Abstract
Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.
Highlights
Begomoviruses are small, circular, single-stranded DNA viral pathogens of plants that infect a wide range of eudicots in the tropics and subtropics
The search result showed that the contigs containing begomoviruses and begomovirus-associated DNA satellite sequences consistently comprised the highest number of assembled reads, indicating that the rolling circle amplification (RCA) successfully enriched for these components
Metagenomics has been used successfully to determine the genome sequence and associated SNPs of selected begomoviral genomes, and their associated satellites from total DNA extracts of symptomatic tomato and okra plants, without cloning or any previous knowledge of the target virus-satellite complexes that were present in naturally-infected plants from the field
Summary
Begomoviruses are small, circular, single-stranded (ss) DNA viral pathogens (family, Geminiviridae; genus, Begomovirus) of plants that infect a wide range of eudicots in the tropics and subtropics. Using sequence-specific (or degenerate) primers, or phi DNA polymerase-mediated rolling circle amplification (RCA) through the use of random primers [19], followed by cloning and capillary DNA sequencing These strategies produce limited information about the presumed predominant genomes, given that the depth of sequencing is low, that precludes assessment of the genetic differentiation and population structure of begomovirus-satellite variants. The amplification of begomoviral genomic and associated components by PCR, followed by cloning and capillary DNA sequencing, are limited by the specificity of the primers, and by the number of variants produced by the earliest amplification steps, and by selection during the molecular cloning step. The enriched begomoviral genomes and satellites were subjected to Illumina-NGS, assembled, and subjected to analysis to detect polymorphisms
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