Study genetic structure and diversity of Afghanistan Chalghoza pine (Pinus gerardiana) populations using SCoT and iPBS molecular markers

The Chalghoza pine (Pinus gerardiana) is grown in the eastern and southeastern regions of Afghanistan and has an important role in the socio-economic progress of rural communities. It serves various purposes, such as providing pine nuts, fuel wood, medicinal plants, grazing areas, and shelter for livestock. In this research, the genetic diversity and structure of different Chalghoza pine populations were examined using molecular markers known as start codon targeted (SCoT) and inter primer binding site (iPBS) markers. Due to the excessive harvesting of its nuts and a decline in its population, the IUCN categorizes the Chilgoza pine as a nearing threat.

39 Chalghozeh pine genotypes were collected from various regions across five provinces in Afghanistan, namely Khost, Paktia, Laghman, Kunar, and Nuristan. To extract genomic DNA, a modified CTAB procedure was employed, utilizing megagametophyte tissue from 4-5 seeds per genotype. For this research, six primers each for SCoT and iPBS markers were utilized. To analyze the data and determine genetic relationships, cluster analysis was conducted using the unweighted pair-group method arithmetic averages.

In this study, 12 SCoT and iPBS primers were utilized, resulting in the generation of 48 and 55 bands for SCoT and iPBS markers, respectively. The percentage of polymorphism was estimated at 20.8% for SCoT markers and 29.1% for iPBS markers. The average values of PIC (polymorphic information content) were determined as 0.026 for SCoT markers and 0.045 for iPBS markers, indicating a higher differentiation power of iPBS markers compared to SCoT markers. The genetic similarity coefficient revealed a relatively low genetic diversity among the populations examined. The UPGMA dendrogram, based on the similarity coefficient, demonstrated that the genotypes did not cluster according to their collection sites. This outcome can be attributed to the species' highly cross-pollinating nature and limited distribution range in the studied area. The observed number of alleles (NA) was 1.08 and 1.09, while the effective number of alleles (NE) was determined as 1.075 for both SCoT and iPBS markers. The Shannon's information index (I) was calculated as 0.055 and 0.060 for SCoT and iPBS markers, respectively. The expected heterozygosity (HE) values were estimated as 0.039 for SCoT markers and 0.042 for iPBS markers. The analysis of molecular variance (AMOVA) indicated that the genetic diversity within populations was higher than among populations. Through a Bayesian model-based STRUCTURE analysis, two groups (K=2) were identified among the five populations of Pinus gerardiana, and admixture was observed within individuals. 

The minimal variations observed in total diversities and levels of population differentiation among the five Chalghoza pine populations suggest that the genetic structure of these populations aligns with the species' long-lived perennial nature and regional distribution. These findings have implications for the conservation and cultivation of this economically significant tree, providing valuable insights for its management and preservation.