Genotyping variability of computationally categorized peach microsatellite markers

Numerous expressed sequence tag (EST) simple sequence repeat (SSR) primers can be easily mined out. The obstacle to develop them into usable markers is how to optimally select downsized subsets of the primers for genotyping, which accordingly reduces amplification failure and monomorphism often occurring in randomly chosen primers of unknown distribution, reliability, and polymorphism. It is essential to computationally align the numerous primer sequences onto a quality reference genome, and to categorize them into subgroups by calculated genomic and expressed amplicon size differences (ASD). In this study, 288 optimally selected peach EST-SSR primers with known chromosomal distribution, SSR types and ASD subgroups were evaluated using representative peach cultivars and Prunus species. The 288 primers were spread on peach genome main scaffolds 1 to 8 (64, 34, 33, 36, 25, 29, 29, and 32 primers, respectively) and minor scaffolds 9 to 12 (1, 2, 2, and 1, respectively). The primers were also categorized by the SSR motif unit lengths into six types: 82 bi-type, 80 tri-type, 37 tetra-type, 16 penta-type, 15 hexa-type, and 58 compound-type; and by the ASD into five subgroups: 110 “deletion” (ASD<0), 50 “same-size” (ASD=0), 45 “insertion” (0<ASD≤20), 34 “insertion/intron” (20<ASD≤100), and 49 “intron” (100<ASD≤466). Analysis of genotyping data showed genotyping variability in the number of detected alleles, number of polymorphic genotypes, gene diversity, heterozygosity, and polymorphic information content for each marker, each genotype, and each primer category, among the six SSR types and the five ASD subgroups. The categorization facilitates optimal selection of considerably downsized subsets with known distribution and predicted performance. Likewise, the genotyping variability allows prioritized utilization of EST-SSR primers of SSR types and ASD subgroups with improved polymorphism in EST-SSR genotyping applications.