High-resolution mapping of Ryd4Hb, a major resistance gene to Barley yellow dwarf virus from Hordeum bulbosum.

KEY MESSAGE: We mapped Ryd4Hb in a 66.5 kbp interval in barley and dissociated it from a sublethality factor. These results will enable a targeted selection of the resistance in barley breeding. Virus diseases are causing high yield losses in crops worldwide. The Barley yellow dwarf virus (BYDV) complex is responsible for one of the most widespread and economically important viral diseases of cereals. While no gene conferring complete resistance (immunity) has been uncovered in the primary gene pool of barley, sources of resistance were searched and identified in the wild relative Hordeum bulbosum, representing the secondary gene pool of barley. One such locus, Ryd4Hb, has been previously introgressed into barley, and was allocated to chromosome 3H, but is tightly linked to a sublethality factor that prevents the incorporation and utilization of Ryd4Hb in barley varieties. To solve this problem, we fine-mapped Ryd4Hb and separated it from this negative factor. We narrowed the Ryd4Hb locus to a corresponding 66.5 kbp physical interval in the barley 'Morex' reference genome. The region comprises a gene from the nucleotide-binding and leucine-rich repeat immune receptor family, typical of dominant virus resistance genes. The closest homolog to this Ryd4Hb candidate gene is the wheat Sr35 stem rust resistance gene. In addition to the fine mapping, we reduced the interval bearing the sublethality factor to 600 kbp in barley. Aphid feeding experiments demonstrated that Ryd4Hb provides a resistance to BYDV rather than to its vector. The presented results, including the high-throughput molecular markers, will permit a more targeted selection of the resistance in breeding, enabling the use of Ryd4Hb in barley varieties.

Rapid evolution of an RNA virus to escape recognition by a rice nucleotide-binding and leucine-rich repeat domain immune receptor.

Viral diseases are a major limitation for crop production, and their control is crucial for sustainable food supply. We investigated by a combination of functional genetics and experimental evolution the resistance of rice to the rice yellow mottle virus (RYMV), which is among the most devastating rice pathogens in Africa, and the mechanisms underlying the extremely fast adaptation of the virus to its host. We found that the RYMV3 gene that protects rice against the virus codes for a nucleotide-binding and leucine-rich repeat domain immune receptor (NLRs) from the Mla-like clade of NLRs. RYMV3 detects the virus by forming a recognition complex with the viral coat protein (CP). The virus escapes efficiently from detection by mutations in its CP, some of which interfere with the formation of the recognition complex. This study establishes that NLRs also confer in monocotyledonous plants immunity to viruses, and reveals an unexpected functional diversity for NLRs of the Mla clade that were previously only known as fungal disease resistance proteins. In addition, it provides precise insight into the mechanisms by which viruses adapt to plant immunity and gives important knowledge for the development of sustainable resistance against viral diseases of cereals.

ATP-Dependent Clp Protease Subunit C1, HvClpC1, Is a Strong Candidate Gene for Barley Variegation Mutant luteostrians as Revealed by Genetic Mapping and Genomic Re-sequencing.

Implementation of next-generation sequencing in forward genetic screens greatly accelerated gene discovery in species with larger genomes, including many crop plants. In barley, extensive mutant collections are available, however, the causative mutations for many of the genes remains largely unknown. Here we demonstrate how a combination of low-resolution genetic mapping, whole-genome resequencing and comparative functional analyses provides a promising path toward candidate identification of genes involved in plastid biology and/or photosynthesis, even if genes are located in recombination poor regions of the genome. As a proof of concept, we simulated the prediction of a candidate gene for the recently cloned variegation mutant albostrians (HvAST/HvCMF7) and adopted the approach for suggesting HvClpC1 as candidate gene for the yellow-green variegation mutant luteostrians.

High-resolution mapping of Rym14Hb, a wild relative resistance gene to barley yellow mosaic disease.

KEY MESSAGE: We mapped the Rym14Hb resistance locus to barley yellow mosaic disease in a 2Mbp interval. The co-segregating markers will be instrumental for marker-assisted selection in barley breeding. Barley yellow mosaic disease is caused by Barley yellow mosaic virus and Barley mild mosaic virus and leads to severe yield losses in barley (Hordeum vulgare) in Central Europe and East-Asia. Several resistance loci are used in barley breeding. However, cases of resistance-breaking viral strains are known, raising concerns about the durability of those genes. Rym14Hb is a dominant major resistance gene on chromosome 6HS, originating from barley's secondary genepool wild relative Hordeum bulbosum. As such, the resistance mechanism may represent a case of non-host resistance, which could enhance its durability. A susceptible barley variety and a resistant H. bulbosum introgression line were crossed to produce a large F2 mapping population (n = 7500), to compensate for a ten-fold reduction in recombination rate compared to intraspecific barley crosses. After high-throughput genotyping, the Rym14Hb locus was assigned to a 2Mbp telomeric interval on chromosome 6HS. The co-segregating markers developed in this study can be used for marker-assisted introgression of this locus into barley elite germplasm with a minimum of linkage drag.

Genome Wide Association Study Pinpoints Key Agronomic QTLs in African Rice Oryza glaberrima.

BACKGROUND: African rice, Oryza glaberrima, is an invaluable resource for rice cultivation and for the improvement of biotic and abiotic resistance properties. Since its domestication in the inner Niger delta ca. 2500 years BP, African rice has colonized a variety of ecologically and climatically diverse regions. However, little is known about the genetic basis of quantitative traits and adaptive variation of agricultural interest for this species. RESULTS: Using a reference set of 163 fully re-sequenced accessions, we report the results of a Genome Wide Association Study carried out for African rice. We investigated a diverse panel of traits, including flowering date, panicle architecture and resistance to Rice yellow mottle virus. For this, we devised a pipeline using complementary statistical association methods. First, using flowering time as a target trait, we found several association peaks, one of which co-localised with a well described gene in the Asian rice flowering pathway, OsGi, and identified new genomic regions that would deserve more study. Then we applied our pipeline to panicle- and resistance-related traits, highlighting some interesting genomic regions and candidate genes. Lastly, using a high-resolution climate database, we performed an association analysis based on climatic variables, searching for genomic regions that might be involved in adaptation to climatic variations. CONCLUSION: Our results collectively provide insights into the extent to which adaptive variation is governed by sequence diversity within the O. glaberrima genome, paving the way for in-depth studies of the genetic basis of traits of interest that might be useful to the rice breeding community.

Allele mining unlocks the identification of RYMV resistance genes and alleles in African cultivated rice.

BACKGROUND: Rice yellow mottle virus (RYMV) is a major rice pathogen in Africa. Three resistance genes, i.e. RYMV1, RYMV2 and RYMV3, have been previously described. RYMV1 encodes the translation initiation factor eIF(iso)4G1 and the best candidate genes for RYMV2 and RYMV3 encode a homolog of an Arabidopsis nucleoporin (CPR5) and a nucleotide-binding domain and leucine-rich repeat containing domain (NLR) protein, respectively. High resistance is very uncommon in Asian cultivated rice (Oryza sativa), with only two highly resistant accessions identified so far, but it is more frequent in African cultivated rice (Oryza glaberrima). RESULTS: Here we report the findings of a resistance survey in a reference collection of 268 O. glaberrima accessions. A total of 40 resistant accessions were found, thus confirming the high frequency of resistance to RYMV in this species. We analysed the variability of resistance genes or candidate genes in this collection based on high-depth Illumina data or Sanger sequencing. Alleles previously shown to be associated with resistance were observed in 31 resistant accessions but not in any susceptible ones. Five original alleles with a frameshift or untimely stop codon in the candidate gene for RYMV2 were also identified in resistant accessions. A genetic analysis revealed that these alleles, as well as T-DNA insertions in the candidate gene, were responsible of RYMV resistance. All 40 resistant accessions were ultimately linked to a validated or candidate resistance allele at one of the three resistance genes to RYMV. CONCLUSION: This study demonstrated that the RYMV2 resistance gene is homologous to the Arabidopsis CPR5 gene and revealed five new resistance alleles at this locus. It also confirmed the close association between resistance and an amino-acid substitution in the leucine-rich repeat of the NLR candidate for RYMV3. We also provide an extensive overview of the genetic diversity of resistance to RYMV in the O. glaberrima species, while underlining the contrasted pattern of diversity between O. glaberrima and O. sativa for this trait. The different resistance genes and alleles will be instrumental in breeding varieties with sustainable field resistance to RYMV.

High Resolution Mapping of a Hordeum bulbosum-Derived Powdery Mildew Resistance Locus in Barley Using Distinct Homologous Introgression Lines.

Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is one of the main foliar diseases in barley (Hordeum vulgare L.; Hv). Naturally occurring resistance genes used in barley breeding are a cost effective and environmentally sustainable strategy to minimize the impact of pathogens, however, the primary gene pool of H. vulgare contains limited diversity owing to recent domestication bottlenecks. To ensure durable resistance against this pathogen, more genes are required that could be unraveled by investigation of secondary barley gene-pool. A large set of Hordeum bulbosum (Hb) introgression lines (ILs) harboring a diverse set of desirable resistance traits have been developed and are being routinely used as source of novel diversity in gene mapping studies. Nevertheless, this strategy is often compromised by a lack of recombination between the introgressed fragment and the orthologous chromosome of the barley genome. In this study, we fine-mapped a Hb gene conferring resistance to barley powdery mildew. The initial genotyping of two Hb ILs mapping populations with differently sized 2HS introgressions revealed severely reduced interspecific recombination in the region of the introgressed segment, preventing precise localization of the gene. To overcome this difficulty, we developed an alternative strategy, exploiting intraspecific recombination by crossing two Hv/Hb ILs with collinear Hb introgressions, one of which carries a powdery mildew resistance gene, while the other doesn't. The intraspecific recombination rate in the Hb-introgressed fragment of 2HS was approximately 20 times higher than it was in the initial simple ILs mapping populations. Using high-throughput genotyping-by-sequencing (GBS), we allocated the resistance gene to a 1.4 Mb interval, based on an estimate using the Hv genome as reference, in populations of only 103 and 146 individuals, respectively, similar to what is expected at this locus in barley. The most likely candidate resistance gene within this interval is part of the coiled-coil nucleotide-binding-site leucine-rich-repeat (CC-NBS-LLR) gene family, which is over-represented among genes conferring strong dominant resistance to pathogens. The reported strategy can be applied as a general strategic approach for identifying genes underlying traits of interest in crop wild relatives.

Fine mapping of RYMV3: a new resistance gene to Rice yellow mottle virus from Oryza glaberrima.

KEY MESSAGE: A new resistance gene against Rice yellow mottle virus was identified and mapped in a 15-kb interval. The best candidate is a CC-NBS-LRR gene. Rice yellow mottle virus (RYMV) disease is a serious constraint to the cultivation of rice in Africa and selection for resistance is considered to be the most effective management strategy. The aim of this study was to characterize the resistance of Tog5307, a highly resistant accession belonging to the African cultivated rice species (Oryza glaberrima), that has none of the previously identified resistance genes to RYMV. The specificity of Tog5307 resistance was analyzed using 18 RYMV isolates. While three of them were able to infect Tog5307 very rapidly, resistance against the others was effective despite infection events attributed to resistance-breakdown or incomplete penetrance of the resistance. Segregation of resistance in an interspecific backcross population derived from a cross between Tog5307 and the susceptible Oryza sativa variety IR64 showed that resistance is dominant and is controlled by a single gene, named RYMV3. RYMV3 was mapped in an approximately 15-kb interval in which two candidate genes, coding for a putative transmembrane protein and a CC-NBS-LRR domain-containing protein, were annotated. Sequencing revealed non-synonymous polymorphisms between Tog5307 and the O. glaberrima susceptible accession CG14 in both candidate genes. An additional resistant O. glaberrima accession, Tog5672, was found to have the Tog5307 genotype for the CC-NBS-LRR gene but not for the putative transmembrane protein gene. Analysis of the cosegregation of Tog5672 resistance with the RYMV3 locus suggests that RYMV3 is also involved in Tog5672 resistance, thereby supporting the CC-NBS-LRR gene as the best candidate for RYMV3.