Development of trait related markers for bacterial wilt resistance in tomato (solanum lycopersicum L)
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Date
2007
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College of Horticulture, Vellanikkara
Abstract
Tomato is one of the commercially important vegetable crops grown throughout the world, both for the fresh market and processed food industries. It is the second most important vegetable in India. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is a major constraint for tomato production in many tropical and sub-tropical regions. The area under tomato cultivation in Kerala is limited due to the incidence of this disease. The warm, humid tropical climate and acidic soil favours the bacterial wilt incidence in Kerala. The crop loss estimated by bacterial wilt attack is to the extent of 90 per cent. Chemical control measures are not effective as the pathogen is soil-borne. The mechanism of bacterial wilt resistance is cryptic and mainly dependent on environment and strain of the pathogen. Molecular level approaches can provide the key to unravel this complex genetic disease and can help a long way in breeding resistant genotypes. The study entitled “Development of trait related markers for bacterial wilt resistance in tomato (Solanum lycopersicum L.)” was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2005-2007 to characterize selected tomato genotypes and to detect markers linked to bacterial wilt resistance/susceptibility. Fifteen tomato genotypes obtained from various centers were raised under open condition and greenhouse condition in earthen pots containing sterile soil medium during the peak period of infection. The seedlings were artificially inoculated to confirm the genotypic response to bacterial wilt. The greenhouse grown seedlings defended the infection. Out of the 15 genotypes evaluated, five (Anagha, Sakthi, Mukthi, Hawaii 7998 and LE-66) were resistant, three (LE-20, LE-474 and LE-1-2) were moderately resistant, three were moderately susceptible (BT-1, BT-218 and BTH-102-1-2-2) and four (Pusa Ruby, Arka Vikas, BL-333-3-1 and PKM-1) were susceptible. Further, the genotypes were subjected to molecular characterization using RAPD and AFLP assay. Genomic DNA was isolated by two protocols and the protocol suggested by Rogers and Bendich (1994) was found to be the ideal one for DNA isolation from tomato leaves. Forty random decamer primers were screened for RAPD profiling and 35 of these were selected for genotype screening. Out of this, only 14 primers that displayed clear, reproducible bands were selected for further analysis. Almost all the primers yielded monomorphic banding pattern, except OPS 1, which yielded a specific fragment unique to resistant genotypes. The dendrogram obtained from the pooled data indicated high genetic similarity among the genotypes studied. A more sensitive assay, AFLP was performed with five combinations of EcoRI and MseI based primer pairs. This technique could display the genetic diversity among the genotypes in a better way compared to RAPD marker analysis. Two markers linked to bacterial wilt susceptibility were obtained using the primer combinations E+ACG/M+CAC and E+AGC/M+CAA. The RAPD specific amplicon obtained in resistant genotypes, with the primer OPS 1 was eluted and cloned in pGEM-T Easy Vector and was transformed into E. coli JM 109 cells. The recombination efficiency was assessed through blue-white screening. Recombination of the insert was confirmed through RAPD reaction and restriction analysis. The cloned fragment was sequenced to obtain the nucleotide sequence information. The sequence obtained after vector screening was named as Tomato seq 2 was subjected to Blast search. It revealed significant levels of homology with genomic DNA of tomato in chromosome 11, 9 and 8, and the sequence for T-2 Type RNase LER gene of tomato deposited in the public domain. The sequence was also subjected to various sequence analyses using bioinformatics tools, which include ORF finder, SOPMA, NEB cutter, Hydropathy plot, NASTATS and AASTATS tools of Biology Workbench. Scientists have already reported QTL conferring resistance to bacterial wilt on tomato chromosome 11 and 8. Moreover, the role of RNase LE in wound healing and defense mechanism is well established. So the future line of works should be focused on development of SCAR marker based on sequence data for its use as a trait related marker. Efforts are also to be made to isolate and characterize the full-length gene.
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