Biology subjects

Dye, A. E.

Publications and source records attributed to Dye, A. E..

2 recordsLinked to original sources

bioRxiv2021

Rapid multilocus adaptation of clonal cabbage leaf curl virus populations to Arabidopsis thaliana

Cabbage leaf curl virus (CabLCV) has a bipartite single-stranded DNA genome and infects the model plant Arabidopsis thaliana. CabLCV serves as a model for the genus Begomovirus, members of which cause tremendous crop losses worldwide. We have used CabLCV as a model for within-plant virus evolution by inoculating individual plants with infectious clones of either a wild-type or mutagenized version of the CabLCV genome. Consistent with previous reports, detrimental substitutions in the Replication-associated gene (Rep) were readily compensated for by direct reversion and/or alternative mutations. A surprising number of common mutations were detected elsewhere in both viral segments (DNA-A and DNA-B) indicating convergent evolution and suggesting that CabLCV may not be as well adapted to A. thaliana as commonly presumed. Consistent with this idea, a spontaneous coat protein variant consistently rose to high allele frequency in susceptible accession Col-0, at a higher rate than in hypersusceptible accession Sei-0. Numerous high-frequency mutations were also detected in a candidate Rep binding site in DNA-B. Our results reinforce the fact that spontaneous mutation of this type of virus occurs rapidly and can change the majority consensus sequence of a within-plant virus population in weeks.

evolutionary biology↗

bioRxiv2020

An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

We present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses. HighlightsO_LIProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primers C_LIO_LIComparison of the sequencing of total DNA versus size-selected DNA C_LIO_LIComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes C_LI

plant biology↗