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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Schematic display of six-dimensional BAC pooling strategy. One hundred twenty eight 384-well microtiter plates containing 49,192 individual BAC clones were conceptually arranged in a cubic stack consisting 32 layers × 48 columns × 32 rows. The stack was pooled on the six unique coordinate axes as shown to generate a total of 208 DNA pools.

Figure 2

Distribution of BAC hits and contig/marker. (a) the distribution of BAC hits/marker using 1470 markers; (b) the distribution of the numbers of identified contigs of each marker.

Figure 3

Example of the integrated map view of a ~8 cM region on LG E. The genetic map was redrawn based on the integrated genetic linkage map [11]. The QTL name and position refer to the Soybean Breeders Toolbox [34]. The dashed lines indicate the discrepancies of marker alignments between the physical map and genetic map. The highlighted FPC contigs are questionable. The number above the lines connecting genetic markers and contigs is the number of BAC hits. The white bars between highlight bars are gaps between the WSS scaffolds.

Figure 4

Distribution of the number of markers according to the number of positive clones for each category of marker. The soybean Unigene set (21,700 unigenes) served as a query entry to blast against the non-redundant BAC end sequences (80,700 reads) of the Williams 82 BstY I library. The cutoff parameters were setup as: 10^-30 e-value, ≥95% sequence identity and minimum aligned sequence length ≥100 bp.

Figure 5

Anchoring paralogous sequences onto the physical map by 6-D pools and confirmed by FISH. (a) Paralogous sequences of PCR product. The pink arrow shows nucleotide variations in a sequence read. (b) Alignment between homoeologous BACs. Blue lines show some regions with inconsistent alignment between the two BACs (c) FISH mapping of homoeologous BACs (Gm_WBb0130B22 and Gm_WBb0060P19) anchored by marker 13617. The leftmost panel shows the entire karyotype of a chromosome spread prepared from mitotic root tip cells. The probe for BAC Gm_WBb0130B22 (abbreviated as 130B22) was labeled with green fluorophore and Gm_WBb0060P19 (abbreviated as 60P19) was labeled with red fluorophore; overlap therefore appears as yellow. Right panels are enlarged fields in which the left panels show the merged channels, the middle panels show the green (60P19) channel and the right panels show the red (130B22) channel. The 60P19 probe localizes to two pairs of strong spots (corresponding to the duplicated chromatids of homologous chromosomes) as well as to two pairs of less intense signal that overlap with the 130B22 loci. The yellow arrows indicate overlapping spots.