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'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

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 (ZREL), berry texture, and 3D bunch architecture. As references, microscopic determined cuticle thickness (MSCT) and infestation of grapes with Botrytis bunch rot were used. ZREL 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 ZREL 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 ZREL value (+317) and a 1-μm thicker berry cuticle. Quantitative trait loci (QTLs) on LGs 2, 6, 11, 15, and 16 were identified for ZREL 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 ZREL and berry texture to predict Botrytis bunch rot resilience revealed McFadden R2 = 0.99. Taken together, this study shows that in addition to loose grape bunch architecture, berry diameter, ZREL, 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.

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

In viticulture, winemakers and the industry demand grape bunches that have a reduced degree of bunch compactness. The major aspect is that a loose bunch compactness reduces the risk of severe Botrytis bunch-rot infections. Grapevine breeders focus hereby on several bunch-architecture-related traits. For specific breeding approaches and breeding-research-related topics, such as Quantitative Trait Locus (QTL) analysis or molecular marker development, the exact and objective phenotyping of such traits is mandatory. In this study, a precise and high-throughput 3D phenotyping pipeline was applied to screen 1514 genotypes from three mapping populations with different genetic backgrounds to investigate its applicability for QTL mapping approaches. In the first step, the phenotypic data of one population containing 150 genotypes were collected and analyzed with the 3D phenotyping pipeline. Additionally, corresponding reference data were obtained. Phenotypic values and results of a QTL analysis were compared with each other. Strongly positive correlations up to r = 0.93 between 3D and reference measurements could be detected for several traits. The ten-times-faster 3D phenotyping pipeline revealed 20, and the reference phenotyping methods revealed 22 QTLs. Eighteen of these QTLs were consistent between both procedures. In the next step, screening was extended to four different mapping populations across several seasons. In total, up to 1500 genotypes were screened during one season (>5000 grape bunches in total). The data analysis revealed significant differences across years and populations. Three bunch-architecture traits, including total berry volume, bunch width, and berry diameter, explained the highest amount of variability in the phenotypic data. A QTL analysis was performed on the phenotypic data of the involved populations to identify comparative genetic loci for bunch-architecture traits. Between 20 and 26 stable and reproducible QTLs for the investigated populations were detected. A common QTL for berry diameter could be identified for all populations. Our results strongly conclude that this locus is co-located on chromosome 17 when mapped to the grapevine reference genome. The results show that the implementation of the 3D phenotyping platform allows for precise and extended screenings of different, genetic diverse mapping populations and thus opens up the possibility to uncover the genomic architecture of this highly complex quantitative grapevine trait.

Mutation analysis is the gold standard for assessing the effectiveness of a test suite to prevent bugs. It involves injecting syntactic changes in the program, generating variants (mutants) of the program under test, and checking whether the test suite detects the mutant. Practitioners often rely on these live mutants to decide what test cases to write for improving the test suite effectiveness. While a majority of such syntactic changes result in semantic differences from the original, it is possible that such a change fails to induce a corresponding semantic change in the mutant. Such equivalentmutants can lead to wastage of manual effort. We describe a novel technique that produces high-quality mutants while avoiding the generation of equivalent mutants for input processors. Our idea is to generate plausible, near correct inputs for the program, collect those rejected, and generate variants that accept these rejected strings. This technique allows us to provide an enhanced set of mutants along with newly generated test cases that kill them. We evaluate our method on eight python programs and show that our technique can generate new mutants that are both interesting for the developer and guaranteed to be mortal.

BackgroundGrapevine 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.ResultsThe 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 designatedVviRTIC1andVviRTIC2(VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP.ConclusionsMany 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.

‘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.

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.

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 (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 &lsquo, Teinturier&rsquo, 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.

Organic acids are an important determinant for wine quality. A mapping population from a cross between the breeding strain Gf.Ga-47-42 (?Bacchus? × ?Seyval?) and the cultivar ?Villard Blanc? (?Seibel 6468? × ?Subereux?) was investigated in detail regarding differences in the acidity level of the must. The acidity profile was recorded in three consecutive years under field conditions at Geilweilerhof (Palatinate, Germany). The obtained data were subjected to QTL analysis in order to identify genetic regions involved in acidity regulation. Investigated traits included total acidity, tartaric and malic acid levels as well as pH values in musts and wines. The identification of genomic regions which carry genes with a significant influence on these traits will give new insights into the acidity characteristics of grape cultivars. Newly identified trait-linked molecular markers are intended for application in marker-assisted selection (MAS) during the breeding process. This will contribute to accelerate grapevine breeding by early selection for quality traits.

Plasmopara viticola causes downy mildew of grapevine, one of the most important diseases in viticulture. Resistance to this oomycete is present in American and Asian Vitis species, while traditional European Vitis vinifera cvs. for wine and table grape production are susceptible. Breeding aims to achieve resistance through introgression, but the molecular mechanisms are still unknown. Therefore, the differential display approach was used to detect grapevine genes involved in defense. P. viticola sporangia were applied to the lower leaf surface of in vitro plants of the resistant Vitis riparia selection ‘Gloire de Montpellier’ and susceptible cv. ‘Riesling’. Controls were treated with sterile water. Messenger RNAs extracted 12 h post infection were subjected to differential display. Seven transcripts appeared specifically induced during the incompatible interaction. Sequencing showed that they build three classes. One of them, named VRP1, represented by three transcripts of almost identical sequence but differing lengths, showed clear homology to resistance genes of the NBS-LRR type from other plants. Northern hybridizations confirmed its elevated expression in the resistant ‘Gloire de Montpellier’. Redundancy of VRP1 PCR products from V. riparia prevented PCR-walking, so the VRP1 genes were isolated from a BAC-library of the resistant cv. ‘Regent’. Three genes matching the original VRP1 sequences were found within a BAC clone carrying a 134,392 bp insertion. These were referred to as VRP1-1, 1-2 and 1-3. They encode proteins of 798, 811 resp. 813 amino acids and exhibit the structure of CC-NBS-LRR resistance genes. They were genetically mapped to linkage group 10.

International audience; 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' x 'Seyval') x '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.

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’.

The oomycete Plasmopara viticola is one of the most important pathogens of grapevine (Vitis vinifera L.). All traditional European cultivars used for wine production are highly susceptible. Therefore a great quantity of fungicides is necessary to enable high quality production of grapes. Newly bred resistant cultivars generated via marker-assisted selection (MAS) are a major contribution toward reducing fungicide applications for more sustainable viticulture. Identification of specific markers linked to resistance loci is crucial to facilitate high throughput screening in a breeding program. Many North American Vitis species carry resistance genes due to co-evolution with Plasmopara viticola. Some Asian Vitis species also exhibit such resistances although their evolutionary origin is not yet understood. Among others, the two loci Rpv3 (American origin, e.g., ?Regent?) and Rpv10 (Asian origin, e.g., ?Solaris?) have been identified in earlier studies. Analysis of the genomic DNA sequence of ?Solaris? at the Rpv10 locus and ?Villard Blanc? at the Rpv3 locus revealed several candidate genes which might be responsible for the expression of resistance against Plasmopara viticola. These candidates include six genes with protein domains similar to resistance proteins known from other plant species. The aim of the study is the elucidation of the cellular defense mechanisms and the development of gene-linked markers for high throughput screening in grapevine breeding.

Marker-assisted selection is an important tool of current grapevine breeding which aims to improve cultivars for more sustainable viticulture. Markers genetically linked to traits of interest are hence required. These can be identified by QTL (quantitative trait loci) analysis scanning genetic maps elaborated from controlled crosses for marker genotypes correlating with segregating phenotypes. Major traits of interest are resistances to pathogens like powdery and downy mildew (Erysiphe necator resp. Plasmopara viticola) of leaves and fruits. In addition, phenological traits have recently acquired special attention. Breeders wish to select grapes with a better defined “window” of ripening time as ripening behavior affects both pathogen susceptibility and fruit quality. Ripening time needs to be considered within the context of the climate change debate. To identify genomic regions carrying loci affecting resistance traits and ripening time we constructed a genetic map using 151 individuals from the cross of grapevine breeding line Gf.GA- 47-42 (‘Bacchus’ × ‘Seyval’) with ‘Villard blanc’ (‘Seibel 6468’ × ‘Subereux’). Both parental lines are resistant to powdery and downy mildew and vary considerably in “véraison” time as an indicator of ripening. While Gf.GA-47-42 is rather early, ‘Villard blanc’ exhibits middle to late “véraison” and this trait segregates clearly in the cross. Linkage/recombination analysis for map construction was based on more than 350 simple sequence repeat (SSR)-derived markers. Many of these loci were newly extracted from the Vitis reference genome sequence of PN40024 (http://www.genoscope.cns.fr/externe/GenomeBrowse r/Vitis/). In addition, the mapping included 210 newly developed SNP (single nucleotide polymorphisms) markers. A subset of these was analyzed with two different techniques permitting to assess the reliability of SNP genotyping with both methods. The resulting map was scanned for QTL influencing powdery and downy mildew disease resistance and the time of “véraison”. Several major QTL were identified.