Genetic diversity analysis and identification of molecular markers associated with leaf blight resistance in taro (Colocasia esculenta (L.) Schott)
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Date
2015
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Department of Plant Biotechnology, College of Agriculture, Vellayani
Abstract
The project titled “Genetic diversity analysis and identification of molecular markers associated with leaf blight resistance in taro (Colocasia esculenta (L.) Schott)” was carried out at the Division of Crop improvement, ICAR-CTCRI, Sreekaryam, Thiruvananthapuram, during 2014-15. Thirty Kerala accessions were employed to study the genetic diversity and 12 genotypes comprising 6 susceptible and 6 resistant which were screened in the fields of ICAR-CTCRI was taken for the study. DNA was isolated from all the 30 varieties by a method standardized by Doyle and Doyle, (1987) and later by Sharma et al., (2008) which was slightly modified by Vinutha, K. B. (2014). A purity level of 1.7-2.0 was achieved by most of the genotypes. PCR conditions already standardized in the molecular biology lab of Crop Improvement lab of ICAR-CTCRI were used to amplify the SSR markers. After preliminary screening, 5 primers were eliminated from the study because of its poor amplification profile. 10 primers out of the 15 screened were utilized for PCR. The PCR products were made to run in 6% acrylamide gel (denaturing urea gel) and the bands were studied after staining with silver nitrate. PIC and cluster analysis was done to analyze its diversity. By calculating polymorphic information content, the highest heterozygosity was obtained for the primer uq 97-256 with a value of 0.871 and the lowest was obtained for the primer uq 84-207 with a value of 0.694. Number of alleles per locus ranged from 1.9-4.56 with the maximum alleles shown by uq 97-256 and the minimum showed by uq 84-207 and uq 201-302. The first cluster comprised of the accessions from C-406 to TCR 450. The second cluster consists of C-218 to C-384 and the third large cluster groups most of the genotypes ranging from TCR 424 to TCR-296 and ADS2014-20 has formed an 126 outlier. The third cluster is further subdivided into four sub clusters and the second sub cluster consists of major number of genoytpes and has an outlier C-273. In the next part of the work, 12 sets of DNA was isolated comprising 6 susceptible and 6 resistant varieties each, to identify molecular markers associated with TLB. 13 ISSR primers were tried to identify the same. The product was amplified at 56⁰C. The PCR products were resolved in 2% agarose Out of the 13 primers, 5 of them gave an extra band for resistant varieties. UBC 825 and (AG)₉AC gave an extra band in all the resistant varieties in 685 bp and 808 bp regions, respectively. The primers UBC 827, UBC 817 and UBC 808 gave bands in 5 resistant varieties in the region, 598, 675 and 667 bp, respectively. The dendrogram analysis using UPMGA clustered resistant and susceptible varieties into two different clusters which gave a more promising result. But the SSR primers employed for the same study using the primers Ce1 A08, Ce1 F04, Ce1 CO3, Ce1 F12, Ce1 H12, uq 97- 256, uq 201- 302, uq 73-164, uq 115-71 and uq 88-94 failed to show any specific bands in the expected product size. However, more number of accessions showing different levels of tolerance and susceptibility to TLB as well as more number of markers could lead to the identification of promising markers associated with TLB resistance.
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