Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
P27
Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108, USA.
DNA libraries with large genomic inserts, such as bacterial artificial chromosome (BACs) libraries, are becoming increasingly popular in genomic research. We previously constructed an EcoRI soybean BAC library with an average insert size of 120 kb for use in positional cloning of a major cyst nematode (SCN) resistance gene in soybean (rhg 1). Since the soybean genome has a high level of repetitive DNA, efficient chromosome walking depends on: 1) establishment of physical contiguity between BAC clones, 2) isolation of BAC ends to re-screen the library and orient the walk, and 3) generation of sub-clones for use in genetic mapping. To simplify this effort, we developed a BAC-end isolation and cloning protocol that uses a single buffer system and requires as little as 5 ng of BAC DNA in a single tube without the need for DNA extraction. PCR and inverse PCR are used to amplify the left and right ends, respectively, which can then be rapidly cloned. Contigs were established by comparative restriction digests of BACs and by Southern blot hybridization of newly uncovered clones probed with the original BAC clone and/or BAC ends. To date, we have isolated and mapped seven BAC clones around the rhg 1 locus spanning 620 kb of the region. End-clones and sub-clones have been used to map 11 RFLP marker loci around rhg 1. Sub-clones from a BAC clone that maps only 0.4 cM away from rhg 1 are also being analyzed by single-pass sequencing of ends. BLAST and GRAIL searches will be used to identify candidate genes.