Hyten et al. BMC Genomics 2010, 11:38 http://www.biomedcentral.com/1471-2164/11/38 smaller unmapped scaffolds could be anchored to the genetic map. In total, 7,108 SNPs were predicted for use in anchoring and orienting additional scaffolds. Ulti- mately, 1,536 SNPs were selected from this pool of 7,108 SNPs to create the higher resolution map needed for validation of scaffold ends, for anchoring of addi- tional scaffolds, and for mitigating the ambiguity of orientations of anchored scaffolds - all of which required a map with markers placed in the existing gaps that are present within the soybean Consensus Map 4.0. These 1,536 SNPs were used to create an Illumina GoldenGate soybean oligo pool all (SoyOPA-4) [Additional file 1]. The SoyOPA-4 produced 1,254 successful GoldenGate assays indicating that the predicted SNPs had a valida- tion and assay conversion rate of 81.6%. High Resolution Genetic Map SoyOPA-4 was used to genotype 470 F5-derived RILs from the Williams 82 × PI 468916 (W82 × 468) popula- tion with the 1,254 polymorphic SNPs. A total of 26 of the 470 RILs were excluded from further analysis due to marker heterozygosity levels > 20%, which suggested that those 26 RILs trace to outcrosses occurring during generation advance, rather than being true F5-derived lines. To tie the newly constructed high resolution genetic map to the existing Soybean Consensus Map 4.0, SoyOPA-3 was used to genotype a subset of 282 RILs from the W82 × 468 population. From these 282 genotyped RILs, the genotype data of 14 RILs were sub- sequently eliminated which were in common with the 26 RILs that had already been eliminated due to high heterozygosity levels. SoyOPA-3 was one of three pre- viously designed custom soybean OPAs developed and tested by Hyten et al. [2,7]. SoyOPA-3 contains 1,396 SNPs that had been mapped on the Soybean Consensus Map 4.0 and had been developed so that all SNPs included in the OPA were polymorphic within at least one of three RIL mapping populations used in the crea- tion of the Soybean Consensus Map 4.0 [2]. Of the 1,396 SNPs on SoyOPA-3 that mapped to the Soybean Consensus Map 4.0, a total of 565 were polymorphic between Williams 82 and PI 468916. A total of 550 of the 565 SNPs from SoyOPA-3 and 1,240 of the 1,254 SNPs from SoyOPA-4 were mapped using 444 RILs to create the 20 linkage groups that cor- respond to the 20 chromosomes of soybean and had an estimated total genetic length of 2,537 cM. The remain- ing 29 SNPs were not linked to any of the 20 linkage groups. The average level of heterozygosity observed in the population was 6.3% which is the expected level of heterozygosity for a RIL population in the F5 generation. Segregation distortion was observed for multiple tightly linked markers in 16 regions throughout the genome [Additional file 2]. Page 4 of 8 To determine if the mapping of SoyOPA-3 on a sub- set of the 444 RILs caused any significant expansions or contractions of the genetic map, a separate map with only the SNPs from SoyOPA-4 was created [Additional file 2]. Comparing the map created using only 1,240 SoyOPA-4 SNPs to the map created using all 1,790 SNPs from SoyOPA-3 and SoyOPA-4 revealed only one substantive change of 22 cM on chromosome 5. This change was due to the elimination of the SoyOPA-3 SNPs leaving a gap of 66 cM between adjacent SoyOPA-4 SNPs. There were a total of 16 discrepancies between the two maps that were 2 to 10 cM with all other discrepancies between the two maps being less than 2 cM in genetic distances between the SoyOPA-4 SNP markers. Anchoring of the Soybean Genome The high-resolution W82 × 468 genetic map with 1,790 SNP markers was successful in anchoring and orienting additional scaffolds in the subsequent 8× scaffold assem- bly using the preliminary 6.5× scaffold assembly for initial SNP discovery and assay development [1]. It added markers to 335 8× assembly scaffolds, of which 23 scaffolds (totaling 7.1 Mb) were previously unmapped in the preliminary 6.5× scaffold assembly. The high- resolution map also oriented 151 scaffolds as a result of positioning markers such that there was at least 1 cM between the most widely separated markers on any given scaffold. The map also helped to evaluate the integrities of the continuous sequence of the scaffolds by adding markers to regions of scaffolds that were pre- viously without markers, and to scaffold ends [1]. Discussion The use of a reduced representation library, coupled with next generation sequencing and the initial release of soybean genome sequence [1], provided a powerful method for the additional discovery of large numbers of SNPs. The validation rate of predicted SNPs varied from 79% up to 92.5%. Not surprisingly, the largest factor for this range in validation was due to the use of one versus multiple short reads to predict a SNP. The decrease in the validation rate arising from the use of only one read for SNP prediction, though modest in percentage terms, may be due to sequencing error from the Genome Ana- lyzer. Still, the validation rate of 79% for SNPs predicted from a single short sequence read was sufficiently high to lead us to ultimately use 63 SNPs predicted by a sin- gle short sequence read to serve as markers in regions that otherwise might have not been anchored or prop- erly oriented in the 8× Glyma1.01 assembly. The production run Maq analysis method resulted in the conversion of 81.6% of the predicted SNPs into working GoldenGate assays. Hyten et al. [7] demon- strated that with high confidence SNPs, the conversion