A Sequence-Independent Strategy for Amplification and Characterisation of Episomal Badnavirus Sequences Reveals Three Previously Uncharacterised Yam Badnaviruses

Abstract

Yam (Dioscorea spp.) plants are potentially hosts to a diverse range of badnavirus species (genus Badnavirus, family Caulimoviridae), but their detection is complicated by the existence of integrated badnavirus sequences in some yam genomes. To date, only two badnavirus genomes have been characterised, namely, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV). A further 10 tentative species in yam have been described based on their partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences, generically referred to here as Dioscorea bacilliform viruses (DBVs). Further characterisation of DBV species is necessary to determine which represent episomal viruses and which are only present as integrated badnavirus sequences in some yam genomes. In this study, a sequence-independent multiply-primed rolling circle amplification (RCA) method was evaluated for selective amplification of episomal DBV genomes. This resulted in the identification and characterisation of nine complete genomic sequences (7.4–7.7 kbp) of existing and previously undescribed DBV phylogenetic groups from Dioscorea alata and Dioscorea rotundata accessions. These new yam badnavirus genomes expand our understanding of the diversity and genomic organisation of DBVs, and assist the development of improved diagnostic tools. Our findings also suggest that mixed badnavirus infections occur relatively often in West African yam germplasm.

Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes

“…Multiple alignments of the partial RT-RNase H sequences can be performed using the CLUSTALW default settings in MEGA version 7, where phylogenetic relationships can also be analysed. This has been successfully performed in our previous study (Bömer et al, 2016).…”

“…germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al, 2008;Bousalem et al, 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al, 1999;Seal and Muller, 2007;Bömer et al, 2016 andSukal et al, 2017;Umber et al, 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm.…”

“…We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam badnaviruses (Bömer et al, 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of badnavirus diversity as well as the identification of potentially integrated badnavirus sequences in the host genome (Turaki et al, 2017).…”

Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

“…(a) Pairwise sequence comparison and (b) phylogenetic analysis (Clustal W alignment using the maximum‐likelihood method [Kimura‐2‐parameter model]) of partial RT/RNase H‐coding nucleotide sequences, showing the relationships of Dioscorea bacilliform AL virus (DBALV) isolates from this study with previously published complete sequences DBALV‐[2ALa], DBALV‐[2ALb] and DBALV‐[3RT] from Nigeria (Bömer et al, ). DBALV2‐PNG10 (KY827395) was used as the outgroup for the analysis [Colour figure can be viewed at wileyonlinelibrary.com]…”

“…, ), while Bömer et al . () also detected and characterized DBRTV2 from D. rotundata originating from Nigeria. Because the SPC‐CePaCT collections do not include D. rotundata from any additional Pacific countries, the presence of DBRTV2 in countries other than Samoa could not be ascertained.…”