Sebastian Szczepański, Bartosz Łabiszak, Witold Wachowiak
Development of a SNaPshot assay for the genotyping of organellar SNPs in four closely related pines
Dendrobiology 2023, vol. 90: 76-85
https://doi.org/10.12657/denbio.090.006
Abstract:
Mitochondrial (mtDNA) and chloroplast (cpDNA) polymorphisms are valuable resources tostudy past demographic changes, phylogenetics and evolution, especially in forest tree species, where thesegenomes are haploid and uniparentally transferred. The organellar markers were usually scored separatelyusing direct sequencing or PCR-based approaches, which can be time-consuming and expensive, especiallyin large-scale population genetics research. In this study, we developed an efficient and cost-effective SNaPshotassay for genotyping preselected mtDNAand cpDNA polymorphism in four closely related pine speciesincluding Scots pine (Pinus sylvestris L.) and three taxa from the Pinus mugo complex. We validated themethod by genotyping the samples derived from 12 populations of the species from their wide geographicaldistribution range in Europe. The results proved high accuracy of the method with a genotyping successrate of 99.7%. The set of assayed markers shows significant genetic variation. By using multiplex SNaPshotassay, we provided an efficient and sensitive molecular tool for intra- and interspecific genetic analyses. Thepresented protocol is useful for fast and relatively cheap SNP genotyping of organelle genome of closely relatedpine species. The assayed SNPs allow studying the species discrimination and detailed investigationsof their population history and structure. Given its numerous benefits and efficient genotyping rate, theSNaPshot method appears to be a valuable and practical resource for studying the genetic makeup of foresttree species. Particularly, it proves to be advantageous for population genetics.
Keywords: genetic diversity, mtDNA, hybridization, Pinus, SNP genotyping, organelle DNA