Circular DNA enrichment sequencing reveals the viral/satellites genetic diversity associated with the third epidemic of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is the most important limiting factor for cotton production in Pakistan. The CLCuD passed through two major epidemics in this region with distinct begomoviruses/satellites complexes. Since 2015 the disease has again started to appear in epidemic form, causing heavy losses to cotton crop, which we termed as the "third epidemic". We applied CIDER-seq (Circular DNA Enrichment Sequencing), a recently developed sequencing method for PCR-free virus enrichment to produce a full length read of a single circular viral genome coupled with Sanger sequencing to explore the genetic diversity of the disease complex. We identified a highly recombinant strain of Cotton leaf curl Multan virus and a recently evolved strain of Cotton leaf curl Multan betasatellite that are dominant in all major cotton growing regions in the country. Moreover, we also identified multiple species of alphasatellites with one distinct species, Mesta yellow vein mosaic alphasatellite (MeYVMA) for the first time in cotton. Relative abundance of virus and associated satellites was also determined by real-time quantitative PCR. To the best of our knowledge, this is the first study that determined the CLCuD complex associated with its third epidemic.