Cloning, Sequencing and Phylogenetic Analysis of 13- DNA of Cotton Leaf Curl virus

Abstract

Viruses are very successful intracellular parasite infecting almost every life on this planet. Plants are also the hosts for many vi ruses including them are circul ar single stranded DNA (cssDNA) viruses, which have been classified in two different families i.c. Gemin;viridae and Nono lliridae, both infecting plants and have cssDNA multicomponcnt genome. Geminiviric/(Ie is a very di verse and include many viruses whi ch lnfect many host plant of agronomi cal importance worldwide , Cotton leaf curl disease (CLeO) is one of the mosl important disease economi cally wh ich has result ed in huge losses in many epidemi cs in cotton growing countries around the worl d. The causative agents of this disease is not yet conclusively determined but so far about nice distinct bipartite begomoviruses are found associated with this disease. The genome of these viruses are composed of DNA-A which contains genes for DNA replicaiion, coat protein and regulation of gene expression ana associated DNA-P wl,ich contains gens l"Or virus movement and plays role in virus pathogenicity. COlloll is an important cash crop o f Pakistan which plays imponanl role III its economy, Many epidemlcs of CLC D have bcen reported whi ch have resulted in huge economical loses despite the introduction of resistant varieties. Much of' the sc ientific attention rema ined on understanding of DNA-A whi ch are associated with CLCO and DNA-~ rerna iul!d puurl y unuerslouu despite its rule in virus muvemenl and pathogenicity, In Clirrent investigation a p eR assay was deve loped and optimi zed to amplify ONA- fTom infec ted conan p lants along with actin gene as positive control from nuclear genome of cotton plants. The primer pair CLCuV DN -~ sllccessfully amplified tlus component from infected plants originated from Bahawalnagar: a conan producing area in Southern Punjab Pakistan. These products from two isolates were cloned and sequenced and sequence data was lIsed for phylogeneti c ana.lysis to understand the evolution of DNA-13 in Pakistan. The phylogenetic analysis of DNA-I) sequenced in this study a long with sequences of DNA-I) publically avai lable in GenBank: till March 20 1 J usi ng NJ me thod revealed two major groups where isolates from Indi an subcontinent and African conlincl1l grouped together in lnclo-African group while other isolates from Jndian slI bcontinent and China where grouped in lndo-Ch ina group. These groups indi cate two distinct vii CHAPTER 1 INTRODUCTION Cotton is one of the most important cash crop of Pakistan. Cotton is a shrubby plant belongs to family Malvaceae and genus Gossypium. It has unique economic value all around the world. Cotton plants are sensitive to many environmental hazards, pests and diseases. Virus diseases of cotton have historically been of only sporadic importance to global cotton production. Worldwide Pakistan has a key role in cotton industry on behalf of its production (4th number in world), exporting of cotton in raw form (3rd) also a huge quantity of cotton is consume in Pakistan and stay as largest 4th consumer in world as well. Strength of Pakistan cotton industry is the exporting of fine yarn which leads the world cotton yarn. Worldwide cotton is produce in large amount other than all other nonfood crops. Cotton is a multipurpose crop which provides 5 principle product including lint, food, oil, seed hulls and linters. The chief use of cotton is in Textile industry. Lint is use to achieve a high quality fiber from the textile industries. The history shows cotton cultivation and use from very past like in the time of Mohenjodaro civilization. The seeds of cotton were also discovered in Mehr Garh Baluchistan which were from about 5th millennium BC (Ahmad et at, 1999). Some other reports show that the cotton growth as domesticated crop started from Africa, Mexico and Peru. Nowaday more than 3.4 billion hectares area is cultivated (occupy about 2.5 % of the cultivated area across the world) on cotton crop and more than 25 billion people are associated with cotton industry accounting for about 7% of the labor in the arising world. Worldwide China and USA is the leading cotton producer followed by India, Pakistan and Uzbekistan. The economy of most growing nations largely depends on cotton production and export. Oil is also extract from the seeds of cotton by crushing which is used for cooking, the seed covering called "hulls" is used for animal feed. Beside these the lint around the cotton seeds is use in plastic and explosive products formation.