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This article in TPG

  1. Vol. 2 No. 2, p. 121-133
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    Received: Jan 14, 2009
    Published: July, 2009


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doi:10.3835/plantgenome2009.01.0002

Large-Scale Discovery of Gene-Enriched SNPs

  1. Michael A. Gore ,
  2. Mark H. Wright*,
  3. Elhan S. Ersoz,
  4. Pascal Bouffard,
  5. Edward S. Szekeres,
  6. Thomas P. Jarvie,
  7. Bonnie L. Hurwitz,
  8. Apurva Narechania,
  9. Timothy T. Harkins,
  10. George S. Grills,
  11. Doreen H. Ware and
  12. Edward S. Buckler
  1. M.A. Gore, Dep. of Plant Breeding and Genetics, Cornell Univ., 175 Biotechnology Bldg., Ithaca, NY 14853; M.H. Wright, Dep. of Genetics and Development, Cornell Univ., 102 Weill Hall, Ithaca, NY 14853; E.S. Ersoz, Institute for Genomic Diversity, Cornell Univ., 175 Biotechnology Bldg., Ithaca, NY 14853; P. Bouffard, E.S. Szekeres, and T.P. Jarvie, 454 Life Sciences, 20 Commercial St., Branford, CT 06405; B.L. Hurwitz and A. Narechania, Cold Spring Harbor Lab., 1 Bungtown Rd., Cold Spring Harbor, NY 11724; T.T. Harkins, Roche Applied Science Corp., 9115 Hague Rd., Indianapolis, IN 46250; G.S. Grills, Life Sciences Core Labs. Center, Cornell Univ., 139 Biotechnology Bldg., Ithaca, NY 14853; D.H. Ware, USDA-ARS, Cold Spring Harbor Lab., 1 Bungtown Rd., Cold Spring Harbor, NY 11724; E.S. Buckler, USDA-ARS, Dep. of Plant Breeding and Genetics, Institute for Genomic Diversity, Cornell Univ., 159 Biotechnology Bldg., Ithaca, NY 14853. All custom code and scripts used in this study are available upon request from M. H. Wright (mhw6@cornell.edu). M.A. Gore and M.H. Wright contributed equally to this work.

Abstract

Whole-genome association studies of complex traits in higher eukaryotes require a high density of single nucleotide polymorphism (SNP) markers at genome-wide coverage. To design high-throughput, multiplexed SNP genotyping assays, researchers must first discover large numbers of SNPs by extensively resequencing multiple individuals or lines. For SNP discovery approaches using short read-lengths that next-generation DNA sequencing technologies offer, the highly repetitive and duplicated nature of large plant genomes presents additional challenges. Here, we describe a genomic library construction procedure that facilitates pyrosequencing of genic and low-copy regions in plant genomes, and a customized computational pipeline to analyze and assemble short reads (100–200 bp), identify allelic reference sequence comparisons, and call SNPs with a high degree of accuracy. With maize (Zea mays L.) as the test organism in a pilot experiment, the implementation of these methods resulted in the identification of 126,683 putative SNPs between two maize inbred lines at an estimated false discovery rate (FDR) of 15.1%. We estimated rates of false SNP discovery using an internal control, and we validated these FDR rates with an external SNP dataset that was generated using locus-specific PCR amplification and Sanger sequencing. These results show that this approach has wide applicability for efficiently and accurately detecting gene-enriched SNPs in large, complex plant genomes.

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