Molecular characterization of chilli(Capsicum annuum L.) genotypes for tagging bacterial wilt resistance gene

Abstract

Investigation on “Molecular characterization of chilli (Capsicum annuum L.) genotypes for tagging bacterial wilt resistance gene” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2006-2008. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is the most important problem of chilli cultivation in warm humid tropics. The loss due to this varies from 30-100 per cent. Use of chemicals and field sanitation are not sufficient for controlling the disease. World wide approach is to use resistant varieties. KAU has developed and released bacterial wilt resistant varieties viz., Ujwala, and Anugraha for cultivation. The present investigation was taken up to develop a molecular marker for bacterial wilt resistance gene through molecular characterization of resistant and susceptible genotypes. The genotypes used for the study were Ujwala, Pusa Jwala, Anugraha and F2 population of the cross between Ujwala and Pusa Jwala. The genotypes were phenotyped for bacterial wilt incidence through artificial inoculation with Ralstonia solanacearum. The varieties Ujwala and Anugraha were resistant. Pusa Jwala, F1 and F2 progenies were susceptible. The molecular marker techniques used were RAPD and SCAR. Genomic DNA was isolated by Rogers and Bendich (1994) method. Forty seven decamer primers belonging to six Operon primer series viz., OPE, OPAH, OPN, OPP, OPS and OPY were used for primer screening. Twenty two primers which gave more than seven bands were used for molecular characterization of selected genotypes through bulk segregant analysis. OPS 1 primer amplified a DNA fragment of 1.24 kb in resistant parent and resistant bulk. Co-segregation analysis was also done with OPS 1 primer with individuals of susceptible and resistant bulk. The polymorphic band produced by OPS 1 primer in resistant parent and resistant bulk was eluted and cloned in pGEM-T vector, and was transformed into E. coli JM 109 cells. Recombination of the insert was confirmed through RAPD reaction with OPS 1 primer. The cloned fragment was sequenced to obtain the nucleotide sequence information and was named as Chilli seq 1. The sequence obtained after vector sequence deletion was named as Chilli seq 2 and it contained only 440 bp in place of 1240 bp. It was subjected to nucleotide blast search. It revealed significant levels of homology with Capsicum annuum ethylene responsive element binding protein C2, lipid transfer protein III gene, clone A1-4 PR-protein gene and ripening regulated protein DDTFR10/A gene of chilli deposited in the public domain. The sequence was also subjected to various sequence analysis using bioinformatics tools, which include ORF finder, SOPMA, NEB cutter, Hydropathy plot, NASTATS and AASTATS tools of Biology Workbench. Based on the end sequences of the cloned RAPD fragment, SCAR primers were designed. The efficiency of SCAR primer to distinguish resistant and susceptible genotypes was tested but no polymorphism was detected. The SCAR primer OPS 11240 amplified a fragment of 396 bp in all the genotypes tested. Full sequence data of the cloned RAPD fragment was not available for SCAR primer designing. More efforts needed to get full-length sequence data.