A high efficient approach used for BAC-contig extension of Oryza sativa with PCR screening the BAC clone pools

To extend 8 BAC contigs, which were previously located in the 56.1-68 cM region of the chromosome 4 of the Oryza sativa indica GuangLuAi4, 14 pairs of primers were designed according to the terminal sequences of the existing seed BACs and were deliberately divided into 3 groups. With the 3 groups of primer mixtures, 233 pools of BAC DNA that represent 22 368 BAC clones from O.sativa indica GuangLuAi4 genomic library were screened. 65 positive clones corresponding to the 8 contigs were isolated and 29 clones of them were confirmed to be extended to the seed BACs by end-sequencing and fingerprinting. The protocol greatly enhanced the efficiency of the contig extension and was also superior for its specificity, sensitivity and reusability to the colony in situ hybridization which is a conventional method employed in contig extension and physical map construction.