Microsatellite Isolation and Characterization in Pomegranate (Punica granatum L.)

Development of microsatellite markers has been an increasing trend in crop genetic studies because oftheir applicability in breeding programs. Here we report the development of inter simple sequencerepeat (SSRs) in pomegranate (Punica granatum L.) using an enrichment method that makes use of magneticbeads. Enriched genomic libraries with AG and ATG microsatellite motifs were constructed, and 60 positive clones were detected by a colony PCR technique, of which 43 clones showed high-quality sequences. Out of these, 32 (74.4%) contained microsatellite sequences and 25 primer pairs were designed, of which 11 (44%) revealed polymorphisms, 12 (48%) showed monomorphic patterns and 2 (8%) generated poor amplification on a set of 20 pomegranate genotypes. Eleven microsatellite primers (two of them amplified two loci) were selected to assess polymorphism in the set of genotypes. There were 44 alleles amplified over 13 loci, with an average of 3.38 alleles per locus. The mean polymorphism information content (PIC) value was 0.433 over 13 loci, which shows that the majority of the microsatellite loci are highly informative. Cluster analysis was able to separate genotypes based on their geographical distribution and type (i.e., wild or domestic). This study shows the isolation efficiency of the magnetic beads technique, the abundance of microsatellites in pomegranate, and their potential application in pomegranate genome mapping and genotyping.