MICROSATELLITE ENRICHMENT IN ORGANISMS WITH LARGE GENOMES (ALLIUM-CEPA L.)

To exploit the polymorphism of repeat numbers in short tandem repent ( STR) sequences (microsatellites) as molecular markers, STRs must be is olated and PCR primers must be developed in flanking sequences. In spe cies with large genomes such as Allium cepa L. (onion and shallot), an efficient selection procedure for genomic fragments containing STRs i s a crucial step. Here we describe a nonradioactive method for microsa tellite isolation based on affinity capture of single-stranded restric tion fragments annealed to biotinylated microsatellite oligonucleotide s (CA)(10), (GAA)(8) and (AAC)(8) followed by adapter-mediated genomic PCR. Cloning of the products in E. coli and plasmid sequencing reveal ed more than 60% positive clones. Primers were designed in STR-flankin g regions, and one or two bands were amplified in 13 diploid onion and five shallot accessions. Allelism of the the bands was confirmed by p roduct sequencing.