ANALYSIS OF GENETIC VARIABILITY AMONG GERMPLASM OF FINGER MILLET BY USING ISSR MARKER (Accession No. T06030)
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Date
2016-05
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DBSKKV., Dapoli
Abstract
PLANT BIOTECHNOLOGY CENTRE
COLLEGE OF AGRICULTURE, DAPOLI
DR. BALASAHEB SAWANT KONKAN KRISHI VIDYAPEETH, DAPOLI
Name of Student : Mr. Kelkar Vipul Ganesh
Registration No. : 0017
Name and Designation : Dr. S. G. Bhave
of Research Guide Director of Extension Education,
Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli.
Title of Thesis : Analysis of genetic variability among germplasm of finger millet by using ISSR marker.
Academic Year : 2014-2016
ABSTRACT
The molecular marker technology has a great potential for assessing genetic variability and relationship among the selected germplasms. In the present study forty germplasm of finger millet showing distinct morphological differences were screened using 15 ISSR markers.
The DNA was extracted from the green leaf samples collected from 15 days old seedlings of finger millet from 40 gremplasms by rapid DNA extraction method. The combination of extraction buffer used was 200 mM Tris-HCl having pH 8.0, 25 mM EDTA, 250 mM NaCl which showed clear and specific banding pattern when subjected to PCR.
Optimum concentration of each component in master mix was used for further ISSR analysis. In which 10 mM (1μl) dNTPs concentration and Taq polymerase 3 U/μl (0.5 μl) gave better amplification. The annealing temperature ranging from 40.40C to 56.70C for 1 minute yielded good results.
The finger millet DNA showed better amplification with 15 ISSR primers studied. A total of 1876 bands were amplified and out of which 1552 were polymorphic which showed 83.32 % polymorphism. The primer UBC-834 showed 29.82 minimum per cent polymorphism while the average bands per primer were 124.8.
The ISSR profile generated by each of the primer was analyzed using standard DNA ladder (1353-310bp) and compared with their respective banding pattern. The average size of amplified fragment ranged from 200bp to 1650bp. The primer UBC-872 recorded minimum PIC value 0.20, whereas primer UBC-841 gave maximum PIC value 0.88 and average polymorphic information content is 0.70 among the all 40 germplasms. It indicates that
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ISSR markers have a great potential to show the polymorphism among the finger millet germplasms.
The data of 40 germplasms of finger millet were used to generate pair-wise matrix based on the Jaccard’s Similarity co-efficient. The genetic distance was calculated on the basis of pooled data and the dendrogram was constructed. The similarity co-efficient ranged from 0.197 (between germplasm Nagali-55 and KMR-204) to 0.679 (between germplasm VR-762 and PR-202) indicating the distinctness and similarities of these germplasm.
Cluster analysis was carried out based on UPGMA analysis and it divided 40 germplasms into two main clusters and each having two sub-clusters. The first sub-clusters of first major cluster comprised of 13 genotypes and the second sub-clusters also comprised of 20 genotypes. The first sub-clusters of second major cluster had 1 genotype. While the second sub-clusters of second major cluster consisted of 6 genotypes.
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