SRAP markers for characterization of the genetic diversity and differentiation of Pinus nigra populations in protected forested areas in Bulgaria, Greece, and Cyprus

Abstract

The present study aimed to evaluate the genetic diversity and differentiation of Pinus nigra Arnold, the European black pine, populations located in protected forested areas in three countries from the Balkan and Mediterranean region: Bulgaria, Greece, and Cyprus. Totally, 175 DNA samples from P. nigra plants collected from eight populations in these countries were analyzed using three SRAP primer pairs. The applied SRAP markers demonstrated high-resolution power, resulting in the identification of an average of 215 ± 67 polymorphic loci per SRAP primer pair. The analysis of molecular variance (AMOVA) showed that 82% of the observed variation was due to intra-population variation, while 18% was due to inter-population variation. The overall analysis of the population structure suggested low (Fst ≤ 0.01) intra-country differentiation for the populations from Bulgaria and Cyprus and moderate (0.15 ≤ Fst ≤ 0.17) inter-country differentiation. On the contrary, high differentiation between the populations (0.06 ≤ Fst ≤ 0.20) and complex genetic structure were characteristic of the three populations from Greece, relatively closely located in the area of the Pindos National Park. Analysis of the population structure also revealed that one of the Pindos populations belonged to the cluster of Bulgarian populations (Fst ≤ 0.01), showing moderate (Fst ≤ 0.11) to high (Fst ≤ 0.20) genetic differentiation from the other analyzed Pindos populations. The further use of SRAP markers for mapping the genetic diversity among P. nigra populations and identification of local populations diverted from the overall phylogeography-driven pattern in the studied region are discussed.

Keywords:

• European black pine
• Pinus nigra arnold
• SRAP markers
• population structure
• mapping genetic diversity

Acknowledgments

The authors would like to thank Rumyana Velcheva, technician at the AgroBioInstitute, Sofia, Bulgaria, for the excellent technical assistance.

Author contributions

Conceptualization, pm, V.F. and I.A.; Sampling, K.R., pm, V.F., A.S., F.U. and I.A. Methodology, T.Z., K.R., pm, V.F. and I.A.; Software, K.R. and T.Z.; Investigation, T.Z., E.B., A.S. and M.R.; Funding Acquisition, pm, V.F., and I.A.; Writing—original draft, review and editing, T.Z., I.A. and K.R. All authors have read and approved the final version of the paper.

Disclosure statement

The authors declare no conflict of interest

Data availability statement

All data included in this study are available from the corresponding author [I.A.] upon request.

Additional information

Funding

This work was supported by the INTERREG Balkan-Mediterranean Project BIOPROSPECT “Conservation and sustainable capitalization of biodiversity in forested areas” (BMP1/2.1/2336/2017), as well as by the Operational Program Science and Education for Smart Growth 2014–2020, co-funded by the European Union through the European Structural and Investment Funds, Grant BG05M2OP001-1.002-0012 Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products.