A Non-redundant Strategy for Identification of a Minimum Tiling Path BAC Contig Spanning Approximately 390 kb of the QTL yld1.1 in Oryza rufipogon

摘要

We describe here a strategy combining the sequence-tagged sites (STS) mapping and selection of non-redundant bacterial artificial chromosomes (BAC) clones and fingerprint analysis for identification of ininimum tiling path (MTP) contig assembly purposes. To facilitate development of MTP BAC contig spanning part of the yield enhancing quantitative trait locus (QTL) yldl.l and linked marker RM5 in Oryza rufipogon, fifteen positive BAC clones identified in the BAC library screening using four-pooled overgos were used for STS mapping of a brief physical map. Based on the STS-content map information, four major clones which are tentatively form a MTP contig were fingerprinted and confirmed their MTP orientation. The four selected core BAC clones were found to be sufficient in generating a MTP contig, suggesting the feasibility of the non-redundant strategy in contig assembly. As a result, we rapidly established a single contig of four MTP BAC clones spanning approximately 390 kb at the yld1.1 region rice on chromosome 1. The RMS microsatellite and 6 newly designed STS markers were anchored by STS-PCR and BLASTN analysis for integration of physical and genetic map. This integrated physical map will facilitate regional sequencing, aid in map-based cloning (MBC) of any agronomically important genes and will provide a powerful means in comparative analysis of rice genome. BLASTN analysis using the RMS and STS markers sequence reveals that the analyzed yldl.l region shares more than 99% sequence similaritywith O. sativa ssp. japonica cv. Nipponbare genome. The genome conservation suggests a pressure of evolutionary selection of the RM5 region and a critical functional role of the genes within the region. The described low-cost physical mapping strategy is fast and less-laborious compared to conventional restriction fingerprint method especially when enter large-scale fingerprint analysis.