Genome-wide mining of potentially-hypervariable microsatellites and validation of markers in Momordica charantia L

Relatively large number of bitter melon microsatellite markers have been reported; however, only few resulted in successful PCR amplification and a small fraction shown polymorphisms. This limited chance of recovering polymorphic markers makes the primer screening a cost-demanding process. To test the hypothesis that microsatellites with longer motifs as well as shorter motifs repeated substantially shall have better prospects to be polymorphic, we performed a genome-wide microsatellite mining. We selected a sample of genome-wide microsatellites with prescribed motif lengths or satisfying a target repeat number, which were considered potentially-hyper variable, for primer designing and validation. Seventy five microsatellites satisfying these criteria were identified, of which 69 were validated through successful PCR amplification. Among them, 40 (53.33% of the markers identified) were polymorphic. This result showed a significantly higher success compared to our initial results of 51 (20.64%) polymorphic markers out of the 188 amplified when 247 previously reported markers were screened. The screening of two cultivars revealed that markers were efficient to identify up to three alleles. The characterization of these 69 new markers with 247 markers previously reported showed that di-nucleotide motifs were most abundant, followed by tri- and tetra-nucleotide motifs. TC motif markers were most polymorphic (12.08%) followed by AG and CT motifs (both 9.89%). Similarly, AGA (6.59%) and TATT (3.29%) were most polymorphic among the tri- and tetra-nucleotide motifs. These 69 hypervariable microsatellite markers along with 188 markers initially validated in this study shall be useful for phylogenetic analyses, studies of linkage, QTL, and association mapping in bitter melon.