Categorization of Expressed Sequence Tag Microsatellites for Marker Optimization and Genotype Evaluation in Peach

Exponential growth of genomic and expressed sequences has allowed thousands of microsatellite motifs and primers to be easily mined out of them by pipelined bioinformatics programs. But it remains economically and practically difficult to synthesize and screen such a great number of primers to develop useful microsatellite markers of desired amplification reliability, allelic polymorphism, and chromosomal distribution. Preselection of desired primers depends on computational prediction and optimized selection criteria. In this study, over ten thousand expressed sequence tag microsatellite primer sequences were generated from Prunusexpressed sequence tags, analyzed by BLAST against the peach reference genome, and categorized into eight subgroups based on the alignment status and the size difference between the genomic and expressed amplicons of each primer. Based on the categorizations and predicted performance and distribution, a subset of three hundred eighty-four primers were optimally chosen and arranged in four ninety-six-well plates for genotyping evaluation of selected peach genotypes. Only two of the primers failed in the peach cultivars evaluated, suggesting the reliability of optimally chosen primers was substantially improved. A majority of the primers also yielded high average allele numbers and polymorphism information content values among the screened peach genotypes. Further utilization of these new gene-based microsatellite markers in peach genetics and breeding studies are discussed.