Development of a SNaPshot assay for the genotyping of organellar SNPs in four closely related pines

Mitochondrial (mtDNA) and chloroplast (cpDNA) polymorphisms are valuable resources to study past demographic changes, phylogenetics and evolution, especially in forest tree species, where these genomes are haploid and uniparentally transferred. The organellar markers were usually scored separately using direct sequencing or PCR-based approaches, which can be time-consuming and expensive, especially in large-scale population genetics research. In this study, we developed an efficient and cost-effective SNaPshot assay for genotyping preselected ­mtDNA and cpDNA polymorphism in four closely related pine species including Scots pine (Pinus sylvestris L.) and three taxa from the Pinus mugo complex. We validated the method by genotyping the samples derived from 12 populations of the species from their wide geographical distribution range in Europe. The results proved high accuracy of the method with a genotyping success rate of 99.7%. The set of assayed markers shows significant genetic variation. By using multiplex SNaPshot assay, we provided an efficient and sensitive molecular tool for intra- and interspecific genetic analyses. The presented protocol is useful for fast and relatively cheap SNP genotyping of organelle genome of closely related pine species. The assayed SNPs allow studying the species discrimination and detailed investigations of their population history and structure. Given its numerous benefits and efficient genotyping rate, the SNaPshot method appears to be a valuable and practical resource for studying the genetic makeup of forest tree species. Particularly, it proves to be advantageous for population genetics.