Evaluation of genetic purity among chickpea (Cicer arietinum) seeds with different colours through SSR markers

Maintaining high genetic purity in crop cultivars is mandatory for achieving certification presenting reasonable agronomic performance in the field, and attracting farmers and investors. DNA-based technologies were developed to provide better discrimination of varieties for breeders (Smith and Register III, ۲۰۰۸). Chickpea genotypes with two different colours along with two reference seeds were investigated to discriminate molecular characterization at the molecular markers Laboratory. Genomic DNA extraction was isolated from leaves as described by Doyle (Doyle, ۱۹۹۱). DNA quantification and qualification were measured using NanoDrop (ND-۱۰۰۰, Thermo Fisher Scientific) at ۲۶۰ nm. Two SSR primer pairs were used to identify genetic purity of the studied chickpea genotypes. PCR amplification and polyacrylamide gel electrophoresis were run through the highly polymorphic primer sets. Both SSR primers exhibited polymorphisms, as shown in Table ۱. Amplified DNA fragments were scored based on the presence and absence of the alleles. The total number of alleles per locus, allele frequency, PCOA (Principal Coordinate Analysis), and molecular variance were calculated using the statistical analysis software GenAlEx version ۶.۵. Polymorphic Information Content (PIC), heterozygosity index (HI), marker Index (MI), and discriminating power (D) were calculated using the software POWERMARKER, version ۳.۲۳ (Table ۱). In this study, two sets of SSR markers (SSR-H۱A۰۶, H۳C۱۱) were employed to determine the genetic purity of two chickpea genotypes introduced by a breeder, along with two reference genotypes from our seed bank. Both markers demonstrated polymorphism in the chickpea genotypes during the purity test, whereas marker SSR-H۱A۰۶ showed the greater efficiency based on polymorphism indices (Table ۱). SSR markers are widespread molecular markers due to their high abundance and polymorphic status (Zane et al, ۲۰۰۲; Xu et al, ۲۰۱۳). SSR polymorphism produced satisfactory results for plant genetic research and molecular breeding due to its multi-allelic nature and variation in DNA segment length (Shu et al, ۲۰۲۱). The highest values of the genetic variation parameters were observed in the dark-coloured chickpea genotype (Figure ۱). The PCOA analysis of SSR data grouped the four chickpea populations into three main clusters: white-coloured chickpea was clustered separately, while the dark-coloured chickpea grouped with the two reference genotypes (Figure ۱). Further, the white and dark-coloured chickpea seeds showed no complete conformity with our seed bank genotypes. Our results revealed ۴۳% molecular variance within populations and ۵۷% among populations (Figure ۲). Thus, this study demonstrates the potential of two SSR markers to distinguish different colours of chickpea genotypes and assess their genetic purity.

کلیدواژه ها:

Chickpea ، Genetic purity ، SSR markers ، Polymorphism ، Genetic variation