1. Objective Assessment of allelic diversity for economic traits by sequencing genes and/or markers associated with traits. Goal Use Pisum core germplasm collection for association mapping studies ASSESSMENT OF ALLELIC DIVERSITY IN USDA PEA CORE COLLECTION Brown, Coyne, Timmerman-Vaughan

3. Why SNPs? Single nucleotide polymorphisms (SNPs) are the most common type of sequence difference between alleles and provide a way to detect direct associations between allelic forms of genes and phenotypes. Human HapMap published in Nature 437, 1299-1320 (27 Oct 2005).

4. SNPs Advantages Low quantities of template DNA required No gel-electrophoresis required Easy and quick to assay Closed tube assay which prevents erratic results due to post-PCR contamination High reproducibility Amenable to automation Disadvantages Sequencing required

5. Preliminary data: average of 3 SNPs and 1 insertion/deletion (INDEL) per sequence, one monomorphic from sequencing 32 (3300) diverse accessions. NamePutative functionGenebank accession sequence length (bp) SNPsIndels P108ThioredoxinAA43091676421 P531Unknown proteinAA43094226400 L109Unknown proteinAA43090934620 P393Unknown proteinAA43091243250 P482ABA regulated proteinAA427337415** P628GAPDHAA430910420** ABR17ABA responsive proteinZ1512858121 DCChit25ChitinaseL37876<50021 FED1FerredoxinX1420754330 K6Unknown proteinAA43094280830 D8BMT-like proteinAA43092337112 D8CGTP-binding proteinAA43091435120 PSODCu/Zn superoxide dismutaseM6300382131 Q407Ligand binding proteinAA430905429**

6. Example of SNP or INDEL in pea gene PI 116944CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGACA PI 117998CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 163129CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 180693CTAGATGAATAATATCAAATTTTATATAATAGTCAACAGAAGAAAAGATA PI 184130CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 203064CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 207508CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGACA PI 209507CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 261677CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 273605CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 314794CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 358613CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 429839CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGACA PI 429843CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI 595933CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGAGA

7. Genotyping SNP genotyping- “TAQMAN” quantitative PCR. Design primers-probe, test on 32 known, run 384 accessions from pea core INDEL genotyping- fluorescence-labeled primers- gel or capillary electrophoresis – ABI 3730 or Beckman CEQ8000 ABI PRISM® 7000 Sequence Detection System

8. Next: Population structure analysis Jonathan K. Pritchard, Matthew Stephens and Peter Donnelly. 2000. Inference of Population Structure Using Multilocus Genotype Data. Genetics 155: 945–959. Daniel Falush, Matthew Stephens and Jonathan K. Pritchard. 2003. Inference of Population Structure Using Multilocus Genotype Data: Linked Loci and Correlated Allele Frequencies. Genetics 164: 1567– 1587. Software STRUCTURE URL =http://pritch.bsd.uchicago.edu. Generation Challenge Program url=http://www.generationcp.org/vw/index.php Cicer: ICARDA and ICRISAT, 3000 accessions, 50 microsatellites Lens: ICARDA, 3000 accessions, 50 microsatellites GLIP software Germinate url http://germinate.scri.sari.ac.uk/cgi- bin/germinate/germinate_presentations.cgi GLIP Pisum: Underway EU GLIP project: 3000 pea accessions – 76 retro. elements, some genes Software: TASSEL-Dr. Buckler’s talk

9. Overcoming SNP Disadvantage of Sequence required Bioinfomatics Genbank accessions EST projects Geneva EST filtering software, contact, Angela Baldo abaldo@pgru.ars.usda.govAngela Baldo HarvEST filtering software, contact Tim Close, timothy.close@ucr.edu timothy.close@ucr.edu http://harvest.ucr.edu/

10. SNP or INDEL data in GRIN Proposal: same as SSR data Provide the experimental detail Cite publication Downloadable to other databases Unique issues of SNP or INDEL molecular data vs. SSR or other marker data Sequence and assay from a single plant from accession Pea solution- created single plant pea core collection