Detection of Cotton Leaf Curl Disease using Betasatellite-based Molecular Marker

Abstract

Cotton leaf curl disease (CLCuD) caused by whitefly-transmitted begomoviruses has hampered cotton production across the Punjab and Sindh provinces of Pakistan and northeastern India. Eight species of begomoviruses in association with a single betasatellite “Cotton leaf curl Multan betasatellite (CLCuMB)” have been reported to cause CLCuD. Objective: To detect early and efficiently Cotton leaf curl disease (CLCuD) using betasatellite-based molecular marker. Methods: 3-7 samples leaves were collected from symptomatic cotton fields in selected five areas of cotton production in Pakistan. Total DNA was extracted from collected leaves using the Cetyl trimethylammonium bromide (CTAB) method. Primers were designed by MUSCLE alignment tool and target region was amplified by PCR and amplification confirmed by performing gel electrophoresis. After DNA sequencing Phylogenetic analysis of the was carried out using software MEGA-X. Results: Amplified target region of483bp was observed by running 1% agarose gel. Comparison of DNA sequences revealed two nucleotide substitutions in DNA sequence from samples collected from Multan, Sakrand, Rahim Yar Khan and, while four nucleotide substations in sample collected from Vehari. High nucleotide substation in DNA sequence from Vehari as compared to other regions. Conclusions: In conclusion all of eight distinct begomoviruses causing CLCuD with CLCuMB is indicative of the fact that CLCuMB based molecular marker can be developed for detection of the disease. Early detection of disease will help the breeders and farmers to manage the disease.