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Addressing the pressing challenges in agriculture necessitates swift advancements in breeding programs, particularly for perennial crops like grapevines. Moving beyond the traditional biparental quantitative trait loci (QTL) mapping, we conducted a genome-wide association study (GWAS) encompassing 588 Vitis vinifera L. cultivars from a Chilean breeding program, spanning three seasons and testing 13 key yield-related traits. A strong candidate gene, Vitvi11g000454, located on chromosome 11 and related to plant response to biotic and abiotic stresses through jasmonic acid signaling, was associated with berry width and holds potential for enhancing berry size in grape breeding. We also mapped novel QTL associated with post-harvest traits across chromosomes 2, 4, 9, 11, 15, 18, and 19, broadening our grasp on the genetic intricacies dictating fruit post-harvest behavior, including decay, shriveling, and weight loss. Leveraging gene ontology annotations, we drew parallels between traits and scrutinized candidate genes, laying a robust groundwork for future trait-feature identification endeavors in plant breeding. We also highlighted the importance of carefully considering the choice of the response variable in GWAS analyses, as the use of best linear unbiased estimators (BLUEs) corrections in our study may have led to the suppression of some common QTL in grapevine traits. Our results underscore the imperative of pioneering non-destructive evaluation techniques for long-term conservation traits, offering grape breeders and cultivators insights to improve post-harvest table grape quality and minimize waste.

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OBJECTIVES: The frequency of GDM and vitamin D insufficiency in Mexico is high. Vitamin D supplementation in GDM patients has shown favorable but non-homogeneous results regarding improvement of glycemic profile. The aim of the study was to assess the effects of supplementing with 5,000 IU of vitamin D on the glycemic profile of women with GDM. METHODS: A randomized clinical trial was conducted on women with GDM who received 5,000 IU of vitamin D (n=27) or a placebo (n=27) for eight weeks. Changes in vitamin D levels and metabolic parameters before and after the intervention were analyzed. RESULTS: Vitamin D vs. placebo: 25-OHD (32 vs. 26 ng/mL, p=0.006), HbA1c (6.0 vs. 6.1%, p=0.29), glucose (99 vs. 87 mg/dL, p=0.29), insulin (14 vs. 13 µIU/mL, p=0.79), HOMA-IR (3.6 vs. 2.6, p=0.55), QUICKI (0.31 vs. 0.33, p=0.55). CONCLUSIONS: Supplementation with 5,000 IU of vitamin D for eight weeks had no significant effect on the glycemic profile.

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Grapevine (Vitis vinifera) is one of the main fruit crops worldwide. In 2020, the total surface area planted with vines was estimated at 7.3 million hectares. Diverse pathogens affect grapevine yield, fruit, and wine quality of which powdery mildew is the most important disease prior to harvest. Its causal agent is the biotrophic fungus Erysiphe necator, which generates a decrease in cluster weight, delays fruit ripening, and reduces photosynthetic and transpiration rates. In addition, powdery mildew induces metabolic reprogramming in its host, affecting primary metabolism. Most commercial grapevine cultivars are highly susceptible to powdery mildew; consequently, large quantities of fungicide are applied during the productive season. However, pesticides are associated with health problems, negative environmental impacts, and high costs for farmers. In paralleled, consumers are demanding more sustainable practices during food production. Therefore, new grapevine cultivars with genetic resistance to powdery mildew are needed for sustainable viticulture, while maintaining yield, fruit, and wine quality. Two main gene families confer resistance to powdery mildew in the Vitaceae, Run (Resistance to Uncinula necator) and Ren (Resistance to Erysiphe necator). This article reviews the powdery mildew resistance genes and loci and their use in grapevine breeding programs.

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Leaf shape in plants plays important roles in water use, canopy structure, and physiological tolerances to abiotic stresses; all important traits for the future development and sustainability of grapevine cultivation. Historically, researchers have used ampelography, the study of leaf shape in grapevines, to differentiate Vitis species and cultivars based on finite leaf attributes. However, ampelographic measurements have limitations and new methods for quantifying shape are now available. We paired an analysis of finite trait attributes with a 17-point landmark survey and generalized Procrustes analysis (GPA) to reconstruct grapevine leaves digitally from five interspecific hybrid mapping families. Using the reconstructed leaves, we performed three types of quantitative trait loci (QTL) analyses to determine the genetic architecture that defines leaf shape. In the first analysis, we compared several important ampelographic measurements as finite trait QTL. In the second and third analyses, we identified significant shape variation via principal components analysis (PCA) and using a multivariate least squares interval mapping (MLSIM) approach. In total, we identified 271 significant QTL across the three measures of leaf shape and identified specific QTL hotspots in the grape genome which appear to drive major aspects of grapevine leaf shape.

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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.

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KEY MESSAGE: 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.

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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. 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.

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Quantitative phenotyping of downy mildew sporulation is frequently used in plant breeding and genetic studies, as well as in studies focused on pathogen biology such as chemical efficacy trials. In these scenarios, phenotyping a large number of genotypes or treatments can be advantageous but is often limited by time and cost. We present a novel computational pipeline dedicated to estimating the percent area of downy mildew sporulation from images of inoculated grapevine leaf discs in a manner that is time and cost efficient. The pipeline was tested on images from leaf disc assay experiments involving two F1 grapevine families, one that had glabrous leaves (Vitis rupestris B38 × 'Horizon' [RH]) and another that had leaf trichomes (Horizon × V. cinerea B9 [HC]). Correlations between computer vision and manual visual ratings reached 0.89 in the RH family and 0.43 in the HC family. Additionally, we were able to use the computer vision system prior to sporulation to measure the percent leaf trichome area. We estimate that an experienced rater scoring sporulation would spend at least 90% less time using the computer vision system compared with the manual visual method. This will allow more treatments to be phenotyped in order to better understand the genetic architecture of downy mildew resistance and of leaf trichome density. We anticipate that this computer vision system will find applications in other pathosystems or traits where responses can be imaged with sufficient contrast from the background.

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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 <10% missing data, and read depth had a median of 220-244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.

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The genomics era brought unprecedented opportunities for genetic analysis of host resistance, but it came with the challenge that accurate and reproducible phenotypes are needed so that genomic results appropriately reflect biology. Phenotyping host resistance by natural infection in the field can produce variable results due to the uncontrolled environment, uneven distribution and genetics of the pathogen, and developmentally regulated resistance among other factors. To address these challenges, we developed highly controlled, standardized methodologies for phenotyping powdery mildew resistance in the context of a phenotyping center, receiving samples of up to 140 grapevine progeny per F1 family. We applied these methodologies to F1 families segregating for REN1- or REN2-mediated resistance and validated that some but not all bioassays identified the REN1 or REN2 locus. A point-intercept method (hyphal transects) to quantify colony density objectively at 8 or 9 days postinoculation proved to be the phenotypic response most reproducibly predicted by these resistance loci. Quantitative trait locus (QTL) mapping with genotyping-by-sequencing maps defined the REN1 and REN2 loci at relatively high resolution. In the reference PN40024 genome under each QTL, nucleotide-binding site-leucine-rich repeat candidate resistance genes were identified-one gene for REN1 and two genes for REN2. The methods described here for centralized resistance phenotyping and high-resolution genetic mapping can inform strategies for breeding resistance to powdery mildews and other pathogens on diverse, highly heterozygous hosts.

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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.

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Vitis rupestris B38 is a North American grapevine resistant to the major pathogen of cultivated grapevines, Erysiphe necator. Sources of powdery mildew resistance, like V. rotundifolia, are widely used in grape breeding but are already threatened, even before commercialization, by isolates that can reproduce on Run1 and other rotundifolia-derived breeding lines. Thus, complementary sources of resistance are needed to improve resistance durability. The segregation of foliar powdery mildew severity in an F1 family, derived from a cross of V. rupestris B38×V. vinifera 'Chardonnay', was observed in the field over three growing seasons and in potted vines following single-isolate inoculation. A pattern of continuous variation was observed in every instance. Mechanisms of resistance were analyzed on the resistant and susceptible parent by using microscopy to quantify the ability of the pathogen to penetrate and to form a colony on detached leaves. While 'Chardonnay' was susceptible in all tested conditions, V. rupestris B38 resistance was characterized by a reduction in pathogen penetration, with an effect of leaf position and significant differences among powdery mildew isolates. Segregation of the ability of the pathogen to penetrate and form a colony in F1 individuals showed a pattern of quantitative penetration resistance with no delay or restriction on colony formation once penetration has been achieved. Moreover, V. rupestris B38 showed an enhanced penetration resistance to a powdery mildew isolate with the ability to overcome the Run1 gene, making it an interesting resistance source to prolong the durability of this gene.

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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.

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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.

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Race-specific resistance against powdery mildews is well documented in small grains but, in other crops such as grapevine, controlled analysis of host-pathogen interactions on resistant plants is uncommon. In the current study, we attempted to confirm powdery mildew resistance phenotypes through vineyard, greenhouse, and in vitro inoculations for test cross-mapping populations for two resistance sources: (i) a complex hybrid breeding line, 'Bloodworth 81-107-11', of at least Vitis rotundifolia, V. vinifera, V. berlandieri, V. rupestris, V. labrusca, and V. aestivalis background; and (ii) Vitis hybrid 'Tamiami' of V. aestivalis and V. vinifera origin. Statistical analysis of vineyard resistance data suggested the segregation of two and three race-specific resistance genes from the two sources, respectively. However, in each population, some resistant progeny were susceptible in greenhouse or in vitro screens, which suggested the presence of Erysiphe necator isolates virulent on progeny segregating for one or more resistance genes. Controlled inoculation of resistant and susceptible progeny with a diverse set of E. necator isolates clearly demonstrated the presence of fungal races differentially interacting with race-specific resistance genes, providing proof of race specificity in the grape powdery mildew pathosystem. Consistent with known race-specific resistance mechanisms, both resistance sources were characterized by programmed cell death of host epidermal cells under appressoria, which arrested or slowed hyphal growth; this response was also accompanied by collapse of conidia, germ tubes, appressoria, and secondary hyphae. The observation of prevalent isolates virulent on progeny with multiple race-specific resistance genes before resistance gene deployment has implications for grape breeding strategies. We suggest that grape breeders should characterize the mechanisms of resistance and pyramid multiple resistance genes with different mechanisms for improved durability.

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In the present study we screened the progeny of Vitis vinifera × V. romanetii populations segregating for resistance to powdery mildew and determined the presence of a single, dominant locus, Ren4, conferring rapid and extreme resistance to the grapevine powdery mildew fungus Erysiphe necator. In each of nine Ren4 pseudo-backcross 2 (pBC(2)) and pBC(3) populations (1,030 progeny), resistance fit a 1:1 segregation ratio and overall segregated as 543 resistant progeny to 487 susceptible. In full-sib progeny, microscopic observations revealed the reduction of penetration success rate (as indicated by the emergence of secondary hyphae) from 86% in susceptible progeny to below 10% in resistant progeny. Similarly, extreme differences were seen macroscopically. Ratings for Ren4 pBC(2) population 03-3004 screened using natural infection in a California vineyard and greenhouse and using artificial inoculation of an aggressive New York isolate were fully consistent among all three pathogen sources and environments. From 2006 to 2010, Ren4 pBC(2) and pBC(3) vines were continuously screened in California and New York (in the center of diversity for E. necator), and no sporulating colonies were observed. For population 03-3004, severity ratings on leaves, shoots, berries, and rachises were highly correlated (R(2) = 0.875 to 0.996) in the vineyard. Together, these data document a powdery mildew resistance mechanism not previously described in the Vitaceae or elsewhere, in which a dominantly inherited resistance prevents hyphal emergence and is non-race-specific and tissue-independent. In addition to its role in breeding for durable resistance, Ren4 may provide mechanistic insights into the early events that enable powdery mildew infection.

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We report Agrobacterium tumefaciens-mediated transformation of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the hypocotyl slice technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supplemented Murashige and Skoog (MS) media reached 11% for 'Angeleno' and 19% for 'Larry Anne' hypocotyl slices. Transformation using Agrobacterium tumefaciens GV3101 harboring a plasmid with the neomycin phosphotransferase II (nptII) and the green fluorescent protein (gfp) genes produced ten independent lines, six from 'Angeleno' and four from 'Larry Anne', representing transformation efficiencies of 0.8 and 0.3%, respectively, relative to the initial number of hypocotyl slices. Plants of six lines were found to produce the transgene encoded mRNAs. DNA blotting demonstrated the presence of transgene sequences in trees from five lines after 18 months of growth in the greenhouse.

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BGLII is a bacterial endoglucanase that hydrolyzes the beta-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.

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