Development of a molecular marker for bacterial wilt resistance in brinjal ( Solanum melongene L.) varieties Surya and Swetha.

Abstract

The study entitled ‘Development of a molecular marker for bacterial wilt resistance in brinjal (Solanum melongena L.) varieties Surya and Swetha’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is one of the important problems of brinjal 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. Worldwide approach is to use resistant varieties. KAU has developed and released bacterial wilt resistant brinjal varieties for cultivation. The Surya and Swetha are two among them and have received bacterial wilt resistance from SM-6 an Annamalai collection. This investigation was taken up to develop a molecular marker for bacterial wilt resistance in Surya by RAPD through bulk segregant analysis as reported by Michelmore et al (1991). It also aimed to test the suitability of the same for identifying bacterial wilt resistance trait of resistant variety Swetha. The genotypes used for the study were Surya, Pusa Purple Long (susceptible variety released from IARI), Swetha and F2 population of the cross between Surya and Pusa Purple Long. To raise segregating F2 population F1 was raised by controlled crossing of Surya with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population. Two different methods viz., stem puncturing and soil drenching with root wounding were compared for the delivery of inoculum of R. solanacearum for bacterial wilt incidence and stem puncture method was found as the best. So stem puncturing method was used for phenotyping of genotypes for bacterial wilt incidence. The F2 population along with Surya, Pusa Purple Long and F1 were phenotyped for bacterial wilt incidence. This was done through artificial inoculation with Ralstonia solanacearum by stem puncture method. Confirmation was done by ooze test. The genotypes were classified according to classification of Mew and Ho (1976). The variety Surya was resistant and Pusa Purple Long was susceptible. F1 population showed 90 per cent susceptibility while F2 population showed 83 per cent susceptibility. They were classified as susceptible. Five resistant and five susceptible genotypes from F2 were selected for bulk segregant analysis. Genomic DNA for RAPD analysis was isolated by Rogers and Bendich method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis.PCR reaction mixtures and conditions for DNA amplification were standardised. Ninty two, 10-12 bp primers belonging to OPA, OPB, OPC, OPF, OPE, OPU, OPH, OPAH, OPAG, OPL, OPM, RY, RN, RA, SC, RF, AG 8, WG 44, GLE11, RF, R10, R6, and PUC101 were initially screened with resistant genotype Surya and susceptible genotype Pusa Purple Long to select primers with polymorphism and good amplification. Out of ninty two primers tested thirty were reported as bacterial wilt specific. The PCR products were electrophoresed and twenty two primers were selected for BSA based on amplification power and polymorphism. They were RY 01, RY 02, RY 03, RN 19, OPF 5, OPL 04, OPA 04, OPA 6, OPA 9, OPA 24, OPA 26, OPA 34, OPA 36, OPS 9, OPS 10, OPS 16, OPS 17, PUC 101, RA 12-41, and RF. Among these only the primer RY 02 recorded polymorphism between resistant and susceptible variety with an amplicon of 1.2kb. In bulk segregant analysis DNA of Surya, Swetha, Pusa Purple Long and bulk DNA from resistant genotypes and susceptible genotypes were amplified with selected primers and products were electrophoresed. All primers produced only monomorphic band. None has produced unique polymorphic bands capable of differentiating resistant and susceptible genotypes. This may be due to low polymorphism at genomic level.