Exploration and characterization of candidate genes for race 4 Fusarium wilt resistance in chickpea (Cicer arietinum L.)
Loading...
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
RPCAU, Pusa
Abstract
The production and productivity of chickpea is significantly hampered by numerous factors of biotic and abiotic nature. Among the biotic stress, wilt disease caused by fungal species i.e. Fusarium oxysporum f. sp. ciceris is a devastating one. Several races of the pathogens already been reported and the researchers are working continuously to develop the robust resistance mechanism against the wilt disease of chickpea. Marker assisted selection is employed in developing chickpea plants resistant against Fusarium wilt using the markers like TA59 and TR19. The TA59 and TR19 are reported to be linked particularly with race 4 (Foc4) of Fusarium oxysporum f. Sp. ciceris, however, the region between TA59 and TR19 in linkage group 2 is significantly wide and the region is largely unexplored for the identification of potential candidate genes that is actually imparting resistance against Foc4 wilt resistance. Therefore, the present study was conducted to explore and characterize the genes present within the region flanked by TA59 and TR19 markers using in silico as well as gene specific marker based wet lab approach. A total of 225 genes were identified to be present in the targeted region among which 51 were found to be showing differential expression under Fusarium wilt stress when assessed from Fusarium wilt specific shoot transcriptome data of two contrasting chickpea genotypes available in NCBI SRA database. Further in silico analysis was carried out for these selected 51 genes. Several cis acting elements such as BIHD1OS, WRKY71OS, SEBFCONSSTPR10A, WBOXATNPR1 etc., which are reported to be involved in response against biotic stresses, were found to be present in the 1kb 5’UTR of the 51 selected genes. Most of the proteins encoded by 51 selected genes were hydrophilic in nature having good solubility and were mostly localized to nucleous and cell membrane. These proteins were found to be interacting with 398 other proteins among which only 30 proteins interacting with query proteins encoded by 15 selected genes of the region were found to be showing differential expression under Fusarium wilt stress when assessed from Fusarium wilt specific shoot transcriptome data of two contrasting chickpea genotypes available in NCBI SRA database. Simultaneously, 246 diverse set of chickpea lines were sown in a Fusarium wilt sick plot and data on different field traits including disease incidence % were recorded. The PCR was done with TA59 and TR19 markers in 246 lines of chickpea and 40 highly resistant and 40 highly susceptible lines of chickpea were selected based on amplification pattern of TA59 and disease incidence% observed in 246 lines of chickpea. The gel based polymorphism survey using primers specific to 51 selected candidate genes revealed low level of polymorphism mostly in term of presence or absence of the amplified products with only 27 gene specific primers showing amplification in 80 selected lines. The polymorphism information content of the gene specific primers ranged from 0.049 to 0.554 with the mean Nei’ Gene Diversity and Shanon Index of 0.2162 and 0.3403 respectively, explaining low level of gene diversity in 80 selected lines of chickpea. Regression analysis revealed 8 out of 27 gene specific primers had R2value of >0.1 with the traits like disease incidence, no. of pods/plant, plant height, shoot dry weight and 100 seed weight. Among 8 gene specific primers showing R2value of >0.1, the amplification of primers specific to 101502928, 101495508, 101505289, 101496712, 101499005 and 101488582 showed positive association with the desired traits and the amplification of primers specific to 101505077 and 101510207 showed negative associations with desired traits. Regression analysis also revealed non-significant association of TA59 and TR19 with the disease incidence %. The comparison of cistron and promoter (5’UTR) sequences of the two potential candidate genes namely 101502928 (WRKY transcription factor 55) and LOC101488582 (CBL-interacting serine/threonine-protein kinase 2-like) in 4 contrasting chickpea genotypes revealed several single nucleotide polymorphisms (SNPs), nucleotide deletions and undetermined nucleotide present in the 5’UTR, exonic and intronic regions. Further studies could be done to understand the pathways of protein-protein interaction, impact of SNPs or nucleotide deletions in the genes leading to susceptibility or resistantance against Fusarium wilt in chickpea. From the findings of the present experiment, it could be concluded that the use of TA59 and TR19 alone is not sufficient in marker assisted selection for developing wilt resistant chickpea lines. Therefore, the use of markers specific to candidate genes like 101502928 (WRKY transcription factor 55) and LOC101488582 (CBL-interacting serine/threonine-protein kinase 2-like) could be more effective in developing robust resistance mechanism in chickpea plants against Fusarium wilt disease.