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MLOs belong to the largest family of seven-transmembrane (7TM) domain proteins found in plants. The Arabidopsis and rice genomes contain 15 and 12 MLO family members, respectively. Although the biological function of most MLO family members remains elusive, a select group of MLO proteins have been demonstrated to negatively regulate defence responses to the obligate biotrophic pathogen, powdery mildew, thereby acting as “susceptibility” genes. Recently we identified a family of 17 putative VvMLO genes in the genome of the cultivated winegrape species, Vitis vinifera. Expression analysis indicated that the VvMLO family members respond differently to biotic and abiotic stimuli. Infection of V. vinifera by grape powdery mildew (Erysiphe necator) specifically upregulates four VvMLO genes that are orthologous to the Arabidopsis and tomato MLOs previously demonstrated to be required for powdery mildew susceptibility. We postulate that one or more of these E. necator responsive VvMLOs may have a role in the powdery mildew susceptibility of grapevine.

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.

Electronic supplementary material

The online version of this article (doi:10.1007/s00122-010-1511-6) contains supplementary material, which is available to authorized users.

Powdery mildew (PM), caused by fungus Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone “Shang-24”. A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material “Shang-24” than in the sensitive V. pseudoreticulata clone “Hunan-1” by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm.

Background

The grape powdery mildew fungus, Erysiphe necator, was introduced into Europe more than 160 years ago and is now distributed everywhere that grapes are grown. To understand the invasion history of this pathogen we investigated the evolutionary relationships between introduced populations of Europe, Australia and the western United States (US) and populations in the eastern US, where E. necator is thought to be native. Additionally, we tested the hypothesis that populations of E. necator in the eastern US are structured based on geography and Vitis host species.

Results

We sequenced three nuclear gene regions covering 1803 nucleotides from 146 isolates of E. necator collected from the eastern US, Europe, Australia, and the western US. Phylogeographic analyses show that the two genetic groups in Europe represent two separate introductions and that the genetic groups may be derived from eastern US ancestors. Populations from the western US and Europe share haplotypes, suggesting that the western US population was introduced from Europe. Populations in Australia are derived from European populations. Haplotype richness and nucleotide diversity were significantly greater in the eastern US populations than in the introduced populations. Populations within the eastern US are geographically differentiated; however, no structure was detected with respect to host habitat (i.e., wild or cultivated). Populations from muscadine grapes, V. rotundifolia, are genetically distinct from populations from other Vitis host species, yet no differentiation was detected among populations from other Vitis species.

Conclusions

Multilocus sequencing analysis of the grape powdery mildew fungus is consistent with the hypothesis that populations in Europe, Australia and the western US are derived from two separate introductions and their ancestors were likely from native populations in the eastern US. The invasion history of E. necator follows a pattern consistent with plant-mediated dispersal, however, more exhaustive sampling is required to make more precise conclusions as to origin. E. necator shows no genetic structure across Vitis host species, except with respect to V. rotundifolia.

Background

Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.

Results

Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.

Conclusions

The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled (θ = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (θ = 0.138, P = 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge.

Background

MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species.

Results

A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism.

Conclusions

Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.

Background

Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead been found to be resistant to this pathogen and have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity is closely related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling × Teroldego cross by profiling the stilbenoid content of the leaves of an entire population and the transcriptome of resistant and susceptible individuals following infection.

Results

A three-year analysis of the population's response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine.

Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response.

A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis.

A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.

Conclusions

This study reports the results of a combined metabolic and transcriptional profiling of a grapevine population segregating for resistance to P. viticola. Some resistant individuals were identified and further characterized at the molecular level. These results will be valuable to future grapevine breeding programs.

Background

Downy mildew, caused by Plasmopara viticola, is one of the most severe diseases of grapevine and is commonly controlled by fungicide treatments. The beneficial microorganism Trichoderma harzianum T39 (T39) can induce resistance to downy mildew, although the molecular events associated with this process have not yet been elucidated in grapevine. A next generation RNA sequencing (RNA-Seq) approach was used to study global transcriptional changes associated with resistance induced by T39 in Vitis vinifera Pinot Noir leaves. The long-term aim was to develop strategies to optimize the use of this agent for downy mildew control.

Results

More than 14.8 million paired-end reads were obtained for each biological replicate of T39-treated and control leaf samples collected before and 24 h after P. viticola inoculation. RNA-Seq analysis resulted in the identification of 7,024 differentially expressed genes, highlighting the complex transcriptional reprogramming of grapevine leaves during resistance induction and in response to pathogen inoculation. Our data show that T39 has a dual effect: it directly modulates genes related to the microbial recognition machinery, and it enhances the expression of defence-related processes after pathogen inoculation. Whereas several genes were commonly affected by P. viticola in control and T39-treated plants, opposing modulation of genes related to responses to stress and protein metabolism was found. T39-induced resistance partially inhibited some disease-related processes and specifically activated defence responses after P. viticola inoculation, causing a significant reduction of downy mildew symptoms.

Conclusions

The global transcriptional analysis revealed that defence processes known to be implicated in the reaction of resistant genotypes to downy mildew were partially activated by T39-induced resistance in susceptible grapevines. Genes identified in this work are an important source of markers for selecting novel resistance inducers and for the analysis of environmental conditions that might affect induced resistance mechanisms.

RING finger proteins comprise a large family and play important roles in regulation of growth and development, hormone signalling, and responses to biotic and abiotic stresses in plants. In this study, the identification and functional characterization of a C4C4-type RING finger protein gene from the Chinese wild grapevine Vitis pseudoreticulata (designated VpRFP1) are reported. VpRFP1 was initially identified as an expressed sequence tag (EST) from a cDNA library constructed from leaves of V. pseudoreticulata inoculated with the grapevine powdery mildew Uncinula necator. Sequence analysis of the deduced VpRFP1 protein based on the full-length cDNA revealed an N-terminal nuclear localization signal (NLS) and a C-terminal C4C4-type RING finger motif with the consensus sequence Cys-X2-Cys-X13-Cys-X1-Cys-X4-Cys-X2-Cys-X10-Cys-X2-Cys. Upon inoculation with U. necator, expression of VpRFP1 was rapidly induced to higher levels in mildew-resistant V. pseudoreticulata plants. In contrast, expression of VpRFP1 was down-regulated in mildew-susceptible V. vinifera plants. Western blotting using an antibody raised against VpRFP1 showed that VpRFP1 was also induced to higher levels in V. pseudoreticulata plants at 12–48 hours post-inoculation (hpi). However, there was only slight increase in VpRFP in V. vinifera plants in the same time frame, even though a more significant increase was observed at 96–144 hpi in these plants. Results from transactivation assays in yeast showed that the RING finger motif of VpRFP1 exhibited some activity of transcriptional activation; however, no activity was seen with the full-length VpRFP1. Overexpression of VpRFP1 in Arabidopsis plants was found to enhance resistance to Arabidopsis powdery mildew Golovinomyces cichoracearum, which seemed to be correlated with increased transcript levels of AtPR1 and AtPR2 in the pathogen-infected tissues. In addition, the Arabidopsis transgenic lines showed enhanced resistance to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Taken together, the results suggested that VpRFP1 may be a transcriptional activator of defence-related genes in grapevines.

Isolates of the causal ascomycete of grapevine powdery mildew, Erysiphe necator, correspond to two genetically differentiated groups (A and B) that coexist on the same host. This coexistence was analyzed by investigating temporal changes in the genetic and phenotypic structures of E. necator populations during three epidemics. Group A was present only at the start of the growing season, whereas group B was present throughout all three epidemics. Group A was less aggressive in terms of germination and infection efficiency but was more aggressive than group B in terms of the latency period, lesion diameter, and spore production. Our results are consistent with a temporal differentiation of niches, preventing recombination, and suggest an association between the disease level and the frequencies of genetic groups.

Background

Grapevine (Vitis vinifera subsp. vinifera) is one of the most important and ancient horticultural plants in the world. Domesticated about 8–10,000 years ago in the Eurasian region, grapevine evolved from its wild relative (V. vinifera subsp. sylvestris) into very diverse and heterozygous cultivated forms. In this work we study grapevine genetic structure in a large sample of cultivated varieties, to interpret the wide diversity at morphological and molecular levels and link it to cultivars utilization, putative geographic origin and historical events.

Results

We analyzed the genetic structure of cultivated grapevine using a dataset of 2,096 multi-locus genotypes defined by 20 microsatellite markers. We used the Bayesian approach implemented in the STRUCTURE program and a hierarchical clustering procedure based on Ward’s method to assign individuals to sub-groups. The analysis revealed three main genetic groups defined by human use and geographic origin: a) wine cultivars from western regions, b) wine cultivars from the Balkans and East Europe, and c) a group mainly composed of table grape cultivars from Eastern Mediterranean, Caucasus, Middle and Far East countries. A second structure level revealed two additional groups, a geographic group from the Iberian Peninsula and Maghreb, and a group comprising table grapes of recent origins from Italy and Central Europe. A large number of admixed genotypes were also identified. Structure clusters regrouped together a large proportion of family-related genotypes. In addition, Ward’s method revealed a third level of structure, corresponding either to limited geographic areas, to particular grape use or to family groups created through artificial selection and breeding.

Conclusions

This study provides evidence that the cultivated compartment of Vitis vinifera L. is genetically structured. Genetic relatedness of cultivars has been shaped mostly by human uses, in combination with a geographical effect. The finding of a large portion of admixed genotypes may be the trace of both large human-mediated exchanges between grape-growing regions throughout history and recent breeding.

Background

Downy mildew is a destructive grapevine disease caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, which can only be controlled by intensive fungicide treatments. Natural sources of resistance from wild grapevine (Vitis) species are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood.

Results

Early transcriptional changes associated with P. viticola infection in susceptible V. vinifera and resistant V. riparia plants were analyzed using the Combimatrix microarray platform. Transcript levels were measured 12 and 24 h post-inoculation, reflecting the time points immediately preceding the onset of resistance in V. riparia, as determined by microscopic analysis. Our data indicate that resistance in V. riparia is induced after infection, and is not based on differences in basal gene expression between the two species. The strong and rapid transcriptional reprogramming involves the induction of pathogenesis-related proteins and enzymes required for the synthesis of phenylpropanoid-derived compounds, many of which are also induced, albeit to a lesser extent, in V. vinifera. More interestingly, resistance in V. riparia also involves the specific modulation of numerous transcripts encoding components of signal transduction cascades, hypersensitive reaction markers and genes involved in jasmonate biosynthesis. The limited transcriptional modulation in V. vinifera represents a weak attempted defense response rather than the activation of compatibility-specific pathways.

Conclusions

Several candidate resistance genes were identified that could be exploited in future biotechnological approaches to increase disease resistance in susceptible grapevine species. Measurements of jasmonic acid and methyl jasmonate in infected leaves suggest that this hormone may also be involved in V. riparia resistance to P. viticola.

Background

Grapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affecting mainly Vitis vinifera cultivated varieties around the world. Breeding for resistance through the crossing with less susceptible species is one of the possible means to reduce the disease incidence and the application of fungicides. The hybrid Bianca and some of its siblings are considered very promising but their resistance level can vary depending on the pathogen strain. Moreover, virulent strains characterized by high fitness can represent a potential threat to the hybrid cultivation.

Results

The host response and the pathogen virulence were quantitatively assessed by artificially inoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived from single germinating oospores collected in various Italian viticultural areas. The host phenotypes were classified as susceptible, intermediate and resistant, according to the Area Under the Disease Progress Curve caused by the inoculated strain. Host responses in cv Bianca and its siblings significantly varied depending on the P. viticola isolates, which in turn differed in their virulence levels. The fitness of the most virulent strain did not significantly vary on the different hybrids including Bianca in comparison with the susceptible cv Chardonnay, suggesting that no costs are associated with virulence. Among the individual fitness components, only sporangia production was significantly reduced in cv Bianca and in some hybrids. Comparative histological analysis revealed differences between susceptible and resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increase in peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis of stomata and cells immediately surrounding the point of invasion and determined alterations in the size of the infected areas and in the number of sporangia differentiated.

Conclusions

Some hybrids were able to maintain an intermediate-resistant behaviour even when inoculated with the most virulent strain. Such hybrids should be considered for further field trials.

Background

Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.

Results

We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation.

Conclusions

The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.

Grape is one of the most important fruit crops worldwide. The suitable geographical locations and productivity of grapes are largely limited by temperature. Vitis amurensis is a wild grapevine species with remarkable cold-tolerance, exceeding that of Vitis vinifera, the dominant cultivated species of grapevine. However, the molecular mechanisms that contribute to the enhanced freezing tolerance of V. amurensis remain unknown. Here we used deep sequencing data from restriction endonuclease-generated cDNA fragments to evaluate the whole genome wide modification of transcriptome of V. amurensis under cold treatment. Vitis vinifera cv. Muscat of Hamburg was used as control to help investigate the distinctive features of V. amruensis in responding to cold stress. Approximately 9 million tags were sequenced from non-cold treatment (NCT) and cold treatment (CT) cDNA libraries in each species of grapevine sampled from shoot apices. Alignment of tags into V. vinifera cv. Pinot noir (PN40024) annotated genome identified over 15,000 transcripts in each library in V. amruensis and more than 16,000 in Muscat of Hamburg. Comparative analysis between NCT and CT libraries indicate that V. amurensis has fewer differential expressed genes (DEGs, 1314 transcripts) than Muscat of Hamburg (2307 transcripts) when exposed to cold stress. Common DEGs (408 transcripts) suggest that some genes provide fundamental roles during cold stress in grapes. The most robust DEGs (more than 20-fold change) also demonstrated significant differences between two kinds of grapevine, indicating that cold stress may trigger species specific pathways in V. amurensis. Functional categories of DEGs indicated that the proportion of up-regulated transcripts related to metabolism, transport, signal transduction and transcription were more abundant in V. amurensis. Several highly expressed transcripts that were found uniquely accumulated in V. amurensis are discussed in detail. This subset of unique candidate transcripts may contribute to the excellent cold-hardiness of V. amurensis.

Background

Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns.

Results

Uncommonly large size of spliceosomal intron was observed in V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars.

Conclusions

In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera genome.

The genus Serangium Blackburn from China is reviewed. The genus Catanella Miyatake is removed from synonymy with Serangium. Serangium baculum Xiao is transferred to Catanella, as Catanella baculum (Xiao), comb. n. Twelve species of Serangium are described, keyed and illustrated, including eight new species, Serangium magnipunctatum Wang & Ren, sp. n., Serangium trimaculatum Wang & Ren, sp. n., Serangium centrale Wang & Ren, sp. n., Serangium leigongicus Wang & Ren, sp. n., Serangium latilobum Wang & Ren, sp. n., Serangium digitiforme Wang & Ren, sp. n., Serangium dulongjiang Wang, Ren & Chen, sp. n., and Serangium contortum Wang & Ren, sp. n. Serangium punctum Miyatake is newly recorded from China.

The genus Vitis is represented by several coexisting species in Europe. Our study focuses on naturalised rootstocks that originate in viticulture. The consequences of their presence to the landscape and to native European species (Vitis vinifera ssp. silvestris) are evaluated. This study compares ecological traits (seven qualitative and quantitative descriptors) and the genetic diversity (10 SSR markers) of populations of naturalised rootstocks and native wild grapevines. 18 large naturalised rootstock populations were studied in the Rhône watershed. Wild European grapevines are present in four main habitats (screes, alluvial forests, hedges, and streamside hedges). In contrast, naturalised rootstock populations are mainly located in alluvial forests, but they clearly take advantage of alluvial system dynamics and connectivity at the landscape level. These latter populations appear to reproduce sexually, and show a higher genetic diversity than Vitis vinifera ssp. silvestris. The regrouping of naturalised rootstocks in interconnected populations tends to create active hybrid swarms of rootstocks. The rootstocks show characters of invasive plants. The spread of naturalised rootstocks in the environment, the acceleration of the decline of the European wild grapevine, and the propagation of genes of viticultural interest in natural populations are potential consequences that should be kept in mind when undertaking appropriate management measures.

Background

The oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni causes downy mildew in grapevine (Vitis vinifera L.). This pathogen is strictly biotrophic, thus completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We have carried out a large-scale cDNA-AFLP analysis to identify grapevine and P. viticola genes associated with the infection process.

Results

We carried out cDNA-AFLP analysis on artificially infected leaves of the susceptible cultivar Riesling at the oil spot stage, on water-treated leaves and on a sample of pure sporangia as controls. Selective amplifications with 128 primer combinations allowed the visualization of about 7000 transcript-derived fragments (TDFs) in infected leaves, 1196 of which (17%) were differentially expressed. We sequenced 984 fragments, 804 of which were identified as grapevine transcripts after homology searching, while 96 were homologous to sequences in Phytophthora spp. databases and were attributed to P. viticola. There were 82 orphan TDFs. Many grapevine genes spanning almost all functional categories were downregulated during infection, especially genes involved in photosynthesis. Grapevine genes homologous to known resistance genes also tended to be repressed, as were several resistance gene analogs and carbonic anhydrase (recently implicated in pathogen resistance). In contrast, genes encoding cytoskeletal components, enzymes of the phenylpropanoid and beta-oxidation pathways, and pathogenesis related proteins were primarily upregulated during infection. The majority of P. viticola transcripts expressed in planta showed homology to genes of unknown function or to genomic Phytophthora sequences, but genes related to metabolism, energy production, transport and signal transduction were also identified.

Conclusion

This study provides the first global catalogue of grapevine and P. viticola genes expressed during infection, together with their functional annotations. This will help to elucidate the molecular basis of the infection process and identify genes and chemicals that could help to inhibit the pathogen.

Background

Most of the grapevine (Vitis vinifera L.) cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project.

Results

We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome.

Conclusions

We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.

Background

Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera 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 fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication.

Results

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 V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. 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 flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera 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 flb region.

Conclusions

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 flb 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 flb gene in a F2 population when available.

Background

The powdery mildew disease represents a valuable patho-system to study the interaction between plant hosts and obligate biotrophic fungal pathogens. Numerous discoveries have been made on the basis of the quantitative evaluation of plant-powdery mildew interactions, especially in the context of hyper-susceptible and/or resistant plant mutants. However, the presently available methods to score the pathogenic success of powdery mildew fungi are laborious and thus not well suited for medium- to high-throughput analysis.

Results

Here we present two new protocols that allow the rapid quantitative assessment of powdery mildew disease development. One procedure depends on quantitative polymerase chain reaction (qPCR)-based evaluation of fungal biomass, while the other relies on the quantification of fungal conidiospores. We validated both techniques using the powdery mildew pathogen Golovinomyces orontii on a set of hyper-susceptible and resistant Arabidopsis thaliana mutants and found that both cover a wide dynamic range of one to two (qPCR) and four to five (quantification of conidia) orders of magnitude, respectively. The two approaches yield reproducible results and are easy to perform without specialized equipment.

Conclusions

The qPCR and spore count assays rapidly and reproducibly quantify powdery mildew pathogenesis. Our methods are performed at later stages of infection and discern mutant phenotypes accurately. The assays therefore complement currently used procedures of powdery mildew quantification and can overcome some of their limitations. In addition, they can easily be adapted to other plant-powdery mildew patho-systems.

Autophagy is a conserved intracellular recycling system that traffics cellular organelles and cytosolic proteins within lysosomes for reuse or breakdown in eukaryotes. Increased evidence indicates that autophagy is involved in programmed cell death and disease resistance in plants. We recently showed that atg2, atg5, atg7 and atg10 displayed early senescence and cell death in later growth stage under nutrient-rich conditions in Arabidopsis thaliana. These mutants also exhibited powdery mildew resistance and mildew-induced cell death. Salicylic acid (SA) signaling is required for atg2-mediated powdery mildew resistance, however, inactivation of SA signaling is not sufficient to fully suppress powdery mildew-induced cell death in atg2 mutant.1 Here, we show that atg18a-2 is also resistant to the powdery mildew pathogen, Golovinomyces cichoracearum, and it shows mildew-induced cell death similar to the atg2 mutant. Taken together, our study reveals that autophagy plays important roles in suppression of cell death and defense response to the biotrophic pathogen, the powdery mildew fungus. Future work on autophagy in plants will shine light on how autophagy is involved in cell death and defense response in plants.