Construction And Screening Of An Aibai-Chinese Spring Line Bacterial Artificial Chromosome (BAC) Library In Wheat

Map-based cloning is a kind of efficient way to clone genes in plants while a large-insert library provides a technical platform for map-based cloning. Aibai-Chinese Spring, a near isogenic wheat line of Chinese Spring is a valuable hexaploid wheat material that carries dominant male sterility gene ms2 and tightly linked dominant reduced height gene Rht10. In order to clone these two genes, a BAC library was constructed using BAC pooling strategy. The library consists of 1,000,000 colonies from three ligations with more than 90% recombinants and less than 3% empty clones. 1,000 to 6,000 BAC clones could be pooled in one bacterial culture, total 399 pools were generated with inserts ranging from 6kb to 312kb, the average insert size 118kb and therefore the library represented approximately 6.5 fold haploid genome equivalents. The BAC clone in BAC pool could be amplified by culturing for 84 hr without changing the structure of BAC pool and was suitable for PCR screening. The whole BAC library screening was performed using allele-specific PCR marker cosegregating with Rht10. By serial PCR screening and fingerprinting identification, three BAC clones containing RhtlO gene candidates were obtained from the Aibai-Chinese Spring BAC library and were placed in the same contig and covering 230kb with tolerance 6 and cutoff le-15. These results agreed with the library's predicted genome coverage since Aibai is a heterozygote of dominant reduced height gene RhtlO and male sterile gene ms2. BAC pooling strategy is a very efficient and economical way for map-based cloning special genes in large complex genomes, such as wheat here.