After screening bacterial artificial chromosome (BAC) libraries by pooled overgo hybridizations, determining the precise relationship between individual overgo probes and positively hybridizing clones is generally time consuming and laborious. To simplify the disconsolation step we developed over go-tagged site (OTS) PCR-based markers and used them to verify BAC-over go relationships and build a local BAC contig. We demonstrate the approach using fourteen OTS-PCR markers and an additional five sequence-tagged site (STS)-PCR markers spanning the yield related QTL yld1.1 from the wild rice species Oryza rufpogon. All OTS and STS tests revealed PCR fragments of the expected size. Comparisons between the OTS-PCR-based BAC contig and the publicly available physical map of yld1.1 indicated that these markers efficiently produced a robust local BAC contig assembly. In the process we reveal strong conservation of synteny within the studied 1.2 Mb region, which potentially harbors yield related genes, when comparing the physical order of the OTS-PCR markers on the O. rufpogon BAC contig with the O. sativa genome sequence. By resequencing amplicons across the region we established that the rice species examined exhibit a very low level of molecular polymorphism, suggestive of strong selection. We discuss how OTS-PCR marker application could be extended to larger genomic regions or play a valuable role in future BAC-based genomic comparisons at the 'local' scale.
Song, B. K., Waugh, R., Marshall, D., Nadarajah, K., & Ratnam, W. (2011). Comparative physical mapping using overgo-tagged site reveals strong conservation of synteny between cultivated and common wild rice in the 1.2 mb yld1.1 region. Asia-Pacific Journal of Molecular Biology and Biotechnology, 19(4), 157–168.