Classical and next generation sequencing approaches unravel Bymovirus diversity in barley crops in France

Abstract

Despite the generalized use of cultivars carrying the rym4 resistance gene, the impact of viral mosaic diseases on winter barleys increased in recent years in France. This change could reflect i) an increased prevalence of the rym4 resistance-breaking pathotype of Barley yellow mosaic virus Y (BaYMV-2), ii) the emergence of rym4 resistance-breaking pathotypes of Barley mild mosaic virus (BaMMV) or iii) the emergence of other viruses. A study was undertaken to determine the distribution and diversity of viruses causing yellow mosaic disease. A collection of 241 symptomatic leaf samples from susceptible, rym4 and rym5 varieties was gathered from 117 sites. The viruses present in all samples were identified by specific RT-PCR assays and, for selected samples, by double-stranded RNA next-generation sequencing (NGS). The results show that BaYMV-2 is responsible for the symptoms observed in varieties carrying the resistance gene rym4. In susceptible varieties, both BaYMV-1 and BaYMV-2 were detected, together with BaMMV. Phylogenetic analyses indicate that the rym4 resistance-breaking ability independently evolved in multiple genetic backgrounds. Parallel analyses revealed a similar scenario of multiple independent emergence events in BaMMV for rym5 resistance-breaking, likely involving multiple amino acid positions in the viral-linked genome protein. NGS analyses and classical techniques provided highly convergent results, highlighting and validating the power of NGS approaches for diagnostics and viral population characterization.

Plant virome reconstruction and antiviral RNAi characterization by deep sequencing of small RNAs from dried leaves

“…Samples of barley leaves exhibiting mosaic symptoms collected during field surveys in 2013–2015 (each year from January to April), dried and kept stored over anhydrous calcium chloride at room temperature, were analyzed by high-throughput dsRNA sequencing to evaluate the diversity of bymoviruses in barley 8 (see below). In August 2016, four of these dried leaf samples were taken for total RNA extraction and quality control using RNA blot hybridization analysis with the plant miRNA miR160-specific probe (as detailed in Methods section below).…”

“…A phylogenetic analysis of BaYMV isolates revealed that the 7563 nt RNA1 sequences reconstructed from HYT-37 (deposited in GenBank as MN107377) and HYT-38 (deposited in GenBank as MN107378) exhibit 98.0% pairwise identity (155 SNPs), and are respectively 97.8% and 98.1% identical to the sequence of an isolate collected in France in 2014 (KX117192) 8 that we used for reference-based reconstruction, with SNPs not disrupting a single polyprotein ORF. Among other BaYMV isolates, the RNA1 of HYT-37 is 99.0% identical to that of the KX117208 isolate from Germany, while the RNA1 of HYT-38 is 99.0% identical to that of the KX117195 isolate from France.…”

“…Among other BaYMV isolates, the RNA1 of HYT-37 is 99.0% identical to that of the KX117208 isolate from Germany, while the RNA1 of HYT-38 is 99.0% identical to that of the KX117195 isolate from France. The 3516 nt BaYMV RNA2 sequences reconstructed from HYT-37 (deposited in GenBank as MN107381) and HYT-38 (deposited in GenBank as MN107380) exhibit 95.6% pairwise identity (154 SNPs), and are respectively 95.2% and 98.1% identical to the RNA2 reference sequence obtained by dsRNA sequencing 8 , with SNPs not disrupting the single polyprotein ORF. Furthermore, they exhibit respectively 94.7% (HYT-37) and 97.4% (HYT-38) identity to RNA2 sequences of the HAT (AJ515487) and EST (AJ515486) isolates from UK 10 .…”

Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV

“…As there has been some variations identified in the virus genome to be crucial to the pathogenesis (Habekuß et al 2008;Kanyuka et al 2004;Kühne et al 2003;Rolland et al 2017), future studies on the interaction of particular sites between virus protein and eIF4E could help understanding the mechanism(s) of barley susceptibility to the soil-borne virus disease.…”

“…Over the last few decades, barley breeding for resistance to the barley yellow mosaic virus disease has mainly relied on alleles of rym4, rym5 or rym1/11 (Kühne 2009), while the diversification and wide distribution of BaYMV/BaMMV in both European (Achon et al 2003;Jezewska and Trzmiel 2009;Rolland et al 2017) and East Asian countries (Chen et al 1999;Hosseini et al 2014;You and Shirako 2013;Zheng et al 1999) remain a constant threat to winter barley production. For example, BaYMV isolates were recently detected in winter barley in Poland and Iran, where the disease was not yet reported (Hosseini et al 2014;Jezewska and Trzmiel 2009).…”