Identification and Isolation of Microsatellite in Pomegranates (Punica granatumL.) for Evaluation of Iranian genotypes

Microsatellites or simple sequence repeats (SSRs,) have become the markers of choice for genetic studies with many plant species. Although the discovery and development of new microsatellites is a costly and technically demanding process, they are widely used in many plant systems. There has been no report of the development and utilization of microsatellite markers in Pomegranate. The objectives of the present study were to construct pomegranate microsatellite-enriched library, to isolate microsatellite sequences and evaluate their level of polymorphism in pomegranate cultivars. Edwards's method was used for this purpose which is based on partial genomic library construction and enrichment in order to capture digested fragments containing microsatellite. Ease and efficiency of enrichment is one of the advantages of this method in comparison to similar isolation methods. A genomic DNA library enriched for dinucleotides (AG)n and (GT)n and trinucleotide (ATT)n microsatellite motives has been developed from "Malase- Yazd" pomegranate. The enrichment level for library was 20-60%, and 27% of clones (4 out of 15) contained microsatellite sequence. Four primer pairs were designed based on flanking regions of the microsatellites using the Oligo software. Among the primer sets two pairs amplified cleanly and produced an easily interpretable PCR product in the pomegranate cultivars. A18 primer pairs displayed polymorphism among 29 pomegranate cultivars. Gorche-Shahvar and Nabati-Ardakan cultivars appeared to be homozygote in this locus while the other cultivars were heterozygote. A10 primer was monomorph and did not show polymorphism among cultivars.

Keywords:

Pomegranate , Microsatellite , Simple Sequence Repeat (SSR) , Enrichment