A High-Throughput DNA Extraction Protocol and its Utilization in Molecular Characterization of Noni (Morinda citrifolia L.) Genotypes.

Order of Publishing in Issue: 
7
Volume :8
Issue :2
April, 2014
Page No: 
166-174
Authors: 
M.N.Patel[1], L.D. Parmer[1], A. Parihar[1, 2], A.K.Singh[3] and W.A. Sheikh[1, 4]*
Address: 
[1]Department of Plant Molecular Biology and Biotechnology, C.P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Gujarat, India
Address: 
[2]Department of Plant Molecular Biology and Biotechnology, B.A. College of Agriculture, Anand Agricultural University, Anand, Gujarat, India
Address: 
[3]Central Horticulture Experiment Station, Vejalpur, Godhra, Gujarat, India
Address: 
[4]International Rice Research Institute- South Asia Breeding Hub, ICRISAT, Patancheru, Andhra Pradesh, India.
Email-ID: 
waseems84@gmail.com

This study describes the standardization of DNA isolation protocol and DNA based molecular characterization of Noni, potentially designated as Morinda citrifolia L. Total Genomic DNA was isolated from fresh and young leaves of M.citrifolia and M.tomentosa following CTAB method with three indispensable modifications which includes methanol and PVP treatment to remove phenolics with PCI and long incubation in ethanol for enhancement of quantity of DNA. Upon gel documentation of isolated DNA by modified method evinced single discrete band of genomic DNA and yielded significantly superior, 441.20 ng/ìl average concentration of DNA over thirteen different genotypes tested with absorbance ratio of DNA at A260/A280 with a mean value of 1.81 as compared to 1.66 by conventional method. Molecular characterization of 13 Noni genotype was done with 40 RAPD and 15 SSR molecular markers. Among 40 RAPD markers, only 20 showed polymorphism among the 13 genotype of Noni. RAPD dendrogram showed two major clusters with coefficient value 0.39. The genotypes of M. tomentosa were found in cluster (A) and (B) whereas, M. citrifolia was observed in cluster (A). Genotypes CHESN11 and CHESN12 had highest similarity with maximum co-efficient value (0.699) which falling under same cluster A while CHESN8 and CHESN1 with least similarity and noted in different Sub cluster A2 and A1.Out of the 15 SSR primers, 5 detected polymorphic with 25 scorable bands among 13 accessions of M. citrifolia and M. tomentosa. The sizes of amplified products ranged from 66 to 5229 bp. Dendogram for SSR based on Jaccard’s similarity coefficients showed two major clusters with coefficient value 0.53. The genotypes of M. tomentosa were found in cluster (A) and cluster (B), while M. citrifolia was observed only in cluster (A). Genotypes CHESN11 and CHESN12 had highest similarity with maximum co-efficient value (0.857) which falling under same cluster A even as CHESN12 and CHESN3 with least similarity observed in two different clusters A and B.

Keywords: 
DNA isolation, M. citrifolia, M. tomentosa, RAPD, SSR
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