Development and transferability of two multiplexes nSSR in Scots pine(Pinus sylvestris L.)

Single sequence repeat(SSR) multiplexing is a semi high-throughput PCR methodology for the analysis of multiple SSRs.We developed two SSR multiplexes selected from SSR loci previously reported in the pine literature and tested the transferability of both SSR multiplexes in nine other pine species.We tested 234 nuclear SSR loci(n SSRs) previously described in the pine literature and selected ten n SSRs following the simple criteria of interpretability and reproducibility.Selected nuclear loci were divided into two n SSRs multiplex sets and their amplification was optimized for three different multiplex PCR methods based on:(a) a custom PCR protocol,(b) a custom protocol with hotstart taq polymerase,and(c) a commercially available kit for SSR multiplexing.To validate their performance,the level of genetic diversity was assessed in three Scots pine natural populations(Hungary,northern Sweden and southern Sweden).In addition,we also tested the transferability of these multiplexes in nine other pine species.We have developed two n SSRs multiplexes of five loci each that will contribute to reduce the costs of n SSR scoring,while increasing the capacity of n SSR loci analysis.Amplification was successful in all three populations(94 % success) and the level of polymorphism(7.1 alleles/marker) was similar to that previously reported for other Scots pine natural populations.Transferability of both multiplexes was successful for those pine species closely related to Scots pine.