Browsing by Author "FATIMA ABBAS"

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    Optimization of rolling circle amplification for the detection of circular Phytopathogenic DNA viruses
    (UMT Lahore, 2019) FATIMA ABBAS
    Begomoviruses is the one of the major threat to the cultivars in different countries especially in third world developing countries including Pakistan. Pakistan’s economy is based on agriculture. Begomovirus is the genus of family Geminiviridae and transfers through single vector white fly (Bemisia tabaci). It is important to identify the begomoviruses not only in cultivated but also in non-cultivated plants as they serve as alternative hosts and provide shelter and reservoirs. Leaves of six different plants Parthenium, Duranta, Ocimum, Malvestrum, Catharanthus and Capsicum displaying leaf curling and yellowing of veins were collected from different areas of Lahore. TDNA was isolated from leaf tissues by using CTAB extraction buffer followed by PCR. 1.5kb band size of DNA –A of Parthenium and Catharanthus sp. was amplified by using a set of degenerative primers WTGF/WTGR. The detection of betasetallite was also done by using abutting primers Beta01/Beta02. Betasatellite was detected in Parthenium. The DNA-A of Parthenium was further amplified by Rolling Circle Amplification (RCA). RCA was followed by restriction digestion using different restriction enzymes. These enzymes included EcoR1, Hindi3, Pst, BamH1, Xho1, Xba1, Kpn1, and Apa1. Digestion with EcoR1 gave two bands. One was 2.8 kb that was DNA-A, and the other was 1.4 kb that is a betasatellite. Upon phylogenetic analysis, partial sequence of DNA-A of Parthenium showed 99% identity with Mesta yellow vein mosaic virus (MYVMV). Parthenuim belongs to Asteraceae family, which shows that begomoviruses can invade non- asteraceous hosts.