Next generation sequencing of the Salix purpurea genome and transcriptome: Tools for the genetic improvement of willow biomass crops
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1 Next generation sequencing of the Salix purpurea genome and transcriptome: Tools for the genetic improvement of willow biomass crops Larry Smart, Michelle Serapiglia, Fred Gouker Cornell University, NYS Ag Experiment Station, Geneva, NY Chris Town and Haibao Tang J. Craig Venter Institute, Rockville, MD Jerry Tuskan, Kerrie Barry, and Erika Lindquist DOE Joint Genome Institute, Walnut Creek, CA Ed Buckler, Sharon Mitchell, and Rob Elshire USDA-ARS, Institute for Genomic Diversity, Cornell University Steve DiFazio and Eli Rodgers-Melnick West Virginia University, Morgantown, WV
2 Shrub willow (Salix spp.) bioenergy crops
3 Shrub willow biomass is a feedstock for biopower, liquid biofuels, and heat Harman Wood Pellet Stove VeraSun Station, Brookings, SD McNeil Generating Station, Burlington, VT Photo: US DOE
4 Site preparation Willow Biomass Production Cycle Three years old after coppice Planting One year old after coppice First-year growth Winter coppice Regrowth after coppice
5 Willow Biomass Production Cycle Three years old after coppice Winter harvest At least 7 harvests Fertilizer: ~100 lbs N per acre after every harvest (every 3 years) Regrowth after coppice One year old after coppice
6 Mean Yield of Top Five Clones in Yield Trials Selections from Toronto breeding and natural collections First harvest three-years post-coppice oven dry tonnes ha -1 yr SV1 0 Sheridan NY '98 Tully NY '93 Massena NY '93 Peter's Tract DE '98 Wolcott NY '98 Burlington VT '97 Canastota NY '98
7 Clones in yield trials:
8 Crosses attempted to capture hybrid vigor Compilation of results from US, Canadian, and European breeding programs
9 Intra- and Inter-specific Hybridizations F 1 S. integra S. viminalis S. cordata S. nigra S. sachalinensis S. cordata x S. eriocephala S. purpurea x S. eriocephala S. purpurea x S. viminalis S. purpurea x S. sachalinensis S. purpurea x S. gilgiana S. viminalis x S. miyabeana S. viminalis x S. eriocephala S. x dasyclados x S. miyabeana S. x dasyclados x S. eriocephala S. x dasyclados x S. viminalis S. eriocephala x S. purpurea S. matsudana x S. alba S. sachalinensis x S. eriocephala S. sericea x S. purpurea S. sericea x S. eriocephala S. sericea x S. sachalinensis S. discolor x S. cinerea S. discolor x S. eriocephala F 1 and F 2 S. purpurea S. eriocephala S. miyabeana S. sachalinensis x S. miyabeana S. purpurea x S. integra Multi-species hybrids S. viminalis x (S. sachalinensis x S. miyabeana) S. viminalis x (S. purpurea x S. miyabeana) S. viminalis x (S. viminalis x S. miyabeana) S. purpurea x (S. sachalinensis x S. miyabeana) S. purpurea x (S. purpurea x S. miyabeana) S. purpurea x (S. viminalis x S. miyabeana) (S. sachalinensis x S. miyabeana) x S. purpurea (S. sachalinensis x S. miyabeana) x S. miyabeana (S. sachalinensis x S. miyabeana) x S. viminalis (S. viminalis x S. schwerinii) x S. cinerea (S. sachalinensis x S. miyabeana) x (S. purpurea x S. miyabeana) (S. sach x S. miya) x (S. viminalis x (S. schwerinii x S. viminalis))
10 Mean Yield of Top Five Clones in Yield Trials New varieties contribute to 38% greater yield overall oven dry tonnes ha -1 yr Sheridan NY '98 Tully NY '93 SV1 Massena NY '93 Peter's Tract DE '98 Wolcott NY '98 Canastota NY '98 Burlington VT '97 Tully NY '05 Belleville NY '05 Constableville NY '06 Waseca MN '06 Middlebury VT '07 Boisbriand QC '07
11 Clones in yield trials: Dennis Rak Double A Willow Photo by Larry Abrahamson
12 Flow Cytometric Estimation of Nuclear DNA Content Clone Species (pg/2c) Diploid Björn S. schwerinii x S. viminalis 0.76 Olof S. viminalis x (S. schwerinii x S. viminalis) 0.80 Jorr S. viminalis 0.83 P63 S. integra S. purpurea 0.94 SV1 S. x dasyclados 0.94 Triploid Nimrod (S. schwerinii x S. viminalis) x S. udensis 1.15 Terra Nova (S. triandra x S. viminalis) x S. udensis 1.11 Tully Champion S. viminalis x S. miyabeana 1.31 Oneida S. purpurea x S. miyabeana X S. viminalis x (S. viminalis x S. miyabeana) 1.26 Tetraploid SX64 S. miyabeana 1.65 SX61 S. sachalinensis (S. udensis) 1.65 Canastota S. sachalinensis x S. miyabeana X S. purpurea x (S. purpurea x S. miyabeana) 1.87 Pentaploid? 05X S. miyabeana x (S. purpurea x S. miyabeana) 2.17
13 2008 Genetic Selection Trial - Geneva, NY 24-plant plots, 3 replicates, 76 clones Biomass harvested from middle 8 plants in Dec Dry biomass (Mg ha -1 yr -1 ) new clones ranked higher than SX61 Top clone produced 21% greater yield 0 Putative TRIPLOIDS (3X) DIPLOIDS (2X) TETRAPLOIDS (4X) Putative PENTAPLOIDS (5X)
14 Macrosynteny between Populus and Salix Berlin et al. (2010) BMC Genomics 11:129
15 Sequencing of the Salix purpurea L. genome Whole genome shotgun sequencing of clone ID diploid female (n = 19, ~485 Mb) - underway at DOE JGI (PIs: Tuskan, Smart, Town) Genomic data to date: Mbp 454 standard - 19 Gb Illumina 76 bp standard - 3 Gb of 76 bp Illumina from 7 kbinsert jumping library - Waiting for ~20 Gb of Illumina 150bp from fragment libraries (250bp, 500bp, 4 kb) Two 5X BAC libraries constructed (third on the way)
16 Draft de novo genome assembly Using CLC de novo assembly tool Number of assembled sequences 290,871 Residue counts: Number of N's % Total 327,266,967 (327 Mbp) Sequence lengths: Minimum 200 Maximum Average 1125 N (50% of bases are in contigs <2224 bp, this is too small) This is a quick and dirty assembly. We will try to reassemble using ALLPATHS-LG and/or Celera Assembler when we have additional Illumina data
17 Comparison to poplar genome Willow assembled size: 327 Mb (real bases) Poplar assembled size: 404 Mb (real bases) Compared willow assembly against poplar genome using LASTZ (like BLAST, nucl based) 65% of the P. trichocarpa genome (261.9 Mb) aligned to the draft S. purpurea genomic assembly: - sequence identity: 88% (coding and non-coding)
18 Transcriptome sequencing Salix purpurea transcriptome data to date: - 5 lanes Illumina 2x76 from 8 tissue libraries done at JGI Shoot tip 44.0 M reads Leaves Day: 16.4 M Nite: 15.3 M Drought: 13.6 Stem Node:18 M reads Internode:21.5 M Roots 51.7 M reads Catkins 46.1M reads Salix sachalinensis SX61 : stem (8.3 M), shoot tip (7.7 M) Salix miyabeana SX64 : stem (6.2 M), shoot tip (7.4 M) - these are tetraploid, sequencing done at JCVI
19 Salix purpurea RNA-Seq assembly All reads from the same genotype were pooled and assembled for S. purpurea 94006, S. sachalinensis SX61, and S. miyabeana SX64. Transcript assembly using CLC assembler, for S. purpurea Number of assembled sequences 201,926 Residue counts: Total 79,555,995 (80 Mb) Sequence lengths: Minimum 100 Maximum Average 394 N % of the poplar transcripts have at least half of their lengths aligned to the willow transcripts S. purpurea coding sequence vs. poplar: 92% (genomic: 88%) S. purpurea coding seq vs. S. sach or S. miya: 98%
20 Salix purpurea transcriptome: GBrowse Salix purpurea de novo transcript assembly aligned to Populus trichocarpa v.2.0 as the reference genome
21 High-density markers: GBS 96-plex protocol Digest DNA with EcoT22I (6-base) or ApeKI (5 base-cutter) Ligate barcoded adapters (may be done simultaneously) Rob Elshire, Sharon Mitchell, Ed Buckler Elshire et al. (2011) PLoS One Pool DNAs Sequence on Illumina Platform ~200,000 reads per sample 75, ,000 SNPs Flowcell 8 channels PCR Primers Solid Phase Oligos Clean-up Optimize fragment sizes & adapter ligation
22 Next generation sequencing of the Salix purpurea genome and transcriptome: Tools for the genetic improvement of willow biomass crops Summary Excellent hybrid pedigrees have been produced and new hybrid combinations are being explored Each round of breeding has generated cultivars with ~20% improved yields over previous standards Triploid progeny have shown hybrid vigor for yield and low fertility A draft sequence of the Salix purpurea genome is nearly complete High density GBS markers are being developed
23 Thanks to
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