Genetic diversity and differentiation of Pinus sylvestris L. cenopopulations growing in bog land and upland ecotopes

Scots pine (Pinus sylvestris L.) forming coenopopulations in diverse soil-hydrological conditions is of utmost interest in studying the environmental mechanisms of divergence among populations of tree species. The analysis of protein polymorphism educed the genetic differentiation between bog land and upland coenopopulations of Scots pine, emerging from phenological isolation. Thus, we find it essential to study further the mechanisms of developing the population structure of tree plants with the use of more meaningful methods of molecular genetic analysis that would allow identifying differences at the DNA level. The aim of the research is to study the indicators of genetic diversity and differentiation of coenopopulations of Scots pine growing in contrastive swamp and dry land conditions, using ISSR markers. The polymerase chain reaction (PCR) method with ISSR primers was used to identify the level of genetic polymorphism and differentiation. DNA for analysis was isolated from the cambial layer of the trunk using the CTAB method. Four upland and three bog coenopopulations were studied in total (See Table 1). Within each coenopopulation, 30 trees were analyzed using (CA)₆AGCT, (CA)₆AG, (CA)₆GT, (CA)₆AC, (AG)₈T and (AG)₈GCT primers. We used the following reaction mixture (total volume of 10 pl) to amplify DNA with ISSR primers: 1 pl of PCR buffer; 0.2 pl 10 mM dNTPs; 0.1 pl of 100 pm primer; 1 pl of DNA sample; 0.1 pl of Taq polymerase (2 u / pl); 7.6 pl of water. PCR mode: 5 min. denaturation at 94 °C, 35 cycles: 0.5 min. denaturation at 94 ° C, 45 sec. annealing at 60 ° C, 45 sec elongation at 72 °C, final elongation for 7 min at 72 °C. The PCR results were visualized by electrophoresis in 1.5% agarose gel in TBE buffer. The results were recorded and data were processed using the GelDoc 2000 gel documentation system (Bio-Rad, USA) and the Quantity One® Version 4.6.3 software package. The date analysis was performed by the software tools POPGENE ver. 1.31., POPTREEW, GenAlEx and PAST 3.25. We revealed that out of 215 ISSR-loci elicited in 240 trees out of eight coenopopulations 208 ISSR-loci were polymorphic. The number of elicited ISSR loci in different coenopopulations varied from 162 to 194 (See Table 2). The studied coenopopulations were characterized by different levels of genetic diversity. Indicators of genetic diversity ranged within the following: the proportion of polymorphic loci was 67.9-88.5; the observed number of alleles was 1.679-1.805; effective number of alleles 1.279-1.331; the expected heterozygosity was 0.174-0.207. However, no dependency of genetic diversity indicators on soil-hydrological conditions has been found. Moreover, we considered that dry and bog coenopopulations differed significantly in the ISSR-markers frequency obtained with five of six ISSR-primers, which indicates a different genetic structure (See Table 3). Nei’s genetic distance between different coenopopulations varied from 0.100 to 0.317. The UPGMA dendrogram and analysis of principal coordinates (PCoA) show that the bog forests make up a separate cluster (See Fig. 1 and 2). This may be a sign that there are genetic processes inducing the divergence of dry and bog coenopopulations of Scots pine. The Mantel test shows that genetic distance between populations had a weak positive correlation with geographic distance (R=0.3085, P=0.1728). Analysis of molecular variance (AMOVA) shows that 9% of total variation was accounted for by differences between groups of upland and bog land coenopopulations (See Table 4). The percentage of genetic variation among coenopopulations within compared groups was 13%. The majority of the variation was found within populations (78%). The overall level of genetic subdivision of coenopopulations was 17% (G_ST=0.17), hence, the main part of genetic variation is inside the coenopopulations (83%) (See Table 5). Thus, we showed a significant impact on the genetic structure formation of P. sylvestris population in this research. The paper contains 5 Tables, 2 Figures and 22 References.

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