Poornaprajna College, Udupi, Karnataka, 576101
Prof. Virendra Kumar Baranwal
Indian Agricultural Research Institute, New Delhi, 110012
A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. The presence of endogenous BSV sequences in the banana genome and their potential to express as episomal Banana Streak Viruses (eBSV) in natural genetic hybrids of banana complicates the diagnosis, characterization and management of BSV. Complete genome sequences of 8 kb, 7.0 kb and 8 kb in the samples by name RCMB 200, RCMB 201 and RCMB 202 respectively were achieved by sequence- independent improved Rolling Circle Amplification( RCA). RCA coupled with Restricted Fragment Length Polymorphism (RFLP) revealed diverse restriction profile with different enzymes for the above samples. RCMB 200 was restricted by a number of enzymes in the single 8 kb amplicon : BamH1, Kpn1 and Sal1 ; RCMB 201 with EcoR1, Sal1, Sac1 ; RCMB 202 with Sph1. This shows that there is different restriction pattern in the BSV infecting different banana crops with different restriction enzymes. The restricted full genome obtained from above mentioned samples were cloned into pUC 18 vector. The improved RCA protocol provided an opportunity to use this technique for the discovery and full genome characterization of novel episomal badnaviruses from banana crop and also a reliable way to distinguish between integrated and episomal sequences.
Keywords: Badnavirus, Methodology, RCA, RCA-RFLP, Molecular cloning.