Xylella fastidiosa (Xf) is the etiological agent of Pierce's disease (PD), a major threat to viticulture worldwide. The Xf prtA- mutant, characterized by its planktonic phenotype and absence of biofilm formation, has been previously labeled as hypervirulent due to its aggressive symptomatology in the PD-susceptible Vitis vinifera 'Thompson Seedless'. This study challenges the hypervirulent concept by demonstrating that a diverse range of grapevine accessions, especially the PD line U0505, exhibited resistance to Xf prtA-. Our findings suggest that different resistance mechanisms are present against the planktonic and biofilm phenotypes of Xf, as represented by the prtA- Xf mutant and the wild-type Temecula1 Xf strain, respectively. This study underscores the complexity of host-pathogen interactions and highlights the importance of multiple host defense mechanisms in countering specific virulence strategies of Xf. The resistance assessed in the U0505 line against Xf prtA- infection provides valuable insights into potential genetic and molecular targets for breeding PD-resistant grapevine cultivars and developing effective disease management strategies.

The Chinese grape accession Vitis piasezkii DVIT2027 carries 2 loci associated with powdery mildew (PM) resistance, Ren6 and Ren7, which differ in timing and strength of response to Erysiphe necator. Both loci are consistent with recognition by intracellular immune receptors. To identify the underlying nucleotide-binding leucine-rich repeat (NLR) genes, we assembled chromosome-scale diploid genomes of DVIT2027 and the susceptible V. vinifera F2-35, parents of a segregating F1 population. We integrated these assemblies with deep resequencing data from 8 F1 siblines carrying different Ren6/Ren7 combinations and generated trio-binned, parent-phased genomes for 6 progeny. This resolved both PM-resistant (PMR) and PM-susceptible (PMS) haplotypes at Ren6 and Ren7. Comparative analyses revealed extensive structural variation and complete haplotype specificity among NLRs, with several candidate genes lacking allelic counterparts in PMS haplotypes. Expression profiling across PMR siblines identified 4 and 2 CC-NBS-LRR genes potentially associated with Ren6 and Ren7, respectively. Sequence graph reconstruction of these loci across multiple V. piasezkii accessions revealed broad intraspecific diversity and DVIT2027-specific nodes, including within candidate NLR genes. These results provide a high-resolution view of Ren6 and Ren7 and support the identification of resistance gene candidates for functional validation and grapevine breeding.

The Chinese grape accession Vitis piasezkii DVIT2027 carries two loci associated with powdery mildew (PM) resistance, Ren6 and Ren7 . Although these loci differ in the timing and strength of their response to Erysiphe necator , both are consistent with recognition by intracellular immune receptors. Here, we aimed to identify the nucleotide-binding leucine-rich repeat (NLR) genes responsible for PM resistance within Ren6 and Ren7 . To do so, we assembled diploid telomere-to-telomere (T2T) genomes of V. piasezkii DVIT2027 and V. vinifera F2-35, the resistant and susceptible parents, respectively, of a segregating F1 population. We then integrated these assemblies with deep resequencing data from eight F1 sib-lines carrying different combinations of Ren6 and Ren7 , and generated trio-binned, parent-phased genomes for six of them. This allowed us to resolve and phase the Ren6 and Ren7 loci and their PM-susceptible alternative haplotypes. Comparative analyses revealed extensive structural variation, particularly duplications, and numerous short polymorphisms between resistant and susceptible haplotypes, leading to complete sequence specificity among all NLRs in Ren6 and Ren7 , with several lacking allelic counterparts in their respective susceptible haplotypes. Expression profiling across PM-resistant sib-lines identified four and two candidate CC-NBS-LRR genes associated with Ren6 and Ren7 , respectively, based on both expression and haplotype specificity. These results provide a high-resolution view of two key PM resistance loci and support the identification of candidate resistance genes for functional validation and targeted grapevine breeding. ### Competing Interest Statement The authors have declared no competing interest. USDA NIFA, 2022-51181-38240

Diversified reproductive systems can be observed in the plant kingdom and applied in crop breeding; however, their impacts on crop genomic variation and breeding remain unclear. Grapevine (Vitis vinifera L.), a widely planted fruit tree, underwent a shift from dioecism to monoecism during domestication and involves crossing, self-pollination, and clonal propagation for its cultivation. In this study, we discover that the reproductive types, namely, crossing, selfing, and cloning, dramatically impact genomic landscapes and grapevine breeding based on comparative genomic and population genetics of wild grapevine and a complex pedigree of Pinot Noir. The impacts are widely divergent, which show interesting patterns of genomic purging and the Hill-Robertson interference. Selfing reduces genomic heterozygosity, while cloning increases it, resulting in a “double U-shaped” site frequency spectrum (SFS). Crossing and cloning conceal while selfing purges most deleterious and structural burdens. Moreover, the close leakage of large-effect deleterious and structural variations in repulsion phases maintains heterozygous genomic regions in 4.3% of the grapevine genome after successive selfing for nine generations. Our study provides new insights into the genetic basis of clonal propagation and genomic breeding of clonal crops by purging deleterious variants while integrating beneficial variants through various reproductive systems. Effects of reproductive systems on crop genomic variation and breeding remain unclear. Here, the authors report that reproductive types impact genomic landscapes and grapevine breeding based on comparative genomic and population genetic analyses of wild grapevine and a complex pedigree of Pinot Noir.

Grapevine is one of the most economically important fruit crops cultivated worldwide. However, grapevine is highly susceptible to virus infections and exposed to the most diverse forms of viral diseases compared to other fruit crops, and virus-induced incompatibility affects plant growth to different degrees ranging from decline to death. The influence of virus-induced incompatibility could be mitigated to an acceptable level by using appropriate rootstocks. However, the viral tolerance of various grapevine rootstocks with diverse genetic backgrounds remains unclear, along with the identification of the specific viral tolerance factors. In this study, the viral tolerance of 21 grapevine rootstocks was evaluated in a green grafting system. Cabernet Franc varieties infected with a single virus [grapevine leafroll associated virus-1 (GLRaV-1)], a co-infection of two viruses (GLRaV-1 plus grapevine virus A—GVA), and no infection were used as the scions, respectively. The vegetative growth and photosynthetic function of the grafts were analyzed 4 months after grafting. The results indicated that some rootstocks could alleviate the influence of the virus infection, with vegetative growth and photosynthetic function sustained at a normal level, whereas other rootstocks were susceptible to the virus infection, resulting in a decline in the growth and photosynthetic function of the grafts. Our research provides evidence for the existence and diversity of viral tolerance among grapevine rootstocks, offering important information for appropriate rootstock selection in the establishment of new vineyards and in the breeding of grapevine rootstocks with enhanced viral tolerance.

BACKGROUND AND AIMS:Many agricultural areas are expected to face hotter, drier conditions from climate change. Understanding the mechanisms that crops use to mitigate these stresses can guide breeding for more tolerant plant material. We tested relationships between traits, physiological function in hot conditions and historical climate associations to evaluate these mechanisms for winegrapes. We expected a more negative leaf osmotic potential at full hydration (πo), which reduces leaf turgor loss during drought, and either a metabolically cheaper or more osmoprotectant leaf chemical composition, to allow cultivars associated with hot, dry regions to maintain greater gas exchange in hot growing conditions. METHODS:We measured πo, gas exchange and leaf chemistry for seven commercially important winegrape cultivars that vary widely in historical climate associations. Vines were grown in common-garden field conditions in a hot wine-growing region (Davis, CA, USA) and measured over the hottest period of the growing season (July-September). KEY RESULTS:The value of πo varied significantly between cultivars, and all cultivars significantly reduced πo (osmotically adjusted) over the study period, although osmotic adjustment did not vary across cultivars. The value of πo was correlated with gas exchange and climate associations, but in the direction opposite to expected. Photosynthesis and πo were higher in the cultivars associated with hotter, less humid regions. Leaf chemical composition varied between cultivars but was not related to climate associations. CONCLUSIONS:These findings suggest that maintenance of leaf turgor is not a primary limitation on grapevine adaptation to hot or atmospherically dry growing conditions. Thus, selecting for a more negative πo or greater osmotic adjustment is not a promising strategy to develop more climate-resilient grape varieties, contrary to findings for other crops. Future work is needed to identify the mechanisms increasing photosynthesis in the cultivars associated with hot, dry regions.

Root-knot nematodes (RKNs) Meloidogyne spp. are extremely polyphagous pests and four species severely affect grapevines throughout the world: M. arenaria, M. incognita, M. javanica and M. ethiopica. Californian populations of M. arenaria and M. incognita are reported to be virulent to widely used rootstocks and to the rootstock ‘Harmony’ in particular. Breeding RKNs-resistant grape rootstocks is a promising alternative to highly toxic nematicides. Muscadine (Vitis rotundifolia syn. Muscadinia rotundifolia) is a resistance (R) source with undercharacterised genetics. To this end, we used a segregating progeny between the RKN-resistant Vitis x Muscadinia accession ‘VRH8771’ from the muscadine source ‘NC184-4’ and the RKN-susceptible V. vinifera cv. Cabernet-Sauvignon. We first phenotyped its resistance to isolates of the i) M. arenaria, ii) M. incognita and iii) M. javanica species, and then to iv) two mixed Harmony-virulent Californian populations of M. arenaria and M. incognita. Finally, we created an isolate of M. arenaria and M. incognita from these Harmony populations and phenotyped the progeny to each of them [v) and vi)], and to vii) an isolate of M. ethiopica. The resistance phenotype of all the progeny’s individuals was independent of the RKN isolates or populations used. Resistance was mapped in a region of chromosome 18 in VRH8771, supporting the hypothesis that it is conferred by a single gene with an unprecedented wide spectrum in grapevine, including Harmony-virulent isolates. This dominant gene, referred to as MsppR1, is linked to the telomeric QTL XiR4 for X. index resistance from the same source. Additionally, plant mortality data showed that MsppR1-resistant material expressed a high-level resistance to the Harmony-virulent isolates. Our results are a first step towards the development of marker-assisted breeding using SSR and SNP markers for resistance to RKNs in accession VRH8771.

Background and goals Pierce's disease (PD) is a bacterial disease that threatens vineyards across the United States and Mexico. Genetic resistance against this disease has been achieved using the resistance locus PdR1, which was found in a wild grapevine from Mexico. To broaden the genetic base, we aimed to identify additional unique sources of resistance. The objec-tive of this study was to characterize PD resistance in accession b46-43, a PD-resistant wild grapevine from Texas. The manifestation of PD resistance in b46-43 is different from b43-17, with even cane lig-nification, minor leaf scorch, and minimum bacterial growth.It was hypothesized that b46-43 possibly carries resistance that differs from the PdR1 locus in b43-17.Methods and key findings A total of 318 seedlings from population 14399 were evaluated for PD resistance and genotyped. The framework genetic map was developed and a quantitative trait locus (QTL) analysis was carried out. The QTL analysis identified a major locus that explained 55.5% of the phenotypic variation on chromosome 14, at the genomic position where PdR1 was mapped in earlier studies. No minor loci were identified on other chromosomes.Conclusions and significance The b46-43 PD resistance locus can be used in the breeding program, but further comparative ge-nomic analysis with b43-17 is needed to understand their genetic differences and how the b46-43 locus can be used in the breeding program. Moreover, the rarity of additional loci indicates that PD resistance may have a common origin and is widespread as a result of gene flow.

Xylella fastidiosa is a bacterium that infects crops like grapevines, coffee, almonds, citrus and olives. There is little understanding of the genes that contribute to plant resistance, the genomic architecture of resistance, and the potential role of climate in shaping resistance, in part because major crops like grapevines ( Vitis vinifera ) are not resistant to the bacterium. Here we study a wild grapevine species, V. arizonica , that segregates for resistance. Using genome-wide association, we identify candidate resistance genes. Resistance-associated kmers are shared with a sister species of V. arizonica but not with more distant species, suggesting that resistance evolved more than once. Finally, resistance is climate dependent, because individuals from low ( < 10 °C) temperature locations in the wettest quarter were typically susceptible to infection, likely reflecting a lack of pathogen pressure in colder climates. In fact, climate is as effective a predictor of resistance phenotypes as some genetic markers. We extend our climate observations to additional crops, predicting that increased pathogen pressure is more likely for grapevines and almonds than some other susceptible crops.

Domesticated grapevines spread to Europe around 3,000 years ago. Previous studies have revealed genomic signals of introgression from wild to cultivated grapes in Europe, but the time, mode, genomic pattern, and biological effects of these introgression events have not been investigated. Here, we studied resequencing data from 345 samples spanning the distributional range of wild (Vitis vinifera ssp. sylvestris) and cultivated (V. vinifera ssp. vinifera) grapes. Based on machine learning-based population genetic analyses, we detected evidence for a single domestication of grapevine, followed by continuous gene flow between European wild grapes (EU) and cultivated grapes over the past ~2,000 y, especially from EU to wine grapes. We also inferred that soft-selective sweeps were the dominant signals of artificial selection. Gene pathways associated with the synthesis of aromatic compounds were enriched in regions that were both selected and introgressed, suggesting EU wild grapes were an important resource for improving the flavor of cultivated grapes. Despite the potential benefits of introgression in grape improvement, the introgressed fragments introduced a higher deleterious burden, with most deleterious SNPs and structural variants hidden in a heterozygous state. Cultivated wine grapes have benefited from adaptive introgression with wild grapes, but introgression has also increased the genetic load. In general, our study of beneficial and harmful effects of introgression is critical for genomic breeding of grapevine to take advantage of wild resources.

Background and goals Pierce's disease (PD) is a bacterial disease that threatens vineyards across the United States and Mexico. Genetic resistance against this disease has been achieved using the resistance locus PdR1, which was found in a wild grapevine from Mexico. To broaden the genetic base, we aimed to identify additional unique sources of resistance. The objective of this study was to characterize PD resistance in accession b46-43, a PD-resistant wild grapevine from Texas. The manifestation of PD resistance in b46-43 is different from b43-17, with even cane lignification, minor leaf scorch, and minimum bacterial growth. It was hypothesized that b46-43 possibly carries resistance that differs from the PdR1 locus in b43-17. Methods and key findings A total of 318 seedlings from population 14399 were evaluated for PD resistance and genotyped. The framework genetic map was developed and a quantitative trait locus (QTL) analysis was carried out. The QTL analysis identified a major locus that explained 55.5% of the phenotypic variation on chromosome 14, at the genomic position where PdR1 was mapped in earlier studies. No minor loci were identified on other chromosomes. Conclusions and significance The b46-43 PD resistance locus can be used in the breeding program, but further comparative genomic analysis with b43-17 is needed to understand their genetic differences and how the b46-43 locus can be used in the breeding program. Moreover, the rarity of additional loci indicates that PD resistance may have a common origin and is widespread as a result of gene flow.

Background and goals Pierce's disease (PD) is an important grapevine disease in California and other warm grapegrowing areas in North America. Farming grape cultivars with natural resistance to the disease that have broader genetic base is an effective approach in high-pressure PD regions. The goal of this study was to genetically characterize PD resistance in three new accessions, b40-14 and b41-14, both collected from Mexico, and T03-16, collected from Texas. Methods and key findings Simple sequence repeat (SSR) marker based genetic maps that cover all 19 grape chromosomes were developed for b40-14 and b41-14, while the genetic map for T0316 was developed only for chromosome 14. Quantitative trait locus (QTL) analysis was carried out by interval mapping, using parental and consensus maps for all three backgrounds. The QTL analysis identified a major locus in the three accessions on chromosome 14, within the genetic window of the previously-identified PD resistance locus PdR1. This increases the total to 13 known accessions with a major QTL for PD resistance in a similar genomic region. Conclusions and significance All three accessions have major, but not complete, effect QTL for PD resistance on chromosome 14, indicating that other minor QTLs may be present in other genomic regions. Larger populations and map resolution are required to identify all minor QTLs.

AbstractXylella fastidiosais a bacterium that infects crops like grapevines, coffee, almonds, citrus and olives, causing economically devastating damage. There is, however, little understanding of the genes that contribute to resistance, the genomic architecture of resistance, and the potential role of climate in shaping resistance, in part because major crops like grapevines (V. vinifera) are not resistant to the bacterium. Here we studied a wild grapevine species,Vitis arizonica, that segregates for resistance toX. fastidiosa. Using genome-wide association, we identified candidate genes that mediate the host response toX. fastidiosainfection. We uncovered evidence that resistance requires genes from multiple genomic regions, based on data from breeding populations and from additionalVitisspecies. We also inferred that resistance evolved more than once in the wild, suggesting that wildVitisspecies may be a rich source for resistance alleles and mechanisms. Finally, resistance inV. arizonicawas climate dependent, because individuals from low (< 10°C) temperature locations in the wettest quarter were typically susceptible to infection, likely reflecting a lack of pathogen pressure in these climates. Surprisingly, climate was nearly as effective a predictor of resistance phenotypes as some genetic markers. This work underscores that pathogen pressure is likely to increase with climate, but it also provides genetic insight and tools for breeding and transforming resistant crops.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries 2 powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the 2 loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat genes. Heterozygous structural variations were also found to impact the domain composition of some nucleotide-binding leucine-rich repeat proteins. By comparing the nucleotide-binding leucine-rich repeat proteins at Run1.2 and Run2.2 loci, we discovered that the 2 loci include different numbers and classes of nucleotide-binding leucine-rich repeat genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the nucleotide-binding leucine-rich repeat genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several nucleotide-binding leucine-rich repeat genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate nucleotide-binding leucine-rich repeat genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.

Adjusting yearly pruning severity is a common vineyard management practice employed to manipulate vegetative and reproductive growth in grapevines. Although the effects of pruning on total vegetative growth are well documented, there is little research on the effects of adjusting shoots meter−1 via dormant season pruning on addressing mid-cordon shoot weakness and developmental delays. Cordon-trained, spur-pruned vines are thought, by many growers, to be especially prone to weaker positions and delayed development at mid-cordon positions. This phenomenon is also thought to become more exaggerated as the vine ages. Therefore, the effects of shoot density manipulation, implemented via dormant pruning practices, to homogenize shoot and cluster development along the length of the cordon were examined. In this research, Cabernet Sauvignon grapevines were pruned to either 5.5 shoots meter−1 (5.5) or 11.1 shoots meter−1 (11.1). To control for variations in light interception into the fruiting zone, a control of 11.1 shoots meter−1 with sensor guided leaf thinning (11.1LT) was implemented at full berry set to match the canopy light of the 5.5 shoots meter−1 treatment. It was found that individual shoot growth and yield were directly impacted by manipulation of pruning severity. Shoot growth response varied primarily by growing season, including shoot length and internode length. Yield components were significantly lower in the 5.5 treatment during the first two years of the study but were not significantly different during the last year of the study. The 5.5 treatment resulted in the highest pH and total soluble solids at harvest in 2016 and 2017.

Multiple grape powdery mildew (PM) genetic resistance ( R ) loci have been found in wild grape species. Little is known about the defense responses associated with each R locus. In this study, we compare the defense mechanisms associated with PM resistance in interspecific crosses segregating for a single R locus from Muscadinia rotundifolia ( Run1, Run1.2b, Run2.1, Run2.2 ), Vitis cinerea ( Ren2 ), V. romanetii ( Ren4D and Ren4U ), and the interspecific hybrid Villard blanc ( Ren3 ). By combining optical microscopy, visual scoring, and biomass estimation, we show that the eight R loci confer resistance by limiting infection at different stages. We assessed the defense mechanisms triggered in response to PM at 1 and 5 days post inoculation (dpi) via RNA sequencing. To account for the genetic differences between species, we developed for each accession a diploid synthetic reference transcriptome by incorporating into the PN40024 reference homozygous and heterozygous sequence variants and de novo assembled transcripts. Most of the R loci exhibited a higher number of differentially expressed genes (DEGs) associated with PM resistance at 1 dpi compared to 5 dpi, suggesting that PM resistance is mostly associated with an early transcriptional reprogramming. Comparison of the PM resistance-associated DEGs showed a limited overlap between pairs of R loci, and nearly half of the DEGs were specific to a single R locus. The largest overlap of PM resistance-associated DEGs was found between Ren3 + , Ren4D + , and Ren4U + genotypes at 1 dpi, and between Ren4U + and Run1 + accessions at 5 dpi. The Ren3 + , Ren4D + , and Ren4U + were also found to have the highest number of R locus-specific DEGs in response to PM. Both shared and R locus-specific DEGs included genes from different defense-related categories, indicating that the presence of E. necator triggered distinct transcriptional responses in the eight R loci.

Pierce’s disease is a deadly disease of grapevines caused by the bacterial pathogen Xylella fastidiosa (Xf). A Pierce’s disease resistance locus from Vitis arizonica/candicans b43-17 segregated as a single dominant gene and mapped as PdR1a and PdR1b in two F1 sibling selections. The physical mapping of the PdR1b allele allowed the identification of five ORFs of the Leucine-Rich Repeat Receptor Kinase gene family. Two ORFs: V.ari-RGA14 and V.ari-RGA18 were used to transform embryogenic callus of V. vinifera Chardonnay (CH) and Thompson Seedless (TS) and V. rupestris St George (SG) via Agrobacterium tumefaciens . Regenerated plants were inoculated with Xf under greenhouse conditions. Genetic transformation with RGA14 and 18 did not generate resistance in CH and TS, although some lines of CH showed significantly lower stem bacterial concentration and/or exhibited reduced symptoms. In transgenic SG14, improved regrowth was accompanied with lower bacterial titers and decreased pectin lyase and ß-1,3-glucanase 3 gene expression. The limited effects of the transgenes on PD resistance could be explained by the lack of suitable partners or the presence of susceptibility factors that could not be overcome under these experimental conditions. The involvement of RGA17 in b43-17 resistance to Xf should not be discarded.