Brief restriction endonuclease digestion of genomic DNA improves PCR amplifiability and reproducibility of SSR loci: Augmenting exploration of hidden genetic variability in an endemic carp of South-East Asia

Abstract

Majority of the microsatellite primers developed through painstaking, resource intensive research often fail to amplify due to secondary structures in the repeat rich target. In the present work, we describe the use of restriction endonuclease digestion of genomic DNA prior to PCR to counter this issue. The methodology, Restriction Endonuclease Digestion of genomic DNA to improve PCR Amplifiability and Repeatability of SSR loci (REDDAR-SSR), was tested on 40 species-specific Simple Sequence Repeat (SSR) loci in Osteobrama belangeri (F: Cyprinidae). Undigested and HpaII digested pooled genomic DNA samples were used as templates. Two primer concentrations (10 pM and 25 pM) and two annealing temperatures (56 °C uniformly and individual primer-specific annealing temperatures) were tested on both template types in sets of three, repeated thrice (360 reactions/condition). The digested templates exhibited significantly higher (P < 0.05) amplifiability (93.9 % against 24.16 % of undigested counterpart) as well as repeatability (94.47 % against 21.56 % of undigested counterpart). Primer concentration had no significant effect on outcomes (P > 0.05). The number of successfully amplified loci in our method was 38 compared to 18 by the conventional protocol. All 38 loci could also be reliably amplified in individual gDNA using the developed protocol out of which 9 loci were observed to be polymorphic. Notably, 4 of the polymorohic loci could be amplified only through REDDAR-SSR. Our protocol can potentially reduce expenses; resources & time vis-a-vis enhance the reach of SSR markers for genotyping species of commercial or conservational significance.