Cloning of Begomoviruses and Betasatellites Associated with Chilli Leaf Curl Disease.

Chilli leaf curl disease (ChiLCD), caused by diverse begomoviruses and associated betasatellites, leads to significant yield losses in chilli crops. This chapter outlines an efficient molecular cloning strategy using rolling circle amplification (RCA) for the full-length amplification of circular viral genomes. Subsequent steps include restriction digestion, ligation into suitable plasmid vectors, transformation into E. coli, screening through RFLP analysis, and sequencing of cloned products. Conventional PCR has been used for begomovirus genome characterization, but it typically needs prior sequence knowledge and often fails to efficiently amplify full-length genomes, leading to incomplete or biased results. However, RCA offers a high-fidelity, isothermal, and sequence-independent amplification. The described protocol enables reliable isolation, characterization, and diversity analysis of begomoviruses and betasatellites, facilitating downstream applications, such as diagnostics, functional genomics, and virus-host interaction studies.