2012-11-23

Annotating the cytochrome P450 CYP720B gene family in white spruceShaun Jackman

http://en.wikipedia.org/wiki/Picea_glauca

1

White sprucePicea glauca

http://en.wikipedia.org/wiki/Picea_glauca 2

Cytochrome P450

✤ Enzymes involved in biosynthesis and metabolism

✤ Catalyze the oxidation of organic compounds

✤ Contain a heme cofactor

✤ Primarily membrane bound

3

CYP720B gene family

✤ 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/Mountain_pine_beetle

4http://en.wikipedia.org/wiki/Resin

CYP720B in sitka sprucePicea sitchensis

✤ Twelve CYP720B genes identified in sitka spruce

✤ Full-length transcript sequences available

✤ Use these sequences to identify orthologs in white spruce (Picea glauca)

http://en.wikipedia.org/wiki/Picea_sitchensis

Katrin Geisler 5

White spruce sequencing dataPicea glauca

✤ RNA-seq of eight tissues:bark, embryo, flush bud, mature needle, megagametophyte, seedling, xylemand young bud

✤ Whole genome sequencing24 lanes of Illumina HiSeq for 64-fold coverage

6Hamberger, Ohnishi et al. (2011) Plant Phys. 157: 1677-1695

Identify white spruce transcripts

✤ Assemble the white spruce RNA-seq datausingTrinity (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

7

8

White spruce CYP720B transcriptsSitka spruceWhite spruce

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

9

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)

10

CYP720B4

11

✤ Expressed in both sitka spruce and white spruce

Sitka spruceWhite spruce

CYP720B7

12

✤ Expressed only in sitka spruce

Sitka spruceWhite spruce

CYP720B15/16/17 related

13

✤ White spruce transcripts align, but no sitka spruce transcripts

Sitka spruceWhite spruce

Cytochrome P450 CYP720B Genes

Sitka spruceEST

White spruce RNA-seq

White spruce genome assembly

CYP720B2 yes yesCYP720B4 yes yesCYP720B5 yes yesCYP720B7 no yesCYP720B8 no partialCYP720B9 no yesCYP720B10 no partialCYP720B12 yes yesCYP720B15 yes partialCYP720B16 no partialCYP720B17 no partial 14

15