Identification of molecular marker linked to the resistance for vascular streak dieback disease in cocoa (Theobroma cacao L.)
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Date
2014
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Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara
Abstract
Cocoa is the third important plantation crop next to coffee and tea and is the third highest traded commodity in the world after coffee and sugar. Current world production of cocoa is about 4 million tonnes but India contributes only 0.02 per cent. In Kerala, area under the cocoa is 12,480 ha. with the production of 6.14 metric tonnes and productivity 490 kg/ ha (Directorate of Cashewnut and Cocoa Development, 2013). Vascular streak dieback (VSD), caused by the fungus Oncobasidium theobromae and first reported in India in 1981, is a very serious disease of cocoa that has already spread to all the cocoa growing tracts of India. The characteristic symptoms include a green-spotted chlorosis and fall of leaves beginning on the second or third flush behind the stem apex, raised lenticels, and darkening of vascular traces at the leaf scars and infected xylem. Eventually complete defoliation occurs and, if the fungus spreads to the trunk, the tree will die. The confirmation of transfer of gene is at most important in crop breeding and availability of a tool to quickly detect the gene will reduce the breeding cycle length. Molecular markers are already proven good to detect the genes of interest. The present study aims at developing a reliable molecular marker linked with the gene offering resistance to VSD in cocoa, using SSR and ISSR marker systems. Extensive germplasm collection and evaluation have been done at Kerala Agricultural University, India and tolerant cocoa clones were identified. Using these clones, 45000 hybrids have been bred and were screened for resistance using natural inoculum under high humidity and 1177 field established resistant hybrids are being evaluated for the past 15 years. Already, KAU has released three hybrids and 7 selections of cocoa with field tolerance to VSD. In the present investigation, twelve genotypes of cocoa, having different responses to VSD namely, VSD I-4.6, VSD I-4.11, VSD I-5.8, VSD I-6.9 (resistant), G VI-50, G VI-52, G VI-82, G VI-100 (Susceptible), G VI-25, G VI-53, G VI-67 and G VI-144 (partially resistant) were employed. The genotypes were selected on the basis of field screening experiments started in 1998-1999 at Cocoa Research Station, Kerala Agricultural University. Genomic DNA isolated using modified CTAB method suggested by Doyle and Doyle (1987). ISSR assay on the total genomic DNA of twelve cocoa clones using 71 primers had shown that the primers UBC811, UBC815, UBC826, UBC857 and UBC866 are capable to yield the polymorphic bands, in relation to VSD resistance, whereas, SSR assay with 46 primer sets has failed to generate any marker for the same. The most distinct polymorphic marker generated in the resistant lines by the ISSR primer UBC857 was eluted. This DNA was further subjected to PCR analysis using the same primer, to verify its suitability for direct sequencing. The direct sequencing has yielded 246 nucleotides, which on BLASTn had shown 94 per cent identity with the Theobroma cacao microsatellite DNA clone of mTcCIR42 SSR (NCBI accession number AJ271944). Subsequently, the clones were subjected to SSR assay using the primer mTcCIR42. The assay has generated distinctly polymorphic banding pattern that differentiated the resistant lines from the susceptible and partially resistant ones. Since highly successful in differentiating the resistant, susceptible and partially resistant cocoa clones, these markers are recommended for use in marker assisted breeding for VSD resistance.
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