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

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, (27 Oct 2005).

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

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 P108ThioredoxinAA P531Unknown proteinAA L109Unknown proteinAA P393Unknown proteinAA P482ABA regulated proteinAA ** P628GAPDHAA ** ABR17ABA responsive proteinZ DCChit25ChitinaseL37876<50021 FED1FerredoxinX K6Unknown proteinAA D8BMT-like proteinAA D8CGTP-binding proteinAA PSODCu/Zn superoxide dismutaseM Q407Ligand binding proteinAA **

Example of SNP or INDEL in pea gene PI CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGACA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTAGATGAATAATATCAAATTTTATATAATAGTCAACAGAAGAAAAGATA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGACA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGACA PI CTTGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGATA PI CTAGATGAATAATATCAAATTTTATATAATAGTCAACCGAAGAAAAGAGA

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

Next: Population structure analysis Jonathan K. Pritchard, Matthew Stephens and Peter Donnelly Inference of Population Structure Using Multilocus Genotype Data. Genetics 155: 945–959. Daniel Falush, Matthew Stephens and Jonathan K. Pritchard Inference of Population Structure Using Multilocus Genotype Data: Linked Loci and Correlated Allele Frequencies. Genetics 164: 1567– Software STRUCTURE URL = Generation Challenge Program url= Cicer: ICARDA and ICRISAT, 3000 accessions, 50 microsatellites Lens: ICARDA, 3000 accessions, 50 microsatellites GLIP software Germinate url 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

Overcoming SNP Disadvantage of Sequence required Bioinfomatics Genbank accessions EST projects Geneva EST filtering software, contact, Angela Baldo Baldo HarvEST filtering software, contact Tim Close,

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