DEVELOPMENT OF OPTIMIZED FIBER FEEDSTOCKS THROUGH APPLIED GENOMICS MICHAEL DEYHOLOS UNIVERSITY OF BRITISH COLUMBIA (OKANAGAN CAMPUS)

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Solomon Short
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1 DEVELOPMENT OF OPTIMIZED FIBER FEEDSTOCKS THROUGH APPLIED GENOMICS MICHAEL DEYHOLOS UNIVERSITY OF BRITISH COLUMBIA (OKANAGAN CAMPUS)

2 OBJECTIVE: Develop novel flax varieties to support development of advanced composites from natural fibers.

3 FLAX FIBERS ARE PHLOEM (BAST) FIBERS xylem (wood, hurd) phloem (bast) stem cross-section

4 BAST FIBER CELL WALL PROPERTIES Properties long (5 80mm) strong (800MPa) thick secondary walls >70% cellulose <2% lignin low S/G ratio low mf angle similar to gelatinous type walls of tension wood

5 CELL WALL STRUCTURE glucose hemicelluloses pectins, lignin crystalline cellulose MFA amorphous cellulose ultimate fiber cellulose microfibrils cellulose molecules

6 BAST FIBERS IN COMPOSITES advantages: high strength/weight low abrasion renewable biodegradable(?) issues: performance supply

7 BAST FIBERS IN COMPOSITES issues: performance supply

8 FLAX IS GROWN FOR EITHER SEEDS OR FIBERS linseed fiber flax abbeybotanicals

9 FLAX IS GROWN FOR EITHER SEEDS OR FIBERS linseed fiber flax J.F. Millett Charles Curran Henry Scott Tuke paints & finishes, linoleum ALA (omega-3 = 18:3 = α-linolenic acid) textiles, fine paper long, strong fibers highly crystalline cellulose, low lignin

10 CANADA GROWS ONLY LINSEED linseed 2.4 Mha fiber flax 0.4 Mha others Egypt others USA Ethiopia Canada China Russia Belarus France China India Crop area harvested (Mha, FAO 2008)

11 EXTRACTABILITY LIMITS SUPPLY Saskatchewan, October 2009 cold, usually dry Ken Jackle Normandy, July 2009 warm, moist conditions allow retting: microbial degradation of pectins

12 YIELD LIMITS SUPPLY fiber flax linseed Sushmita Nandy & Gordon Rowland

13 LINSEED FIBERS HAVE NEGATIVE VALUE tractor fire caused by linseed straw Ryan Adams

14 TUFGEN (TOTAL UTILIZATION OF FLAX GENOMICS) Whole genome sequence Physical maps SSRs SNPs ESTs L. usitatissimum 81% N 50 scaffold = 694kb 407 accessions resequenced L. bienne (draft & 125 accessions) ~99% coverage, 43,776 clones 1,066 markers 1,755M discovered 286,852 S. Cloutier G. Rowland C. Barker F. You R. Datla B. Soto unigenes 43,384 EMS mutants >16,000 lines

15 GENE EXPRESSION ANALYSIS APEX A 4-5 cm whole stem stem peel no apical sections B 12-13cm C cm D cm E cm 15

16 CHARACTERIZING GENE & PROTEIN FUNCTION galactan rich β-galactosidase (BGAL) T. Gorshkova (Kazan) crystalline cellulose rich

17 COMPARATIVE GENOMICS ~25-30 Mya ~5-9 Mya Cronk, Sveinsson, Grassa, Geraldes (UBC)

18 FEEDSTOCK IMPROVEMENT TARGETS yield extractability quality matrix adhesion strength uniformity application specific traits

19 FEEDSTOCK IMPROVEMENT TARGETS yield extractability quality matrix adhesion strength uniformity NOVEL GERMPLASM application specific traits phenotyping at FibreCity Composites Innovation Centre Winnipeg, MB

genetics: screen for phenotype of interest novel germplasm M 1 M 2 M 3 grown

20 NOVEL GERMPLASM (FORWARD & REVERSE GENETICS) EMS collect seed from individuals collect seed and DNA from individuals store seeds forward genetics: screen for phenotype of interest novel germplasm M 1 M 2 M 3 grown in field grown in lab store DNA reverse genetics: screen for mutation in sequence of interest

21 2-6% lignin / CWR S/G 0.17 EMS MUTANTS WITH LIGNIFIED BAST FIBER WT mut 14-21% lignin / CWR S/G 0.37 Hawkins, Chantreau (U Lille)

REVERSE GENETICS: THREE APPROACHES AMPLICON SEQUENCING EXOME CAPTURE WHOLE GENOME RE-SEQ PCR amplify 250bp target regions from barcoded pools of mutant DNA ~5 genes, 768

22 REVERSE GENETICS: THREE APPROACHES AMPLICON SEQUENCING EXOME CAPTURE WHOLE GENOME RE-SEQ PCR amplify 250bp target regions from barcoded pools of mutant DNA ~5 genes, 768 individuals per sequencing run capture ~250bp target regions using custom oligos ~40,000 genes, <24 individuals per seq run no complexity reduction >40,000 genes, 1 individuals per lane

23 REVERSE GENETICS: RESULTS Average number of genes mutated per line: 73 genes: very deleterious non-sense, splice-site and frameshift mutations 349 genes: deleterious non-synonymous mutations 0.05 > SIFT score 68 genes: weakly deleterious 0.05 < SIFT score < ,054 mutations: neutral non-synonymous with SIFT score > 0.1, synonymous, introns, intergenic 20 mutations/mb ~7500 /line

24 CONCLUSIONS: Natural fibers (especially flax) have unique advantages in composites TUFGEN and related programs have generated resources sufficient to address quality & supply issues in feedstock genomic knowledge + mutant population can generate novel germplasm through reverse & forward genetics