PCRBased Markers for Oil b glucan and Protein

PCR-Based Markers for Oil, b -glucan, and Protein Content Steve Molnar, Winson Orr, Julie Chapados, Anissa Lybaert, Ismahane Elouafi, Shan He, Charlene Wight, Diane Mather, and Nick Tinker Eastern Cereal and Oilseed Research Centre, Ottawa and Mc. Gill University Presented to American Oat Workers’ Conference Fargo, North Dakota July 23 -26, 2006

Why Are We Interested? • Oil, b-glucan, and protein content are three major quality traits in oats. • One strategy for improving these traits is to use molecular marker assisted selection (MAS) breeding. • For MAS, PCR-based markers are currently the best choice balancing low cost with high reliability.

The Challenge • Identify the genetic loci that are responsible for variation in these 3 traits. • Identify or develop PCR-based markers linked to these loci. • Preferably, identify or develop PCR-based markers within the gene itself, as these can not be unlinked by recombination and as these could be allele specific.

Oil QTLs Reported in Mapping Populations KO LG KO QTL KM QTL KO 11 KM 11 KO 37 KO ? TM QTL OM 3 Bcd 1829 y Unlinked Coleoptal KO 6 (KO 6) KM 22 KO ? KM 5 X Gene ? bg, protein Accase A protein TM 15 protein bg KO 24_26_34 TM 5 KO 32 TM 4_16 KO ? TM 30 bg, protein KO 2 OM 6 KO ? OM 13 KO ? OM 19 KO ? OM 20 KO ? OM 24 KO 30 OT 16 45 -48 % Linked QTLs bg KO 22 % Explained Iowa 22 -28 % 39 % 42 -52 % N 1 naked

The Challenge • Identify the genetic loci that are responsible for variation in these traits • Identify or develop PCR-based markers linked to these loci • Preferably, identify or develop PCR-based markers within the gene itself, as these can not be unlinked by recombination and as these could be allele specific

PCR-Based Markers Proven as Linked to Oil, b. G, or Protein QTLs in Map. Pops. or as Candidate Genes Trait Oil Pop # RFLP QTLs SCAR AFLP KO 3 -4 Many KM 3 Many TM 5 Many OM 3 -6 Many FR 2+ 1 DE 2+ 1 Other ? RAPD 2 RAPD SSR & Gene EST SCAR ISSR Primers 1 1 6 2 Total 1 2 >2 > 19 b-g KO Many KM Many TM Many 2 2 5 3 5 1 18 Pro TM Many OM Many 1 6 1 1 9

PCR Based Markers Linked to Oil QTL T T M RAPD M RRRRRTTMM SCAR Ethidium bromide-stained gel (2% agarose) of PCR amplification products after electrophoresis. RAPD UBC 121 and derived SCAR on Terra, Marion and their RILs. Orr and Molnar, submitted

QTL Linked RAPD Markers and Derived SCARs A B TM 15(KO 22) 0. 0 TM 11 acc_cta 222 Oil 13. 4 umn 575 14. 0 14. 3 14. 4 bcd 327 ubc 167 a ubc 198 15. 0 15. 2 15. 3 15. 5 ubc 186, ubc 167 ts ubc 186 s ubc 121 ms ubc 167 ms ubc 198 s aco 139 x 16. 1 TM 5(KO 24, 34) bcd 327 x 0. 0 aco 139 y 4. 9 ubc 121 18. 9 58. 3 cdo 482 x 62. 3 62. 6 ubc 185 s ubc 185 64. 6 n 1 C TM 30 cdo 460 b 33. 9 umn 5214 43. 0 cdo 54 48. 4 51. 6 ubc 364 b bcd 115 c Oil, Protein, etc. D KO 15 19. 3 ß-glucan acc_cat 259 Oil 0. 0 isu 1507 xp 12. 8 bcd 115 x 32. 0 KO 11_41+20 umn 5112_2 6. 2 7. 0 7. 1 7. 8 ACCase 1 cdo 665 b ubc 364 os 10. Orr and Molnar, submitted 1 cdo 1436 c Oil

SCAR UBC 364 Os Tested On Dal (High Oil) x Exeter (Low Oil) Recombinant Inbred Lines Kanota Ogle 2 -11 High Oil RILs; 12 -19 Low Oil LDD EE KK OO

PCR-Based Markers Proven as Linked to Oil, b. G, or Protein QTLs in Map. Pops. or as Candidate Genes Trait Oil Pop # RFLP QTLs SCAR AFLP KO 3 -4 Many KM 3 Many TM 5 Many OM 3 -6 Many FR 2+ 1 DE 2+ 1 Other ? RAPD 2 RAPD SSR & Gene EST SCAR ISSR Primers 1 1 6 2 Total 1 2 >2 > 19 b-g KO Many KM Many TM Many 2 2 5 3 5 1 18 Pro TM Many OM Many 1 6 1 1 9

Microsatellite (SSR) Markers • Microsatellite or SSR (Simple Sequence Repeat) markers target variation in small repeat units, typically 2 or 3 bp in size. • Graham Scoles’ lab and Les Domier’s lab isolated oat SSRs. Polymorphism is extremely low (5%). However, they and we have tested over 100 and mapped all polymorphic ones. Of those, most are +/SCAR type markers, not typical co-dominant SSRs. This reflects variation in primer binding site, not in the number of repeat units. Two link to oil QTLs and 6 to b-glucan.

Microsatellite (SSR) Markers • Heidi Kaeppler’s lab has developed 2 SSRs linked to oil and protein QTLs respectively • Graham Scoles’ lab has identified 2 ISSR (Inter SSR) markers, linked to b-glucan QTLs. • Studying the Iowa high oil selection program, Jean-Luc Jannink has identified 2 SSRs (1 from Jim Holland 1 from Graham Scoles) associated with high oil. • Recently, Nick Tinker’s lab has developed new SSRs of which 2 are linked to oil, 2 to bglucan and 1 to protein.

Challenge • Identify the genetic loci that are responsible for variation in these traits • Identify or develop PCR-based markers linked to these loci • Preferably, identify or develop PCR-based markers within the gene itself, as these can not be unlinked by recombination and as these could be allele specific

PCR Cloning of Candidate Genes • In this approach, we proposed candidate genes based on metabolic pathways. Available grass DNA sequences for each gene were compared and PCR primers designed. These were used to isolate the oat homolog, which can then be mapped and its map location compared to that of known QTL.

Candidate Gene Primers • Anissa Lybaert in our lab found that most PCR primers developed this way are monomorphic. • Catherine Howarth et al have had success with this strategy, in part by going one step further to identify sequence differences in those PCR products and to design SNP (Single Nucleotide Polymorphism) markers targeting them. They have developed new PCR-based markers for ACCase and KAS and other oil biosynthesis enzymes.

EST Primers • EST (Expressed Sequence Tag) libraries developed from tissues actively involved in oil, b-glucan or protein synthesis might be a rich source of genes. • PCR-primers for such ESTs is a potential strategy. • Ismahane Elouafi in my lab recently developed 73 primer pairs to oat ESTs. We targeted those oat ESTs with homology to rice genomic sequences with introns, in an effort to increase polymorphism. However, only 1 was polymorphic on Kx. O, even after restriction digestion. • By chance, that EST mapped near a b-glucan QTL. • However, low polymorphism may limit this approach.

PCR-Based Markers Proven as Linked to Oil, b. G, or Protein QTLs in Map. Pops. or as Candidate Genes Trait Oil Pop # RFLP QTLs SCAR AFLP KO 3 -4 Many KM 3 Many TM 5 Many OM 3 -6 Many FR 2+ 1 DE 2+ 1 Other ? RAPD 2 RAPD SSR & Gene EST SCAR ISSR Primers 1 1 6 2 Total 1 2 >2 > 19 b-g KO Many KM Many TM Many 2 2 5 3 5 1 18 Pro TM Many OM Many 1 6 1 1 9

Conclusions • This is a very conservative inventory of markers since it is limited to PCR-based markers mapped in the same population as the QTL or derived from candidate genes or ESTs. Excellent markers in homologous regions of other mapping populations or in homeologous regions of all populations have not been added to this inventory. • We need more common markers to better define homologous and homeologous relationships and ideally a consensus map • A good selection of PCR-based markers have been developed for oil, b-glucan, and protein content by numerous laboratories. • We need databases to manage these and make them readily available to the community.

Conclusions • These markers are of many different types, that have different features. This increases flexibility but is problematic for automation. • Further, the low frequencies of polymorphism in oats mean that multiple markers must be developed per locus to ensure that an informative marker is available in the breeder’s germplasm of choice. • We need a single type of marker that is robust, numerous and polymorphic.

Conclusions • It is unclear if sufficient markers exist because it is unclear how many loci are important and need to be tagged with markers. • To determine the number of unique loci we need to better define homologous and homeologous relationships. • Regarding the identification of important loci, it is noteworthy that QTLs identified for b-glucan explain only 20 -25% of the parental variation. Values are better for protein (20 -50%) and best for oil (35 -70%).

Major Conclusions • It is clear that sufficient high quality markers exist to initiate MAS for these three traits, when appropriate. • It is also clear that the mapping of QTLs and candidate genes has increased our knowledge of the biology and genetics of these traits, which in turn affects the design of all oat improvement strategies, whether they use markers for MAS or not.

Acknowledgements We thank: • Quaker Oats (USA), a division of Pepsico, and QTG (Quaker/Tropicana/Gatorade) Canada for many years of generous financial support. • In particular, Fran Webster, Sam Weaver, Bruce Roskens and Grant Morrison, our coordinators from Quaker. • Members of the oat team at ECORC, AAFC, Ottawa for their dedication to oat improvement. • as well as Agriculture and Agri-Food Canada’s Matching Investment Initiative.

Oat Team and Lab-Mates