A New Set of Nuclear Microsatellites for an Ecologically and Economically Important Conifer: the European Black Pine (Pinus nigra Arn.)

The European black pine (Pinus nigra Arn.) is an ecologically and economically important conifer discontinuously and often patchily distributed across different ecological environments and climatic conditions around the Mediterranean Basin. Many studies aiming at understanding the evolutionary history of the species have been carried out, particularly in the hope of resolving the taxonomy of this collective species where many subspecies have been described. So far, neither morphology, enzyme, and metabolite polymorphism nor DNA marker studies have been able to converge on a single taxonomy, and the evolutionary history of the European black pine remains unclear. To date, nuclear microsatellites (nSSRs) are not available for P. nigra. In this work we designed two multiplexes of eight and six nSSRs each, both characterized by high amplification quality and polymorphism. Nine nSSRs were specifically developed for the European black pine starting from six transcriptomes sequenced using Next Generation Sequencing (NGS) techniques following the Illumina method, while five previously developed for other pines were successfully transferred to P. nigra. The mean number of alleles per locus was 5.9, and the observed heterozygosity ranged between 0.341 and 0.875. F ST was statistically significant for all loci, and Bayesian clustering approaches revealed a geographically meaningful population structure. We thus show this set of 14 nSSRs to be highly informative for population genetic studies and of significant interest to resolve the taxonomy of the European black pine.