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Pomegranate Punica granatum was first introduced to Sri Lanka, possibly through ancient trade routes, thousands of years ago. However, there is no information about the diversity of the pomegranate germplasm in the country, which is important both for breeding new varieties and for conservation efforts. We used inter‐simple sequence repeat (ISSR) regions to investigate the genetic diversity and population structure of pomegranate on the island of Sri Lanka. Hundred and twenty accessions representing seven populations from all pomegranate growing regions of the country were analyzed using 20 ISSR primers. A total of 107 loci were amplified with an average polymorphism information content of 0.3. While the average inter‐population genetic distance was 0.141, it was 0.149 between populations, indicating moderate genetic diversity both within and among populations. Analysis of molecular variance and Nei's genetic diversity revealed higher genetic variation within populations than among populations, and low genetic differentiation (GST) in pair‐wise comparison of populations also suggested limited population differentiation. A considerable level of among‐population gene flow (Nm) was indicated, irrespective of geographical structure and distances. The results of cluster analysis was also in agreement with above analysis and suggest human mediated gene flow and migration patterns. Cluster analysis revealed two main population clusters with several sub‐clusters. While these clusters did not show any correlation with geography, all red peeled accessions clustered into a small sub‐cluster. The results indicate that analysis of ISSR variability is sufficiently informative and powerful to assess the genetic diversity of P. granatum landraces in Sri Lanka.

Abstract:The genetic studies of tropical tree species containing high amount of phenols are greatly hampered by the inability to extract sufficient quantities of high quality DNA and the presence of PCR inhibitors in extracted DNA samples. While there are some DNA extraction and PCR protocols for such species, no consistency in results. Hence, optimization of such protocols is a prerequisite for conducting research studies on tropical flora. This study reports the optimization of cheaper DNA extraction and PCR procedures for plants having high amount of phenolic compounds and PCR inhibitors. Three fruit crop species, Bael (Aegle marmelos L. Correa), Pomegranate (Punica granatum L.) and Mango (Mangifera indica L.), with distinctly diverse secondary metabolic profiles, were used as examples. The CTAB DNA extraction method, with some modifications, was compared with two commercially available DNA extraction kits namely; Promega Wizard® Genomic DNA purification kit and QIAGEN DNeasy® Plant Mini kit. DNA from three to five genotypes from each species was extracted from each method and the quality and quantity were assessed. Spermidine was added to the PCR mix at the rate of 0.8 µM to block the PCR inhibitors and the DNA samples were amplified using universal plant barcoding primer pair rbcL, and SSR or ISSR primers. The modified CTAB method resulted significantly higher quantity of quality DNA in all samples compared to two commercial kits. Henceforth DNA extracted from CTAB method, and the two commercial kits were used to precede PCR. However, expected bands were not generated in regular PCR. Interestingly, the inclusion of spermidine amplified the relevant band/s in relatively easy PCR reactions such as rbcL, as well as trickier reactions such as SSR and ISSR. These results suggest that cheaper alternative procedures used in this research study could be used successfully for the range of applications in plants with array of secondary metabolic profiles.