Hyten et al. BMC Genomics 2010, 11:38 http://www.biomedcentral.com/1471-2164/11/38 selected to internal marker-poor islands (regions greater than 2 Mb without markers), and the remainder were selected near scaffold ends to help assess scaffold integ- rity. The 1,536 SNPs were selected from SNPs predicted using the production run Maq analysis method described previously. In the case of 63 SNPs, the SNP discovery protocol was relaxed to allow SNPs to be called with a single short sequence read in an effort to target markers to more of the smaller, harder-to-map scaffolds. For GoldenGate assay design all SNPs were required to have a designability rank score >0.4, which is predic- tive of a moderate to high success rate for the conver- sion of the SNP into a working GoldenGate assay. The 1,536 chosen SNPs made up the custom Oligo Pool All (OPA) which was given the name SoyOPA-4. To help anchor and align the genetic map created by the SNPs on SoyOPA-4, the previously designed and tested SoyOPA-3 [2] was tested on 282 RILs of the Williams 82 × PI 468916 population per the manufacturer’s pro- tocol and as described by Fan et al. [18] and Hyten et al. [7]. SoyOPA-4 was tested on 470 RILs per the manu- facturer’s protocol and as described by Fan et al. [18] and Hyten et al. [7]. The Illumina BeadStation 500G (Illumina Inc., San Diego, CA) was used for genotyping the GoldenGate assay. The automatic allele calling for each locus and the calculation of minor allele frequen- cies were accomplished with the GenCall software (Illu- mina Inc., San Diego, CA). All GenCall data were visually inspected and re-scored if any errors in calling the homozygous or heterozygous clusters were evident. JoinMap 3.0 software [19] was used for creation of the high resolution genetic maps. Genotype data for 26 RILs were excluded from the full genotype data set due to high heterozygosity levels detected when genotyped with SoyOPA-3 and SoyOPA-4. The exclusion of these 26 RILs left genotype data for 268 RILs genotyped with SoyOPA-3 and 444 RILs genotyped with SoyOPA-4 to create the high resolution genetic map. Genetic dis- tances were calculated using the Kosambi mapping function. A minimum LOD ≥ 10.0 and a maximum dis- tance ≤ 50 cM were used to test linkages among markers. Additional file 1: SoyOPA-4 SNPs. The 1,536 SNPs along with the flanking sequence to design the GoldenGate assay SoyOPA-4. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 38-S1.XLS ] Additional file 2: Williams 82 × PI 468916 genetic map. The high- resolution Williams 82 × PI 468916 genetic map created with 1,790 SNPs genotyped on 268 or 444 recombinant inbred lines (RILs). Also shown is the high-resolution map resulting when only using the 1,254 SNPs genotyped with SoyOPA-4 on 444 RILs and the Consensus Map 4.0 position for any SNPs in common between the two genetic maps. Click here for file Page 7 of 8 [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 38-S2.XLS ] Additional file 3: Williams 82 × PI 468916 comparison to the soybean Consensus Map 4.0. The charts of the 20 linkage groups from the Consensus Map 4.0 (left chart) compared to the same 20 linkage groups produced from the 444 recombinant inbred lines of Williams 82 × PI 468916 (right chart). Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 38-S3.PPT ] Acknowledgements We thank Christian D. Haudenschild from Illumina, Inc for the sequencing on the Genome Analyzer and Ronghui Yang and Chuck Quigley for assistance with the genomic STS sequencing of SNP validation. The authors gratefully acknowledge the early pre-release access to the draft DOE-JGI Williams 82 preliminary 4× and 6.5× scaffold assembly and the opportunity to assist Jeremy Schmutz et al. [1] in assembling the 8× soybean genome sequence. This work was partially supported by United Soybean Board Projects 7268 and 8212. The support of the United Soybean Board is greatly appreciated. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval of a product to the exclusion of others that may be suitable. Author details 1Soybean Genomics and Improvement Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA. 2Department of Agronomy, U.S. Department of Agriculture, Agricultural Research Service, Iowa State University, Ames, IA 50011, USA. 3Department Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA. 4Department of Agronomy and Horticulture, University of Nebraska Lincoln, Lincoln, Nebraska, NE 68583, USA. 5National Center for Genome Resources, Santa Fe, NM 87505, USA. Authors’ contributions DLH designed and oversaw the study, developed the mapping population, designed the GoldenGate OPA, performed genotyping analysis of the GoldenGate assay, and drafted the manuscript. SBC, NW and QS performed the SNP discovery, designed the GoldenGate OPA, and performed additional analysis of the short read data. SBC also assisted in preparing the manuscript. QS created the high resolution linkage map. ECF created the reduced representation library, performed sequencing of the validation sets, helped to develop the mapping population, and assisted in preparation of the manuscript. RCS helped conceive the study. JES assisted in the analysis of the genetic map data and in preparing the manuscript. ADF and GDM assisted in an initial analysis of the genome analyzer short reads. PBC designed and oversaw the study, developed the mapping population, and assisted in preparing the manuscript. All authors read and approved the final manuscript. Received: 4 September 2009 Accepted: 15 January 2010 Published: 15 January 2010 References 1. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, et al: Genome seqeunce of the paleopolyploid soybean. Nature 2010, 463:178-183. 2. 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