Genetic structure in populations of the fungus Fomitiporia punctata associated with the esca syndrome in grapevine

Six populations of Fomitiporia punctata (Fr.) Murrill were sampled in France and Italy from vines showing symptoms of esca syndrome. Genetic variation within and among populations was studied by using 34 random amplified polymorphic DNA (RAPD) markers. All the 192 isolates analyzed were distinguished, and the haplotypic diversity was similar in each population. The hypothesis that markers were randomly associated was not rejected for 5 populations and for the total sample. Comparison of marker frequency showed significant differences among populations for only 4 markers, indicating a low level of genetic differentiation. The analysis of molecular variance (AMOVA) confirmed that most of the variance in RAPD banding patterns was present within populations (98.5 %). These data strongly suggest that the fungus spreads by means of airborne basidiospores and regularly outcrosses in nature. The prospected regions seem to form an epidemiological unit with a panmictic population of F. punctata. However, a very low but significant differentiation was detected between the populations in western France and those in Mediterranean locations.

Do we need to consider grape phyllosphere microbiome in breeding schemes?

The aerial surface of the plant (phyllosphere) is the habitat of complex microbial communities. These communities may have profound effects on host plant health and its performance traits. When breeding new cultivars, i.e. the aerial component of a grape plant, one can simply ignore the phyllosphere in breeding schemes if its composition is mainly dependent on the environment. It is considered an important component if the genotype is the main driver of the phyllosphere composition. In order to answer this question, we have analysed several factors influencing the structure of the phyllosphere microbial community. Using amplicon sequencing of the 16S rRNA gene and of the internal transcribed spacer (ITS), we explored the microbial diversity at genus level for both bacteria and fungi present in the phyllosphere of leaves and grape berries. We analysed it on different grape taxonomic level (between five Vitis species or a set of Vitis vinifera cultivars chosen to represent the three genetic pools of the species), for different years and on five commercially important varieties of Vitis vinifera that were sampled from three different French terroirs. Our results indicated the presence of complex microbial diversity and assemblages in the phyllosphere. A significant effect of several factors (organ, grape species, growing year and terroir) on taxa abundance was observed with varying degrees of effect. At a given location, genotypes have an impact on microbial assemblage in the phyllosphere of leaf and berries, most pronounced on fruits but the effect of terroir was much stronger than the cultivar identity when the leaf phyllosphere of five grapevine varieties grown in different agro-climatic zones was compared. Limitations of the study as well as implied consequences of this work will be discussed

Phaeoacremonium species associated with Eutypa dieback and esca of grapevines in Algeria

Algerian grapevines showing symptoms of Eutypa dieback and esca were examined for the presence of Phaeoacremonium species. Species were identified on the basis of morphological and cultural characteristics as well as DNA sequence data (β-tubulin and actin). From a total of 200 vines sampled, 61 Phaeoacremonium isolates were obtained corresponding to four different species. Pm. aleophilum was the most frequently isolated (n=42) followed by Pm. parasiticum (n=10) and Pm. venezuelense (n=8). Pm. hispanicum was also found but only once. Phaeoacremonium species were more frequently associated with Eutypa dieback than esca symptoms. This correlates with their frequent association with sectorial brown necrosis (V-shaped necrosis).Algerian grapevines showing symptoms of Eutypa dieback and esca were examined for the presence of Phaeoacremonium species. Species were identified on the basis of morphological and cultural characteristics as well as DNA sequence data (β-tubulin and actin). From a total of 200 vines sampled, 61 Phaeoacremonium isolates were obtained corresponding to four different species. Pm. aleophilum was the most frequently isolated (n=42) followed by Pm. parasiticum (n=10) and Pm. venezuelense (n=8). Pm. hispanicum was also found but only once. Phaeoacremonium species were more frequently associated with Eutypa dieback than esca symptoms. This correlates with their frequent association with sectorial brown necrosis (V-shaped necrosis)

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions

<p>Abstract</p> <p>Background</p> <p>Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (<it>Vitis vinifera </it>L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the <it>fleshless berry </it>mutation (<it>flb</it>) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication.</p> <p>Results</p> <p>In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both <it>V. vinifera cv</it>. Chardonnay and a genotype carrying the <it>flb </it>mutation, <it>V. vinifera cv</it>. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that <it>flb </it>could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the <it>flb </it>region, representing 48,874 bp in a highly diverse set of cultivated and wild <it>V. vinifera </it>genotypes, to identify possible signatures of domestication in the cultivated <it>V. vinifera </it>compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the <it>flb </it>region.</p> <p>Conclusions</p> <p>We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the <it>flb </it>region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the <it>flb </it>gene in a F2 population when available.</p

Taxonomy and pathology of Togninia (Diaporthales) and its Phaeoacremonium anamorphs.

The genus Togninia (Diaporthales, Togniniaceae) is here monographed along with its Phaeoacremonium (Pm.) anamorphs. Ten species of Togninia and 22 species of Phaeoacremonium are treated. Several new species of Togninia (T.) are described, namely T. argentinensis (anamorph Pm. argentinense), T. austroafricana (anamorph Pm. austroafricanum), T. krajdenii, T. parasitica, T. rubrigena and T. viticola. New species of Phaeoacremonium include Pm. novae-zealandiae (teleomorph T. novae-zealandiae), Pm. iranianum, Pm. sphinctrophorum and Pm. theobromatis. Species can be identified based on their cultural and morphological characters, supported by DNA data derived from partial sequences of the actin and ß-tubulin genes. Phylogenies of the SSU and LSU rRNA genes were used to determine whether Togninia has more affinity with the Calosphaeriales or the Diaporthales. The results confirmed that Togninia had a higher affinity to the Diaporthales than the Calosphaeriales. Examination of type specimens revealed that T. cornicola, T. vasculosa, T. rhododendri, T. minima var. timidula and T. villosa, were not members of Togninia. The new combinations Calosphaeria cornicola, Calosphaeria rhododendri, Calosphaeria transversa, Calosphaeria tumidula, Calosphaeria vasculosa and Jattaea villosa are proposed. Species of Phaeoacremonium are known vascular plant pathogens causing wilting and dieback of woody plants. The most prominent diseases in which they are involved are Petri disease and esca, which occur on grapevines and are caused by a complex of fungi, often including multiple species of Phaeoacremonium. Various Phaeoacremonium species are opportunistic fungi on humans and cause phaeohyphomycosis. The correct and rapid identification of Phaeoacremonium species is important to facilitate the understanding of their involvement in plant as well as human disease. A rapid identification method was developed for the 22 species of Phaeacremonium. It involved the use of 23 species-specific primers, including 20 primers targeting the ß-tubulin gene and three targeting the actin gene. These primers can be used in 14 multiplex reactions. Additionally, a multiple-entry electronic key based on morphological, cultural and ß-tubulin sequence data was developed to facilitate phenotypic and sequence-based species identification of the different Phaeoacremonium species. Separate dichotomous keys are provided for the identification of the Togninia and Phaeoacremonium species. Keys for the identification of Phaeoacremonium-like fungi and the genera related to Togninia are also provided. The mating strategy of several Togninia species was investigated with ascospores obtained from fertile perithecia produced in vitro. Togninia argentinensis and T. novae-zealandiae have homothallic mating systems, whereas T. austroafricana, T. krajdenii, T. minima, T. parasitica, T. rubrigena and T. viticola were heterothallic.

Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding

A limited genetic mapping strategy based on simple sequence repeat (SSR) marker data was used with five grape populations segregating for powdery mildew (Erysiphe necator) resistance in an effort to develop genetic markers from multiple sources and enable the pyramiding of resistance loci. Three populations derived their resistance from Muscadinia rotundifolia ‘Magnolia’. The first population (06708) had 97 progeny and was screened with 137 SSR markers from seven chromosomes (4, 7, 9, 12, 13, 15, and 18) that have been reported to be associated with powdery or downy mildew resistance. A genetic map was constructed using the pseudo-testcross strategy and QTL analysis was carried out. Only markers from chromosome 13 and 18 were mapped in the second (04327) and third (06712) populations, which had 47 and 80 progeny, respectively. Significant QTLs for powdery mildew resistance with overlapping genomic regions were identified for different tissue types (leaf, stem, rachis, and berry) on chromosome 18, which distinguishes the resistance in ‘Magnolia’ from that present in other accessions of M. rotundifolia and controlled by the Run1 gene on chromosome 12. The ‘Magnolia’ resistance locus was termed as Run2.1. Powdery mildew resistance was also mapped in a fourth population (08391), which had 255 progeny and resistance from M. rotundifolia ‘Trayshed’. A locus accounting for 50% of the phenotypic variation mapped to chromosome 18 and was named Run2.2. This locus overlapped the region found in the ‘Magnolia’-based populations, but the allele sizes of the flanking markers were different. ‘Trayshed’ and ‘Magnolia’ shared at least one allele for 68% of the tested markers, but alleles of the other 32% of the markers were not shared indicating that the two M. rotundifolia selections were very different. The last population, 08306 with 42 progeny, derived its resistance from a selection Vitis romanetii C166-043. Genetic mapping discovered a major powdery mildew resistance locus termed Ren4 on chromosome 18, which explained 70% of the phenotypic variation in the same region of chromosome 18 found in the two M. rotundifolia resistant accessions. The mapping results indicate that powdery mildew resistance genes from different backgrounds reside on chromosome 18, and that genetic markers can be used as a powerful tool to pyramid these loci and other powdery mildew resistance loci into a single line

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies

UMR-AGAP Equipe DAVV (Diversité, adaptation et amélioration de la vigne) ; équipe ID (Intégration de Données)International audienceAbstractBackgroundAs for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies.ResultsStarting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance).ConclusionsOur association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment

Identification of stable QTLs for vegetative and reproductive traits in the microvine (Vitis vinifera L.) using the 18 K Infinium chip

UMR AGAP - équipe DAAV - Diversité, adaptation et amélioration de la vigne[b]Background[/b] [br/]The increasing temperature associated with climate change impacts grapevine phenology and development with critical effects on grape yield and composition. Plant breeding has the potential to deliver new cultivars with stable yield and quality under warmer climate conditions, but this requires the identification of stable genetic determinants. This study tested the potentialities of the microvine to boost genetics in grapevine. A mapping population of 129 microvines derived from Picovine x Ugni Blanc flb, was genotyped with the Illumina® 18 K SNP (Single Nucleotide Polymorphism) chip. Forty-three vegetative and reproductive traits were phenotyped outdoors over four cropping cycles, and a subset of 22 traits over two cropping cycles in growth rooms with two contrasted temperatures, in order to map stable QTLs (Quantitative Trait Loci). [br/][b]Results[/b] [br/]Ten stable QTLs for berry development and quality or leaf area were identified on the parental maps. A new major QTL explaining up to 44 % of total variance of berry weight was identified on chromosome 7 in Ugni Blanc flb, and co-localized with QTLs for seed number (up to 76 % total variance), major berry acids at green lag phase (up to 35 %), and other yield components (up to 25 %). In addition, a minor QTL for leaf area was found on chromosome 4 of the same parent. In contrast, only minor QTLs for berry acidity and leaf area could be found as moderately stable in Picovine. None of the transporters recently identified as mutated in low acidity apples or Cucurbits were included in the several hundreds of candidate genes underlying the above berry QTLs, which could be reduced to a few dozen candidate genes when a priori pertinent biological functions and organ specific expression were considered. [br/][b]Conclusions[/b] [br/]This study combining the use of microvine and a high throughput genotyping technology was innovative for grapevine genetics. It allowed the identification of 10 stable QTLs, including the first berry acidity QTLs reported so far in a Vitis vinifera intra-specific cross. Robustness of a set of QTLs was assessed with respect to temperature variatio