Genotyping-by-sequencing-based high-resolution mapping reveals a single candidate gene for the grapevine veraison locus Ver1.

Veraison marks the transition from berry growth to berry ripening and is a crucial phenological stage in grapevine (Vitis vinifera): the berries become soft and begin to accumulate sugars, aromatic substances, and, in red cultivars, anthocyanins for pigmentation, while the organic acid levels begin to decrease. These changes determine the potential quality of wine. However, rising global temperatures lead to earlier flowering and ripening, which strongly influence wine quality. Here, we combined genotyping-by-sequencing with a bioinformatics pipeline on ∼150 F1 genotypes derived from a cross between the early ripening variety 'Calardis Musqué' and the late-ripening variety 'Villard Blanc'. Starting from 20,410 haplotype-based markers, we generated a high-density genetic map and performed a quantitative trait locus analysis based on phenotypic datasets evaluated over 20 years. Through locus-specific-marker-enrichment and recombinant screening of ∼1000 additional genotypes, we refined the originally postulated 5 Mb veraison locus, Ver1, on chromosome 16 to only 112 kb, allowing us to pinpoint the ethylene response factor (ERF) VviERF027 (VCost.v3 gene ID: Vitvi16g00942, CRIBIv1 gene ID: VIT_16s0100g00400) as veraison candidate gene. Furthermore, the early veraison allele could be traced back to a clonal 'Pinot' variant first mentioned in the 17th century. 'Pinot Precoce Noir' passed this allele over 'Madeleine Royale' to the maternal grandparent 'Bacchus Weiss' and, ultimately, to the maternal parent 'Calardis Musqué'. Our findings are crucial for ripening time control, thereby improving wine quality, and for breeding grapevines adjusted to climate change scenarios that have a major impact on agro-ecosystems in altering crop plant phenology.

Determination of Sugars and Acids in Grape Must Using Miniaturized Near-Infrared Spectroscopy.

An automatic determination of grape must ingredients during the harvesting process would support cellar logistics and enables an early termination of the harvest if quality parameters are not met. One of the most important quality-determining characteristics of grape must is its sugar and acid content. Among others, the sugars in particular determine the quality of the must and wine. Chiefly in wine cooperatives, in which a third of all German winegrowers are organized, these quality characteristics serve as the basis for payment. They are acquired upon delivery at the cellar of the cooperative or the winery and result in the acceptance or rejection of grapes and must. The whole process is very time-consuming and expensive, and sometimes grapes that do not meet the quality requirements for sweetness, acidity, or healthiness are destroyed or not used at all, which leads to economic loss. Near-infrared spectroscopy is now a widely used technique to detect a wide variety of ingredients in biological samples. In this study, a miniaturized semi-automated prototype apparatus with a near-infrared sensor and a flow cell was used to acquire spectra (1100 nm to 1350 nm) of grape must at defined temperatures. Data of must samples from four different red and white Vitis vinifera (L.) varieties were recorded throughout the whole growing season of 2021 in Rhineland Palatinate, Germany. Each sample consisted of 100 randomly sampled berries from the entire vineyard. The contents of the main sugars (glucose and fructose) and acids (malic acid and tartaric acid) were determined with high-performance liquid chromatography. Chemometric methods, using partial least-square regression and leave-one-out cross-validation, provided good estimates of both sugars (RMSEP = 6.06 g/L, R2 = 89.26%), as well as malic acid (RMSEP = 1.22 g/L, R2 = 91.10%). The coefficient of determination (R2) was comparable for glucose and fructose with 89.45% compared to 89.08%, respectively. Although tartaric acid was predictable for only two of the four varieties using near-infrared spectroscopy, calibration and validation for malic acid were accurate for all varieties in an equal extent like the sugars. These high prediction accuracies for the main quality determining grape must ingredients using this miniaturized prototype apparatus might enable an installation on a grape harvester in the future.

A 69 kbp Deletion at the Berry Color Locus Is Responsible for Berry Color Recovery in Vitis vinifera L. Cultivar 'Riesling Rot'.

'Riesling Weiss' is a white grapevine variety famous worldwide for fruity wines with higher acidity. Hardly known is 'Riesling Rot', a red-berried variant of 'Riesling Weiss' that disappeared from commercial cultivation but has increased in awareness in the last decades. The question arises of which variant, white or red, is the original and, consequently, which cultivar is the true ancestor. Sequencing the berry color locus of 'Riesling Rot' revealed a new VvmybA gene variant in one of the two haplophases called VvmybA3/1RR. The allele displays homologous recombination of VvmybA3 and VvmybA1 with a deletion of about 69 kbp between both genes that restores VvmybA1 transcripts. Furthermore, analysis of 'Riesling Weiss', 'Riesling Rot', and the ancestor 'Heunisch Weiss' along chromosome 2 using SSR (simple sequence repeat) markers elucidated that the haplophase of 'Riesling Weiss' was inherited from the white-berried parent variety 'Heunisch Weiss'. Since no color mutants of 'Heunisch Weiss' are described that could have served as allele donors, we concluded that, in contrast to the public opinion, 'Riesling Rot' resulted from a mutational event in 'Riesling Weiss' and not vice versa.

Effects of Defoliation Treatments of Babica Grape Variety(Vitis vinifera L.) on Volatile Compounds Content in Wine.

The aim of this study was to determine the effects of defoliation performed in the Babica red grape variety on the volatile compounds in produced wine. Three treatments were performed during 2017 and 2018: the removal of six leaves before flowering (FL) and at the end of veraison (VER), as well as control (C). Volatile compounds were analyzed using a gas chromatograph coupled to a mass spectrophotometric detector. Results were statistically evaluated by analysis of variance (ANOVA at the p = 0.05 level) and principal component analysis (PCA). Defoliation treatments were affected by the concentration of several compounds, but only in one year. The VER2017 treatment significantly increased the concentration of three aliphatic esters up to 8 C atoms and octanoic acid ethyl ester. The FL2017 treatment increased the concentration of three aliphatic alcohols. The FL2018 treatment has significantly enhanced the concentration ethyl cinnamate but decreased the concentrations of eugenol and dihydro-2-methyl-3(2H)-thiophenone. Both defoliation treatments reduced the concentration of γ-decanolactone in 2017. Aldehydes, monoterpenoles, and monoterpenes remained unaffected by the defoliation treatments. Vintage was found to be the largest source of variability for most volatile compounds under investigation, which was confirmed by PCA. The effect of defoliation in the mild-Mediterranean climate was found to mostly depend on seasonal weather conditions.

PhenoApp: A mobile tool for plant phenotyping to record field and greenhouse observations.

With the ongoing cost decrease of genotyping and sequencing technologies, accurate and fast phenotyping remains the bottleneck in the utilizing of plant genetic resources for breeding and breeding research. Although cost-efficient high-throughput phenotyping platforms are emerging for specific traits and/or species, manual phenotyping is still widely used and is a time- and money-consuming step. Approaches that improve data recording, processing or handling are pivotal steps towards the efficient use of genetic resources and are demanded by the research community. Therefore, we developed PhenoApp, an open-source Android app for tablets and smartphones to facilitate the digital recording of phenotypical data in the field and in greenhouses. It is a versatile tool that offers the possibility to fully customize the descriptors/scales for any possible scenario, also in accordance with international information standards such as MIAPPE (Minimum Information About a Plant Phenotyping Experiment) and FAIR (Findable, Accessible, Interoperable, and Reusable) data principles. Furthermore, PhenoApp enables the use of pre-integrated ready-to-use BBCH (Biologische Bundesanstalt für Land- und Forstwirtschaft, Bundessortenamt und CHemische Industrie) scales for apple, cereals, grapevine, maize, potato, rapeseed and rice. Additional BBCH scales can easily be added. The simple and adaptable structure of input and output files enables an easy data handling by either spreadsheet software or even the integration in the workflow of laboratory information management systems (LIMS). PhenoApp is therefore a decisive contribution to increase efficiency of digital data acquisition in genebank management but also contributes to breeding and breeding research by accelerating the labour intensive and time-consuming acquisition of phenotyping data.

Grapevine and Wine Metabolomics-Based Guidelines for FAIR Data and Metadata Management.

In the era of big and omics data, good organization, management, and description of experimental data are crucial for achieving high-quality datasets. This, in turn, is essential for the export of robust results, to publish reliable papers, make data more easily available, and unlock the huge potential of data reuse. Lately, more and more journals now require authors to share data and metadata according to the FAIR (Findable, Accessible, Interoperable, Reusable) principles. This work aims to provide a step-by-step guideline for the FAIR data and metadata management specific to grapevine and wine science. In detail, the guidelines include recommendations for the organization of data and metadata regarding (i) meaningful information on experimental design and phenotyping, (ii) sample collection, (iii) sample preparation, (iv) chemotype analysis, (v) data analysis (vi) metabolite annotation, and (vii) basic ontologies. We hope that these guidelines will be helpful for the grapevine and wine metabolomics community and that it will benefit from the true potential of data usage in creating new knowledge being revealed.

Transcriptomic analysis of temporal shifts in berry development between two grapevine cultivars of the Pinot family reveals potential genes controlling ripening time.

BACKGROUND: Grapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars 'Pinot Noir' (PN) and 'Pinot Noir Precoce' (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years. RESULTS: The difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designated VviRTIC1 and VviRTIC2 (VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP. CONCLUSIONS: Many of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.

Towards Sensor-Based Phenotyping of Physical Barriers of Grapes to Improve Resilience to Botrytis Bunch Rot.

Botrytis bunch rot is one of the economically most important fungal diseases in viticulture (aside from powdery mildew and downy mildew). So far, no active defense mechanisms and resistance loci against the necrotrophic pathogen are known. Since long, breeders are mostly selecting phenotypically for loose grape bunches, which is recently the most evident trait to decrease the infection risk of Botrytis bunch rot. This study focused on plant phenomics of multiple traits by applying fast sensor technologies to measure berry impedance (Z REL ), berry texture, and 3D bunch architecture. As references, microscopic determined cuticle thickness (MS CT ) and infestation of grapes with Botrytis bunch rot were used. Z REL hereby is correlated to grape bunch density OIV204 (r = -0.6), cuticle thickness of berries (r = 0.61), mean berry diameter (r = -0.63), and Botrytis bunch rot (r = -0.7). However, no correlation between Z REL and berry maturity or berry texture was observed. In comparison to the category of traditional varieties (mostly susceptible), elite breeding lines show an impressive increased Z REL value (+317) and a 1-µm thicker berry cuticle. Quantitative trait loci (QTLs) on LGs 2, 6, 11, 15, and 16 were identified for Z REL and berry texture explaining a phenotypic variance of between 3 and 10.9%. These QTLs providing a starting point for the development of molecular markers. Modeling of Z REL and berry texture to predict Botrytis bunch rot resilience revealed McFadden R 2 = 0.99. Taken together, this study shows that in addition to loose grape bunch architecture, berry diameter, Z REL , and berry texture values are probably additional parameters that could be used to identify and select Botrytis-resilient wine grape varieties. Furthermore, grapevine breeding will benefit from these reliable methodologies permitting high-throughput screening for additional resilience traits of mechanical and physical barriers to Botrytis bunch rot. The findings might also be applicable to table grapes and other fruit crops like tomato or blueberry.

Color Intensity of the Red-Fleshed Berry Phenotype of Vitis vinifera Teinturier Grapes Varies Due to a 408 bp Duplication in the Promoter of VvmybA1.

Grapevine (Vitis vinifera) teinturier cultivars are characterized by their typical reddish leaves and red-fleshed berries due to ectopic anthocyanin formation. Wines of these varieties have economic importance as they can be used for blending to enhance the color of red wines. The unique and heritable mutation has been known for a long time but the underlying genetic mechanism still is not yet understood. Here we describe the association of the red-fleshed berry phenotype with a 408 bp repetitive DNA element in the promoter of the VvmybA1 gene (grapevine color enhancer, GCE). Three different clones of 'Teinturier' were discovered with two, three and five allelic GCE repeats (MybA1t2, MybA1t3 and MybA1t5). All three clones are periclinal chimeras; these clones share the same L1 layer, but have distinct L2 layers with different quantities of GCE repeats. Quantitative real time PCR and HPLC analysis of leaf and berry samples showed that the GCE repeat number strongly correlates with an increase of the expression of VvmybA1 itself and the VvUFGT gene regulated by it and the anthocyanin content. A model is proposed based on autoregulation of VvmybA1t to explain the red phenotype which is similar to that of red-fleshed apples. This study presents results about the generation and modes of action of three MybA1t alleles responsible for the red-fleshed berry phenotype of teinturier grapevines.

Effects of Defoliation on Phenolic Concentrations, Antioxidant and Antibacterial Activity of Grape Skin Extracts of the Varieties Blaufränkisch and Merlot (Vitis vinifera L.).

Vitis vinifera L. has been highlighted by its many positive effects on human health, including antibacterial, anti-inflammatory and antioxidant activity due to its rich phytochemical content. Removing six basal leaves has great potential to influence the production of phytochemicals. The purpose of this study was to determine the impact of different terms of defoliation on the antioxidant and antibacterial activity of grape skin extracts of the Blaufränkisch and Merlot varieties. The total phenolic concentration, total and individual anthocyanin concentration, antioxidant activity and antibacterial activity on gram-positive and gram-negative human pathogens have been determined. The study was conducted on three treatments (control treatment without defoliation, defoliation immediately after bloom and defoliation before the start of the véraison phase). The results showed statistically significant enhancement of the total phenolic concentration as well as the antioxidant and antibacterial activity in both studied cultivars. Defoliation just after blooming was the preferable defoliation term in the Merlot variety for achieving the highest total anthocyanin concentration, antioxidant activity and significant increase of antibacterial activity against all four investigated bacteria. Defoliation before the start of the véraison phase was the preferable defoliation term for achieving the highest total anthocyanin concentration in the Blaufränkisch variety. In general, treatment of defoliation immediately after bloom was more beneficial compared with the defoliation before the start of the véraison phase.

Quantitative trait loci affecting pathogen resistance and ripening of grapevines.

Grapevines (Vitis vinifera L.) form the basis of viticulture, and are susceptible to diseases such as downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necator). Therefore, successful viticulture programs require the use of pesticides. Breeding for resistance is the only eco-friendly solution. Marker-assisted selection is currently widely used for grapevine breeding. Consequently, traits of interest must be tagged with molecular markers linked to quantitative trait loci (QTL). We herein present our findings regarding genetic mapping and QTL analysis of resistance to downy and powdery mildew diseases in the progenies of the GF.GA-47-42 ('Bacchus' × 'Seyval') × 'Villard blanc' cross. Simple sequence repeats and single nucleotide polymorphisms of 151 individuals were analyzed. A map consisting of 543 loci was screened for QTL analyses based on phenotypic variations observed in plants grown in the field or under controlled conditions. A major QTL for downy mildew resistance was detected on chromosome 18. For powdery mildew resistance, a QTL was identified on chromosome 15. This QTL was replaced by a novel QTL on chromosome 18 in 2003 (abnormally high temperatures) and 2004. Subsequently, both QTLs functioned together. Additionally, variations in the timing of the onset of veraison, which is a crucial step during grape ripening, were studied to identify genomic regions affecting this trait. A major QTL was detected on linkage group 16, which was supplemented by a minor QTL on linkage group 18. This study provides useful information regarding novel QTL-linked markers relevant for the breeding of disease-resistant grapevines adapted to current climatic conditions.

Rpv10: a new locus from the Asian Vitis gene pool for pyramiding downy mildew resistance loci in grapevine.

A population derived from a cross between grapevine breeding strain Gf.Ga-52-42 and cultivar 'Solaris' consisting of 265 F1-individuals was genetically mapped using SSR markers and screened for downy mildew resistance. Quantitative trait locus (QTL) analysis revealed two strong QTLs on linkage groups (LGs) 18 and 09. The locus on LG 18 was found to be identical with the previously described locus Rpv3 and is transmitted by Gf.Ga-52-42. 'Solaris' transmitted the resistance-related locus on LG 09 explaining up to 50% of the phenotypic variation in the population. This downy mildew resistance locus is named Rpv10 for resistance to Plasmopara viticola. Rpv10 was initially introgressed from Vitis amurensis, a wild species of the Asian Vitis gene pool. The one-LOD supported confidence interval of the QTL spans a section of 2.1 centi Morgan (cM) corresponding to 314 kb in the reference genome PN40024 (12x). Eight resistance gene analogues (RGAs) of the NBS-LRR type and additional resistance-linked genes are located in this region of PN40024. The F1 sub-population which contains the Rpv3 as well as the Rpv10 locus showed a significantly higher degree of resistance, indicating additive effects by pyramiding of resistance loci. Possibilities for using the resistance locus Rpv10 in a grapevine breeding programme are discussed. Furthermore, the marker data revealed 'Severnyi' × 'Muscat Ottonel' as the true parentage for the male parent of 'Solaris'.