Genetic marker anchoring by six-dimensional pools for development of a soybean physical map

Integrated genetic and physical maps are extremely valuable for genomic studies and as important references for assembling whole genome shotgun sequences. Screening of a BAC library using molecular markers is an indispensable procedure for integration of both physical and genetic maps of a genome. Molecular markers provide anchor points for integration of genetic and physical maps and also validate BAC contigs assembled based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy and an in silico approach to anchor molecular markers onto the soybean physical map.

A total of 1,470 markers (580 SSRs and 890 STSs) were anchored by PCR on a subset of a Williams 82 BstY I BAC library pooled into 208 pools in six dimensions. This resulted in 7,463 clones (approximately 1x genome equivalent) associated with 1470 markers, of which the majority of clones (6,157, 82.5%) were anchored by one marker and 1106 (17.5%) individual clones contained two or more markers. This contributed to 1184 contigs having anchor points through this 6-D pool screening effort. In parallel, the 21,700 soybean Unigene set from NCBI was used to perform in silico mapping on 80,700 Williams 82 BAC end sequences (BES). This in silico analysis yielded 9,835 positive results anchored by 4152 unigenes that contributed to 1305 contigs and 1624 singletons. Among the 1305 contigs, 305 have not been previously anchored by PCR. Therefore, 1489 (78.8%) of 1893 contigs are anchored with molecular markers. These results are being integrated with BAC fingerprints to assemble the BAC contigs. Ultimately, these efforts will lead to an integrated physical and genetic map resource.

We demonstrated that the six-dimensional soybean BAC pools can be efficiently used to anchor markers to soybean BACs despite the complexity of the soybean genome. In addition to anchoring markers, the 6-D pooling method was also effective for targeting BAC clones for investigating gene families and duplicated regions in the genome, as well as for extending physical map contigs.