Molecular characterization and development of trait related markers for aphid resistance in cowpea (Vigna unguiculate (L) Walp.)
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Date
2007
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Centre for Plant Biotechnology and Molicular Biology, College of Horticulture, Vellanikkara
Abstract
Cowpea is one of the most important grain legumes that has been grown throughout the tropics. It is a rich source of proteins (25 percent). Their ability to fix nitrogen is of great advantage on small farms. However their production is greatly limited by many pests, especially aphids. Keeping in view of the importance of this crop, a study was conducted on ‘Molecular characterization and development of trait related markers of aphid resistance in cowpea (Vigna unguiculata (L) Walp.)’.
The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology and Radio Tracer Laboratory, College of Horticulture during the period 2005-2007. The objectives of the study were to identify the sources of resistance to aphids and to develop markers for aphid resistance in cowpea. The cowpea accessions which were susceptible, moderately resistant and resistant were identified based on the average count of aphids. Five susceptible (VS 1177, VS 1179, VS 1173, VS 1208, VS 1034) and five resistant (VS 1230, VS 1231, VS 1201, VS 1248, VS 1263) accessions were used for the present study. These 10 accessions were subjected to molecular characterization using RAPD and AFLP markers.
For RAPD and AFLP analysis, the protocol for genomic DNA isolation from cowpea was standardized. The protocol suggested by Doyle and Doyle (1987) was found to be the most appropriate.
Thirty random primers were screened and ten were selected for RAPD profiling of cowpea accessions. The primer OPA 4 was found to have the highest resolving power. A total of 75 scorable amplification products were generated by 10 random primers of which 48 bands were polymorphic. Specific bands were generated for the resistant accessions VS 1230 and VS 1201 with OPA 2, VS 1248 and VS 1263 using OPA 3 and VS 1248 alone with OPA 10. In the dendrogram, the 10 accessions were grouped into two major clusters of 8 and 2 accessions each. The resistant accessions VS 1230 and VS 1231 were the most closely related with 90 percent similarity. Another two resistant accessions VS 1248 and VS 1263 were also grouped together in the dendrogram.
The cowpea accessions were subjected to AFLP analysis with 5 primer combinations of EcoR1 and Mse1. A total of 237 scorable amplification products were produced by the primer pairs of which 93 bands were polymorphic. The highest resolving power was obtained for EACT+MCAA. Specific bands were produced for EAAG+MCAA in resistant accessions VS 1230, VS 1263 and VS 1248. The dendrogram obtained for AFLP showed that VS 1177 and VS 1179 were most closely related at 91 percent similarity. Similarly the resistant accessions VS 1263 and VS 1248, VS 1230 and VS 1201 were grouped in different sub clusters.
Thus RAPD and AFLP markers were utilized to characterize cowpea accessions for aphid resistance. The specific bands identified in the resistant accessions could be treated as trait related markers for aphid resistance. Further studies should be conducted on screening the cowpea accessions with more number of primers to develop more specific markers with reference to the aphid resistance. The studies should also be focused on converting these RAPD and AFLP markers linked to aphid resistance to a more specific amplification, a technique called Sequence Characterized Amplified Regions (SCAR).
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172714