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Rapid characterization of novel NB-LRR-associated resistance to Phomopsis cane spot on grapevine using high-throughput sampling and low-coverage sequencing for genotyping, locus mapping and transcriptome analysis provides insights into genetic resistance to a hemibiotrophic fungus. Phomopsis cane and leaf spot, caused by the hemibiotrophic fungus Diaporthe ampelina (syn = Phomopsis viticola), reduces the productivity in grapevines. Host resistance was studied on three F1 families derived from crosses involving resistant genotypes ‘Horizon’, Illinois 547-1, Vitis cinerea B9 and V. vinifera ‘Chardonnay’. All families had progeny with extremely susceptible phenotypes, developing lesions on both dormant canes and maturing fruit clusters. Segregation of symptoms was observed under natural levels of inoculum in the field, while phenotypes on green shoots were confirmed under controlled inoculations in greenhouse. High-density genetic maps were used to localize novel qualitative resistance loci named Rda1 and Rda2 from V. cinerea B9 and ‘Horizon’, respectively. Co-linearity between reference genetic and physical maps allowed localization of Rda2 locus between 1.5 and 2.4 Mbp on chromosome 7, and Rda1 locus between 19.3 and 19.6 Mbp of chromosome 15, which spans a cluster of five NB-LRR genes. Further dissection of this locus was obtained by QTL mapping of gene expression values 14 h after inoculation across a subset of the ‘Chardonnay’ × V. cinerea B9 progeny. This provided evidence for the association between transcript levels of two of these NB-LRR genes with Rda1, with increased NB-LRR expression among susceptible progeny. In resistant parent V. cinerea B9, inoculation with D. ampelina was characterized by up-regulation of SA-associated genes and down-regulation of ethylene pathways, suggesting an R-gene-mediated response. With dominant effects associated with disease-free berries and minimal symptoms on canes, Rda1 and Rda2 are promising loci for grapevine genetic improvement.

Key message Downy mildew resistance across days post-inoculation, experiments, and years in two interspecific grapevine F1 families was investigated using linear mixed models and Bayesian networks, and five new QTL were identified. Abstract Breeding grapevines for downy mildew disease resistance has traditionally relied on qualitative gene resistance, which can be overcome by pathogen evolution. Analyzing two interspecific F1 families, both having ancestry derived from Vitis vinifera and wild North American Vitis species, across 2 years and multiple experiments, we found multiple loci associated with downy mildew sporulation and hypersensitive response in both families using a single phenotype model. The loci explained between 7 and 17% of the variance for either phenotype, suggesting a complex genetic architecture for these traits in the two families studied. For two loci, we used RNA-Seq to detect differentially transcribed genes and found that the candidate genes at these loci were likely not NBS-LRR genes. Additionally, using a multiple phenotype Bayesian network analysis, we found effects between the leaf trichome density, hypersensitive response, and sporulation phenotypes. Moderate–high heritabilities were found for all three phenotypes, suggesting that selection for downy mildew resistance is an achievable goal by breeding for either physical- or non-physical-based resistance mechanisms, with the combination of the two possibly providing durable resistance. Electronic supplementary material The online version of this article (doi:10.1007/s00122-018-3065-y) contains supplementary material, which is available to authorized users.

Este documento fue originado durante la “Primera Reunión del Núcleo de Estudio de Nuevas Técnicas de Mejoramiento Genético” del PROCISUR, realizada en Montevideo entre el 25 y 26 de agosto de 2017 y redactado, en versiones corregidas, entre esa fecha y el 16 de noviembre de 2018. Especialistas de los INIAs que integran el PROCISUR alientan el debate en torno a la ingeniería genética, a partir de los avances que se están alcanzando con la edición génica. Analizan el potencial de las tecnologías de modificación genética que tiene un consecuente impacto en el aumento de la variabilidad. La incidencia actual y futura en la generación de animales y plantas mejoradas, debido a su menor costo y mayor accesibilidad, presenta oportunidades en cultivos de reproducción agámica como la papa, el banano, la yuca, la caña de azúcar o la vid, entre otros. Reconocen que la utilización de esta herramienta puede modificar sustancialmente el esquema de los programas de mejoramiento, ya que permitiría realizar mejoras incrementales sobre genotipos establecidos y adaptados. EEA Balcarce Fil: Feingold, Sergio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Bonnecarrère, Victoria. Instituto Nacional de Investigación Agropecuaria (INIA); Uruguay Fil: Nepomuceno, Alexandre. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); Brasil Fil: Hinrichsen, Patricio. Instituto de Investigaciones Agropecuarias (INIA); Chile. Fil: Cardozo Tellez, Lourdes Maria. Instituto Paraguayo de Tecnología Agrícola (IPTA); Paraguay. Fil: Molinari, Hugo. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); Brasil Fil: Barba, Paola. Instituto de Investigaciones Agropecuarias (INIA); Chile Fil: Eyherabide, Guillermo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino; Argentina Fil: Ceretta, Sergio Eduardo. Instituto Nacional de Investigación Agropecuaria (INIA); Uruguay Fil: Dujack, Christian. Instituto Paraguayo de Tecnología Agrícola (IPTA); Paraguay.

The abundance of predatory phytoseiid mites, Typhlodromus pyri, important biological control agents of spider mite pests in numerous crops, is positively influenced by the density of leaf trichomes and tuft-form domatia in vein axils. Identification of the genetic regions controlling both trophic levels could facilitate the improvement of predatory mite habitat in breeding programs. The abundance of T. pyri and non-glandular trichomes was measured in a segregating F1 family derived from the cross of the complex Vitis hybrid, ‘Horizon’, with Illinois 547-1 (V. rupestris B38 × V. cinerea B9), finding positive correlation among traits. High density genetic maps were used to localize one major quantitative trait locus (QTL) on chromosome 1 of Illinois 547-1 associated with both predatory mite abundance and leaf trichomes. This QTL explained 23% of the variation in phytoseiid abundance and similar amounts of variance in domatia rating (21%), domatia size (16%), leaf bristle density (37% in veins and 33% in blades), and leaf hair density (20% in veins and 15% in blades). Another nine QTL distributed among chromosomes 1, 2, 5, 8, and 15 were associated solely with trichome density, and explained 7–17% of the phenotypic variation. Combined, our results provide evidence of the genetic architecture of non-glandular trichomes in Vitis, with a major locus influencing trichome densities, domatia size and predatory mite abundance. This information is relevant for breeding grapevines with a more favorable habitat for biological control agents.

Improved efficacy and durability of powdery mildew resistance can be enhanced via knowledge of the genetics of resistance and susceptibility coupled with the development of high-resolution maps to facilitate the stacking of multiple resistance genes and other desirable traits. We studied the inheritance of powdery mildew (Erysiphe necator) resistance and susceptibility of wild Vitis rupestris B38 and cultivated V. vinifera 'Chardonnay', finding evidence for quantitative variation. Molecular markers were identified using genotyping-by-sequencing, resulting in 16,833 single nucleotide polymorphisms (SNPs) based on alignment to the V. vinifera 'PN40024' reference genome sequence. With an average density of 36 SNPs/Mbp and uniform coverage of the genome, this 17K set was used to identify 11 SNPs on chromosome 7 associated with a resistance locus from V. rupestris B38 and ten SNPs on chromosome 9 associated with a locus for susceptibility from 'Chardonnay' using single marker association and linkage disequilibrium analysis. Linkage maps for V. rupestris B38 (1,146 SNPs) and 'Chardonnay' (1,215 SNPs) were constructed and used to corroborate the 'Chardonnay' locus named Sen1 (Susceptibility to Erysiphe necator 1), providing the first insight into the genetics of susceptibility to powdery mildew from V. vinifera. The identification of markers associated with a susceptibility locus in a V. vinifera background can be used for negative selection among breeding progenies. This work improves our understanding of the nature of powdery mildew resistance in V. rupestris B38 and 'Chardonnay', while applying next-generation sequencing tools to advance grapevine genomics and breeding.

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had

International audience; Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV:: GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV:: GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

Genotyping by sequencing (GBS) provides opportunities to generate high-resolution genetic maps at a low genotyping cost, but for highly heterozygous species, missing data and heterozygote undercalling complicate the creation of GBS genetic maps. To overcome these issues, we developed a publicly available, modular approach called HetMappS, which functions independently of parental genotypes and corrects for genotyping errors associated with heterozygosity. For linkage group formation, HetMappS includes both a reference-guided synteny pipeline and a reference-independent de novo pipeline. The de novo pipeline can be utilized for under-characterized or high diversity families that lack an appropriate reference. We applied both HetMappS pipelines in five half-sib F1 families involving genetically diverse Vitis spp. Starting with at least 116,466 putative SNPs per family, the HetMappS pipelines identified 10,440 to 17,267 phased pseudo-testcross (Pt) markers and generated high-confidence maps. Pt marker density exceeded crossover resolution in all cases; up to 5,560 non-redundant markers were used to generate parental maps ranging from 1,047 cM to 1,696 cM. The number of markers used was strongly correlated with family size in both de novo and synteny maps (r = 0.92 and 0.91, respectively). Comparisons between allele and tag frequencies suggested that many markers were in tandem repeats and mapped as single loci, while markers in regions of more than two repeats were removed during map curation. Both pipelines generated similar genetic maps, and genetic order was strongly correlated with the reference genome physical order in all cases. Independently created genetic maps from shared parents exhibited nearly identical results. Flower sex was mapped in three families and correctly localized to the known sex locus in all cases. The HetMappS pipeline could have wide application for genetic mapping in highly heterozygous species, and its modularity provides opportunities to adapt portions of the pipeline to other family types, genotyping technologies or applications.