Major Thrust: Genomics January 14, 2013 Santosh Kumar Agriculture and Agri-Food Canada & University of Manitoba, Winnipeg, Manitoba, Canada
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1 Total Utilization Flax GENomics (TUFGEN) Major Thrust: Genomics January 14, 2013 Santosh Kumar Agriculture and Agri-Food Canada & University of Manitoba, Winnipeg, Manitoba, Canada
2 Flax: versatile total utilization crop
3 Flax seeds have a unique fatty acid composition 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% flaxseed oil canola oil soybean oil sunflower oil corn oil cottonseed oil peanut oil safflower oil palm oil olive oil lard butter coconut oil saturated fat oleic acid Linoleic acid linolenic acid Source: Canola Council of Canada
4 Flax competition Competition Flax Canola Yield (t/ha) 1.35 ( ) 1.9 Area ( 000 Ha) 273 ( 22%) 7,471 ( ) Production 368 ( 13%) 14,493 ( ) Price ($/T) Export Forecast ( 000 T) 410 ( ) 8,729 ( 23%) Agriculture and Agri-Food, Canada, 2012
5 The genomic approach of the TUFGEN team Exploration Understand the genetics of the traits Develop markers linked to the traits Characterize genes responsible for the traits Application Improve breeding efficiency Improve flax as a crop (yield and oil content for example) Develop new products from flax fiber Develop new markets Increase yield Increase production Increase value
6 Examples of traits under study Seed yield Seed quality Fibre yield Pods/plant Fatty acid Height Seeds/pod Palmitic acid Lodging Seed size Stearic acid Straw weight Seed weight Linoleic acid Cellulose Oil content Linolenic acid Cell wall Lignin Shive Straw moisture
7 Trait Complexity Affects Breeding Strategies Trait Mendelian Quantitative Heritability High Low Environment Stable GxE Breeding Fast, easy Long term, difficult example Seed color yield For quantitative traits affected by the environments and with potential GxE: QTL or AM analyses
8 5 QTL on a linkage group Lu2286 Lu2287 Lu2076 Lu2235 Lu2230 Lu2237 Lu2239 Lu2031 Lu2054 Lu2043 Lu2981 Lu851 Lu2984 Lu1049 Lu833 Lu2983 Lu3113 Lu207 Lu989 Lu2943 Lu2396 Lu2399 Lu2207 Lu3252 Lu2059 Lu2025 Lu2008 Lu3228 Lu3229 Lu722B Lu2073 Lu2006 Lu2004 Lu2002 Lu2968 Lu3281
9 Genetic mapping CDC Bethune X Macbeth Viking X E1747 F 1 F 1 SSD SSD F 6 F 6 X X 254 RILs 95 RILs SP2047 X UGG5-5 Solin 2-3% ALA Yellow seed High-lin 60-70% ALA Brown seed F 1 78 DH lines
10 SSR consensus map of flax (n=15) 770 SSR markers resolved into 15 linkage groups (Cloutier et al., TAG, 2012)
11 Sequencing, SNP discovery and genotyping Eight varieties sequenced 55 K SNPs discovered SNPs between CDC Bethune and Macbeth: 5,436 SNPs discovered from core collection: 96 varieties sequenced 1.9 million SNPs discovered
12 SNP maker development in the three mapping populations Population Genome coverage (X) No. of SNPs discovered No. of SNPs mapped No. of SSRs mapped Total no. markers mapped BM ,591 12, ,844 EV ,841 12, ,913 SU ,672 7, ,159
13 cm (Kosambi) High-density SNP map of the BM population No. SSRs No. SNPs 0 LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG LG No. markers per 1cM bins
14 Improvement in QTL analysis using core collection Core collection:408 accessions Morden Dr. Axel Diederichsen, PGRC, Saskatoon.
15 Association mapping (AM) QTL analysis on a large scale using collection of diverse accessions Allows simultaneous identification of large number of QTLs To fine map QTLs. To speed up selection in breeding programs. Group 1 Group 2 South Asia Western Europe South America North America/Europe North America Neighbor-joining tree Eastern Europe
16 Number of plants/area Number of bolls/plant Number of seeds/boll Thousand seed weight (TSW) Yield Yield components
17 -Log 10 (P) AM: three QTL for 1000 seed weight c187-s76_lu c160-s260_lu747b c160-s377_lu c187-s76_lu c160-s377_lu c187-s76_lu c160-s420_lu c537-s76_lu c160-s~179_lu c560-s76_lu c160-s120_lu c187-s76_lu c1-s543_lu c187-s76_lu c1-s454_lu c0-s605_lu c1-s454_lu c187-s605_lu c1-s475_lu c s123_lu c28-s475_lu c0-s123_lu49b 51.0 c28-s475_lu c285-s123_lu c28-s475_lu c161-s1143_lu c44-s235_lu c51-s464_lu c1108-s127_lu c161-s~1143_lu c4-s289_lu c161-s1143_lu c14-s577_lu c0-s1982_lu c10-s483_lu c161-s~810_lu c239-s89_lu c161-s1595_lu2802 c0-s810_lu seed 58.5 weight c4-s876_lu2135 c4-s108_lu c586-s1143_lu1160b c4-s108_lu c0-s310_lu c568-s9_lu c325-s360_fad2a 59.3 c771-s108_lu c0-s6_lu c676-s108_lu c887-s1311_lu c286-s59_lu c51-s376_lu2392a 59.6 c286-s108_lu c51-s376_lu2392b c286-s108_lu c51-s~94_lu c0-s0_lu2067a c4-s50_lu c51-s376_lu299 c0-s701_lu c4-s50_lu c51-s376_lu c44-s235_lu c51-s464_lu c4-s876_lu c51-s464_lu c237-s108_lu c51-s376_lu c0-s0_lu926ba 83.4 c512-s464_lu c206-s208_lu U 86.3 c512-s1143_lu c206-s208_lu c586-s1143_lu1160a 70.2 c206-s208_lu c93-s3_lu c206-s208_lu c94-s~947_lu c39-s43_lu c93-s59_lu c39-s35_lu c203-s145_lu2895b 74.6 c39-s43_lu c119-s361_lu c39-s43_lu c275-s297_lu c39-s43_lu c126-s361_lu c39-s43_lu c119-s112_lu c313-s743_lu c119-s112_lu c39-s43_lu c45-s480_lu2374b 89.5 c39-s107_lu c0-s885_lu c39-s107_lu c12-s1202_lu2184a 98.9 c90-s373_lu c12-s584_lu2183a c475-s917_lu2021a c475-s917_lu c297-s584_lu c475-s917_lu c0-s669_lu2010b c475-s917_lu c550-s669_lu c51-s917_lu c550-s722_lu c134-s1313_lu c550-s1099_lu c134-s3229_lu c856-s722_lu c475-s917_lu c0-s722_lu c134-s617_lu c0-s722_lu c112-s103_lu c101-s1376_lu342 c542-s281_lu445 c413-s230_lu318 c0-s280_lu1039 c287-s280_lu3024 c101-s164_lu1161 c101-s164_lu2628 c0-s462_lu452 c101-s462_lu2625a c150-s30_lu899 c150-s156_lu2764 c150-s281_lu2767 c150-s281_lu774 * c150-s156_lu821 c729-s156_lu3262 c0-s156_lu64* c16-s156_lu373* c16-s156_lu139 c0-s156_lu787a c16-s1080_lu2194* c113-s103_lu2689 c7-s471_lu2161 c0-s471_lu2047 c7-s471_lu2044 c7-s471_lu2040 c7-s471_lu2049 c374-s411_lu3117 c0-s138_lu2063 c7-s138_lu2163 c519-s149_lu3223 c7-s398_lu2164 c444-s701_lu3199a c102-s711_lu2635 c102-s711_lu105 c102-s175_lu2633 c102-s711_lu2631 c444-s325_lu3195 c102-s711_lu104 c369-s325_lu3111 c401-s488_lu3151 c401-s488_lu3153 c401-s488_lu3152 c0-s488_lu933 c401-s488_lu638 c401-s488_lu639 c0-s488_lu1144 c119-s182_lu2706 c1078-s216_lu3290 c400-s216_dgata c0-s216_lu2038 c400-s349_lu658 c400-s216_lu3148 c400-s216_lu3150 c400-s196_lu3146 c400-s196_lu3144 c177-s243_lu2838b c118-s196_lu509 c118-s196_lu558 c115-s524_lu2693 c156-s792_lu2775b c115-s196_lu450 c115-s359_lu422a 8.91% of TSW variation c214-s863_lu2917a c0-s765_lu14 c78-s701_lu628 * c78-s701_lu927 c78-s701_lu891 c267-s906_lu140 * c267-s292_lu756* c267-s292_lu2991 c0-s292_lu2041 c0-s0_lu2042 c115-s888_lu2694 c267-s292_lu2997 c36-s25_lu2992 c156-s196_lu2779 c156-s153_lu2780 c0-s25_lu2096 c0-s25_lu867 c197-s1558_lu2880 c156-s596_lu2778 c156-s792_lu2775a c97-s25_lu2985 c73-s29_lu2482 c73-s29_lu2081 c0-s288_lu2090 c73-s29_lu2486 c156-s292_lu2773 c156-s~288_lu2787 c73-s315_lu2485 c2-s200_lu2127a c12-s584_lu2183b c12-s584_lu2183c * c97-s306_lu263 c0-s306_lu896 c73-s165_lu1135 * c161-s19_lu728a c73-s165_lu439 c97-s25_lu2612* c207-s27_fad3b c207-s23_lu2914 c257-s1158_lu58b c0-s218_lu275 c306-s98_lu206b c0-s98_lu1052 c306-s98_lu203b c306-s98_lu3063 c306-s98_lu765bb c306-s98_lu3064 c84-s27_lu2574 c207-s27_lu2911 c0-s27_lu803 c201-s924_lu381 c0-s3956_lu1151 c1064-s1533_lu3289 * Associated SSRs to 1000-seed weight (TSW) based on MLM * 27.22% of TSW variation 17.94% of TSW variation
18 Fatty acid biosynthesis 16:0 Palmitic (~6%) Stearic SAD1, SAD2 Oleic FAD2A,FAD2B Linoleic (~4%) (18-20%) (14-15%) 15 FAD3A,FAD3B fad2a, fad2b Linolenic (55-57%)
19 sad1 Gene forms Frequency Proteins Frequency
20 Summary Gene Gene forms No. of proteins proteins sad1 6 4 A B C D sad2 6 2 A B fad2a 21 3 A B C fad2b 5 4 A B C D fad3a 15 6 A B C D* E* F fad3b 18 7 A B* C # D E F G * Stop codon # Mutation in active site of protein
21 Percentage of LIN Functional characterization of desaturases a a a a a a a b c c c 10 0 CD AA AG AD AE BD FD DD EF DC EB # * * * ** FAD3A/FAD3B protein combinations
22 Sequenced Plant Genomes Species Genome Size (Mb) Year Reference Arabidopsis Arabidopsis Genome Initiative Rice International Rice genome sequencing Project Poplar Tuskan et al Grape French-Italian consortium for grapevine genome characterization Papaya Ming et al Maize Schnable et al Sorghum Paterson et al Cucumber Huang et al Apple Velasco et al Cacao Argout et al Castor bean Chan et al Brachypodium International Brachypodium Initiative Strawberry Shulaev et al Flax Wang et al
23 Correcting the draft sequence map
24 What we have done State of the art in flax: Consensus map of 770 SSRs & 10K+ SNP maps Draft genome sequencing and improvements Association mapping Fatty acid biosynthesis: gene form diversity and functional characterization Secondary metabolites: cyanogenic glucosides
25 What we intend to do Merging phenotype with genotype Improve trait-marker association: QTL mapping and AM Finalize and polish the draft genome Disseminate relevant information to breeders Marker-trait based variety selection for breeding programs Bioinformatics tools for easy access to all data
26 Molecular genetics lab Cereal Research Center Winnipeg, Manitoba, Canada Braulio Soto (Association mapping) Raja Ragupathy (physical map) Frank You (bioinformatics) Santosh Kumar (SNP discovery & maps) Elsa Reimer (mapping) Natasa Radovanovic (yeast assays) Andrzej Walichnowski (mapping) Debbie Miranda (mapping, sequencing) Mitali Banik (consensus map) Adam Chin Fatt (Cyanogenic glucosides) Dinushika Thambugala (FA composition)
27 TUFGEN funding Genome Canada Genome Prairie Genome Alberta Government of Saskatchewan Government of Alberta Government of Manitoba Agriculture Development Fund Canada Foundation for Innovation Alberta Department of Energy Agricultural Bioproducts Innovation Program Flax Council of Canada Saskatchewan Flax Development Commission Manitoba Flax Growers Association Western Economic Diversification University of Alberta Indian Agricultural Research Institute Department of Biotechnology India Thank You
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