Characterization of genetic diversity in chickpea (Cicer arietinum) genotypes using ISSR markers

Introduction

Cultivated chickpea (Scientific name-Cicer arietinum L.) is a self-pollinated, diploid (2n = 16) annual pulse crop with a genomic size of 740 Mbp. Chickpea is an important agricultural crop and 4 th most important grain-legume crop after soybean, bean, and pea (Upadhyaya et al. 2008) [7] . Chickpea is a cheap protein source in most developing countries in Asia and Africa. (Saeed et al. 2011; Jannatabadi et al. 2014) [4,2] . The development of high-yielding and stress-resistant chickpea varieties requires a thorough understanding of genetic diversity among available genotypes. The conservation and use of diverse collections of plant genetic resources is the backbone in plant breeding programs, so this genetic variability is the raw material for crop breeding industry on which selection acts to evolve superior genotypes (Saeed et al. 2011; Upadhyaya et al. 2008) [4,7] . Traditional methods based on morphological characters are often influenced by environmental conditions and may not accurately reflect genetic variation. Therefore, molecular characterization has become an effective and reliable approach for studying genetic diversity in chickpea. Molecular genetic markers have been widely used in the last decades for both assessment of original material and search for valuable plant phenotypes. Genetic diversity in the chickpea has been explored using a range of molecular markers such as SSR (Saeed et al. 2011; Ghaffari et al. 2014) [4,1] , RAPD (Talebi et al. 2008b) [6] and AFLP (Talebi et al. 2008a) [5] . Among various molecular marker techniques, Inter-Simple Sequence Repeat (ISSR) markers are widely used because they are highly reproducible, cost-effective, and capable of detecting high levels of polymorphism without prior sequence information. ISSR markers help in identifying genetic relationships, assessing variability, and supporting marker-assisted breeding programs for the development of improved chickpea cultivars with better yield, quality, and resistance to biotic and abiotic stresses.

Materials and Methods

Plant Materials

The seven different genotypes of chickpea (Cicer arietinum L.) were collected from Pulses improvement Project, Mahtma Phule Krishi Vidyapeeth, Rahuri-413722 (MS) (Table 1). Seeds were thoroughly rinsed with sterile water and sown in a sterilized soil mixture to ensure healthy seedling development.

PCR Amplification

The ISSR markers amplified by polymerase chain reaction (PCR) in a volume of 20μl, containing 20 ng genomic DNA, 1U of Taq DNA polymerase (DSS Takara Biolab) 2 μM of primer (Eurofins Laboratories) (Table 2), 100 μM of each dNTP, 2μl (10×) PCR buffer (DSS Takara Biolab), 2.5 mM MgCl2 (DSS Takara Biolab) and ddH2O, using a BIO-RAD Thermal Cycler (T100 Bio-Rad Laboratories, Inc., United States). Amplification was carried for 38 cycles, each consisting of a denaturation step at 95 °C for 1 min, annealing at 46-53 °C for 1 min and extension step at 72 °C for 2 min. PCR products were analyzed using 2.5% agarose electrophoresis gels stained with ethidium bromide.

Scoring of amplified fragments

Frequencies of incidence of all polymorphic alleles for each ISSR markers were calculated and used for determining statistical parameters. The amplified profiles for all the primers was compared with each other and bands of DNA fragments was scored as 1 for presence and 0 for absence, generating 0 and 1 matrix. Percent polymorphism was calculated by using the formula-Number of polymorphic bands Percent polymorphism = × 100 Total number of bands

Analysis of the Amplified Fragment

Pair-wise genetic similarities between chickpea genotypes and polymorphic band was estimated by Jaccard's similarity coefficient. Clustering was done using the symmetric matrix of similarity coefficient and cluster obtained was based on unweighted pair group method for arithmetic mean (UPGMA) using sequential agglomerative hierarchical nested (SAHN) cluster analysis of NTSYS-PC version 2.02 (Rohlf, 1998) [3] .

Results and Discussion

Extraction of Genomic DNA and Quantification Genomic DNA was extracted using CTAB-DNA extraction method. The quantity of DNA was checked by using 1 µl of DNA of each 7 genotype by using Bio-Spectrometer. The quality of the DNA isolated from all the seven varieties were obtained by calculating the ratio of absorbance at 260 and 280 nm. The value varied from 1.763 to 1.964; however most of the value fell in the range of 1.8-2.0, indicating good quality of the isolated DNA. Quantity of all samples was found to be in the range of 150ng/µl l to 490 ng/µl. The results of 0.8% agarose gel electrophoresis showed good quality of DNA (Fig. 1).

Analysis of genetic divergence

Allelic diversity data was used to produce a dendrogram in order to elucidate the relationship among seven chickpea genotypes. Presence or absence of each band on the gel was scored and similarity between every pair of entries was estimated. Genetic Similarity estimate (Jaccard's coefficient) based on ISSR banding pattern used for cluster analysis to present genetic relationship in the form of dendrogram (Fig. 4). Jaccard's coefficient value for 7 chickpea genotypes are presented in following table 4. The large range of similarity coefficient revealed by ISSR markers provided greater confidence for the assessment of genetic divergence and interrelationship. A perusal of similarity coefficients clearly reflected that a very high degree of similarity exists between chickpea varieties Phule Vikram and Phule Vikrant (0.62). The analysis further revealed that Phule Vishwaraj is the most genetically distinct genotype, as it formed a standalone cluster (Cluster II) and joined the rest of the group at the minimum similarity coefficient of 0.42. This early separation highlights Phule Vishwaraj as a significant source of genetic diversity among the tested samples. The study demonstrates that ISSR markers are highly effective for detecting polymorphism and estimating genetic distance among the selected chickpea genotypes. The large range of similarity coefficient revealed by ISSR markers provided greater confidence for the assessment of genetic divergence and interrelationship. Such characterization is essential for future breeding strategies, as it helps in selecting diverse parents to enhance genetic variability in chickpea improvement programs.

Conclusion

The molecular analysis and dendrogram interpretation of the seven chickpea genotypes lead to several significant conclusions regarding their genetic architecture.