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Geographical distribution and diversity of current plant species have been strongly shaped by climatic oscillations during the Quaternary. Analysing the resulting divergence among species and differentiation within species is crucial to understand the evolution of taxa like the Vitis genus, which provides very useful genetic resources for grapevine improvement and might reveal original recolonization patterns due to growth habit and dispersal mode. Here, we studied the genetic structure in natural populations of three species from eastern North America: Vitis aestivalis, V. cinerea and V. riparia using different marker types. Vitis aestivalis and V. cinerea showed higher diversity than V. riparia. The two former species are less differentiated, confirming an earlier divergence of V. riparia. V. aestivalis and V. riparia exhibited different genetic groups on both sides of the Appalachian Mountains that could mirror different recolonization routes from southern refugia. Genetic structure was stronger in V. cinerea, for which two varieties (var. berlandieri and var. cinerea) are morphologically recognized. Our results confirm this distinction and suggest the existence of three other lineages within var. cinerea. These discontinuities appear linked to adaptation of var. berlandieri to dry and limy areas of Texas and partially to the Mississippi River Valley. Rapid range expansions from refugia upon climate warming are also suggested by the low linkage disequilibrium values observed. Furthermore, large variation for downy mildew resistance was observed in the three species. Our findings appeared consistent with the vegetation history of eastern North America.

Crop breeding involves two selection steps: choosing progenitors and selecting offspring within progenies. Genomic prediction, based on genome-wide marker estimation of genetic values, could facilitate these steps. However, its potential usefulness in grapevine (Vitis viniferaL.) has only been evaluated in non-breeding contexts mainly through cross-validation within a single population. We tested across-population genomic prediction in a more realistic breeding configuration, from a diversity panel to ten bi-parental crosses connected within a half-diallel mating design. Prediction quality was evaluated over 15 traits of interest (related to yield, berry composition, phenology and vigour), for both the average genetic value of each cross (cross mean) and the genetic values of individuals within each cross (individual values). Genomic prediction in these conditions was found useful: for cross mean, average per-trait predictive ability was 0.6, while per-cross predictive ability was halved on average, but reached a maximum of 0.7. Mean predictive ability for individual values within crosses was 0.26, about half the within-half-diallel value taken as a reference. For some traits and/or crosses, these across-population predictive ability values are promising for implementing genomic selection in grapevine breeding. This study also provided key insights on variables affecting predictive ability. Per-cross predictive ability was well predicted by genetic distance between parents and when this predictive ability was below 0.6, it was improved by training set optimization. For individual values, predictive ability mostly depended on trait-related variables (magnitude of the cross effect and heritability). These results will greatly help designing grapevine breeding programs assisted by genomic prediction.

To cope with the challenges facing agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennial species such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait loci in bi-parental crosses, we exploited a diversity panel of 279 Vitis vinifera L. cultivars planted in 5 blocks in the vineyard. This panel was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms by combining an 18K microarray and genotyping-by-sequencing. The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via genotyping-by-sequencing markedly increased the proportion of genetic variance explained by single nucleotide polymorphisms, and 2 multi-single nucleotide polymorphism models identified quantitative trait loci not found by a single nucleotide polymorphism-by-single nucleotide polymorphism model. Overall, 489 reliable quantitative trait loci were detected for 41% more response variables than by a single nucleotide polymorphism-by-single nucleotide polymorphism model with microarray-only single nucleotide polymorphisms, many new ones compared with the results from bi-parental crosses. A prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as quantitative trait locus and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application into breeding are discussed.

Breeding new cultivars is an important lever to cope with the various challenges facing viticulture. As a prerequisite, it is necessary to assess how much genetic variation is available for the traits of interest, and to understand their genetic architecture. To go beyond quantitative trait locus (QTL) mapping in biparental crosses, we exploited the panel of 279 cultivars capturing most of the genetic and phenotypic diversity of Vitis vinifera L. present within the French collection of genetic resources (INRA Vassal). This panel was planted in the vineyard in five randomized complete blocks and numerous traits (berry weight, cluster weight, length and compactness, organic acids, δ13C, polyphenols, etc.) were phenotyped over several years. In parallel, the panel was genotyped with the GrapeReSeq microarray as well as with restriction-assisted DNA sequencing. This allowed us to conduct a genome-wide association study per trait, either by testing each single-nucleotide polymorphism (SNP) separately or by modeling all SNPs jointly. Focusing here on berry weight, we showed that genotyping by sequencing provided enough SNPs to explain most of the genetic variance, and that, compared with SNP-by-SNP approaches, a multi-SNP statistical model led to a gain in power. Several significant associations were detected, some being new compared with the QTLs detected in biparental crosses. Future prospects include setting up an international consortium to plant the panel on various sites to help to decipher genotype-environment interactions.

International audience; Key message A half-diallel population involving five elite grapevine cultivars was generated and genotyped by GBS, and highly-informative segregation data was used to construct a high-density genetic map for Vitis vinifera L. Abstract Grapevine is one of the most relevant fruit crops in the world. Deeper genetic knowledge could assist modern grapevine breeding programs to develop new wine grape varieties able to face climate change effects. To assist in the rapid identification of markers for crop yield components, grape quality traits and adaptation potential, we generated a large Vitis vinifera L. population (N = 624) by crossing five red wine cultivars in a half-diallel scheme, which was subsequently sequenced by an efficient GBS procedure. A high number of fully informative genetic variants was detected using a novel mapping approach capable of reconstructing local haplotypes from adjacent biallelic SNPs, which were subsequently used to construct the densest consensus genetic map available for the cultivated grapevine to date. This 1378.3-cM map integrates 10 bi-parental consensus maps and orders 4437 markers in 3353 unique positions on 19 chromosomes. Markers are well distributed all along the grapevine reference genome, covering up to 98.8% of its genomic sequence. Additionally, a good agreement was observed between genetic and physical orders, adding confidence in the quality of this map. Collectively, our results pave the way for future genetic studies (such as fine QTL mapping) aimed to understand the complex relationship between genotypic and phenotypic variation in the cultivated grapevine. In addition, the method used (which efficiently delivers a high number of fully informative markers) could be of interest to other outbred organisms, notably perennial fruit crops.

The major Grapevine Trunk Diseases (GTD) gather diseases associated with either one particular fungal species, e.g., eutypiose, or with fungal species complexes, e.g., Botryosphaeriae dieback. GTD-associated fungi are part of different fungal classes, depending on their substrate degradation mechanisms or on their lifestyles. They are identified in grapevine wood and they lead to wood typical symptoms. They are sometimes associated to foliar symptoms too, despite not found in aerial parts, probably acting through fungal toxins or through physiological disorders, such as non-functional wood vessels. Grapevine is a liana, and as such shows structural and chemical differences compared to forest tree wood. However, knowledge on wood-degrading fungi growing on tree wood can contribute to a better understanding of grapevine wood adaptation mechanisms to fungal attack. For example, wood density, xylem vessels size and number, or lignin content and composition can play a role in wood sensitivity to fungi. Also, some fungal detoxification mechanisms against wood extractibles are host-specific. In a first part of this chapter, structure and chemical composition of grapevine wood are described, pointing out properties that might play a role in fungal growth. In a second part, GTD main associated fungi and their known mechanisms for wood degradation or wood chemical detoxification are discussed. Extracellular and intracellular enzymatic and non-enzymatic mechanisms are considered. These mechanisms, well described for their role in fungal adaptation to tree wood, have been much less studied for GTD associated fungi. Further studies about those mechanisms would contribute to understand fungal adaptation to grapevine.

To cope with the challenges faced by agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennials such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait locus (QTL) in bi-parental crosses, we exploited a diverse panel of 279 Vitis vinifera L. cultivars. This panel planted in five blocks in the vineyard was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms (SNPs) by combining an 18K microarray and genotyping-by-sequencing (GBS). The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via GBS markedly increased the proportion of genetic variance explained by SNPs, and two multi-SNP models identified QTLs not found by a SNP-by-SNP model. Overall, 489 reliable QTLs were detected for 41% more response variables than by a SNP-by-SNP model with microarray-only SNPs, many new ones compared to the results from bi-parental crosses. Prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as QTL and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application in breeding are discussed.

The MybA1 gene in the genus Vitis encodes a transcription factor, belonging to the R2R3 Myb family, that controls the last steps in the anthocyanins biosynthesis pathway. Polymorphism within MybA1 has been associated with color variation in berries of V. vinifera and other Vitis species. In this work, we analyzed the sequence variation in MybA1 both in the subg. Muscadinia and in an extended set of Asian, American and European genotypes of subg. Vitis. Our aims were to infer the evolution of this gene during the speciation process and to identify polymorphisms that could potentially generate changes in gene regulation. The results show that MybA1 experienced many insertions and deletions in non-coding regions but also in the third exon sequence. Owing to the larger set of Vitis species compared here, new indels were identified and the origin of previously described indels was reconsidered. A large number of single nucleotide polymorphisms were found in non-coding regions but also in the sequence coding for the R2R3 domain and the C terminal part of the protein. Some of these changes led to amino acid substitutions and therefore could have modified MybA1 protein activity. Bayesian phylogenetic analysis of all polymorphisms did not provide a consensus tree depicting the geographical partitioning of the species but allowed highlighting several species relationships within subgenus Vitis. Finally, the evolutionary events described could be useful to gain more insight into the role of MybA1 for anthocyanin biosynthesis in grapevine.

UMR AGAP Equipe Diversité, adaptation et amélioration de la vigne (DAAV); Eutypa lata is a fungal pathogen causing severe dieback in vineyards worldwide. This fungus colonizes vines through pruning wounds, eventually causing a brown sectorial necrosis in wood as well as stunted vegetative growth. Several years may pass between infection and the expression of external symptoms, hindering the rapid evaluation of both grapevine cultivars susceptibility and E. lata variation in aggressiveness. We aimed to develop a rapid quantitative method for the assessment of wood colonization after inoculation of cuttings in controlled conditions. We used several grape cultivars varying in susceptibility in the vineyard and fungal isolates with different levels of aggressiveness to monitor wood colonization during a maximum period of 2 months. Re-isolation allowed demonstration of the effects of both cultivars and fungal isolates on the rate of wood colonization. We also developed a real-time PCR method that was efficient in measuring fungal biomass, which was found to be correlated with isolate aggressiveness based on foliar symptom severity. The real-time PCR approach appears to be a useful technology to evaluate grapevine susceptibility to E. lata, and could be adapted to other pathogens associated with grapevine trunk diseases.

Transcriptome sequences of the grape powdery mildew fungus Erysiphe necator were used to develop microsatellite markers (EST-SSRs) to study its relatively unexplored population structure in its centre of diversity in eastern North America. Screening the transcriptome sequences revealed 116 contigs with candidate microsatellites, from which 11 polymorphic microsatellite markers were developed from 31 markers tested. Eight of these markers were used to genotype isolates from different regions and hosts in the eastern USA and compare them to samples from southern France and Italy. Genetic diversity in the eastern USA is much greater than in Europe. Bayesian cluster analyses showed that 10 isolates from North America have high affinities with, but differ from, European group A; these are referred to as A-like isolates. No isolates with close affinity to European group B were found in the eastern USA. Bayesian analyses also detected genetic differentiation between isolates from Vitis rotundifolia and isolates from other Vitis hosts. Genetic differentiation detected between the northeastern and southeastern USA was mostly attributable to the A-like isolates in the southeast, which are significantly more aggressive than the other populations. This research demonstrates that transcriptome sequencing of fungal pathogens is useful for developing genetic markers in protein-coding regions and highlights the role of these markers in population biology studies of E. necator.

The generalist ascomycete fungus Eutypa lata causes Eutypa dieback of grapevine (Vitis vinifera) worldwide. To decipher the cosmopolitan distribution of this fungus, the population genetic structure of 17 geographic samples was investigated from four continental regions (Australia, California, Europe and South Africa), based on analysis of 293 isolates genotyped with nine microsatellite markers. High levels of haplotypic richness (R = 0·91–1) and absence of multilocus linkage disequilibrium among loci supported the preponderance of sexual reproduction in all regions examined. Nonetheless, the identification of identical multilocus haplotypes with identical vegetative compatibility groups, in some vineyards in California and South Africa, suggests that asexual dispersal of the fungus among neighbouring plants could be a rare means of disease spread. The greatest levels of allelic richness (A = 4·89–4·97) and gene diversity (H = 0·66–0·69) were found in Europe among geographic samples from coastal areas surrounding the Mediterranean Sea, whereas the lowest genetic diversity was found in South Africa and Australia (A = 2·78–3·74; H = 0·49–0·57). Samples from California, Australia and South Africa, which had lower genetic diversity than those of Europe, were also characterized by demographic disequilibrium and, thus, may represent founding populations of the pathogen. Low but significant levels of genetic differentiation among all samples (DEST = 0·12, P = 0·001; FST = 0·03, P = 0·001) are consistent with historical gene flow preventing differentiation at continental scales. These findings suggest that global, human-mediated spread of the fungus may have resulted in its current global distribution.

Aim : Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location: Asia, Europe, North America. Methods: We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra-specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH-psbA-trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions: We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re-examine certain aspects of the current subgeneric classification

In this study, 18 microsatellite loci were used to examine the genetic structure and mode of reproduction of Phaeomoniella chlamydospora, the fungus that causes Petri disease of grapevine and is involved in another grapevine disease, esca. A total of 60 southeastern Australian isolates were tested and compared with 64 isolates from southern France. The French population possessed relatively high genotypic diversity (G = 29·2) whilst the Australian population showed low genotypic diversity (G = 11·1), consistent with a limited founder population. Haplotypes from four different grapevine cultivars were not significantly differentiated (Φpt values effectively zero). Likewise, genetic differentiation of haplotypes from different regions within each country was not significant, although small but significant genetic differentiation (9%) was identified between Australia and France. Based on bootstrapped cluster analysis, there did not appear to be any genetic groups within the overall sample of isolates. Significant linkage disequilibrium identified in both countries, together with overrepresented, widespread identical haplotypes, indicated limited genetic recombination and a largely clonal structure consistent with the absence of an observed sexual cycle in this species.

We analysed symptoms, wood lesions and fungi associated with esca in mature organic vineyards in the Languedoc-Roussillon region. Most previous surveys concerning esca syndrome were conducted in conventionally managed vineyards in other regions. We first found that esca may be present at a very low level in vineyards that were not treated with sodium arsenite. Affected vines displayed three types of symptoms: leaves with interveinal necrosis, wilt of entire branches and these two symptoms in combination. During the 3-year survey, not all affected vines displayed symptoms every year and the same vine rarely displayed the same symptom or combination of symptoms in successive years. The incidence of esca appeared to be correlated with the percentage of vines that died during the survey but no correlation was found with either mortality before the survey or with the age of the vineyard. Observation of cross-sections of a total of 210 vines with esca symptoms and isolation of fungi from the wood lesions led to similar results to those obtained in conventionally managed vineyards. Four different lesions were identified: a white rot lesion, a brown lesion in a central position, a brown lesion in a sectorial position and a scattered black spotting in healthy wood. The most frequently observed fungal species were Fomitiporia mediterranea (Fom), Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Eutypa lata. The white rot lesion caused by Fom was generally accompanied by one or more other lesions in the same vine. Similarly, Fom was generally isolated with one or several other fungi. No relationship was detected between the expression of external symptoms and any specific lesion or fungus. Our study also showed that esca and Eutypa dieback are often superimposed in our region of sampling.

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.

Isolates of Phaeomoniella chlamydospora (Phc) and Phaeoacremonium aleophilum (Pha), two haploid, deuteromycetous fungi, were obtained from vines showing symptoms of esca disease in different localities in two French regions, and within a single vineyard in one of these regions. The population genetic structure was determined in both fungi using random amplified polymorphic DNA (RAPD) analysis. Populations of Phc showed similar levels of diversity at local and regional levels. The most frequent Phc haplotypes were found in every population, and the frequencies of positive alleles of markers were similar across populations. The hypothesis that recombination had occurred was rejected for the full set of samples, but not for the samples reduced to haplotypes, indicating that Phc may be a recombining species. Different features were identified in Pha populations. First, the southern population of Pha appeared more diverse than the south-western populations. Second, genetic differentiation was identified between Pha populations from southern and south-western regions for several RAPDs. Finally, in the southern population of Pha no evidence for recombination was obtained, even by reducing the sample to haplotypes. Within the single vineyard surveyed, several haplotypes of both fungi were recovered and randomly distributed. Thus different infection events appeared to have occurred on a low spatial scale. Data from this study showed that haplotypes of both fungi were distributed over long distances geographically, and that most of the vineyards surveyed were infested by more than one haplotype of Phc and Pha.

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots. (Résumé d'auteur)

Eutypa latais the causal fungal agent ofEutypadieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detectE. latain grapevine wood samples. We designed six pairs of PCR primers for diagnosis ofE. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of theE. lataisolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identifyE. latafrom pure or mixed cultures as well as from grapevine wood samples. Identification ofE. latain wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.

Ustilago scitaminea synthesizes specific carbohydrates, mainly erythritol and mannitol, plus smaller quantities of arabitol, inositol, and trehalose. Intensive secretion of erythritol is obtained when the fungus is grown in liquid culture. Glucose, fructose, or sucrose can be used interchangeably as carbon sources by the fungus, sucrose being quickly inverted.