Development of molecular markers linked to yellow vein mosaic resistance in okra (Abelmoschus esculentus (L.) Moench)

Abstract

Yellow vein mosaic is the most serious disease leading to 50-94 per cent yield loss in okra. None of the chemical measures are successful to save an infected plant and breeding the resistant varieties is the most accepted strategy in the management of this disease. Molecular breeding through marker assisted selection is more advantageous over the conventional breeding since it will help to assure the presence of the gene in the breeding lines and avoid the selection of pseudo-resistant line. As of now, there are no markers for the identification of YVMV resistance gene in okra. Hence the study was undertaken with the objective to identify ISSR and RAPD markers linked with the gene governing resistance to the yellow vein mosaic virus disease in okra through bulked segregant analysis to enable marker assisted selection. In the present investigation, two okra genotypes namely Parbhani Kranthi (YVMV resistant) and Salkeerthi (YVMV susceptible, early bearing, excellent fruit quality) were used. Field screening of both the parental lines was done simultaneously in the same field during February-May 2014. No artificial inoculation methods were followed since the heavy population of white flies observed in the summer months was sufficient to ensure the disease occurrence and crossing was done between the selected plants (Salkeerthi X Parbhani Kranthi). The seeds were harvested from the crossed plants and subsequently used to raise F 1 population. The raising of F 1 plants was carried out in the month of September- December 2014 and the morphological characteristics and disease response of 180 F 1 plants were recorded. All the F 1 plants were free from disease symptoms. The F 1 plants were selfed to produce F 2 seeds. The F 2 population with 200 plants was field screened during January-April 2015. Seven highly susceptible and 7 highly resistant plants were identified, DNA isolated from each, resistant and susceptible DNAs bulked separately and used for Bulked Segregant Analysis (BSA). For theextraction of good quality DNA, the CTAB method (Doyle and Doyle, 1987) may be modified by avoiding the liquid nitrogen while grinding the plant tissue and additionally washing the DNA pellet with wash buffer to remove the mucilage content. Evaluation of quantitative characters on F 1 in comparison with parental lines showed variation for the traits such as plant height, petiole length, days to flowering, days to first harvest, number of fruits per plant and yield per plant. Two molecular marker systems namely, RAPD and ISSR were employed for identification of markers linked with YVMV resistance. A total of 84 RAPD primers and 82 ISSR primers were initially screened for their ability to amplify the DNA fragments. Out of these, 39 RAPD primers and 24 ISSR primers were selected based on the number of bands and nature of amplification. In BSA, two RAPD primers OPB11 and OPL 18 yielded markers linked with resistance to YVMV. OPB11 produced distinct polymorphic bands of 800, 1000 and 1100 bp sizes whereas, OPL 18 produced polymorphic bands of 1000 and 1100bp. Two ISSR primers ISSR 8 and UBC 873 yielded distinct polymorphic bands in relation to YVMV resistance. The ISSR 8 and UBC 873 yielded the markers at 500 and 900 bp, respectively. Another primer ISSR 22 gave a distinct marker at 1100 bp size, linked to susceptibility to YVMV. Co-segregation analysis was performed using individual DNA of resistant parent, susceptible parent, resistant F 2 and susceptible F 2 using RAPD primers OPB 11, OPL 18 and ISSR primers namely ISSR 8, ISSR 22 and UBC 873. OPB 11 produced three distinct markers in all resistant F 2 individuals. ISSR 8 produced distinct marker of 500 bp in all resistant F 2 individuals. ISSR markers were found to be reproducible and they are recommended for use in marker assisted selection for resistance to YVMV in okra.