Molecular characterization of tomato (Solatium lycopersicum L.) with special reference to tomato leaf curl virus (ToLCV) resistance

Abstract

Tomato (Solarium lycopersicum L.) is one of the major vegetable crops in the world. India ranks sixth in the production of tomatoes worldwide with a total area of 0.50 million hectares and productivity of 17.4 MT per hectare. Tomato leaf curl disease caused by the Tomato Leaf Curl Virus (ToLCV) and transmitted by whiteflies (Bemisia tabaci) is one of the most important diseases affecting this crop. The disease causes losses in yield to the tune of 70 to 100 per cent. ToLCV is severe under conditions prevalent in Kerala also. Identification of resistant sources of the disease and development of trait-related markers from these sources would be an important approach to overcome the problem of ToLCV. With this objective in mind, an investigation was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara from the year 2005 to 2007 to characterize the reaction of tomato genotypes to ToLCV under conditions prevalent in the area and to identify molecular markers (RAPD and AFLP) linked to disease resistance.Fifteen genotypes were raised in sterile soil in earthen pots and field during the peak season of ToLCV infection (December - February) and their reaction to the disease was categorized based on the coefficient of infection. Out of 15 genotypes, eight were observed to be highly resistant to ToLCV under both pot culture and field experiments. Observations of biometric characters of the genotypes grown in pots and field were made. All genotypes showed significant difference in all the characters observed both in pot culture experiments and field study. Plant height was the most striking character of difference observed in the two different culture conditions. Genotypes were subjected to molecular characterization using RAPD and AFLP markers. Genomic DNA required for these assays was isolated by two protocols. The protocol suggested by Rogers and Bendich (1994) with modifications was found to be most appropriate for DNA isolation from tomato leaves. Forty random decamer primers were screened for RAPD assay. Thirty-six of these were used for further RAPD profiling of the tomato genotypes. Out of this, 12 primers displaying good and reproducible patterns were selected for molecular characterization. The primer OPS 8 recorded the highest resolving power. A total of 116 amplicons were generated by the 12 selected primers of which 71 were polymorphic. The dendrogram constructed separated the genotypes into two groups. ToLCV resistant genotypes Anagha and H-24 with 92 per cent similarity were found to be most related. RAPD analysis did not reveal any trait- related marker in the present study. AFLP assay was carried out with five combinations of Eco&l and Msel based primers. A total of 241 amplicons were detected, out of which 122 were polymorphic. Three markers linked to ToLCV susceptibility were obtained using the primer combination EAAG/MCAC. All genotypes studied showed genetic uniformity in RAPD and AFLP assay except with respect to a few primers. Trait-related marker was detected in a single primer pair in AFLP assay, while RAPD assay did not give any clear demarcation with respect to ToLCV resistance/susceptibility. The markers identified could be further exploited for obtaining nucleotide sequence information and level of specific gene expression in susceptible/resistant genotypes.