silentc Synonymous Non-synonymous bp per Reference
SNP
Cryptomeria 7 25 (0.4–52) 38 (2–81) 42 (0–86) 7 (0–20) 118 Kado et al. 2003d
japonica (sugi)
10 16 (2–55) 38 (0–100) 5 (0–25) 188 Kado et al. 2008
5 36 (10–76) 46 (17–95) 44 (10–85) 12 (0–35) 50 Fujimoto et al. 2008e
244
Species Loci All Coding Non-codingb Silentc Synonymous Non-synonymous bp per Reference
SNP
246
Species Loci All Coding Non-codingb Silentc Synonymous Non-synonymous bp per Reference
SNP
Figure 6-1 Nucleotide diversity estimates for all and silent (synonymous and non-coding)
sites, and number of base pairs per SNP for studies where 10 or more loci were studied. The
number of loci is in parentheses. Estimates for Pinus taeda and Pinus sylvestris were averaged
for two or more studies. See Table 6.1 for references.
Color image of this figure appears in the color plate section at the end of the book.
Patterns of Nucleotide Diversity and Association Mapping 249
unpubl. data). In addition, recent work has suggested that there is evidence
for continuously ongoing sweeps at some loci, which would have required
substantial LD. A thorough examination of the potential for background
selection and selective sweeps remains to be done in conifers.
Direct estimates of mutation rate at the nucleotide level in conifers
are not available yet. Moreover, indirect estimates inferred from observed
nucleotide differentiation between closely related pine species are often
based on poorly characterized divergence times and on the assumption
that silent sites are neutral. Paleobotanical data are also incomplete and
inconclusive, although recent work suggests large effective population
sizes of conifers during the Holocene or the Pleistocene (Birks and Willis
2008).
<0.01 <0.01
<0.001 <0.001
<0.0001 <0.0001
Figure 6-2 LD plots for dhn1 (left) and sod-chl (right) candidate genes for drought in loblolly
pine. A LD block is apparent in the lower right part of dhn1 while LD is distributed more
regularly in sod-chl.
Color image of this figure appears in the color plate section at the end of the book.
genome scale will be available when intergenic and repetitive regions are
sequenced in population samples. New initiatives on high-throughput
sequencing of bacterial artificial chromosome (BAC) libraries in pines are,
thus, very encouraging.
events (such as, for instance, bottlenecks and fast expansion) affect all or
most genes and have genome-wide effects.
Within-species methods include traditional Tajima’s D, Fu and Li’s D
and F, Fu’s Fs, and Fay and Wu H tests, which assess different properties
of the SFS, often by comparing different estimates of θ (Tajima 1989; Fu and
Li 1993; Fu 1997; Fay and Wu 2000; reviewed in Biswas and Akey 2006).
Among traditional methods that use data from different species are the
Hudson-Kreitman-Aguadé (HKA) and the McDonald Kreitman (MK) tests.
The HKA compares levels of nucleotide polymorphisms within species and
divergence between species across different loci, which should be positively
correlated under neutral expectations (Hudson et al. 1987), and allows
detecting loci with either increased or reduced levels of polymorphisms or
divergence compared to others. A recent version of the test uses a maximum
likelihood analysis of multilocus polymorphism and divergence (Wright and
Charlesworth 2004). The MK test also uses divergence and polymorphism
data. It compares the ratio of non-synonymous to synonymous mutations
for sites that are polymorphic within species, and for sites that are fixed
between species, the neutral prediction being that both ratios are the
same. New tests that explore the joint distribution of different statistics,
i.e., “compound” tests, have also been proposed (Zeng et al. 2007). The
underlying rationale is that the combined test would altogether be more
robust to demography because the distinct statistics differ in their sensitivity
to particular demographic assumptions.
Other types of neutrality tests using multiple species are based on Ka/Ks
ratios (non-synonymous/synonymous substitution rates ratios, as described
above). Those genes that are affected by functional constraints are expected
to have Ka/Ks ratios less than 1, unlike positive selection that is expected to
lead to Ka/Ks > 1, being the neutral expectation around 1. The basic test has
been extended to more complex methods accounting for variation in ratios
along lineages and across genomic regions (Bielawski et al. 2000; Yang and
Nielsen 2000). These methods are free of demographic assumptions and
thus more powerful for detecting longer-time scale selection events, but
they are a priori less powerful for detecting more recent selection events
and their diverse signals (Nielsen 2005).
Population genetic signatures of selection can also be detected among
populations as local adaptation can increase their degree of differentiation
(Charlesworth et al. 1997; Slatkin and Wiehe 1998). Building from the original
Lewontin and Krakauer (1973) test based on the genetic differentiation
variance (FST) among loci, various coalescent-based approaches have been
developed to detect loci showing outlier patterns (i.e., that deviate from
the simulated neutral distribution) for diversity, differentiation or other
summary statistics (e.g., Bowcock et al. 1991; Beaumont and Nichols 1996;
Schlötterer 2003; Beaumont and Balding 2004; Nielsen 2005; reviewed in
Patterns of Nucleotide Diversity and Association Mapping 255
2000). More recent methods, for instance those based on the joint distribution
of different test statistics (e.g., Zeng et al. 2007) or using Approximate
Bayesian Computation (ABC) for adjusting the null neutral model, have
been rarely applied in conifer species as yet. In Picea abies, however, a recent
study used the joint distribution of Tajima’s D and Fay and Wu’s H after
adjustment to demography through Approximate Bayesian Computation
demonstrated that the circadian clock gene PRR3 departs significantly
from neutrality (Källman 2009). A similar approach has also been used in
Douglas-fir by Eckert et al. (2009b).
Earlier studies were often limited in terms of sampling and unknown
demographic history. In contrast, recent studies are based on larger samples
and readily incorporated demography in test statistics and interpretation
of results. Specific models of demographic history suitable for neutrality
testing have been developed for a few conifers. For instance, Heuertz et al.
(2006) and Pyhäjärvi et al. (2007) showed that ancient bottlenecks followed
by expansion could explain the polymorphism patterns observed at 22 loci
in Picea abies and 16 loci in Pinus sylvestris, both cold-tolerant species, and
their deviations from the neutral model at equilibrium. Grivet et al. (2009)
found recent bottlenecks in a Mediterranean, drought-adapted conifer
(Pinus halepensis) using the same approach. Current work also suggests
that, in P. pinaster, we observe the effect of an ancient and possibly recurrent
bottleneck of medium severity, but not followed by expansion (Lepoittevin
2009). Tested against the best fitting demographical models, patterns of
polymorphism for some candidate genes have proved to be indeed caused
by natural selection. For example, a multilocus HKA test pointed to the
supposed action of selection on an abiotic stress-related dehydrin gene
in Scots pine (Wachowiak et al. 2009). The application of several different
neutrality tests, including those that incorporated explicit demographic
models, revealed a suite of six genes consistent with selective sweeps in a
large study of 121 cold-hardiness related candidate genes in coastal Douglas-
fir (Pseudotsuga menziesii var. menziesii) (Eckert et al. 2009b).
Ideally designed for genome-wide scans and detection of local selection,
FST-based outlier approaches have been applied to conifers either on a
limited number of candidate genes or on a wider (but still very limited)
scale with markers derived from expressed sequence tag (EST) databases
[cleaved amplified polymorphisms (CAPS) or SNPs]. Tsumura et al. (2007a)
identified four outliers out of 37 CAPS markers using the Beaumont and
Nichols (1996) approach in one cypress species (Chamaecyparis obtusa).
Using a similar approach, Krutovsky et al. (2009) found that among 25
allozyme loci in coastal Douglas-fir only the PGM-1 locus demonstrated
an unusually high level of differentiation as an apparent outlier. In Pinus
taeda, 7% of 55 SNPs showed a level of population differentiation that was
seven-fold the species average, possibly because of diversifying selection
Patterns of Nucleotide Diversity and Association Mapping 257
genomic regions are still largely unknown, and LD observed so far in the
gene space of conifers is very variable.
traits (Xiong et al. 1998; Abecasis et al. 2000a, b), and is therefore suitable
for many traits of interest in conifers. This test is also appropriate when
large numbers of small families are available (with one or more sibs),
but not always appropriate in trees due to their long generation times
and anonymity or unavailability of both parent genotypes in existing
populations.
the single SNP from α-tubulin associating with earlywood MFA accounted
for up to 4% of the phenotypic variance. These estimates are in line with
expectations, and implore the screening of large numbers of genes and SNPs
to dissect a significant proportion of the genetics underlying quantitative
traits. Still, these effects are larger than those reported in other organisms
such as humans (where the largest effects, with sample sizes of tens of
thousands, were less than 0.5%; Weedon et al. 2008). An obvious outcome
of association genetics in conifers, and other species, is the application of
multiple QTN for gene-assisted breeding (GAS). Not only are QTNs with
larger effects more easily detected in association tests, they will also be more
appealing to breeders wishing to improve a trait of interest as an equivalent
gain can be achieved using fewer loci. Furthermore, interactions among
QTNs from different genes might explain more variation and be useful in
a particular genetic background.
resources and developing new ones, as was recently done in maize (Myles et
al. 2009), will be instrumental to the success of association mapping efforts
in conifers. In particular, this information can be combined with information
arising from the candidate gene and surveys of nucleotide polymorphisms.
As we have shown above, even if numbers are still modest, both approaches
have yielded interesting genes. Co-locating those with QTLs should help
confirm their status. Similarly, when possible, expression studies and
transformation will bring additional information on those genes.
Third, conifer research is being conducted on many different species,
some of which are very closely related at the sequence level, and at the
genome structure level (in terms of map positions of homologous loci, e.g.,
Brown et al. 2001, Chapter 5, this book). It will be important to leverage this
in developing association tests and in using information from other species
on candidate genes and genome structure. Such studies will also provide
very interesting information on genome evolution in conifers.
Fourth, conifers are key organisms in terrestrial ecosystems and
the study of landscape patterns of ecologically important traits/genetic
variation is relevant for local adaptation of conifers in a changing world.
Comparative analysis in this framework is developing rapidly in conifers
and they are likely to become some of the best model systems available for
landscape genetics.
Finally, there are many lessons for conifer researchers in association
studies in humans and crops. Conifers and humans share some biological
features –both are random mating and long lived organisms, both have gone
through recent exponential growth– and a lot will be gained by carefully
following the successes and failures of human geneticists in their quest to
understand the genetic architecture of complex traits. At the same time, as
recent studies in maize illustrate beautifully (Buckler et al. 2009; McMullen
et al. 2009), plant biologists also benefit from more degrees of freedom in
this quest than human geneticists.
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1
Department of Forest Ecology and Genetics, Center of Forest Research, INIA, 28040 Madrid,
Spain;
a
e-mail: santiago@inia.es
b
e-mail: alia@inia.es
c
e-mail: concettaburgarella@hotmail.com
d
e-mail: dgrivet@inia.es
2
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia;
e-mail: shannon.dillon@csiro.au
3
INRA, UMR1202 Biodiversity Genes & Communities, 69 route d’Arcachon, 33612 Cestas
Cedex, France; e-mail: pauline@pierroton.inra.fr
4
Department of Ecosystem Science & Management, Texas A&M University, College Station
TX77843-2138, USA; e-mail: k-krutovsky@tamu.edu
5
Section of Evolution and Ecology and Center for Population Biology, University of California
at Davis, Davis, CA 95616, USA; e-mail: ajeckert@ucdavis.edu
6
Umeå Plant Science Center, Swedish University of Agricultural Science, SE 901 83 Umeå,
Sweden; e-mail: m.rosario.garcia@genfys.slu.se
7
Université Libre de Bruxelles, Faculté des Sciences, Behavioural and Evolutionary Ecology
cp160/12, av. F.D. Roosevelt 50, 1050 Brussels, Belgium; e-mail: mheuertz@ulb.ac.be
8
Department of Evolutionary Ecology, Ecology Institute, Universidad Nacional Autónoma de
México, Ciudad Universitaria, Tercer circuito Exterior, Apartado Postal 70-275, México, D.F.;
e-mail: jaramillo@miranda.ecologia.unam.mx
9
Program in Evolutionary Functional Genetics, Evolutionary Biology Centre, Uppsala
University, 75326 Uppsala, Sweden; e-mail: Martin.Lascoux@ebc.uu.se
10
Department of Plant Sciences, University of California at Davis, Davis, CA 95616, USA;
e-mail: dbneale@ucdavis.edu
11
Institute of Forest Genetics, Pacific Southwest Research Station, U.S. Department of
Agriculture Forest Service, Placerville, CA 95667, USA.
12
Department of Biology, University of Oulu, 90014 Oulu, Finland;
e-mail: outi.savolainen@oulu.fi
13
Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba,
Ibaraki 305-8687, Japan; e-mail: ytsumu@ffpri.affrc.go.jp
14
Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, Via Madonna del Piano 10,
50019 Sesto Fiorentino (Firenze), Italy; e-mail: giovanni.vendramin@igv.cnr.it
†
These four authors have contributed equally to this paper.
Authors in alphabetical order, except leading authors.
*Corresponding author
7
Integration of Molecular
Markers in Breeding
Rowland D. Burdon1,a and Phillip L. Wilcox 1,b,*
ABSTRACT
Applications of genetic markers in conifer breeding fall mostly in two
main areas: population management and selection.
Conifers pose problems, for selection in particular, on account of
their size and typical generation intervals, their typically outbreeding
behavior, the essentially wild state of their genetic systems, and their
huge genomes.
Applications for population management are various. For obtaining
background information, they include studying breeding systems, and
characterizing populations among and within taxa, doing the latter
wherever possible in conjunction with common-garden field trials.
More specifically, use of markers (1) can be used to inform structuring
of breeders’ metapopulations, in terms of sizes and subdivisions, and
(2) offers the prospect of retaining the benefits of full pedigree without
having to incur the costs and effort of exhaustive controlled crossing.
Full practical application, however, is being widely achieved in using
markers to verify genotype and parentage, thereby safeguarding the
capture of genetic gain.
Use of markers for selection in conifers is in principle attractive,
because of the time frames of conventional conifer breeding and the
problems of obtaining good phenotypic expression. Yet developing
marker-trait associations for marker-assisted selection (MAS) is greatly
hampered by the same features, plus extremely limited population-wide
linkage disequilibrium and an evident paucity of major genes. Scope
for selection using linkage between quantitative trait loci (QTL) and
1
Scion: New Zealand Forest Research Institute Ltd;
a
e-mail: rowland.burdon@scionresearch.com
b
e-mail: phillip.wilcox@scionresearch.com
*Corresponding author
Integration of Molecular Markers in Breeding 277
7.1 Introduction
The practical applications of molecular markers lie essentially in two broad
areas (cf Burdon and Wilcox 2007):
• Providing knowledge whereby the breeder can structure the
metapopulation as appropriately as possible. This structuring includes
choice and representation of base populations, the stratification of the
metapopulation and the appropriate sizes of the population strata, and
the crossing schemes.
• Providing knowledge to better target and accelerate and/or enhance
genetic gain. Applications involving faster and more efficient selection
are crucial here. However, the same knowledge can also be important
in eventually using genetic engineering to supplement the breeder’s
efforts in order to produce material for commercial deployment.
278 Genetics, Genomics and Breeding of Conifers
7.2.2 Ploidy
Almost all conifers that are appreciably domesticated are strictly diploid
(Williams 2009; Gernandt et al. 2011), with a haploid chromosome
complement of around 12. This feature facilitates genomic mapping and the
detection of quantitative trait loci (QTL) and quantitative trait nucleotides
(QTN), which will be considered later. Indeed, even minor departures from
perfect diploidy, such as trisomics, typically have catastrophic effects on field
fitness (Mergen 1963). The classic exception is the hexaploid coast redwood
(Sequoia sempervirens [D. Don] Endl.) which, while economically significant,
is not subject to large-scale domestication, let alone to intensive breeding.
Another, less-known exception is the tetraploid Fitzroya cupressoides (Molina)
I.M. Johnstone which, while it has also been commercially significant,
appears to have almost no prospects for significant domestication. The
Podocarpaceae show more variable chromosome numbers than other conifer
families (Gernandt et al. 2011), but they are essentially undomesticated.
280 Genetics, Genomics and Breeding of Conifers
data will remain very challenging. Along with no general LD, the genome
size creates a major obstacle to direct gene discovery, given the limitations
of foreseeable bioinformatics searches. Meanwhile, we will be forced to
leverage genomic information from angiosperm species, with heavy use
of candidate genes for gene discovery.
The very high proportion of non-coding DNA might be expected to favor
finding high-quality selectively-neutral markers. However, reservations
exist concerning neutrality or genetic stability of the markers. There are
sometimes problems with null alleles and hypermutability at some loci.
Moreover, additional problems may well arise with duplication of loci.
An important complication is that functional genetic diversity can
arise in various ways. Classically, the study of functional genetic diversity
has focused on coding-region polymorphisms, which involve amino-
acid substitutions in protein chains. The pathways of expression for
such polymorphisms can be followed by studying proteomics, involving
activities of either enzyme variants or the impacts of variants of “structural”
proteins. Yet it is becoming increasingly clear in biology that at least some
functional variation can be governed by polymorphisms outside the coding
regions (e.g., Morgante and Salamini 2003; Paran and Zamir 2003). These
can certainly involve regulatory sequences, but the effects of such variants
lack even the predictability of amino-acid substitutions. Moreover, study
of regulatory-sequence polymorphisms can be complicated by many such
sequences not even adjoining the coding regions, although the prevalence
of such sequences remains to be quantified. And there is the still-obscure
role of various RNA sequences that are transcribed from non-coding DNA
but somehow help orchestrate developmental processes. The nature and
importance in forest trees of functionally significant polymorphisms in
such sequences is still conjectural, but such a polymorphism evidently
governs a radical difference between the human and chimpanzee brains
(Pollard et al. 2006).
It is unknown whether the very high proportion of non-coding DNA,
which makes the conifer genomes so large, means a comparatively high
proportion of functional variation being governed by polymorphisms in
non-coding regions. This could have a significant impact on the exact nature
of selection applications.
to perform the same roles, showing close synteny; and the locations of
particular genes on specific chromosomes are very similar among species,
representing high levels of colinearity. Furthermore, there are evidently
widespread orthologies, extending to angiosperm species (Krutovsky
et al. 2006). Because of these features, obtaining genomic information
for individual conifer species should eventually allow a rapid spread of
genomic knowledge among other species.
additional cost and time delay, but also to build a repository of genotypic
information regarding breeding-population genotypes that could be used
for other purposes.
Marker System1
B. Applications (suitability2)
Characterizing breeding system •••• •• •• •••• ••• •• ••
Taxonomic studies ••• •• •• •••• ••• •• ••
Clonal/parental verification ••• •• ••••• ••••• •••• •• ••••
Pedigree reconstruction •• •• •• ••••• ••• •• •••
Selection • • • •• ••••• •••• •••
1
Acronyms: RFLP = restriction fragment length polymorphisms, AFLP = amplified fragment length polymorphisms, RAPD = random amplified
polymorphic DNA, SSR = simple sequence repeat (also referred to as ‘microsatellite’), SNP = single nucleotide polymorphism, Indels= sequence
insertion/deletion, DArT = diversity array.
2
Taking into account cost-efficiency of information, i.e., value in relation to cost of obtaining it.
N/A denotes not applicable.
Integration of Molecular Markers in Breeding 285
interest the breeder. Genetic markers are now a powerful adjunct, which
can shed much additional light on species hierarchies (Gernandt et al. 2005)
and population differences (Burdon and Wilcox 2007; Bagnoli et al. 2011;
González-Martínez et al. 2011 and references therein). Often involving
selectively neutral alleles, and purely cryptic variation, they need to be
used warily, in conjunction with other measures of variation. However, they
can be a powerful tool for inferring the evolutionary history of species and
populations thereof. Genetic divergence revealed by markers, for instance,
can give pointers to the prospects of achieving hybrid vigor, or heterosis, in
various interpopulation crosses; this application is called Diversity Index
Breeding (White et al. 2007). For this purpose, neutral markers may give an
early guide. Moreover, when such information is to be used, neutral-marker
data may be the only available marker information to go on.
In principle, much can be inferred from DNA makers concerning the
genetic history of populations and what DNA sequences have been subject
to selection (Gernandt et al. 2011 and references therein; González-Martínez
et al. 2011 and references therein). Aspects of the genetic history include the
timing and sizes of past population bottlenecks and population coalescences,
which tend to be manifested in patterns of linkage disequlibrium. Influences
of selection pressures will be manifested in various ways, such as the
ratios of synonymous to non-synonymous polymorphisms and differences
among chromosome regions in nature and levels of polymorphism. Such
information, however, typically allows only very tentative inferences on
population history, given the simultaneous uncertainties concerning the
rates and nature of mutations. All this allows the breeder to make only
slightly more educated guesses in various areas of population management.
Also, evidence of selection having operated on certain DNA sequences is
subject to the reservation that the selective forces operating in domesticated
populations may be quite different from those operating in the wild.
Furthermore, levels of conservation of DNA sequences, while often thought
to be a guide to significance of the sequences for evolutionary fitness, can
be unreliable in this respect (Monroe 2009).
compared with phenotypic data, are that they not reliant upon common-
garden studies, can provide data faster, and/or are applicable to natural
populations in situ. On the other hand markers can be expensive and time
consuming to develop—particularly SSR markers—or in some cases are
not completely informative due to dominance (Table 7-1).
Breeding systems are of interest for several reasons. First, knowledge of
a breeding system gives some indication of the potential and likely avenues
for genetic improvement. Where a species is fully self-fertile, as in Pinus
resinosa, within-population functional genetic diversity may be too small
to warrant selective breeding (Fowler and Lester 1970; Mullin et al. 2011),
although useful genetic variation may exist among populations. On the other
hand, in western red cedar (Thuja plicata D. Don), which is largely inbreeding,
there is appreciable tree-to-tree genetic variation (Russell et al. 2003) which,
along with other factors (addressed later), has allowed a breeding program
to proceed (Russell and Ferguson 2008; Mullin et al. 2011).
Second, knowledge of the breeding system is important for studying
heritability and testing of progenies. For instance, with a mixed mating
system, with significant but variable rates of selfing, open-pollinated
progenies can give seriously biased estimates of heritability (Borralho
1994) and parental breeding values. In these cases, effects of inbreeding
depression, which typically does occur but with variable severity, can
actually be much greater than the effects of inbreeding inflating coefficients
of relationship. Indeed, open-pollinated progeny performance can even
be poorly correlated in eucalypts with breeding-value estimates from
controlled outcrossing (Hodge et al. 1996). While such situations will almost
certainly be the exception in conifers, they cannot always be ruled out.
Third, care may have to be taken to minimize (e.g., by rigorous nursery
culling) the contribution of self- (or other inbred) seedlings to offspring
performance.
Fourth, strong outbreeding can lead to population-wide linkage
disequilibrium (LD) being negligible, typically ≤ 2 kb in conifers (e.g., Brown
et al. 2004; Wilcox et al. 2007; González-Martínez et al. 2011). This means
that any method of QTL detection or gene discovery based on LD between
marker loci and loci governing functional variation requires information
specific to the various individual pedigrees.
Fifth, markers can differentiate nuclear and organellar inheritance.
In most conifers, chloroplast DNA is paternally inherited (e.g., Neale and
Sederoff 1989; Cato and Richardson 1996; Ahuja 2001), allowing direct
evaluation of paternity and pollen flow. This has also facilitated paternity
assignment in seed orchards, and associated development of paternity-
specific markers (e.g., Cato and Richardson 1996).
288 Genetics, Genomics and Breeding of Conifers
• Large study populations will be needed, unless there are QTLs of very
large effect.
• Associations detected will relate to quite broad chromosome regions
rather than to single-nucleotide polymorphisms (SNPs).
• Where QTN exerting individual effects are in tight repulsion linkage,
there may be no detectable QTL.
• With conifers, the extent of linkage disequilibrium tends to be extremely
limited, typically in the order of ≤ 2 kb (see earlier), meaning that almost
all QTL-marker associations will be pedigree-specific.
• Genetic segregation will also mean that certain loci containing important
population-level polymorphisms will be uninformative in pedigrees
that are not polymorphic for both the marker- and QTL alleles.
• The number of loci involved can lead to large numbers of random
spurious associations (false positives), for which statistical correction
needs to be attempted.
• This also means that errors of estimating QTL effects will tend to be
distributed normally about zero, creating “selection bias”.
• Where epistatic effects, which represent interactions between
phenotypic effects of alleles at different loci, are involved the main
genetic effects of alleles at individual loci will tend to be diluted.
However, the number of potential inter-locus combinations gives huge
scope for false positives. Very stringent genome-wide thresholds for
detection statistics are needed to control the rate of false positives, and
power of valid detection can be very low unless individual epistatic
effects are large (Wei et al. 2010).
• The maximum theoretical efficiency of MAS being greatest with large-
effect QTL combined with low heritability represents a difficult case
for achieving proof of concept in terms of efficacy.
• A lack of QTL of genuinely large effect, which appears to be the rule
for conifers, exacerbates these various problems.
These considerations largely militate against success of QTL detection
and MAS, so it is not surprising that there has been very little use of MAS
in conifers. Putative QTLs have often not been confirmed upon resampling
study populations (e.g., Wilcox et al. 1997), although there are reports
of independently verified QTLs (e.g., Brown et al. 2003). Nonetheless,
independent verification in unrelated genotypes is expensive but necessary
(see Wilcox et al. 2001). Moreover, Bayesian analysis, which tests more
rigorously for genuine QTL, has tended to overturn positive findings (Ball
2001, 2007). On the other hand, results of the classic simulation study of
Meuwissen et al. (2001) indicate that “false negatives”, i.e., genuine QTL
that are not detected or else discounted for want of robust evidence for their
existence, can cause greater loss of selection efficiency.
Integration of Molecular Markers in Breeding 297
appears to be real potential for use of DNA makers as a selection tool. Should
that succeed, operational use of DNA polymorphisms for selection would
be combined with use of Thuja as a model species, almost as a “conifer
Arabidopsis” (JH Russell pers. comm.). An impediment, however, is the
absence of a commitment to intensive, large-scale breeding.
7.5.2.3.4 Pleiotropy
This relates to where particular genes affect phenotypes for more than one
trait. To some degree, this is probably almost universal. The tree breeder
almost certainly encounters it in most of the observed between-trait genetic
correlations, which must lead one to be prepared for individual genes to
exercise pleiotropic effects. Pleiotropy may make it easier for the breeder to
detect QTL or even QTN, because of simultaneous phenotypic associations
among traits known to be genetically correlated.
Adverse genetic correlations, however, pose special problems for the
breeder, limiting the genetic gain simultaneously achievable in the traits
involved, and creating a great need for accurate economic-worth information
on the respective traits (e.g., Burdon 2004). With polymorphisms of large
phenotypic effect, which are likely to be used in MAS or GAS, adverse
pleiotropic effects can pose a special danger. Adverse side-effects on field
fitness of quality-related alleles are likely to be far more serious in conifers
than in annual field crops, which will typically grow in more tightly
controlled environments. Quite prolonged field testing may therefore be
needed for using genes of major effects, which as such pose special risks of
adverse side-effects. Even genes that are desirable for some wood processing
(e.g., chemical pulping) or end-products may be undesirable for other
processes or products, e.g., solid-wood products.
Given these preconditions are not yet in place for most conifers, MARG
is unlikely to be implemented now, but may be useful for the future in
specific circumstances, particularly as association genetics experiments
worldwide are developing catalogs of polymorphisms that affect trait
variation.
outside specific pedigrees. That, and a general paucity of large QTL effects,
severely limits the scope for effective marker-assisted (or marker-based)
selection (MAS). Similarly, an apparent general paucity of large QTN effects,
along with the problems posed by the enormous size of conifer genomes,
creates major difficulties for developing association genetics as a basis
for gene-assisted (or gene-based) selection (GAS). Moreover, institutional
challenges arise in both allocation of resources and communication between
the conventional breeders and those researching selection applications of
genetic markers.
Despite these problems with selection applications, there is reason for
optimism. Results described in Chapter 6 indicate markers can be identified,
although the design of association tests will need to be addressed in the
context of tree improvement imperatives. As with conventional breeding, the
essentially wild state of conifer genomes means that populations typically
have abundant genetic variability. The remarkable levels of orthology among
distantly related plants, and the strong synteny within conifers, will surely
help the breeder identify worthwhile candidate genes for gene discovery
which will then provide a basis for GAS. Leveraging such knowledge from
other species should also contribute to phenomics, whereby the pathways
of initial gene action (transcriptomics—see Mackay and Dean 2011 and
references therein) through proteomics and metabolomics to phenotypic
expression can be elucidated. Breeding for disease resistance is a specific
area where application of genetic markers holds special promise, given
increasing evidence of existence resistance factors of large effect, and the
fact that ”pyramiding” genes for different resistance mechanisms can make
resistance durable against genetic shifts in pathogens.
Finally, future applications of markers for selection in forest tree
breeding may benefit from lessons learned in the interim from experience
with smaller, shorter-lived plants.
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8
Transcriptomics
John J. Mackay1,* and Jeffrey F.D. Dean2
ABSTRACT
RNA transcripts are the first discrete products on the path linking
genomes to function and phenotype. Thus, characterization of the
transcriptome, which is the sum total of all transcripts produced from
the genome, establishes the cast of players working on the cellular
stage to create a biological outcome. The enormity of conifer genomes
has so far constrained researchers to focus their genomic aspirations
on transcriptomes, and substantial bodies of sequence information
have been accumulated to identify many if not most of the more
abundant genes contributing to conifer growth and development. This
chapter reviews the current status of our understanding of conifer
transcriptomes and discusses how this information is being used to
quantify the dynamics of the gene expression changes that define conifer
responses to developmental cues, as well as environmental challenges.
Instrumentation for nucleic acid sequencing and quantitation is
changing rapidly, creating opportunities to study aspects of conifer RNA
metabolism that were previously inaccessible. These new technologies,
and some still on the horizon, are reviewed with respect to their current
application to problems in conifer biology, and possibilities for future
uses are discussed.
Keywords: Pinaceae, cDNA sequencing, expressed sequence tag,
EST clustering, bioinformatics, transcriptional profiling, microarray,
digital gene expression, wood formation, abiotic and biotic stress,
gene families
1
Center for Forest Research, Laval University, Québec City, Québec, Canada, G1V 0A6;
e-mail: John.mackay@sbf.ulaval.ca
2
Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602,
USA; e-mail: jeffdean@uga.edu
*Correspondind author
324 Genetics, Genomics and Breeding of Conifers
Species Name Common Name Family (f) dbEST entries dbEST entries dbEST entries
Order (o) (non-conifers) (2010-08-01)1 (2009-08-20) (2006-10-27) 2
Chamaecyparis formosensis Formosan or Taiwan cypress Cupressaceae 805 805 503
Chamaecyparis obtusa Hinoki Cypress or Hinoki Cupressaceae 5897 5,897 5,830
Cryptomeria japonica Japanese cedar, Sugi Cupressaceae 56,645 56,645 16,230
Cycas rumphii Cycads, Sumo Cycadaceae (f) 21,997 21,997 8,058
Cycadales (o)
Ginkgo biloba Maidenhair tree, ginkgo Ginkgoales (f) 21,590 21,590 6,248
Ginkgoaceae (o)
Gnetum gnemon Melinjo or Belinjo Bago Peesae Gnetaceae (f) 10,724 10,724 4,234
Gnetales (o)
Picea abies Norway spruce Pinaceae 14,224 10,217 7,935
Picea engelmannii x Picea glauca BC Interior spruce (hybrid) Pinaceae 28,174 28,170 28,170
Picea glauca White spruce Pinaceae 313,110 298,657 132,624
Picea sitchensis Sitka spruce Pinaceae 186,637 168,675 80,789
Transcriptomics 325
Pinus pinaster Maritime Pine Pinaceae 34,044 27,847 27,283
Pinus pinea Stone pine Pinaceae 289 290 289
Pinus radiata Radiata pine Pinaceae 7,538 6,757 —
Pinus rigida x Pinus x rigitaeda Pitch x Loblolly Pine (hybrid) Pinaceae 1,002 — —
Pinus sylvestris Scots pine Pinaceae 666 660 —
Table 8-1 contd....
Table 8-1 contd....
326
Species Name Common Name Family (f) dbEST entries dbEST entries dbEST entries
(2010-08-01)1 (2006-10-27) 2
while the genus with the largest number of ESTs is Picea (505,719) (Table
8-1). These entries are for ESTs obtained using Sanger sequencing methods,
and did not include any data obtained using next-generation sequencers.
There are also several species, such as Pinus pinaster, Cryptomeria japonicum,
and Douglas-fir, for which EST datasets ranging from a few hundred up
to approximately 30,000 sequences are available. Among the research
projects currently working to add to the publicly available EST resources
for conifers is an effort at the US DOE Joint Genome Institute that has
targeted the release of 2 million new sequences for P. taeda, in addition to 1
million for Pseudotsuga menziesii, and 0.5 million each for Cedrus atlantica,
Cephalotaxus harringtonia, Gnetum gnemon, Picea abies, Pinus lambertiana,
Pinus palustris, Podocarpus macrophylla, Sciadopitys verticillata, Sequoia
sempervirens, Taxus baccata, Wollemia nobilis (J Dean, unpubl. data). While the
JGI effort will generate relatively long sequences using the Roche GS-XLR
sequencing platform (average read-length of 400–500 nt), the 1KP Project
(http://www.1kp-project.com/) plans to use the Illumina GAII short-read
platform (>75 nt paired-end reads) to generate complete transcriptome
reconstructions for at least one species from every conifer family.
However, as sampling depth and sequence quality has increased, so too has
the reliability of clusters for representing the true transcriptional products
of expressed genes. Cluster analyses usually yield a set of “consensus”
or “representative” sequences that are the result of concatenating the
unique sequence information from all reads in a given cluster. Consensus
sequences obtained through these in silico reconstructions are not acceptable
for submission to public databases, like GenBank (NCBI) or Swissprot.
Therefore, smaller, independent databases dedicated to conifer or forest
tree genome analysis have been developed to provide researchers access to
these consensus sequences as well as datamining tools with which to explore
them. Examples of these databases include ConiferGDB (Liang et al. 2007),
ForestTreeDB (Pavy et al. 2007), and TreeGenes (Wegrzyn et al. 2008).
Figure 8-1 Histogram of cluster size’s from NCBI Unigene database (builds from Table 2A).
Color image of this figure appears in the color plate section at the end of the book.
in Pinus contorta (Morin et al. 2008), Taxus chinensis (Qiu et al. 2009) and
several other conifers (Dolgosheina et al. 2008).
A unique feature of conifer genome biology was highlighted by
Dolgosheina et al. (2008) who showed that the populations of small
RNAs present in tissue samples from a panel of gymnosperms (including
several conifers) were structurally distinct from those that had been widely
observed in angiosperm plants. These researchers showed that whereas
angiosperm miRNAs accumulated in two predominant size-classes of 21
and 24 nt (Axtell et al. 2007), only the 21 nt size class of miRNAs was found
to accumulate in gymnosperms. Large-scale sequencing of small RNAs in
P. contorta (Morin et al. 2008) and T. chinensis (Qiu et al. 2009) confirmed
that 21 nt miRNAs were the only size class to accumulate in these species.
Since distinct mechanisms are employed to produce the different size classes
of miRNAs (e.g., different members of the Argonaute protein family are
involved), these results indicate that the miRNA synthesis mechanisms
have diverged between these two plant phyla. The studies by Morin et
al. (2008) and Qiu et al. (2009) identified additional novel sequences not
encountered in angiosperms that could potentially represent gymnosperm-
specific miRNAs. Among the apparent target sequences for these novel
miRNAs were cellulose synthases and glutathione peroxidases among
others. Limited EST datasets for these species and lack of a reference genome
for conifers have restricted characterization of miRNAs that are putatively
conifer-specific. Nonetheless, 51 novel small RNAs having the secondary
structure typical of miRNAs were identified in P. contorta (Morin et al. 2008).
Taken together, these findings depict a distinct evolutionary path for miRNA
synthesis in conifers compared to angiosperms. The potential functional
implications with respect to gene regulation remain largely to be described.
In conifers, miRNAs have been implicated as potential regulators of gene
expression during embryo growth and development (Oh et al. 2008), as
well as during defense responses (Lu et al. 2007; Qiu et al. 2008). It stands
to reason that these putative miRNAs could be linked to these and many
other important biological processes in conifers.
Pavy et al. 2005b). However, targeted searches using hidden Markov model
(HMM) methods have uncovered conserved domains of unknown function
(DUFs) among conifer transcripts that are preferentially expressed in xylem
tissues. For example, sequences harboring DUF547 (AT5G60720) and
DUF579 (AT5G67210) were linked to xylem tissues in both spruce (Pavy
et al. 2008a) and Arabidopsis (Ko et al. 2006).
Ultimately, transcript profiling using developmental series has the
potential to reveal the full spectrum of xylem-expressed genes and shed light
on the timing of their expression relative to cellular differentiation events.
A wood formation roadmap was developed for Populus using microarray
analyses of finely dissected tangential microsections along an axis running
from the cambial zone to an area of fully differentiated xylem (Hertzberg
et al. 2001). The study established that genes encoding lignin and cellulose
biosynthetic pathway enzymes, as well as several potential regulators of
xylogenesis, were under strict stage-specific transcriptional control. A follow
up analysis using a more comprehensive cDNA microarray comprised
of over 13,000 unique sequences identified a broader set of stage-specific
markers and potential regulators of cambial cell identity (Schrader et al.
2004). No similar studies have yet been reported for conifers, but Friedmann
et al. (2007) compared gene expression in different portions of the terminal
stem of Sitka spruce, contrasting regions of stem consisting mainly of
primary xylem to those containing a well-defined layer of secondary
xylem. Their study showed that the base segment containing secondary
xylem accumulated higher levels of many of the transcripts associated
with preferential xylem expression, and were similar to transcripts that
accumulated in the later stages of Populus wood formation.
digital gene expression approaches (Whetten et al. 2001; Pavy et al. 2005a)
in addition to being used for the construction microarrays.
In conifers, tracheid cell wall thickness and wood density vary
significantly across the seasonal developmental gradient from earlywood
to latewood formed within a single growth ring, regardless of the cambial
age (Plomion et al. 2001). Transcript profiling experiments have been
performed on samples of differentiating xylem collected at different time
points throughout the growth seasons for P. taeda (Egertsdotter et al. 2004)
and P. pinaster (Le Provost et al. 2003). Both experiments followed only a
few hundred sequences and identified relatively small sets of differentially
expressed genes. Transcripts for a number of cell wall-related enzymes and
structural proteins were found to accumulate during latewood formation,
including P. taeda enzymes involved in lignin or cellulose biosynthesis,
as well as a glycine-rich protein and an alpha-tubulin in P. pinaster.
A transcript encoding a putative low molecular weight heat shock proteins
that was preferentially expressed during P. pinaster latewood formation had
a homolog identified in a P. taeda latewood library using digital profiling
(Pavy et al. 2005a). This latter study also found that two putative dehydrin
transcripts were more abundant in P. taeda latewood samples, but the water
stress-inducible gene, lp3, accumulated to higher levels in earlywood.
Overall, fewer transcripts were preferentially expressed in earlywood, and
the transcripts from earlywood represented a greater diversity of functional
categories, ranging from glyceraldehyde-3-phosphate dehydrogenase to
proline-rich cell wall proteins, as well as numerous sequences without
similarity to known proteins. Yang and Loopstra (2005) used microarrays
and quantitative RT-PCR to show that timing of the earlywood to latewood
transition, as well as expression patterns for many of the genes involved,
were strongly influenced by seed source genetics. All of this suggests that
transcript profiling with large gene sets may be needed to fully delineate
the molecular events of the earlywood to latewood transition.
A developmental gradient related to cambial age was investigated in
P. pinaster by collecting xylem samples at different points along the main
stem of a 30 year-old tree (Paiva et al. 2008a). Six different xylem samples
estimated to represent cambial ages ranging from 3 to 21 years were
analyzed using a custom cDNA microarray comprised of 3,512 different
gene sequences and in parallel using two-dimesional polyacrylamide
gel electrophoresis (2-D PAGE). Differential transcript accumulation
patterns identified four general profile classes, including two major classes
comprised of transcripts preferentially expressed in base wood (93 genes)
and crown wood (71). In addition to sequences already discussed, such as
cell wall-related proteins and biosynthetic enzymes, a number of transcripts
encoding products not usually associated with xylem formation were also
recovered. These included many enzymes related to protein synthesis and
Transcriptomics 341
identified a variety of genes that had not previously been associated with
such responses. More recently proteomic work has demonstrated significant
concordance between transcriptional changes in these responses and the
production of specific proteins (Lippert et al. 2007).
Drought stress is a significant concern for conifer forestry since it is
frequently a primary contributor to losses during reforestation efforts. The
amplified fragment length polymorphism (AFLP)-cDNA approach was
used to identify a few hundred genes that showed differential expression
in P. pinaster roots and hydroponically grown seedlings subjected to
drought conditions (Dubos and Plomion 2003; Dubos et al. 2003). Lorenz
et al. (2006) used digital tagging of ESTs from P. taeda cDNA libraries that
varied by genotype and water status to identify genes that displayed
genotype-specific changes in expression pattern under drought conditions.
Interestingly, one genotype in the study, whose parents were selected from
a region that seldom experiences drought conditions, showed lower overall
tolerance to drought, and this was reflected in stronger up-regulation of
drought stress-associated genes under drought conditions. This suggests
that adaptive alleles for drought tolerance may not be well represented in
this particular genotype or its parents. Watkinson et al. (2003) also looked
at drought responses in P. taeda using a 2,173-element cDNA microarray
and reported on physiological and metabolic responses observed during
photosynthetic acclimation. Paiva et al. (2008b) used a microarray comprised
of 3,512 unique cDNA elements from P. pinaster to examine the effect of
drought conditions on gene expression over the earlywood to latewood
transition. The authors noted a great deal of plasticity in the response of
genes related to wood formation when confronted by the challenge posed
by drought. Sathyan et al. (2005) used quantitative RT-PCR to investigate
the expression of drought-responsive genes in Aleppo pine under varied
levels of water stress.
Another area of conifer research where transcriptional profiling is in
active use is the study of cold acclimation. Joosen et al. (2006) used a P. taeda
cDNA microarray to assess differences in gene expression levels of apical
bud or root tissues in P. sylvestris that had either been acclimated to cold
conditions or subjected to an abrupt shift to near-lethal cold temperatures.
Though limited in terms of the number of genes assessed, significant
differences were seen in a number of genes and several appeared to be
homologs of genes that had previously been associated with cold tolerance.
Holliday et al. (2008) recently used a much larger (21,840 unique element)
spruce cDNA array to study acclimation in three populations of P. sitchensis.
More than 2000 genes were significantly up or down-regulated in response
to cold acclimation and there was evidence that at least some of the responses
possibly reflected local adaptations for the populations studied.
344 Genetics, Genomics and Breeding of Conifers
However, the various platform biases do not for the most part overlap,
and hybrid approaches pairing the strengths of different platforms are
already showing great promise for the rapid production of de novo genome
sequences (DiGuistini et al. 2009)
These next-generation sequencer (NGS) or high-throughput sequencing
(HTS) technologies are all characterized by the use of massively parallel
approaches in which millions of sequencing reactions are performed
simultaneously on specialized nano-scale fabricated supports. The scale
of these reactions has reduced the price per base by orders of magnitude.
For example, whereas current best costs for Sanger sequencing run about
US$2.00/kB (US$0.50–1.00 per 500 nt read), the Roche GS-FLX Titanium
platform (based on the original 454 Life Sciences technology and yielding
approximately 1 million reads of 400 nt from a single run) delivers data at
about US$0.03/kB. The GAII and SOLiD sequencers, both of which generate
sequence in the range of up to 50 million reads of 70–120 nt during a single
run, currently deliver data at less than US$0.0025/kB, which is almost a
thousand-fold less than the cost delivered by Sanger sequencing. All three
of these technologies continue to improve by extending read lengths and
increasing the density of parallel reactions, which is a good thing since the next
wave of technology platforms approaching the market have the purported
potential to drop costs per base by another order of magnitude or more. These
prospects are particularly exciting for the conifer community since they signal
a rapidly approaching time when we will have completed reference genome
sequences in hand for multiple members of the conifer family.
Vega-Sanchez et al. 2007; Morrissey et al. 2009), and they should prove
particularly useful for studies in species where the community is insufficient
to support the development costs of microarrays.
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9
Recent Advances in Proteomics
and Metabolomics in
Gymnosperms
Rebecca Dauwe,1,a,# Andrew Robinson1,b,# and
Shawn D. Mansfield1,c,*
ABSTRACT
Functional genomics in forestry is a research area in its infancy that has
the capacity to significantly impact our understanding of the genetic
and environmental control of tree development, and provide new
methods to exploit the genetic variation in tree phenotypes. Tools made
available through the analysis of tree genomes have the potential to
have a profound effect on the capacity to improve forest productivity
and monitor forest health, and revolutionize in the selection of trees
lines for the future. This chapter discusses the emerging application of
proteomics and metabolomics in gymnosperms.
Keywords: 2-DE; SDS-PAGE; genetic mapping; developmental
biology; metabolite profiling; metabolic markers; GC-MS; LC-MS; high
throughput; automation, somatic embryogenesis, wood formation
9.1 Introduction
In this era of plant systems biology, “high-throughput” and genome-
wide research aims at establishing an integrated understanding of
the biological processes occurring in all plant tissues. In that respect,
1
Department of Wood Science, Faculty of Forestry, 4030-2424 Main Mall, Vancouver, BC, V6T
1Z4, Canada;
a
e-mail: rebecca.dauwe@u-picardie.fr
b
e-mail: andrewrobinsonnz@gmail.com
c
e-mail: shawn.mansfield@ubc.ca
#
these authors contributed equally
*Corresponding author
Recent Advances in Proteomics and Metabolomics in Gymnosperms 359
9.2 Proteomics
The term “proteome” defines the expressed protein complement of a genome
and was, according to a review by Thiellement et al. (2002), first introduced
in 1994 by Wilkins at a conference. The roots of this concept, however,
date back to 1975 with the development of high resolution 2-dimensional
polyacrylamide gel electrophoresis, abbreviated as 2-D PAGE or 2-DE.
The first plant large-scale proteomic work was published on Arabidopsis
thaliana (Kamo et al. 1995). During the following years, proteomics emerged
as a complementary approach to the analysis of genome expression at the
mRNA level.
9.2.1.1 Platforms
Two-dimensional polyacrylamide gel electrophoresis (2-DE) coupled with
MS has been the most extensively used platform in plant proteome analyses.
To analyze simple proteomes, such as the proteome of juniper pollination
drops, even 1-dimensional sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), proved to generate good quality data
(Wagner et al. 2007). 2-DE, which separates denatured proteins based
on their charge (pI), by isoelectric focusing (IEF) in the first dimension,
and based on their relative molecular weight (MW), by SDS-PAGE in
the second dimension, results in a high resolution, quantitative image of
intact proteins that provides a good overview of different isoforms and
post-translational modifications. However, the technique cannot meet the
“full-genome” objectives of proteomics, analogous to, for example, what
microarray analysis presents in transcriptomics. 2-DE covers only a subset
of the most abundant proteins, with a poor representation of membrane,
high molecular weight, and basic proteins. Furthermore, the fraction of
the proteome that is studied is determined by the precipitation protocol
and the 2-DE separation technique (for example the pH range of the first
dimension). A major disadvantage of the use of 2-DE as a high-throughput
method, is that this technique is difficult to automate, and the technical
variation is therefore difficult to control. In an attempt to realize automated
high-throughput proteomics, gel-free protein separation methods (Berg et
al. 2006; Pirondini et al. 2006; Roe and Griffin 2006; Whitelegge et al. 2006;
Zolla 2006) and “second generation” proteomic techniques are currently
being developed and refined. These include MudPIT (multidimensional
protein identification technology) and quantitative proteomics techniques
such as DIGE (differential in gel electrophoresis), ICAT (isotope-coded
affinity tags), iTRAQ (isobaric tag for relative and absolute quantitation),
and SILAC (stable isotope labeling by amino acids in cell culture) (Amme
et al. 2006; Basu et al. 2006; Bayer et al. 2006; Jones et al. 2006; Komatsu
et al. 2006; Lilley and Dupree 2006), all of which have been developed
and applied recently in plant biology research but remain unexploited
Recent Advances in Proteomics and Metabolomics in Gymnosperms 361
buffers optimized for beech roots, containing two chaotropes (urea and
thiourea) and a sugar-based detergent, could be used for the extraction
and separation of proteins from spruce roots only after optimizations (not
specifically reported). Valcu and Schlink (2006) also reported an extraction
buffer containing two chaotropes and two detergents (7 M urea, 2 M
thiourea, 2% CHAPS, 2% SB3–10) that effectively separated spruce needle
proteins.
62% when compared with using GenBank data alone (Lippert et al. 2005).
For the interpretation of protein sequence data collected in a following
proteomic study of sitka spruce defense against white pine weevils (Pissodes
strobi Peck), Lippert et al (2007) relied on a translated protein database that
contained 164,621 predicted peptide sequences, derived from a total of
249,149 nucleotide sequences from sitka spruce, white spruce and interior
spruce, available from previous research programs (Pavy et al. 2005; Ralph
et al. 2006), and an additional 7,317 conifer proteins from various species
obtained from NCBI. This database allowed the identification of 68.5% of
the queried proteins, as compared to 31.7% when using the NCBI database,
and, in contrast to the previous study, there appeared no additional benefit
in employing the larger NCBI database. The improved protein identification
results of the second, weevil response study, which focused on bark tissue, in
comparison with the somatic embryogenesis study, was attributed not only to
the size of the database used (5-times more ESTs), but also to the fact that ESTs
from bark tissue were enriched in the database, whereas embryonic tissue was
not represented in the EST database at the time of the somatic embryogenesis
study. In a proteomic study of wood forming tissue in maritime pine, the use
of an EST database of modest size but highly enriched in pine xylem ESTs
(18,254 Pinus pinaster ESTs and 59,447 Pinus taeda xylem ESTs) resulted in a
67.9% identification of proteins by LC ESI-MS/MS (Gion et al. 2005). These
examples illustrate the importance of deep and biologically relevant EST and
FL-cDNA sequencing as an essential resource for proteome analysis when
no whole genome sequence is available. The importance of having sequence
data from the same species as studied by proteomics, results from the fact
that a single amino acid substitution will, in most cases, change the mass of
a peptide enough to prevent its being identified by MS data interpretation
software. On the other hand, a drawback of the use of EST databases for
identification is that, because of their restricted length, often only 400–600
bp (of which up to a third can represent untranslated regions), ESTs cannot
be used to identify proteins by their peptide mass fingerprints (Lahm and
Langen 2000), and peptide sequencing by MS/MS must be used. This has been
confirmed in a proteome analysis of maritime pine xylem (Gion et al. 2005).
In that study, the success rate of identification by MS/MS was high (68%),
whereas identification by MALDI-TOF MS, using the same EST databases as
used for the identification by MS/MS, had a success rate of only 16%.
In most gymnosperm spp., the situation is much less straightforward:
with poorly characterized genomes and no considerable EST sequence
collection available, cross-species identification becomes the only option for
protein identification. If the corresponding gene of the investigated organism
is unknown, actual identification of the protein senso stricto is not possible,
and the goal is to find the most similar gene in a closely related organism.
In such cases, sequence databases from closely related organisms can be
364 Genetics, Genomics and Breeding of Conifers
from the biosynthesis of anthocyanins, and some proteins that were under-
expressed in compression wood suggested that molecular mechanisms
determining cell shape and cell size are disturbed in gravity-stimulated
tissue. Interestingly, this study also showed that seasonal effect dominated
the tissue type effect on the control of protein accumulation. A more detailed
physiological study was performed using the analysis of the proteome
changes, in parallel with lignin and cellulose content changes, which
accompanied the formation of compression wood in a range of Maritime
pine xylem samples, subject to gradually increasing growth strain (Plomion
et al. 2000). Functional relationships between proteins were traced via
correlation analysis clustering of similar protein expression patterns along
the gradient of gravity-stimulated stressed xylem tissue. A small cluster of
traits, showing the strongest positive correlation with the growth strain,
comprised the phenotypic trait, lignin content, an ethylene forming enzyme.
A larger cluster, positively correlated with growth strain, included mainly
lignification proteins. These results support the suggestion that ethylene
plays a role in compression wood differentiation by inducing the expression
of enzymes involved in lignification. Furthermore, a transcription factor
identified in the protein cluster positively correlated with growth strain,
was established as a candidate protein for controlling compression wood
differentiation. Such proteomic expression analyses can thus lead to detailed
functional hypotheses, which are, taking into account the direct involvement
of the proteins in the biological processes, more relevant as compared to
transcriptomics based hypotheses.
Only a few proteomic studies have focused on the changes associated
with gymnosperm growth and development (Fernando et al. 2005; Lippert
et al. 2005). A study of the molecular programs involved in pollen tube
development was performed in eastern white pine (Pinus strobus) via a
comparative analysis of the proteomes in 2-day-old pollen tubes and in
ungerminated grains (Fernando et al. 2005). The differential proteome
in the developing pollen tubes revealed the involvement of both cell
wall formation and stress/defense responses. A study of the embryo
developmental process during somatic embryogenesis was performed in
white spruce, by recording the proteomic changes occurring across four
stages of somatic embryo maturation, and revealed the involvement of a
broad variety of biological processes (Lippert et al. 2005).
9.2.4 Databases
Along with the development of reproducible high-throughput techniques,
an enormous amount of data is expected to be produced via functional
proteomics programs. The exploitation of these data will largely depend
on the development and organization of databases.
The Maritime pine database constitutes the first public proteome
database dealing with forest trees (http://www.pierroton.inra.fr/genetics/2D)
(Costa et al. 1999). This database consists of scanned gels of Maritime pine
needle and xylem tissue with hyperlinked spots, which allow one to retrieve
sequence data and, for certain proteins, the location on a linkage map and
the behavior in drought environment, from the position of protein markers
in 2-DE gels, and vice versa.
In 2005, a web-based plant 2-DE database “PROTICdb” (http://moulon.
inra.fr/~bioinfo/PROTICdb) was established to store, track, query and
compare plant proteome data, and is freely available upon request (Ferry-
Dumazet et al. 2005). Maritime pine proteomes of differentiating xylem,
corresponding to different developmental stages and treatments, have been
stored in “PROTICdb” and are also publicly available on the website http://
cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/index.php, with the information
concerning plant material and experimental conditions, protocols for
extraction, electrophoresis, staining and digitalization, mass spectrometry
techniques, and details concerning the identification of the protein spots
and the query databases (Gion et al. 2005). On the other hand, all protein
sequences derived from the translation of the coding sequences that have
been submitted to the public nucleic acid database (EMBL/GenBank/DDBJ)
are integrated into the UniProt Knowledgebase (UniProtKB) (http://www.
uniprot.org). A query for gymnosperm (Coniferopsida) proteins, performed
in UniProtKB as of 28 August 2008, resulted in only 108 gymnosperm
proteins for which the existence has been proven on the protein level.
Proteins in the database were identified in Pinaceae (78), Taxus (17), and
Cupressaceae (13). This corresponds to less than 1% of the total of 14,104
predicted gymnosperm proteins in the database. The gymnosperm proteins
represent only a very small fraction (less than 2%) of the 6,052 plant proteins
(Spermatophyta), with proven existence on the protein level, in the database.
For illustrative comparison, 2,497 (41%) of the proven plant proteins in the
database are derived from Arabidopsis thaliana.
Recent Advances in Proteomics and Metabolomics in Gymnosperms 371
9.3 Metabolomics
In systems biology, the term “metabolome” refers to the complete set of
small molecules (i.e., metabolites) that participate in or are products of
metabolic reactions within an organism or tissue. Hence, “metabolomics”
is concerned with the identification and quantification of those molecules
in order to advance biological understanding and the development of
novel biomarkers. As an eventual product of gene expression under the
influence of environment, cellular metabolism is the immediate progenitor
of phenotype and, as such, the relationships between phenotypic and
metabolomic traits are potentially more coherent than for the genomic,
transcriptomic and proteomic counterparts. However, in comparison to the
other “omics”, for which rapid technological advances have been seen, the
emergence of analytical and software tools for the comprehensive analysis
of the metabolome has been slow. Whereas the genome, transcriptome and
372 Genetics, Genomics and Breeding of Conifers
resolved from metabolite profiles are not present in these libraries, and
continue to elude identification. The ongoing expansion of mass spectral
library resources is of paramount importance, as at present, the process of
compound identification constitutes a major limiting factor in the plant
metabolomics field.
Nuclear magnetic resonance spectroscopy is a popular alternative to
chromatography/mass spectrometry for resolving compounds from complex
mixtures, with the sub-class of metabolomics employing this technique
being known as “metabonomics”. Biological NMR spectroscopy usually
exploits the magnetic properties of 1H or 13C nuclei. The different proton
or carbon nuclei in a molecule resonate at slightly different frequencies
due to differences in local chemical environment, so particular compounds
have characteristic nuclear resonance patterns for specific nuclei. Thus, a
1D NMR spectrum can provide information on the number and type of
1
H or 13C nuclei in a mixture of metabolites, and from this the identity and
relative contributions of individual metabolites may be resolved. One of
the major benefits of NMR spectroscopy is that it is non-destructive, and
as such, samples may be analyzed repeatedly over the course of a study,
or studied in other ways once NMR analysis is complete.
of any given metabolite is never one exact value across all runs. When an
experiment involves a large set of samples and metabolites, manual collation
becomes impractical, and the task must be handed to automated software
that decides whether peaks in multiple samples represent the same or
different compounds, based on retention time windows and mass spectral
matching. The need to resolve these issues has led to the development of
a number of commercial and free software tools that are capable of these
tasks, with the more accomplished of these capable of both deconvolution
and collation. Notable non-commercial examples include NIST AMDIS
(for deconvolution only), MetAlign (Tikunov et al. 2005) and the complex
yet highly capable XCMS (Smith et al. 2006). While there are reports
of automated peak collation (AR Robinson et al. 2008 unpubl.), profile
deconvolution has not yet been reported in the gymnosperm metabolomics
literature, although it is certainly only a matter of time before it is.
Data analysis in metabolomics has advanced at a considerable rate,
with the ongoing introduction of statistical analyses and other calculative
tools to the field. Most statistical tools have been applied with a reductive
perspective. Classic, univariate tests between means, such as Student’s t-test,
the F-test and more robust incarnations like Tukey’s “honestly significant
difference” (HSD) test have been used to individually identify metabolites
exhibiting genotype- or treatment-related differences in abundance.
Although useful, these tests deal with each metabolite as an isolated
entity, and are unable to take the interdependence of the components of
metabolite profiles into account. Multivariate analyses are better suited
to this task. The default statistical tools of metabolomics are principal
components analysis (PCA) and hierarchical cluster analysis (HCA), and
many analyses are limited to the use of these two techniques. Both are
useful for comparing complete profiles from multiple samples, and generate
diagrammatic outputs that are visually appealing and easily interpreted.
Although PCA does provide some information regarding the particular
metabolites responsible for any distinction between sample classes, neither
PCA nor HCA are very diagnostic, because they do not provide calculated
measures of the relationships between metabolite profiles and, for example,
phenotypic traits. Canonical correlation analysis (CCA) is one method
that can assist in defining the relationships between two sets of variables,
such as metabolites and quantitative phenotypic traits. Essentially, CCA
identifies groups of variables in one set that are correlated to groups of
variables in the other, and indicates the relative contributions of individual
variables to the relationship. However, in cases where diagnostics are an
objective, techniques that generate models for the prediction of specific
traits on the basis of metabolite profiles are required. To this end, multiple
discriminate analysis (MDA) is useful for distinguishing samples by class
(e.g., genotype, species), while partial least squares regression (PLSR) and
378 Genetics, Genomics and Breeding of Conifers
The quantity and quality of the reserve held by a seed at the time of
germination is critical to seedling emergence and vigor, and unfortunately
the seeds of many conifers exhibit severe storage-related degradation.
As a continuation of their earlier work on the metabolic composition of
coniferous seeds, Terskikh et al. (2008) worked to demonstrate a correlation
between changes in 13C NMR spectra and the rate of deterioration in
stored seed batches of western red cedar. They observed that a decline in
germination capacity due to storage was accompanied by a correlative
decrease and broadening of the resonances associated with triglyceride
reserves, and furthermore that the proportion of polyunsaturated fatty acids
in the triglyceride oil mixture decreased sharply. It was contended that lipid
peroxidation, oxidative polymerization, and consequent solidification of
storage oils was responsible for these changes, and at least partly responsible
for the observed decrease in seed viability. The general relationship between
NMR intensity and germination rate was determined to be hyperbolic, and
modeled accordingly.
The foundation-laying research described not only demonstrates that
metabonomics can help to improve our understanding of conifer seed
biology, but also that it is a very promising diagnostic tool for effective
breeding and propagation of valuable gymnosperm species. Within this
context, the non-destructive nature of NMR makes it an ideal tool for
analyzing precious plant material and monitoring stored germplasm.
Research in this field has an exciting future, with a clear need and an
enormous potential for the industrial application of NMR-based seed
analysis in the coming years.
9.3.2.5 Fragrance
The extractives content of wood has implications for the survival of a tree, but
also for human utilization. One interesting study investigated the fragrance
compounds of six, highly prized odorous and durable coniferous woods
grown in Taiwan. Wang et al. (2006a) used solid-phase microextraction
(SPME) and GC/MS to generate non-biased fragrance composition profiles
for these woods at room temperature (30°C). With the aid of mass spectra
databases, GC retention indices based on alkane standards, and authentic
standard compounds, a total of 46 compounds were identified in the
fragrances, across these species. It was found that fragrance compositions
varied considerably from solvent-extracted essential oil compositions
previously reported in the literature—to the extent that many extractive
compounds previously believed to be important components of fragrance
in specific species (e.g., δ-cardinene, δ-cardinol, β-eudesmol and copaene
in Cryptomeria japonica (Chang et al. 2003)) were not detected as volatiles
at all. Species-related variation in odor was associated with variation
in fragrance chemical composition, and principal components analysis
and the nearest neighbor cluster analysis were performed to determine
the similarity/disparity between species. These multivariate approaches
resolved three groups of two species, with each group based on shared
chemical skeleton classes, and in doing so demonstrated the potential for
further chemotaxonomic classification of gymnosperm species on the basis
of volatile emissions.
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10
Toward the Conifer Genome
Sequence
Michele Morgante1,2,* and Emanuele De Paoli1,3
ABSTRACT
Large genome size and highly repetitive DNA content have thus
far posed considerable difficulties for both structural and functional
genomic studies in coniferous species. As a result, the understanding
of conifer genome structure and evolution is still deficient compared to
the enormous progress in angiosperms genomics since the sequencing of
the Arabidopsis genome. However, the development of high-throughput
DNA sequencing technologies has enabled to complete large-scale
sequencing efforts more cost-effectively and in a shorter time than
previously possible, making the sequencing of a conifer genome an
attractive opportunity to fill the gap. While two different conifer genome
projects have been recently embarked, emerging data from preliminary
studies are providing interesting insights into the characteristics
of conifer genomes, especially with respect to the composition and
evolution of transposable elements that populate them. This chapter
will review the state of the art of DNA sequence analysis in conifers
and other gymnosperms with emphasis on the interesting deviations
from the current model of higher-plant genome evolution that these
species are revealing.
Keywords: genome sequencing, genome evolution, transposable
elements, conifer, gymnosperms, Picea, Pinus, Ginkgo
1
Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, Via delle Scienze 208,
33100 Udine, Italy; e-mail: michele.morgante@uniud.it.
2
Istituto di Genomica Applicata, Parco Scientifico e Tecnologico di Udine, Via Linussio 51,
33100 Udine, Italy.
3
Current address: Istituto Agrario di San Michele all’Adige, Vie E. Mach 1, 38010 San Michele
all’Adige, Italy; e-mail: emanuele.depaoli@iasma.it.
*Corresponding author
390 Genetics, Genomics and Breeding of Conifers
10.1 Introduction
Our understanding of plant biology and evolution has been greatly aided by
the recent advances in DNA sequencing technology and the development of
comparative methods for genome analysis. Since year 2000, when the first
genomic sequence from a plant species, Arabidopsis, was released (Lin et al.
1999; Mayer 1999; Salanoubat et al. 2000; Tabata et al. 2000; Theologis et al.
2000), the nucleotide sequence of other 14 plant organisms has been made
publicly available, albeit with different degrees of completion. Moreover, at
the time this manuscript is being written, a total of 80 genome sequencing
projects aimed at determining the nuclear genome sequences of as many
land plant species or varieties have been publicly announced. While only
two of them are deemed thoroughly completed (Arabidopsis and rice), 15
are at the assembly stage and the remaining 65 are in progress (NCBI data
updated on March 2nd 2010, http://www.ncbi.nlm.nih.gov/genomes/static/
gpstat.html). Notably, despite the immense ecological role and economical
value of conifers, only one of these ongoing projects is committed to the deep
characterization of a conifer genome, namely the Pine Genome Initiative
(PGI) (http://pinegenomeinitiative.org/), which has become a reality almost a
decade after the beginning of the genomic era in plant science. In parallel,
however, a European consortium led by Sweden has recently been granted
to sequence the genome of Norway spruce (http://www.upsc.se/Networks/
Networks/sprucegenome.html) and our group at the University of Udine has
developed extensive genomic resources for spruce, by the deep sequencing
of genomic libraries from the genomes of four Picea species, the annotation
of bacterial artificial chromosome (BAC) clones from the genome of Norway
spruce and the characterization of its repetitive components.
Unquestionably, a major reason for such a delayed effort and at the
same time one of the principal challenges facing gymnosperm genomics is
the large size (Fig. 10-1) and repetitiveness of their genomes, which poses
considerable difficulties for both structural and functional genomic studies.
In consideration of the large costs of de novo sequencing, which until a few
years ago could not take advantage of high-throughput parallel sequencing
methods, the decision to sequence a large plant genome has been always a
serious question that needed to carefully balance scientific interest, social and
economic benefits with the impact on public funds and human resources.
Before the development of next-generation DNA sequencing technologies,
accompanied by technical simplification and drop in the sequencing costs,
gymnosperms did not meet these common sense criteria. As a result, while
reduced-representation approaches (mainly expressed sequence tag (EST) and
cDNA sequencing efforts described in Chapter 8) have been used to alleviate
the redundant nature of their genomes and focus on gene discovery and
gene expression, many essential questions about the origin of gymnosperm
genomes, their organization and evolution are yet to be answered.
Toward the Conifer Genome Sequence 391
Figure 10-1 Selection of plant genome sizes. Genome size is provided for a subset of species
that are object of genomic sequencing. Size information is from NCBI (http://www.ncbi.nlm.
nih.gov/genomes/static/gpstat.html) with the exception of gymnosperm data from the Kew Plant
DNA C-values Database (http://data.kew.org/cvalues/).
Color image of this figure appears in the color plate section at the end of the book.
intervals. One of the five major orders, namely the LTR retrotransposons,
are the most abundant and widespread class of eukaryotic transposable
elements and represent the major constituents of plant genomes as well
(Flavell et al. 1992; Kumar and Bennetzen 2000; Wicker and Keller 2007).
Using a reduced-representation shotgun sequencing approach, De Paoli
and collaborators have estimated that 77% of a spruce genome (Picea abies)
sequence is redundant and 42% of it consists of LTR-retrotransposons (E
De Paoli et al. unpubl. data), although this may be an underestimate of the
actual retrotransposon content, due to the difficulty to annotate transposable
elements de novo from limited amount of contiguous sequence. Indeed,
a large fraction of the genomic library analyzed, consisting of both
repetitive and supposedly single-copy elements, is yet to be characterized
(Fig. 10-2).
Figure 10-2 Estimate of Norway spruce genome composition. Principal genomic components
of Norway spruce genome estimated based on the annotation of a 9 Mbp small insert genomic
library (E. De Paoli et al. unpubl. data).
Color image of this figure appears in the color plate section at the end of the book.
the minimal amount of DNA sequences that represents a genome or, in other
words, the combined length of all of the single-copy DNA sequences plus one
copy of each repetitive sequence (Peterson et al. 2002). Cot analyses, which
enable the separation of low copy genomic features (mainly protein-coding
genes) from high-copy sequences (e.g., transposable elements), were used
to assess sequence complexity before the introduction of high-throughput
parallel sequencing approaches. These early studies revealed that genome
complexity values varied between 13 and 77% among the angiosperm
species analyzed and similarly between 24 and 71% among gymnosperms
(Morse et al. 2009). This result is remarkable if one considers that the
large sizes of gymnosperm genomes imply absolute values of complexity
(or single-copy component) equal to several thousand megabases. Reasoning
that this fraction of the genome should be relatively consistent in size among
species, the values found in gymnosperm seem to be much larger than the
minimum amount of genic components and regulatory elements required
for biological functions. Interestingly, Gymny retrotransposons were found
both in the high-copy and low-copy fraction of the loblolly pine genome
and derivatives or rearranged copies of this retrotransposon family were
deemed more frequent than full-length elements (Morse et al. 2009). This
observation suggests that extensive rearrangements of repetitive sequences
might have contributed to the apparent excess of low-repetitive kinetic
component of the genome, an hypothesis already proposed by Elsik and
Williams ( 2000).
during the transposition process. Within the two LTRs there is a region
that encodes two proteins, specified by two genes called gag and pol.
The gag gene encodes capsid-like proteins involved in maturation and
packaging of retrotransposon RNA into a form suitable for integration into
the genome. Pol encodes a multidomain protein and the single domains
are arranged in different order in the Ty1-copia and the Ty3-gypsy elements
respectively. These domains include in particular a reverse transcriptase
and a RNase H activity that are required for replication/transposition
of the retrotransposon as well as an integrase that allows the DNA form of
the retrotransposon to insert into a new chromosomal location.
Both Ty1-copia and the Ty3-gypsy superfamilies have been detected in
conifers and in other gymnosperm orders, but their relative proportions
are generally not known. In our knowledge, the genome of Norway spruce
(Picea abies) is the only one whose composition has been estimated by low-
coverage shotgun sequencing (E De Paoli et al. unpubl. data). In this case,
Ty3-gypsy elements were found to be twice as abundant as the Ty1-copia
elements (25% vs. 13%). Thus far, no single prominent retrotransposon
family has been recognized that may account for much of the extraordinary
expansion of this genome in a fashion similar to what happened in some
angiosperm species, i.e., in the cotton genus (Hawkins et al. 2006). On the
other hand, the contribution of a single family to the sequence space can be
impressive in absolute terms even if relatively modest relative to the overall
genome size. For instance, by screening a collection of ~18,000 BAC clones
from loblolly pine, Morse et al. (2009) estimated that the Gymny Ty3-gypsy
retrotransposon family accounts for a genomic space at least as large as the
Arabidopsis genome, namely ~135 Mb but corresponding to only ~0.6% of
the host genome.
A few other retrotransposon families have been described in detail
thus far, including TPE1 in Pinus elliottii var. elliottii (Kamm et al. 1996),
IFG7 in Pinus radiata (Kossack and Kinlaw 1999), PpRT1 in Pinus pinaster
(Rocheta et al. 2007), Gymny in Pinus taeda (Morse et al. 2009) and Alisei
in Picea abies (E De Paoli et al. unpubl. data). Notably, TPE1 is the only
representative of copia-like elements. Besides, non-LTR retrotransposons like
long interspersed nuclear elements (LINEs) and DNA (class II) transposons
have also been found in the spruce and pine genomes (Friesen et al. 2001;
E De Paoli et al. unpubl. data), albeit at low frequency as in other large-
genome plants.
Figure 10-3 Annotation of four genomic regions (BAC) randomly selected from the genome
of Norway spruce. Green boxes, LTR-retrotransposons of the copia superfamily; yellow boxes,
LTR-retrotransposons of the gypsy superfamily; light-blue boxes, LTRs of copia elements;
dark-blue boxes, LTRs of gypsy elements; white boxes, uncharacterized LTR-retrotransposons
(with black LTRs when annotated), grey boxes, non-autonomous LTR-retrotransposons
(with dark-grey LTRs); black box, non-LTR retrotransposon. The red bar indicates repetitive
sequences; PARE, Picea abies repetitive element; GY, gypsy; CO, copia; UN, unidentified; NA,
non-autonomous. From E. De Paoli et al. (unpubl. data).
Color image of this figure appears in the color plate section at the end of the book.
of sequence conservation, not only within conifers, but also between conifers
and Ginkgo. Moreover, in most cases the nucleotide differences between
species were similar or even lower than the within-species divergences
(E De Paoli et al. unpubl. data).
In consideration of the very distant evolutionary relationship between
the species investigated in these studies, it is surprising to find a high
degree of sequence conservation in putatively non-functional repetitive
DNA. It is a matter of fact that there is still no conclusive evidence about
the origin of this similarity. Germ line vertical transmission of retroelement
lineages that arose early in plant evolution, strong purifying selection or
the less supported horizontal transmission are non-mutually excluding
mechanisms that have been proposed (Friesen et al. 2001; Stuart-Rogers
and Flavell 2001). Vertical transmission is the regular manner in which
eukaryotes inherit their retroelements; then transposon extinction normally
occurs through sequence degeneration by point mutations and/or removal
mechanisms that may result in interspecific differences with respect to copy
number and genomic location. Indeed, the monophyletic grouping of all
types of retrotransposons analyzed by Friesen et al. (2001) and the timing of
insertions revealed by De Paoli et al. (unpubl. data) suggest that the current
diversity of retrotransposons in conifers and other gymnosperms may be
the consequence of common ancestry and early rapid radiation before the
split that generated the extant orders. According to this model of early
generation, the differential amplification and loss of retroelements, possibly
not accompanied by substantial lineage-specific differentiation, would
be the major factor shaping diversity among the repetitive components
of gymnosperm genomes. Until now, interspecific DNA hybridization
analyses have proved consistent with this scenario, showing that it is the
relative abundance of single retrotransposon families rather than the general
composition of these genomes to change. A notable exception to this is the
restriction of the Gymny retrotransposon family to the subgenus Pinus of
the loblolly pine (Morse et al. 2009), which might anticipate more similar
cases to be found by genome sequencing.
Though vertical transmission is a parsimonious explanation and is
well established in all eukaryote branches, it is still not yet thoroughly
understood why there should be such a limited sequence variation among
noncoding repetitive elements compared to the scenario of much higher
diversification among angiosperms (Flavell et al. 1992a; Flavell et al. 1992b;
Gribbon et al. 1999). An important clue to address this question comes from
the slow mutation rate found in conifers. Fossil calibration of molecular
divergence over 11 nuclear loci yielded an absolute silent mutation rate
estimate in the range of 0.70–1.31×10–9 for the Pinus nuclear genome, which
is approximately 4- to 20-fold slower than in angiosperms (Willyard et
al. 2007). Although this rate is apparently not slow enough to justify the
400 Genetics, Genomics and Breeding of Conifers
10.7 Conclusions
Despite the limited amount of genomic information from gymnosperms
made available thus far, a few pioneering studies reviewed in this chapter
have clearly delineated a series of genomic features that make these species,
in particular conifers, very attractive for a deep sequencing analysis:
1) The overall architecture of these genomes appears to be extraordinarily
conserved with a fairly constant chromosome number across distantly-
related taxonomic groups. Polyploidy has played a minor role in their
evolution and major chromosome rearrangements are unlikely to have
occurred, despite the long evolutionary history of these species.
2) The redundant component of the genome, mainly consisting of LTR-
retrotransposons, is clearly the major factor underlying the large
genome size, but no prominent repetitive families that alone could
account for the great expansion of the genome have been identified
yet.
3) Cytological analyses and the detailed annotation of large genomic
regions has revealed a dispersed distribution of repetitive elements
over the chromosomes and a complex genomic landscape dominated
by LTR-retrotransposons arranged in multilayer nested structures.
4) The low-repetitive and single-copy component largely exceeds the
amount of genomic information that is sufficient for full biological
functionality in known small-genome angiosperms. Old and diverged
copies of retrotransposon elements seem to significantly contribute to
this fraction.
5) Compared to the scenario observed in angiosperms, LTR-retrotransposons
present an unusual degree of conservation across large taxonomic
divides. Whether the limited differentiation was caused only by the slow
mutation rate, by selective restrains or a combination of the two, is still
unclear. However, the extent of sequence conservation, which involves
a large fraction of the conifer genomes, rules out horizontal transfer
as a likely explanation and support the simple mechanism of vertical
transmission accompanied by a slow rate of molecular evolution.
6) Timing of insertions demonstrates the retention of very ancient
retrotransposon elements, which is a rare event in angiosperms. This
suggests a much slower turnover of repetitive features in gymnosperm
species, possibly caused by insufficient removal of redundant
elements.
402 Genetics, Genomics and Breeding of Conifers
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11
Future Prospects
Jeffrey F.D. Dean
ABSTRACT
Rapid advancement of the “omic” sciences has resulted from the recent
development of new technologies driven in large part by investment
from the human biomedical arena. Thus, in considering what might lie
ahead for conifer genomics, current and near-future genomic research
in human biomedicine provides a good yardstick. This chapter relates
current expectations for near-future development and improvements
in technology platforms for nucleic acid sequencing, high-throughput
genotyping, microarrays, and metabolomics. Subsequent discussion
covers how these new technologies might be used to improve our
understanding of fundamental conifer biology. Prospects for using the
findings from conifer genomics studies to address practical problems
for the forest products industry as well as problems related to the
health and management of conifer forests are also discussed. Finally,
some potential barriers to progress are noted along with suggestions
for overcoming them.
Keywords: advanced breeding; biofuels; bioinformatics; carbon
sequestration; comparative genomics; ecological genomics; forest
health; gene conservation; phylogenetics; secondary products;
technology platforms; third-generation sequencing
11.1 Introduction
Niels Bohr once said, “Prediction is very difficult, especially of the future”,
but in thinking about what possibilities the future might hold for improving
our understanding of conifer biology, particularly as viewed through the
twin lenses of genomics and bioinformatics, it might be better to think in
terms of a quotation widely attributed to the science fiction writer, William
Gibson: “The future is here. It’s just not evenly distributed yet.”
Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602,
USA; e-mail: jeffdean@uga.edu
Future Prospects 405
The modern field of genomics may be said to have originated with the
1988 National Research Council study entitled, “Mapping and Sequencing
the Human Genome” (NRC 1988), which laid groundwork for US
contributions to the international efforts that culminated in the release of
two draft human genome sequences in 2001 (IHGSC 2001; Venter et al. 2001).
From that start, the development and first application of novel genomic
technologies has been driven, not surprisingly, by the plans and needs of
human biomedical research. Over time, these futuristic technologies become
more evenly distributed, eventually finding their way to applications in
plant and agricultural research, including forestry and conifer biology. As a
result of this natural progression and redistribution, predictions and plans
set forth in the recent past to guide the application of genomics to future
biomedical research and now coming to fruition provide a realistic glimpse
of what we should expect ahead in the field of conifer genomics.
Collins et al. (2003) distilled the input from numerous meetings and panels
of experts in preparing a roadmap for future genomics research in human
biomedicine for the US National Human Genome Research Institute
(NHGRI). This chapter draws heavily on themes set forth in that report in
considering future directions and prospects for research in conifer genomics.
With this in mind, it may be useful to note some of the hopes for “quantum
leaps” mentioned in that report as technological touchstones to “provoke
creative dreaming” (Collins et al. 2003):
• the ability to determine a genotype at very low cost, allowing an
association study in which 2,000 individuals could be screened with
about 400,000 genetic markers for US$10,000 or less;
• the ability to sequence DNA at costs that are lower by four to five orders
of magnitude than the current cost, allowing a human genome to be
sequenced for US$1,000 or less;
• the ability to synthesize long DNA molecules at high accuracy for
US$0.01 per base, allowing the synthesis of gene-sized pieces of DNA
of any sequence for as little as US$10–20;
• the ability to determine the methylation status of all the DNA in a single
cell; and
• the ability to monitor the state of all proteins in a single cell in a single
experiment.
Some of these quantum leaps are well on their way to becoming fully
realized only six years after the Collins report was released, which serves
to emphasize just how fast the discipline is evolving and just how difficult
it is to make truly bold and insightful predictions.
406 Genetics, Genomics and Breeding of Conifers
11.2.4 Proteomics
Despite the fact that high-throughput proteomics analysis is significantly
more difficult than HTS, substantial improvements in the technology have
been made, as discussed elsewhere in this volume (see Chapter 8). However,
proteomics remains a technology platform that has not yet been fully
exploited with respect to conifer genomics. Efforts to integrate proteomic
analyses with genomic and phenomic data look promising for building
gene network models that can explain the emergence of specific phenotypes
(Gstaiger and Aebersold 2009). Such approaches should improve the
predictability of genotype-phenotype relationships, which, if applied to
conifers, would enable tree improvement programs to make more efficient
crosses and selections. There can be little doubt that the future will bring
greater activity in the area of proteomics with regard to conifer biology.
While advanced mass-spectrometry platforms are garnering the most
attention with respect to proteomics approaches in plant biology, protein
microarrays are just beginning to see application in these fields. A review
by Joos and Bachmann (2009) provides an overview of recent developments
in the use of protein microarrays. The use of a protein microarray to probe
mitogen-activated protein kinase (MPK) target networks in Arabidopsis
provides a fine example of how this technology could be used to study
signaling pathways in conifers (Popescu et al. 2009). Similarly, Espina et
al. (2009) coupled laser-microdissection of specific cell types with reverse-
phase protein microarrays to measure activated signal pathway molecules
in miniscule samples. It would be very interesting to see this approach
applied to the differentiation events in conifer xylem cells undergoing
transition from earlywood to latewood or normal to compression wood.
Protein microarrays have also started to gain some attention as a platform
Future Prospects 409
11.2.5 Metabolomics
As noted in reviews by Harrigan et al. (2007) and Fernie and Schauer (2009),
metabolomics is fast becoming a critical tool for the rapid assessment of
chemical characteristics that can be used as the basis for selective breeding.
Thus, metabolomic, transcriptomic, and phenotype profiles may all be
interrogated with respect to genetic markers in a segregating population
to identify valuable quantitative trait loci (QTLs) (Kliebenstein 2009b). And
even though these approaches have only been largely applied to a handful
of model species, their use for the study of naturally occurring variants is
providing new insights into plant adaptation (Alonso-Blanco et al. 2009;
Bundy et al. 2009). It will be truly illuminating to see the findings of such
studies when the organisms under study are perennial and genetically
diverse, keystone species, such as the conifers that dominate ranges as
large as the boreal forests of the Northern Hemisphere. The recent study
of metabolite profiles in Douglas-fir by Robinson et al. (2007), which
demonstrated a stronger link to environment than to genetics, suggests how
this type of research may have important implications for our understanding
of conifer biology.
Metabolomics and transcriptomics have also been fruitfully paired
as the basis for generating putative biosynthetic pathways and gene
networks through a correlative reasoning process sometimes called “guilt-
by-association” (Saito et al. 2008). This approach has started to see broad
application to the study of secondary products production in a variety of
species for which little or no genomic sequence information is available
(Yonekura-Sakakibara and Saito 2009). Studies in which this approach
is being used to probe the multitude of terpenoid and oleoresin defense
compounds produced in conifers are underway, and the results from such
studies will undoubtedly prove useful for the selection of trees that are
more resistant to insects and disease.
evolution (Jobson and Qiu 2008; Gommans et al. 2009). While progress has
been made in developing algorithms to predict RNA editing target sites in
silico (e.g., Du et al. 2009), one exciting prospect for the near future is the
potential to use the RNA sequencing capability of the Helicos sequencing
platform to directly detect modified nucleotides, such as inosine (Ozsolak
et al. 2009). Because the Helicos system can sequence RNA transcripts
directly and does not require prior cDNA production, it will almost certainly
identify previously unappreciated transcriptional products that can not be
efficiently reverse-transcribed. There is similar interest in the potential for
direct sequencing of methylated genomic DNA on the Helicos platform
as a means to study its role in epigenetic regulation of the genome (Gupta
2008; Ansorge 2009).
11.3.2.2 Phylogenetics
Conifers hold an important place in the evolution of seed plants, and for
many years were considered one of four distinct paraphyletic groups (the
others being Gnetales, Cycads, and Gingko) comprising the gymnosperms
(Doyle 1998; Palmer et al. 2004). However, placement of the Gnetales
within this group has always been controversial (as reviewed in Burleigh
and Matthews 2004). In recent years, molecular sequence data has led
researchers to propose some highly controversial rearrangements of older
phylogenies that were based solely on morphometric and developmental
process characters (Chaw et al. 1997, 2000; Bowe et al. 2000; Burleigh and
Mathews 2004). One of the most surprising of these new phylogenies places
the Gnetales as a sister group to the Pinaceae, fully within the Coniferales
(Gugerli et al. 2001). Although this particular phylogenetic topology has
gained acceptance in some quarters of the systematics community (Doyle
2006), several groups have urged caution because the proposed topologies
are highly sensitive to errors and bias in both the molecular and the
quantitative characters on which they are based (Rydin and Källersjö 2002;
Palmer et al. 2004). As an example of one potential source of error for DNA-
based phylogenies that may be confounding this case, work in loblolly pine
suggests the Pinaceae have somewhat lower rates of nucleotide substitution
than is typical of most other plants (Brown et al. 2004 and Chapter 5 in this
418 Genetics, Genomics and Breeding of Conifers
book), while the Gnetales have been characterized (at least in comparison to
other gymnosperms) as having relatively high rates of substitution (Chaw
et al. 1997; Burleigh and Mathews 2004). Differential rates of nucleotide
substitutions between species have been identified as a potential major
source of error for phylogenies based on DNA sequence information (Rydin
and Källersjö 2002). In another example, Won and Renner (2003) have
documented horizontal transfer of mitochondrial gene sequences between
members of the angiosperms and the Gnetales in the past five million years,
which calls into question characters based on mitochondrial DNA sequence
from these species, such as were used in the study by Gugerli et al. (2001).
Because sequence data is at present so sparse for plant species outside the
angiosperms, many taxonomists recommend reliance on morphological
and developmental characters of both extant and extinct species to establish
broad phylogenies, while limiting use of DNA sequence information to
tests of fine structure within well-recognized groups (Stace 2005; Hilton
and Bateman 2006). Given this evidence, increased taxon sampling and the
development of additional characters, both molecular and morphometric,
will be needed to clarify whether or not the Gnetales should be placed within
the Coniferales as a sister group to the Pinaceae or returned to their more
familiar phylogenetic position(s) somewhere outside the Coniferales. Efforts
to expand the DNA sequence information available for a broader spectrum
of the gymnosperms should help identify the rarer genomic changes that
can clarify broad-scale phylogenetic relationships (Rokas and Holland 2000;
Burleigh and Matthews 2004).
greater insight into the secondary product pathways and their function in
a wide variety of conifers.
Provocative experimental results have recently emerged concerning
the abilities of plants to recognize kin and how this impacts competition
for resources (e.g., Milla et al. 2009). None of the experiments reported
to date appear to have used genomic or even molecular approaches to
understand the genetic components of this phenomenon. Given the obvious
implications for conifer plantation forestry, particularly with the prospect
of using varietals (clonal) versus outcrossed seedlings, this would appear
to be a line of investigation that could produce interesting and practical
results if applied to conifers.
Soil ecology plays a fundamental role in the sustainability of forest
ecosystems, but the research on rhizosphere dynamics in forests has so
far been insufficient to provide a predictive level of understanding of how
rhizosphere communities operate and interact with host trees (Johnston and
Crossley 2002). Transcriptomic, proteomic and metagenomic techniques
are currently being applied to rhizosphere characterization from the level
of single cells to entire microbial communities (Sorensen et al. 2009). The
recent completion of a genome sequence for the ectomycorrhizal fungus,
Lacaria bicolor (Martin et al. 2008), opens the door to a multitude of genomic
approaches for studies of the ectomycorrhizal interactions between
conifers and these beneficial fungi (Martin and Nehls 2009). Similarly, the
completion of genome sequences for a variety of phytopathogens sets the
stage for increased studies of the molecular mechanisms that fungi employ
for causing diseases of conifers (Bhadauria et al. 2009). Tan et al. (2009)
have recently reviewed the impact that transcriptomic, proteomic and
metabolomic technologies are having on fungal phytopathology research
(Tan et al. 2009).
Conifers, like other organisms, benefit from an internal ecosystem.
We are only just beginning to appreciate the importance of these internal
ecosystems for organismal biology, and the National Institutes of Health
(NIH) recently launched the Human Microbiome Project to identify and
characterize the roles that endosymbiotic organisms play in human health
(Phillips 2008; Proal et al. 2009). Endosymbiotic bacteria and fungi also
comprise complex communities in plants (Rosenblueth and Martinez-
Romero 2006; Andreote et al. 2009), and have been recognized as the
source of a variety of bioactive compounds whose formation was once
attributed solely to plant metabolism (as noted in Guo et al. 2008). There
have been studies describing endosymbiont communities in conifers
(Chanway et al. 2000; Izumi et al. 2008); and in one recent piece of work,
endosymbiont inoculation was demonstrated to help protect western
white pine (P. monticola) against the pathogen that causes white pine blister
rust (Cronartium ribicola) (Ganley et al. 2008). Just as the latest genomic
420 Genetics, Genomics and Breeding of Conifers
technologies for microarrays and DNA sequencing are opening new areas of
research into human-microflora interactions (Huyghe et al. 2008; Guazzaroni
et al. 2009; Petrosino et al. 2009), so too are these technologies likely to
modify our perception and appreciation of the microflora that inhabit the
interior and exterior of conifers.
11.4.1.4 Biofuels
After a recent series of oil price hikes, the likes of which had not been seen
since the early 1970s, there has again been renewed interest in the potential
424 Genetics, Genomics and Breeding of Conifers
specifically in the roots of Populus trees, which the authors felt might have
potential for improving carbon sequestration.
organize, and query the data and the biologists who frame the questions and
attempt to interpret the output from datamining exercises (Lee et al. 2009a).
For bioinformaticians, Baxter et al. (2006) have provided concise and cogent
advice on preparations for new software projects, and Bolchini et al. (2009)
describe how usability metrics can be used to evaluate and improve software
interfaces. At the same time, biologists are reminded that the language and
thought processes employed by bioinformatics professionals are in many
ways distinct from those of biological researchers, and it is imperative that
they take this into consideration as they share information and ideas during
software or query development (Penders et al. 2008).
provenance that will grow in a specific locale. Even if these parameters can
be satisfied, it will be 10 to 20 years before the effect can be verified, not to
mention any profit realized. The assumption that great profits will result
from the wholesale patenting of tree genes is bankrupt from the outset.
Thus, to the extent that researchers can overcome reflexive self-interest and
release datasets to the public repositories, we will all gain from speedier
advancement of genomic science.
11.5.3 Annotation
Attwood (2000) noted some time ago that our ability to make sense of
functional genomics data was being compromised by poor annotation and
conflicting nomenclature. This is not a problem that will disappear rapidly,
given the exponential growth of new sequence information, the retention
of legacy datasets and databases, and the tight state of research funding.
Some intermediary databases and software tools have been developed to
address the problem in part (Benoit 2005), while some have proposed new
conventions for naming proteins and genes that would eliminate many
of the uncertainties that now exist (Schluter et al. 2009). Yet problems
would still exist because pipelines developed to handle gene annotation
in an automated fashion so often draw their information from the public
repositories in which poorly annotated legacy genes reside (Liu et al. 2008;
Liang et al. 2009). At some point in the near future, the conifer genomics
community will need to establish a supervised database of high-quality
gene models that are manually curated with respect to their assigned names
and functions.
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438 Genetics, Genomics and Breeding of Conifers
Chapter 3
a) b)
Figure 3-1 Fluorescent in situ hybridization images of ribosomal DNAs (18S-28S rDNA, 5S
rDNA) and telomere (ATRS) probes on Pinus echinata somatic metaphase chromosomes: a)
superimposed images of DAPI, Cy3 (red signals, 18S and 5S rDNA sites) and FITC (green
signals, ATRS sites) filters; b) super imposed images of DAPI and FITC filters.
Figure 3-2 Diagrammatic representation of 18S and 5S rDNA loci in different Pinaceae genera.
All 18S and 5S rDNA patterns reported in the less extensively studied genera (i.e., Picea, Abies,
Pseudotsuga and Larix) are present in the more extensively studied genus Pinus (subgenera,
Pinus and Strobus).
Color Plate Section 451
Figure 3-3 Fluorescent in situ hybridization image of Pinus taeda somatic metaphase
chromosomes probed with 18S-28S rDNA (red signals) and Arabidopsis-type telomere repeat
sequence (green signals). Numbers from 1 to 12 enumerate homologous chromosome pairs.
The ideogram of Pinus taeda in the right hand side box is based on 108 readings of each
measurement (see Islam-Faridi et al. 2007 for details).
452 Genetics, Genomics and Breeding of Conifers
Chapter 5
Figure 5-3 Comparison of homologous linkage groups between white spruce (Picea glauca)
and black spruce (species complex Picea mariana × P. rubens).
Color Plate Section 453
Chapter 6
Figure 6-1 Nucleotide diversity estimates for all and silent (synonymous and noncoding)
sites, and number of base pairs per SNP for studies where 10 or more loci were studied. The
number of loci is in parentheses. Estimates for Pinus taeda and Pinus sylvestris were averaged
for two or more studies. See Table 6.1 for references.
<0.01 <0.01
<0.001 <0.001
<0.0001 <0.0001
Figure 6-2 LD plots for dhn1 (left) and sod-chl (right) candidate genes for drought in loblolly
pine. A LD block is apparent in the lower right part of dhn1 while LD is distributed more
regularly in sod-chl.
454 Genetics, Genomics and Breeding of Conifers
Chapter 8
Figure 8-1 Histogram of cluster size’s from NCBI Unigene database (builds from Table 2A).
Chapter 10
Figure 10-1 Selection of plant genome sizes. Genome size is provided for a subset of species
that are object of genomic sequencing. Size information is from NCBI (http://www.ncbi.nlm.
nih.gov/genomes/static/gpstat.html) with the exception of gymnosperm data from the Kew Plant
DNA C-values Database (http://data.kew.org/cvalues/).
Color Plate Section 455
Figure 10-2 Estimate of Norway spruce genome composition. Principal genomic components
of Norway spruce genome estimated based on the annotation of a 9 Mbp small insert genomic
library (E. De Paoli et al. unpubl. data).
456 Genetics, Genomics and Breeding of Conifers
Figure 10-3 Annotation of four genomic regions (BAC) randomly selected from the genome
of Norway spruce. Green boxes, LTR-retrotransposons of the copia superfamily; yellow boxes,
LTR-retrotransposons of the gypsy superfamily; light-blue boxes, LTRs of copia elements;
dark-blue boxes, LTRs of gypsy elements; white boxes, uncharacterized LTR-retrotransposons
(with black LTRs when annotated), grey boxes, non-autonomous LTR-retrotransposons
(with dark-grey LTRs); black box, non-LTR retrotransposon. The red bar indicates repetitive
sequences; PARE, Picea abies repetitive element; GY, gypsy; CO, copia; UN, unidentified; NA,
non-autonomous. From E. De Paoli et al. (unpubl. data).
ABOUTABOUT ABOUT
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reviewing Editors
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genetics, genomics
genetics, genomics and
genetics, genomics and breeding of conifers. breeding
and breedingof conifers.
of conifers. Christophe Plomion • Jean Bousquet
Christophe
Christophe Plomion Plomion
• Jean • Jean Bousquet
Bousquet
ABOUTABOUT ABOUT
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EDITORSEDITORS
THE EDITORS Chittaranjan KoleKole
Chittaranjan
Chittaranjan Kole
Christophe
Christophe Christophe Plomion
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and Plant and Breeding
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AgroCampus
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Ouest, Rennes, Rennes,
Ouest, France. France.
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“Forest, Grassland
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and Fresh Fresh
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unit
in Bordeaux,Bordeaux,
in Bordeaux,
France.France. France.
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and Research
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in Forest in
and and and
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at Laval University
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is co-director theofspruce
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genomics genomicsgenomics
projectproject ARBOREA.
project
ARBOREA. ARBOREA.
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Christophe Plomion
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Chittaranjan Kole
Jean Bousquet
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Bousquet
Science
Science Publishers
Science
Publishers Publishers
Plomion
Plomion
Kole
Science
Science Science
Publishers
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