Differentiation of susceptible and resistant chickpea genotypes to Ascochyta blight using the ARMS molecular method

Chickpea, cultivated in rainfed and irrigated fields across many countries, faces biological stresses such as Ascochyta blight caused by the pathogenic fungus Ascochyta rabiei. This disease poses a severe threat to chickpea fields, resulting in substantial annual damage in various countries. Identifying resistance sources through molecular methods, specifically genomic tracking of the resistance trait, holds the potential to expedite and enhance control measures effectively. The use of the Amplification Refractory Mutation System (ARMS) method, involving functional point mutations and resistance-related alleles, proves highly efficient in advancing selection programs targeted at combating Ascochyta blight.

The fragment harboring the SNP18-Pos57723 point mutation within the GSh118 gene sequence underwent amplification and sequencing using PSh118-F/R specific primers. This process utilized DNA extracted from both resistant (MCC113) and susceptible (ILC263) chickpea genotypes. The identification of alleles associated with resistance and sensitivity was established. Distinct differentiating primers were formulated for resistant (PSh118-Fc and PSh118-R) and sensitive (PSh18-Fg and PSh18-R) genotypes. The specific annealing temperature for each reaction was determined through a temperature gradient analysis. The efficacy of the designed primers in distinguishing between resistant and sensitive genotypes was assessed by conducting PCR and comparing their electrophoresis patterns.

The presence of alleles linked to GSh18-2773C resistance and GSh18-2773g sensitivity was confirmed in resistant and sensitive chickpea genotypes, respectively. The specific primers PSh18-Fc and PSh18-R, designed for detecting the resistance allele, successfully amplified the expected 330 bp fragment in the resistant chickpea genotype MCC133. The study results affirmed the accurate amplification of alleles using these designated primers.

In the present study, the ARMS method, known for its high sensitivity, specificity, rapidity, cost-effectiveness, multiplexing capabilities, compatibility with high throughput methods, and non-destructive nature, was employed to differentiate chickpea genotypes resistant to Ascochyta blight. The ARMS method's effectiveness in selecting resistance sources among various plant lines and cultivars has been demonstrated through allele tracking related to the resistance gene in prior studies. The results of this study indicated that the ARMS method accurately distinguishes chickpea genotypes resistant and sensitive to Ascochyta blight based on alleles associated with the GSh18-2773 position's point mutation. Thus, employing specific primers for the resistance allele is recommended in molecular selection studies of chickpea sources resistant to Ascochyta blight.