Transcript Document
Discovery of Genes for Improved Cellulose and Cellulose-Extractability from Poplar Secondary Xylem David B. Neale1, Brian J. Stanton2, Mark F. Davis3, Chung-Jui Tsai4, Jill L Wegrzyn1, Andrew J. Eckert1 2Genetic 1Department of Plant Sciences, University of California at Davis, Davis, CA, Resources Conservation Program, Greenwood Resources, Portland, OR, 3National Renewable Energy Lab, Golden, CO 4School of Forest Resouces, Michigan Technical University, Hougton, MI INTRODUCTION The DOE’s “Breaking the Biological Barriers to Cellulosic Ethanol” report identifies poplar as one of the key feedstock species for cellulosic ethanol production in many regions of the country. The goal was to perform SNP discovery using high throughput DNA sequencing (Agencourt Biosciences) and SNP genotyping (Illumina) to associate genetic variation in genes involved in cellulose and lignin biosynthesis with phenotypic variation in cellulose quantity, quality and extractability in a large clonal black cottonwood (Populus trichocarpa) genetic test plantation belonging to GreenWood Resources. A set of 40 genes known to be highly expressed and associated with the desired phenotypes were sequenced using a panel of 15 unrelated poplar clones. Genomic sequences of ~179,000 bp covering the entire proteincoding regions, including introns, and 1,000bp upstream and 300bp downstream were retrieved from JGI. 200 non-overlapping amplicons were selected to cover the length of the genes and were sequenced by Agencourt in both directions and submitted to an automated pipeline developed in-house for sequence alignment and SNP discovery. Utilizing Illumina’s Golden Gate Assay, 456 poplar clones were genotyped for ~1536 SNPs. Wood samples were collected from 1,100 trees and came from the 456 poplar clones. High such as lignin, cellulose, and hemicellulose on the cores collected. Association genetics analysis will be used to identify genes controlling cellulose quantity and quality phenotypic variation in poplar. PROJECT GOALS Resequence 40 candidate genes using a discovery panel of 15 unrelated poplar clones Identify SNPs in the 40 genes using an automated alignment and SNP calling bioinformatics pipeline SNP genotype 456 poplar clones for ~1536 SNPs (Illumina Golden Gate assay) Harvest wood increment cores from 2-3 ramets of each of 456 poplar clones (1100 trees in total) Molecular Beam Mass Spectrometry (MBMS) analysis on all 1100 wood cores to develop secondary xylem metabolomic profiles Association genetics analyses to identify genes controlling cellulose quantity and quality phenotypic variation in poplar Gene Family phenylalanine ammonia -lyase (PAL) cinnamate 4 -hydroxylase (C4H) 4-coumarate:CoA ligase (4CL) hydroxycinnamoyl -CoA quinate/shikimate hydroxycinnamoyltransferase (HCT) coumarate 3 -hydroxylase (C3H) ferulate 5 -hydroxylase (F5H) caffeate O -methyltransferase (COMT) caffeoyl CoA O -methyltransferase (CCoAOMT) cinnamoyl -CoA reductase (CCR) cinnamyl alcool dehydrogenase (CAD) laccase (LAC) alpha -tubulin (TUA) beta-tubulin (TUB) cellulose synthase (CesA) sucrose synthase (SUSY) cellulase (KOR) glycine decarboxylase complex, H subunit (gdcH) glycine decarboxylase complex, T subunit (gdcT) S-adenosylmethionine synthetase (SAMS) Serine hydroxymethyltransferase (SHMT) Gene Names PAL2, PAL4, PAL5 C4H1, C4H2 4CL1, 4CL3, 4CL5 HCT1, HCT6 C3H3 F5H1, F5H2 COMT1, COMT2 CCoAOMT1, CCoAMT2 CCR CAD LAC1a, LAC2, LAC90a TUA1, TUA5 TUB15, TUB9, TUB16 CesA1A, CesA2A, CesA1B, CesA2B, CesA3A SUSY1 KOR1 gdcH1 gdcT2 SAMS1 SHMT1, SHMT3, SHMT6 mRNA sequences were used to direct custom software to use 1000 bp upstream along with intronic sequence from the poplar genome 517 primers were designed across 40 genes 202 non-overlapping primers were finally selected based on quality score Goal: Fully re-sequence 40 candidate genes to facilitate SNP discovery ~ between 3 and 12 amplicons/gene ~ forward and reverse sequencing Autosampler Mass spectrometer Transfer line and interface 48 sample tray cell wall chemistry Automated alignment and SNP calling pipeline processes the re-sequenced amplicons from Agencourt Total Amplicons = 202 Total SNPs from 40 Candidate Genes = 946 Average SNPs/Amplicon ~ 7 Additional SNPs selected from remaining genes of interest to complete the 1536 Illumina Golden Gate Assay.. .High-throughout phenotyping performed with pyrolysis molecular beam mass spectrometry to analyze wood chemistry lignin hemicellulose cellulose High-throughput alignment and automated SNP calling pipeline