PCR amplification and cloning of betasatellite molecule associated with cotton leaf curl disease
2014
Khalid, A.
Cotton (Gossypium hirsutum L.) is the World's leading fiber crop. It presented as a white gold of lndo-Pak and has a central role in the economy of Pakistan. Cotton leaf curl disease (CLCuD) is a major threat for cotton production in lndo-Pak sub-continent. CLCuD first appeared in epidemic form in 1980s was shown to be caused by monopartite begomovirus which carry the DNA-A component and betasatellite molecule transmitted by Bemisia tabaci. After heavy losses to economy of Pakistan during epidemics, breeders developed the resistant varieties of cotton in late 1990s but after few years in 2001, resistance breaking strain named as Burewala strain appeared as honey-bees dispersal across many cotton producing areas of Pakistan. Further studies suggested that a species, Cotton Leaf Curl Burewala Virus (CLCuBuV) is associated with resistance breakdown. Research was designed to clone and analysis the betasatellite molecule of begomovirus associated with CLCuD in District Kasur (Punjab). Total genomic DNA from these samples was isolated by using CT AB method. Full length betasatellite was polymerized into millions of copies through PCR by using universal primers beta Oland beta 02 which polymerized the complete viral component. By using ligase enzyme, ligation of amplified PCR product was done in pTZ57R/T vector and was transformed into competent Escherichia coli cells by heat shock method. Clone was confirmed by Kpnl and analysis with EcoRl, Pstl and SaIl restriction endonucleases. Molecular analysis revealed that different type of betasatellite molecule is associated with CLCuD in District Kasur, Punjab.
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