Going beyond consensus genome sequences: An innovative SNP-based methodology reconstructs different Ugandan cassava brown streak virus haplotypes at a nationwide scale in Rwanda.

Cassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in thirteen cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in twelve districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 per cent divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNPs) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Furthermore, the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNP patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100 per cent occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes' associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars.

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis.

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors.

Prospecting of local grapevine (Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. 'Hencha' were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1-1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1-1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% 'Hencha' somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.