Host range and molecular variability of the sadwavirus dioscorea mosaic associated virus.

Dioscorea mosaic associated virus (DMaV) is a member of the genus Sadwavirus, family Secoviridae, that is associated with mosaic symptoms in Dioscorea rotundata in Brazil. The genome of a DMaV isolate detected in D. trifida in Guadeloupe was sequenced by high-throughput sequencing. Using an RT-PCR-based detection assay, we found that DMaV infects D. alata, D. bulbifera, D. cayenensis-rotundata, D. esculenta, and D. trifida accessions conserved in Guadeloupe and Côte d'Ivoire and displays a very high level of molecular diversity in a relatively small region of the genome targeted by the assay. We also provide evidence that DMaV is also present in D. rotundata in Benin and in D. alata in Nigeria.

Identification of quantitative trait nucleotides and candidate genes for tuber yield and mosaic virus tolerance in an elite population of white guinea yam (Dioscorea rotundata) using genome-wide association scan.

BACKGROUND: Improvement of tuber yield and tolerance to viruses are priority objectives in white Guinea yam breeding programs. However, phenotypic selection for these traits is quite challenging due to phenotypic plasticity and cumbersome screening of phenotypic-induced variations. This study assessed quantitative trait nucleotides (QTNs) and the underlying candidate genes related to tuber yield per plant (TYP) and yam mosaic virus (YMV) tolerance in a panel of 406 white Guinea yam (Dioscorea rotundata) breeding lines using a genome-wide association study (GWAS). RESULTS: Population structure analysis using 5,581 SNPs differentiated the 406 genotypes into seven distinct sub-groups based delta K. Marker-trait association (MTA) analysis using the multi-locus linear model (mrMLM) identified seventeen QTN regions significant for TYP and five for YMV with various effects. The seveteen QTNs were detected on nine chromosomes, while the five QTNs were identified on five chromosomes. We identified variants responsible for predicting higher yield and low virus severity scores in the breeding panel through the marker-effect prediction. Gene annotation for the significant SNP loci identified several essential putative genes associated with the growth and development of tuber yield and those that code for tolerance to mosaic virus. CONCLUSION: Application of different multi-locus models of GWAS identified 22 QTNs. Our results provide valuable insight for marker validation and deployment for tuber yield and mosaic virus tolerance in white yam breeding. The information on SNP variants and genes from the present study would fast-track the application of genomics-informed selection decisions in breeding white Guinea yam for rapid introgression of the targeted traits through markers validation.

Molecular Viral Diagnosis and Sanitation of Yam Genetic Resources: Implications for Safe Yam Germplasm Exchange.

Yam (Dioscorea spp.) is an important crop in tropical and subtropical regions. Many viruses have been recently identified in yam, hampering genetic conservation and safe international exchanges of yam germplasm. We report on the implementation of reliable and cost-effective PCR-based detection tools targeting eight different yam-infecting viruses. Viral indexing of the in vitro yam collection maintained by the Biological Resources Center for Tropical Plants (BRC-TP) in Guadeloupe (French West Indies) unveiled a high prevalence of potyviruses, badnaviruses, Dioscorea mosaic associated virus (DMaV) and yam asymptomatic virus 1 (YaV1) and a high level of coinfections. Infected yam accessions were subjected to a combination of thermotherapy and meristem culture. Sanitation levels were monitored using PCR-based and high-throughput sequencing-based diagnosis, confirming the efficacy and reliability of PCR-based detection tools. Sanitation rates were highly variable depending on viruses. Sixteen accessions were successfully sanitized, paving the way to safe yam germplasm exchanges and the implementation of clean seed production programs worldwide.

Yam asymptomatic virus 1, a novel virus infecting yams (Dioscorea spp.) with significant prevalence in a germplasm collection.

A novel virus infecting yams (Dioscorea spp.), tentatively named "yam asymptomatic virus 1" (YaV1), was characterized and sequenced from an asymptomatic D. alata plant from Vanuatu. Sequence comparisons and phylogenetic analysis showed that YaV1 is a novel ampelovirus and has the smallest genome among "subgroup 1" members. RT-PCR-based screening of a yam germplasm collection conserved in Guadeloupe showed that YaV1 is prevalent in D. alata, D. bulbifera, D. cayennensis subsp. rotundata, D. esculenta and D. trifida accessions but causes no apparent symptoms. Additional phylogenetic analysis revealed a low variability of YaV1 in Guadeloupe in a limited part of the genome, and suggested the occurrence of plant-to-plant transmission.

Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses.

The genus Badnavirus is characterized by members that are genetically and serologically heterogeneous which presents challenges for their detection and characterization. The presence of integrated badnavirus-like sequences in some host species further complicates detection using PCR-based protocols. To address these challenges, we have assessed and optimized various RCA protocols including random-primed RCA (RP-RCA), primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-RCA) and specific-primed RCA (SP-RCA). Using Dioscorea bacilliform AL virus (DBALV) as an example, we demonstrate that viral DNA amplified using the optimized D-RCA and SP-RCA protocols showed an 85-fold increase in badnavirus NGS reads compared with RP-RCA. The optimized RCA techniques described here were used to detect a range of badnaviruses infecting banana, sugar cane, taro and yam demonstrating the utility of RCA for detection of diverse badnaviruses infecting a variety of host plant species.

Air potato (Dioscorea bulbifera) plants displaying virus-like symptoms are co-infected with a novel potyvirus and a novel ampelovirus.

Air potato (Dioscorea bulbifera) plants being grown at the Florida Department of Agriculture and Consumer Services Division of Plant Industry Biological Control Laboratory II in Alachua County, Florida were observed exhibiting foliar mosaic symptoms characteristic of virus infection. A double-stranded RNA library generated from a symptomatic plant underwent high-throughput sequencing to determine if viral pathogens were present. Sequence data revealed the presence of two viral genomes, one with properties congruent with members of the genus Potyvirus (family Potyviridae), and the other with members of the genus Ampelovirus (family Closteroviridae). Sequence comparisons and phylogenetic placement indicate that both viruses represent novel species. The names "dioscorea mosaic virus" and "air potato virus 1" are proposed for the potyvirus and ampelovirus, respectively.

Nanopore-based detection and characterization of yam viruses.

We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.

Characterization of a novel member of the family Caulimoviridae infecting Dioscorea nummularia in the Pacific, which may represent a new genus of dsDNA plant viruses.

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic analysis, DNUaV appears to be a distinct novel member of the family and may represent a new genus.

Complete genome sequence of yam chlorotic necrosis virus, a novel macluravirus infecting yam.

The complete genome sequence of a novel member of the genus Macluravirus was determined from yam plants with chlorotic and necrotic symptoms in China. The genomic RNA consists of 8,261 nucleotides (nt) excluding the 3'-terminal poly(A) tail, containing one long open reading frame (ORF) encoding a large putative polyprotein of 2,627 amino acids. Its genomic structure is typical of macluraviruses, which lack the P1 protein, N-terminal HC-Pro, and D-A-G motif for aphid transmission that are found in potyviruses. The virus shares 56.3-63.8% sequence identity at the genome sequence level and 49.7-63.9% at the polyprotein sequence level with other members of the genus Macluravirus. Phylogenetic analysis based on the complete polyprotein sequence of representative members of the family Potyviridae clearly places the virus within the genus Macluravirus. These results suggest that the virus, tentatively named "yam chlorotic necrosis virus" (YCNV), should be considered a member of a novel species in the genus Macluravirus.

Rapid detection of potyviruses from crude plant extracts.

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named 'Direct RT-RPA', detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP).

A closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP) assay was developed for the detection of yam mosaic virus (YMV, genus Potyvirus) infecting yam (Dioscorea spp.). The assay uses a set of six oligonucleotide primers targeting the YMV coat protein region, and the amplification products in YMV-positive samples are visualized by chromogenic detection with SYBR Green I dye. The CT-RT-LAMP assay detected YMV in leaf and tuber tissues of infected plants. The assay is 100 times more sensitive in detecting YMV than standard RT-PCR, while maintaining the same specificity.

Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam badnaviruses.

Yams (Dioscorea spp.) host a diverse range of badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam badnaviruses. This has important implications for yam breeding programmes globally.

PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm.

Badnaviruses (family Caulimoviridae, genus Badnavirus) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (Dioscorea spp.) plants are hosts to a diverse range of badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex 'fingerprints' composed of multiple sequences of Dioscorea bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm.

Characterization of badnaviruses infecting Dioscorea spp. in the Pacific reveals two putative novel species and the first report of dioscorea bacilliform RT virus 2.

The complete genome sequences of three new badnaviruses associated with yam (Dioscorea spp.) originating from Fiji, Papua New Guinea and Samoa were determined following rolling circle amplification of the virus genomes. The full-length genomes consisted of a single molecule of circular double-stranded DNA of 8106bp for isolate FJ14, 7871bp for isolate PNG10 and 7426bp for isolate SAM01. FJ14 and PNG10 contained three open reading frames while SAM01 had an additional open reading frame which partially overlapped the 3' end of ORF 3. Amino acid sequence analysis of ORF 3 from the three isolates confirmed the presence of conserved motifs typical of other badnaviruses. Phylogenetic analysis revealed the sequences to be closely related to other Dioscorea-infecting badnaviruses. FJ14 and PNG10 appear to be new species, which we have tentatively named dioscorea bacilliform ES virus (DBESV) and dioscorea bacilliform AL virus 2 (DBALV2), respectively, while SAM01 represents a Pacific isolate of the recently published dioscorea bacilliform RT virus 2 and is described as dioscorea bacilliform RT virus 2-[4RT] (DBRTV2-[4RT]).

Complete genome sequence of a putative new secovirus infecting yam (Dioscorea) plants.

The complete genome sequence of a new virus infecting yam plants exhibiting mosaic symptom in Brazil was determined. The genome of this virus is composed of two molecules of positive-sense RNAs of 5979 and 3809 nucleotides in length, excluding the poly(A) tails. One large open reading frame (ORF) in each genomic segment (RNA1-ORF1 and RNA2-ORF2) was predicted. The highest amino acid sequence similarity in the Pro-Pol core region of RNA1 and the CP region of RNA2 was observed with chocolate lily virus A (a putative member of the family Secoviridae), with 54.6 and 27.7 % identity, respectively. This virus is thus likely to be a new member of the family Secoviridae, and we propose the tentative name "dioscorea mosaic-associated virus" (DMaV) for this virus.

The genome sequence of Dioscorea bacilliform TR virus, a member of the genus Badnavirus infecting Dioscorea spp., sheds light on the possible function of endogenous Dioscorea bacilliform viruses.

The complete genome sequence of Dioscorea bacilliform TR virus (DBTRV) was determined. The closest relatives of DBTRV are Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform RT virus 1 (DBRTV1). Specific primers were designed and used to determine the prevalence of DBTRV in a yam germplasm collection. It was found that this virus infects Dioscorea alata and D. trifida plants in Guadeloupe and French Guyana. DTRBV was not detected in any of the tested D. cayenensis-rotundata accessions. In silico analysis provided evidence for the presence of DBTRV-like endogenous sequences in the genome of D. cayenensis-rotundata, pointing to a possible role of these sequences in antiviral defense.

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

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.

Complete genome sequence of yam chlorotic necrotic mosaic virus from Dioscorea parviflora.

The complete genome sequence of yam chlorotic necrotic mosaic virus (YCNMV) was determined. It is a monopartite ssRNA 8208 nucleotides in length (excluding the poly(A) tail) and encoding a polyprotein of 2622 amino acids. Sequence analysis showed that the P1 region and some conserved motifs, such as the typical potyvirus aphid-transmission motifs DAG, PTK and KITC, are absent. Phylogenetic analysis based on the complete polyprotein sequences of YCNMV and selected members of the family Potyviridae clearly showed that this virus should be assigned to the genus Macluravirus and suggest that YCNMV is a new member of the genus Macluravirus.

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification.

Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/µl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories.

Metagenomics Approaches Based on Virion-Associated Nucleic Acids (VANA): An Innovative Tool for Assessing Without A Priori Viral Diversity of Plants.

This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.