cpSSR and High-Resolution Melting Analysis (HRM) for Pinus pseudostrobus Lindl. Variety Genotyping and Discrimination

The unambiguous identification of varieties within the Pseudostrobus complex is a key step to facilitate tree selection and monitoring in the wild as well as in plantations. Molecular tools provide a powerful approach for species delimitation; however, the use of DNA barcodes in this group has met limited success due to widespread haplotype sharing from lineage sorting, hybridization and introgression. Here, we evaluate the utility of real-time PCR coupled with high-resolution melting (HRM) to discriminate among Pinus pseudostrobus Lindl. var. pseudostrobus, apulcensis and oaxacana, from wild populations in central and southern Mexico, using chloroplast DNA sequence variants located within the clpP, ycf2, trnL(UAA)-trnT(UGU) and trnI(CAU)-trnF(GAA) loci. The markers ycf2/trnL(UAA)-trnT(UGU) produced clear melting patterns that separated the varieties pseudostrobus and oaxacana from type var. apulcensis, whereas clpP discriminated over 60% of var. oaxacana individuals. This assay underlines the usefulness of these less-used DNA regions as potential biological markers and exhibits the effect of geography on allele distribution and the likely presence of hybrids among the species and varieties.