Hyten et al. BMC Genomics 2010, 11:38 http://www.biomedcentral.com/1471-2164/11/38 5) SNPs had to be supported by at least two short sequence reads, 6) filter out any remaining sequence containing an N. In addition, Genome Analyzer sequence reads with matches to chloroplast sequence [GenBank:NC_007942] or Nicotiana mitochondrion sequence [GenBank:NC_006581] were discarded. While the above work was in progress, the Maq map- ping and assembly software [14] was tested as an alter- native method to GMAP for alignment of Illumina Genome Analyzer sequence reads with the Williams 82 preliminary 4× scaffold assembly and later when the preliminary 6.5× scaffold assembly became available for SNP discovery. Three rounds of SNP predictions were made using Maq: one for validation using multiply-sup- ported SNPs (VMS); one for validation using singly-sup- ported SNPs (VSS); and a production run. The runs for validation used the same filtering steps described below for the production run, except where noted. The follow- ing constraints were used in the filtering for the identifi- cation of putative SNPs using Maq software: 1) Occurrences of two or more SNPs within a 25-base win- dow were eliminated, 2) SNPs with ambiguous consen- sus bases (i.e., bases other than A, C, T or G) were eliminated, 3) a minimum consensus-base quality score of 20 for VMS and VSS (27 for the production run) was required, 4) an average read-copy of 1.00 i.e., the SNP maps to one place in the Williams 82 reference genome, 5) a minimum Maq mapping score of 30 was required, and 6) with VMS a minimum of two short sequence reads were required to support each putative SNP. In addition, Genome Analyzer reads with matches to chlor- oplast sequence [GenBank:NC_007942] or Nicotiana mitochondrion sequence [GenBank:NC_006581] were discarded. Additionally, we removed SNPs from repeti- tive regions, using the following protocols. For VMS and VSS, extract the 121 nt region centered on the SNP and exclude the SNP if this region contained an “N”. If either 25-mer flanking a SNP hit any other place in the genome with at most two mismatches using an ungapped BLAST it was considered repetitive; if both were repetitive, the SNP was excluded. For the produc- tion run, extract a 601 nt region centered on the SNP and exclude the SNP if this region contained an “N”. If any 25-mer in the 300 nt up- or downstream region hit five or more other positions in the genome with at most two mismatches using an ungapped BLAST, it was con- sidered repetitive; SNPs whose flanking regions were more than 1/3 repetitive were also excluded. SNP Validation through Resequencing Based upon the underlying Williams 82 preliminary 4× scaffold assembly, polymerase chain reaction (PCR) pri- mers were designed using Primer 3 software [15]. The primers were designed to the flanking sequence of 620 putative SNPs for the GMAP method and 96 putative Page 6 of 8 SNPs for the Maq method. Electronic PCR [16] was used to select primer pairs that, based upon the Wil- liams 82 preliminary 4× scaffold assembly, would be anticipated to be locus specific. The predicted amplicon lengths ranged from approximately 100 to 700 bp in length. Initial amplification, sequence analysis and align- ment for validation of putative SNPs between Williams 82 and PI 468916 were performed as described by Choi et al. [3]. Population Development Four hundred and seventy F5-derived recombinant inbred lines (RIL) were created at the Beltsville Agricul- tural Research Center, Beltsville, MD, USA, from a cross made in 2004 between the soybean cultivar Williams 82 [17] and the wild soybean PI 468916 followed by plant- to-row descent. A total of 2,000 F2 seeds were planted in the field at Beltsville, MD the summer of 2005. Approximately 10 to 15 F3 seeds were harvested from each of 1,690 individual F2 plants. In the summer of 2006, five F3 seeds harvested from each of 1,000 ran- domly selected F2 plants were planted in hill plots and subsequently thinned to one F3 plant two weeks after planting. A total of 10 to 15 F4 seeds were harvested from each of 932 F3 plants. In the winter of 2006-2007, three F4 seeds from 600 randomly selected F3 plants were planted in the greenhouse for the next generation advance and subsequently thinned to one plant after two weeks. Ten to fifteen F5 seeds were harvested from each of 583 F4 plants. In the summer of 2007, five F5 seeds from each of the 583 F4 plants were planted in hill plots and thinned to one F5 plant two weeks after planting. DNA was isolated from 470 RILs randomly selected from the 583 F5 plants by taking leaf tissue from the single F5 plant from which the RIL was derived. The Qiagen DNeasy 96 Plant DNA extraction kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol was used to obtain purified DNA for the Gold- enGate Assay analysis. All seeds were harvested from each F5 plant to create an F5-derived line. To help mini- mize segregation distortion in creating the population, seeds in all generations were harvested as soon as indivi- dual plants became mature to avoid loss of seeds due to shattering. GoldenGate assay and Genetic Map Development A total of 1,536 SNPs were selected to develop a 1,536 GoldenGate assay [Additional file 1]. The SNPs were selected from scaffolds of the Williams 82 preliminary 6.5× scaffold assembly to help validate scaffold ends, anchor additional scaffolds, and improve orientations of anchored scaffolds. Of the 1,536 SNPs selected, 242 SNPs were selected to anchor scaffolds not previously anchored with the Consensus Map 4.0, 227 SNPs were selected to orient scaffolds with too little marker separa- tion from the Consensus Map 4.0, 217 SNPs were