Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes

Abstract

 Simple sequence repeats (SSRs) are versatile DNA markers that are readily assayed and highly informative. Unfortunately, non-targeted approaches to SSR development often leave large genomic regions without SSR markers. In some cases these same genomic regions are already populated by other types of DNA markers, especially restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and amplified fragment length polymorphisms (AFLPs). To identify SSR markers in such regions, bacterial artificial chromosome (BAC) clones can be used as intermediaries. First, one or more BAC clones in a region of interest are identified through the use of an existing DNA marker. BAC clones uncovered in this initial step are then used to create a small insert DNA library that can be screened for the presence of SSR-containing clones. Because BAC inserts are often 100-kb pairs or more in size, most contain one or more SSRs. This strategy was applied to two regions of the soybean genome near genes that condition resistance to the soybean cyst nematode on molecular linkage groups G and A2. This targeted approach to identifying new DNA markers can readily be extended to other types of DNA markers, including single nucleotide polymorphisms.