This document summarizes research annotating cytochrome P450 CYP720B genes in white spruce (Picea glauca). The CYP720B gene family is involved in conifer defense and was previously studied in Sitka spruce. The researcher identified CYP720B transcripts in white spruce RNA-seq data and genome assembly by aligning Sitka spruce CYP720B sequences. Some genes were found to be expressed in both white and Sitka spruce, while others were only expressed in one or had partial sequences identified.
3. Cytochrome P450
✤ Enzymes involved in
biosynthesis and
metabolism
✤ Catalyze the oxidation of
organic compounds
✤ Contain a heme cofactor
✤ Primarily membrane
bound
3
4. CYP720B gene family
http://en.wikipedia.org/wiki/Mountain_pine_beetle
✤ Conifer-specific gene family
✤ Involved in the biosynthesis of
diterpene resin acids, important in
conifer defence against insects and
fungus
http://en.wikipedia.org/wiki/Resin 4
5. CYP720B in sitka spruce
Picea sitchensis
✤ Twelve CYP720B genes
identified in sitka spruce http://en.wikipedia.org/wiki/Picea_sitchensis
✤ Full-length transcript
sequences available
✤ Use these sequences to
identify orthologs in white
spruce (Picea glauca)
Katrin Geisler 5
6. White spruce sequencing data
Picea glauca
✤ RNA-seq of eight tissues:
bark, embryo, flush bud,
mature needle,
megagametophyte,
seedling, xylem
and young bud
✤ Whole genome sequencing
24 lanes of Illumina HiSeq
for 64-fold coverage
Hamberger, Ohnishi et al. (2011) Plant Phys. 157: 1677-1695
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7. Identify white spruce transcripts
✤ Assemble the white spruce RNA-seq data
usingTrinity (Grabherr et al. 2011) (assembled by Mack Yuen)
✤ Identify the white spruce CYP720B transcripts by aligning
the sitka spruce transcripts to the white spruce RNA-seq
contigs using BWA-SW (Li and Durbin 2010)
✤ Cluster the sitka spruce and white spruce transcripts
using CLC Genomics Workbench
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9. Assemble the white spruce genome
✤ Assemble the white spruce genome sequence using
ABySS (Simpson et al. 2009)
✤ Assembled 8.4 billion reads using 996 processors on 83
machines with an aggregate 4 TB of RAM
✤ Assembled 18 Gbp in 5 million scaffolds larger than 500
bp with a scaffold N50 of 6 kbp
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10. Identify white spruce genes
✤ Identify CYP720B genomic contigs by aligning the sitka
spruce and white spruce transcripts to the white spruce
genome assembly using BWA-SW
✤ Align the sitka spruce and white spruce transcripts to the
CYP720B genome contigs using gmap (Wu and Watanabe 2005)
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11. CYP720B4
✤ Expressed in both sitka spruce and white spruce
White spruce
Sitka spruce
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12. CYP720B7
✤ Expressed only in sitka spruce
White spruce
Sitka spruce
12
13. CYP720B15/16/17 related
✤ White spruce transcripts align, but no sitka spruce transcripts
White spruce
Sitka spruce
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14. Cytochrome P450 CYP720B Genes
Sitka spruce White spruce White spruce
EST RNA-seq genome assembly
CYP720B2 yes yes
CYP720B4 yes yes
CYP720B5 yes yes
CYP720B7 no yes
CYP720B8 no partial
CYP720B9 no yes
CYP720B10 no partial
CYP720B12 yes yes
CYP720B15 yes partial
CYP720B16 no partial
CYP720B17 no partial 14