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1.  Identification of mildew resistance in wild and cultivated Central Asian grape germplasm 
BMC Plant Biology  2013;13:149.
Background
Cultivated grapevines, Vitis vinifera subsp. sativa, evolved from their wild relative, V. vinifera subsp. sylvestris. They were domesticated in Central Asia in the absence of the powdery mildew fungus, Erysiphe necator, which is thought to have originated in North America. However, powdery mildew resistance has previously been discovered in two Central Asian cultivars and in Chinese Vitis species.
Results
A set of 380 unique genotypes were evaluated with data generated from 34 simple sequence repeat (SSR) markers. The set included 306 V. vinifera cultivars, 40 accessions of V. vinifera subsp. sylvestris, and 34 accessions of Vitis species from northern Pakistan, Afghanistan and China. Based on the presence of four SSR alleles previously identified as linked to the powdery mildew resistance locus, Ren1, 10 new mildew resistant genotypes were identified in the test set: eight were V. vinifera cultivars and two were V. vinifera subsp. sylvestris based on flower and seed morphology. Sequence comparison of a 620 bp region that includes the Ren1-linked allele (143 bp) of the co-segregating SSR marker SC8-0071-014, revealed that the ten newly identified genotypes have sequences that are essentially identical to the previously identified mildew resistant V. vinifera cultivars: ‘Kishmish vatkana’ and ‘Karadzhandal’. Kinship analysis determined that three of the newly identified powdery mildew resistant accessions had a relationship with ‘Kishmish vatkana’ and ‘Karadzhandal’, and that six were not related to any other accession in this study set. Clustering procedures assigned accessions into three groups: 1) Chinese species; 2) a mixed group of cultivated and wild V. vinifera; and 3) table grape cultivars, including nine of the powdery mildew resistant accessions. Gene flow was detected among the groups.
Conclusions
This study provides evidence that powdery mildew resistance is present in V. vinifera subsp. sylvestris, the dioecious wild progenitor of the cultivated grape. Four first-degree parent progeny relationships were discovered among the hermaphroditic powdery mildew resistant cultivars, supporting the existence of intentional grape breeding efforts. Although several Chinese grape species are resistant to powdery mildew, no direct genetic link to the resistance found in V. vinifera could be established.
doi:10.1186/1471-2229-13-149
PMCID: PMC3851849  PMID: 24093598
Powdery mildew resistance; Vitis vinifera subsp. sativa; Vitis vinifera subsp. sylvestris; Gene flow
2.  Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii 
BMC Plant Biology  2016;16:170.
Background
Grapevine powdery mildew Erysiphe necator is a major fungal disease in all grape growing countries worldwide. Breeding for resistance to this disease is crucial to avoid extensive fungicide applications that are costly, labor intensive and may have detrimental effects on the environment. In the past decade, Chinese Vitis species have attracted attention from grape breeders because of their strong resistance to powdery mildew and their lack of negative fruit quality attributes that are often present in resistant North American species. In this study, we investigated powdery mildew resistance in multiple accessions of the Chinese species Vitis piasezkii that were collected during the 1980 Sino-American botanical expedition to the western Hubei province of China.
Results
A framework genetic map was developed using simple sequence repeat markers in 277 seedlings of an F1 mapping population arising from a cross of the powdery mildew susceptible Vitis vinifera selection F2-35 and a resistant accession of V. piasezkii DVIT2027. Quantitative trait locus analyses identified two major powdery mildew resistance loci on chromosome 9 (Ren6) and chromosome 19 (Ren7) explaining 74.8 % of the cumulative phenotypic variation. The quantitative trait locus analysis for each locus, in the absence of the other, explained 95.4 % phenotypic variation for Ren6, while Ren7 accounted for 71.9 % of the phenotypic variation. Screening of an additional 259 seedlings of the F1 population and 910 seedlings from four pseudo-backcross populations with SSR markers defined regions of 22 kb and 330 kb for Ren6 and Ren7 in the V. vinifera PN40024 (12X) genome sequence, respectively.
Both R loci operate post-penetration through the induction of programmed cell death, but vary significantly in the speed of response and degree of resistance; Ren6 confers complete resistance whereas Ren7 confers partial resistance to the disease with reduced colony size. A comparison of the kinetics of induction of powdery mildew resistance mediated by Ren6, Ren7 and the Run1 locus from Muscadinia rotundifolia, indicated that the speed and strength of resistance conferred by Ren6 is greater than that of Run1 which, in turn, is superior to that conferred by Ren7.
Conclusions
This is the first report of mapping powdery mildew resistance in the Chinese species V. piasezkii. Two distinct powdery mildew R loci designated Ren6 and Ren7 were found in multiple accessions of this Chinese grape species. Their location on different chromosomes to previously reported powdery mildew resistance R loci offers the potential for grape breeders to combine these R genes with existing powdery mildew R loci to produce grape germplasm with more durable resistance against this rapidly evolving fungal pathogen.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-016-0855-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-016-0855-8
PMCID: PMC4966781  PMID: 27473850
Disease resistance; Powdery mildew; Grape breeding; Vitis piasezkii; Ren6; Ren7
3.  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.
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.
doi:10.1007/s00122-010-1511-6
PMCID: PMC3056998  PMID: 21188350
4.  Comparative transcriptomics of Central Asian Vitis vinifera accessions reveals distinct defense strategies against powdery mildew 
Horticulture Research  2015;2:15037-.
Grape powdery mildew (PM), caused by the biotrophic ascomycete Erysiphe necator, is a devastating fungal disease that affects most Vitis vinifera cultivars. We have previously identified a panel of V. vinifera accessions from Central Asia with partial resistance to PM that possess a Ren1-like local haplotype. In this study, we show that in addition to the typical Ren1-associated late post-penetration resistance, these accessions display a range of different levels of disease development suggesting that alternative alleles or additional genes contribute to determining the outcome of the interaction with the pathogen. To identify potential Ren1-dependent transcriptional responses and functions associated with the different levels of resistance, we sequenced and analyzed the transcriptomes of these Central Asian accessions at two time points of PM infection. Transcriptomes were compared to identify constitutive differences and PM-inducible responses that may underlie their disease resistant phenotype. Responses to E. necator in all resistant accessions were characterized by an early up-regulation of 13 genes, most encoding putative defense functions, and a late down-regulation of 32 genes, enriched in transcriptional regulators and protein kinases. Potential Ren1-dependent responses included a hotspot of co-regulated genes on chromosome 18. We also identified 81 genes whose expression levels and dynamics correlated with the phenotypic differences between the most resistant accessions ‘Karadzhandahal’, DVIT3351.27, and O34-16 and the other genotypes. This study provides a first exploration of the functions associated with varying levels of partial resistance to PM in V. vinifera accessions that can be exploited as sources of genetic resistance in grape breeding programs.
doi:10.1038/hortres.2015.37
PMCID: PMC4591678  PMID: 26504579
5.  Historical Introgression of the Downy Mildew Resistance Gene Rpv12 from the Asian Species Vitis amurensis into Grapevine Varieties 
PLoS ONE  2013;8(4):e61228.
The Amur grape (Vitis amurensis Rupr.) thrives naturally in cool climates of Northeast Asia. Resistance against the introduced pathogen Plasmopara viticola is common among wild ecotypes that were propagated from Manchuria into Chinese vineyards or collected by Soviet botanists in Siberia, and used for the introgression of resistance into wine grapes (Vitis vinifera L.). A QTL analysis revealed a dominant gene Rpv12 that explained 79% of the phenotypic variance for downy mildew resistance and was inherited independently of other resistance genes. A Mendelian component of resistance–a hypersensitive response in leaves challenged with P. viticola–was mapped in an interval of 0.2 cM containing an array of coiled-coil NB-LRR genes on chromosome 14. We sequenced 10-kb genic regions in the Rpv12+ haplotype and identified polymorphisms in 12 varieties of V. vinifera using next-generation sequencing. The combination of two SNPs in single-copy genes flanking the NB-LRR cluster distinguished the resistant haplotype from all others found in 200 accessions of V. vinifera, V. amurensis, and V. amurensis x V. vinifera crosses. The Rpv12+ haplotype is shared by 15 varieties, the most ancestral of which are the century-old ‘Zarja severa’ and ‘Michurinets’. Before this knowledge, the chromosome segment around Rpv12+ became introgressed, shortened, and pyramided with another downy mildew resistance gene from North American grapevines (Rpv3) only by phenotypic selection. Rpv12+ has an additive effect with Rpv3+ to protect vines against natural infections, and confers foliar resistance to strains that are virulent on Rpv3+ plants.
doi:10.1371/journal.pone.0061228
PMCID: PMC3625174  PMID: 23593440
6.  The Novel Gene VpPR4-1 from Vitis pseudoreticulata Increases Powdery Mildew Resistance in Transgenic Vitis vinifera L. 
Pathogenesis-related proteins (PRs) can lead to increased resistance of the whole plant to pathogen attack. Here, we isolate and characterize a PR-4 protein (VpPR4-1) from a wild Chinese grape Vitis pseudoreticulata which shows greatly elevated transcription following powdery mildew infection. Its expression profiles under a number of abiotic stresses were also investigated. Powdery mildew, salicylic acid, and jasmonic acid methyl ester significantly increased the VpPR4-1 induction while NaCl and heat treatments just slightly induced VpPR4-1 expression. Abscisic acid and cold treatment slightly affected the expression level of VpPR4-1. The VpPR4-1 gene was overexpressed in 30 regenerated V. vinifera cv. Red Globe via Agrobacterium tumefaciens-mediated transformation and verified by the Western blot. The 26 transgenic grapevines exhibited higher expression levels of PR-4 protein content than wild-type vines and six of them were inoculated with powdery mildew which showed that the growth of powdery mildew was repressed. The powdery mildew-resistance of Red Globe transformed with VpPR4-1 was enhanced inoculated with powdery mildew. Moreover, other powdery mildew resistant genes were associated with feedback regulation since VpPR4-1 is in abundance. This study demonstrates that PR-4 protein in grapes plays a vital role in defense against powdery mildew invasion.
doi:10.3389/fpls.2016.00695
PMCID: PMC4882328  PMID: 27303413
grapevine; pathogenesis-related protein-4 (PR-4); transformation; powdery mildew; qRT-PCR
7.  A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence 
Journal of Experimental Botany  2016;67(19):5841-5856.
Highlight
The resistance of the Chinese wild grapevine Vitis pseudoreticulata to powdery mildew is shown to differ from effector-triggered immunity by recruiting salicylic acid into phytoalexin synthesis based on a specific stilbene synthase promoter.
Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. ‘Carigane’ (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis.
doi:10.1093/jxb/erw351
PMCID: PMC5066501  PMID: 27702992
Basal immunity; defence; grapevine (Vitis pseudoreticulata); powdery mildew; promoter activity; SA; signalling; stilbene synthase.
8.  Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease 
Horticulture Research  2015;2:15020-.
The most economically important disease of cultivated grapevines worldwide is powdery mildew (PM) caused by the ascomycete fungus Erysiphe necator. The majority of grapevine cultivars used for wine, table grape, and dried fruit production are derived from the Eurasian grape species Vitis vinifera because of its superior aroma and flavor characteristics. However, this species has little genetic resistance against E. necator meaning that grape production is highly dependent on the frequent use of fungicides. The integration of effective genetic resistance into cultivated grapevines would lead to significant financial and environmental benefits and represents a major challenge for viticultural industries and researchers worldwide. This review will outline the strategies being used to increase our understanding of the molecular basis of V. vinifera susceptibility to this fungal pathogen. It will summarize our current knowledge of different resistance loci/genes that have evolved in wild grapevine species to restrict PM infection and assess the potential application of these defense genes in the generation of PM-resistant grapevine germplasm. Finally, it addresses future research priorities which will be important in the rapid identification, evaluation, and deployment of new PM resistance genes which are capable of conferring effective and durable resistance in the vineyard.
doi:10.1038/hortres.2015.20
PMCID: PMC4595975  PMID: 26504571
9.  Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations 
The Plant Journal  2013;76(3):530-544.
Summary
RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ∼80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines.
doi:10.1111/tpj.12307
PMCID: PMC3935411  PMID: 23937694
NB-LRR; pathogen resistance; Solanaceae; target enrichment; next-generation sequencing; Solanum tuberosum Group Phureja clone DM1-3 516 R44; Solanum ruiz-ceballosii; Solanum berthaultii; Solanum lycopersicum; technical advance
10.  Genetic structure in cultivated grapevines is linked to geography and human selection 
BMC Plant Biology  2013;13:25.
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.
doi:10.1186/1471-2229-13-25
PMCID: PMC3598926  PMID: 23394135
11.  Whole genome comparison between table and wine grapes reveals a comprehensive catalog of structural variants 
BMC Plant Biology  2014;14:7.
Background
Grapevine (Vitis vinifera L.) is the most important Mediterranean fruit crop, used to produce both wine and spirits as well as table grape and raisins. Wine and table grape cultivars represent two divergent germplasm pools with different origins and domestication history, as well as differential characteristics for berry size, cluster architecture and berry chemical profile, among others. ‘Sultanina’ plays a pivotal role in modern table grape breeding providing the main source of seedlessness. This cultivar is also one of the most planted for fresh consumption and raisins production. Given its importance, we sequenced it and implemented a novel strategy for the de novo assembly of its highly heterozygous genome.
Results
Our approach produced a draft genome of 466 Mb, recovering 82% of the genes present in the grapevine reference genome; in addition, we identified 240 novel genes. A large number of structural variants and SNPs were identified. Among them, 45 (21 SNPs and 24 INDELs) were experimentally confirmed in ‘Sultanina’ and six SNPs in other 23 table grape varieties. Transposable elements corresponded to ca. 80% of the repetitive sequences involved in structural variants and more than 2,000 genes were affected in their structure by these variants. Some of these genes are likely involved in embryo development, suggesting that they may contribute to seedlessness, a key trait for table grapes.
Conclusions
This work produced the first structural variants and SNPs catalog for grapevine, constituting a novel and very powerful tool for genomic studies in this key fruit crop, particularly useful to support marker assisted breeding in table grapes.
doi:10.1186/1471-2229-14-7
PMCID: PMC3890619  PMID: 24397443
‘Sultanina’ genome; Vitis vinifera L; Structural variants
12.  Identification and Characterization of Erysiphe necator-Responsive MicroRNAs in Chinese Wild Vitis pseudoreticulata by High-Throughput Sequencing 
Grapevine powdery mildew is one of the most damaging fungal diseases. Therefore, a precise understanding of the grapevine disease resistance system becomes a subject of significant importance. Plant microRNAs(miRNAs) have been implicated to play regulatory roles in plant biotic stress responses. In this study, high-throughput sequencing and miRDeep-P were employed to identify miRNAs in Chinese wild Vitis pseudoreticulata leaves following inoculation with Erysiphe necator. Altogether, 126 previously identified microRNAs and 124 novel candidates of miRNA genes were detected. Among them, 43 conserved miRNAs belong to 20 families and 23 non-conserved but previously-known miRNAs belong to 15 families. Following E. necator inoculation, 119 miRNAs were down-regulated and 131 were up-regulated. Furthermore, the expression changes occurring in 32 miRNAs were significant. The expression patterns of some miRNAs were validated by semi-quantitative RT-PCR and qRT-PCR. A total of 485 target genes were predicted and categorized by Gene Ontology (GO). In addition, 14 vvi-miRNAs were screened with 36 targets which may be involved in powdery mildew resistance in grape. Highly accumulated vvi-NewmiR2118 was detected from accession “Baihe-35-1,” whose targets were mostly NBS-LRR resistance genes. It was down-regulated rapidly and strongly in “Baihe-35-1” leaves after inoculated with E. necator, indicating its involvement in grape powdery mildew resistance. Finally, the study verified interaction between vvi-NewmiR2118 and RPP13 by histochemical staining and GUS fluorescence quantitative assay.
doi:10.3389/fpls.2016.00621
PMCID: PMC4885880  PMID: 27303408
Chinese wild Vitis pseudoreticulata; high-throughput sequencing; miRNA identification; powdery mildew resistance; target genes
13.  Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq 
BMC Plant Biology  2014;14:120.
Background
The availability of draft crop plant genomes allows the prediction of the full complement of genes that encode NB-LRR resistance gene homologs, enabling a more targeted breeding for disease resistance. Recently, we developed the RenSeq method to reannotate the full NB-LRR gene complement in potato and to identify novel sequences that were not picked up by the automated gene prediction software. Here, we established RenSeq on the reference genome of tomato (Solanum lycopersicum) Heinz 1706, using 260 previously identified NB-LRR genes in an updated Solanaceae RenSeq bait library.
Result
Using 250-bp MiSeq reads after RenSeq on genomic DNA of Heinz 1706, we identified 105 novel NB-LRR sequences. Reannotation included the splitting of gene models, combination of partial genes to a longer sequence and closing of assembly gaps. Within the draft S. pimpinellifolium LA1589 genome, RenSeq enabled the annotation of 355 NB-LRR genes. The majority of these are however fragmented, with 5′- and 3′-end located on the edges of separate contigs. Phylogenetic analyses show a high conservation of all NB-LRR classes between Heinz 1706, LA1589 and the potato clone DM, suggesting that all sub-families were already present in the last common ancestor. A phylogenetic comparison to the Arabidopsis thaliana NB-LRR complement verifies the high conservation of the more ancient CCRPW8-type NB-LRRs. Use of RenSeq on cDNA from uninfected and late blight-infected tomato leaves allows the avoidance of sequence analysis of non-expressed paralogues.
Conclusion
RenSeq is a promising method to facilitate analysis of plant resistance gene complements. The reannotated tomato NB-LRR complements, phylogenetic relationships and chromosomal locations provided in this paper will provide breeders and scientists with a useful tool to identify novel disease resistance traits. cDNA RenSeq enables for the first time next-gen sequencing approaches targeted to this very low-expressed gene family without the need for normalization.
doi:10.1186/1471-2229-14-120
PMCID: PMC4036795  PMID: 24885638
RenSeq; NB-LRR; cDNA; Gene model; Disease resistance; Paralogous; Plant breeding; Solanum lycopersicum; Solanum pimpinellifolium; Arabidopsis thaliana
14.  Grapevine MLO candidates required for powdery mildew pathogenicity? 
Plant Signaling & Behavior  2009;4(6):522-523.
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.
PMCID: PMC2688300  PMID: 19816131
MLO; powdery mildew; resistance; susceptibility; grapevine
15.  A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance 
BMC Plant Biology  2008;8:66.
Background
Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.
Results
The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.
Conclusion
Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.
doi:10.1186/1471-2229-8-66
PMCID: PMC2442077  PMID: 18554400
16.  VitisNet: “Omics” Integration through Grapevine Molecular Networks 
PLoS ONE  2009;4(12):e8365.
Background
Genomic data release for the grapevine has increased exponentially in the last five years. The Vitis vinifera genome has been sequenced and Vitis EST, transcriptomic, proteomic, and metabolomic tools and data sets continue to be developed. The next critical challenge is to provide biological meaning to this tremendous amount of data by annotating genes and integrating them within their biological context. We have developed and validated a system of Grapevine Molecular Networks (VitisNet).
Methodology/Principal Findings
The sequences from the Vitis vinifera (cv. Pinot Noir PN40024) genome sequencing project and ESTs from the Vitis genus have been paired and the 39,424 resulting unique sequences have been manually annotated. Among these, 13,145 genes have been assigned to 219 networks. The pathway sets include 88 “Metabolic”, 15 “Genetic Information Processing”, 12 “Environmental Information Processing”, 3 “Cellular Processes”, 21 “Transport”, and 80 “Transcription Factors”. The quantitative data is loaded onto molecular networks, allowing the simultaneous visualization of changes in the transcriptome, proteome, and metabolome for a given experiment.
Conclusions/Significance
VitisNet uses manually annotated networks in SBML or XML format, enabling the integration of large datasets, streamlining biological functional processing, and improving the understanding of dynamic processes in systems biology experiments. VitisNet is grounded in the Vitis vinifera genome (currently at 8x coverage) and can be readily updated with subsequent updates of the genome or biochemical discoveries. The molecular network files can be dynamically searched by pathway name or individual genes, proteins, or metabolites through the MetNet Pathway database and web-portal at http://metnet3.vrac.iastate.edu/. All VitisNet files including the manual annotation of the grape genome encompassing pathway names, individual genes, their genome identifier, and chromosome location can be accessed and downloaded from the VitisNet tab at http://vitis-dormancy.sdstate.org.
doi:10.1371/journal.pone.0008365
PMCID: PMC2791446  PMID: 20027228
17.  Cloning and characterization of XiR1, a locus responsible for dagger nematode resistance in grape 
The dagger nematode, Xiphinemaindex, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) × V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks.
doi:10.1007/s00122-010-1349-y
PMCID: PMC2909427  PMID: 20490447
18.  A comprehensive survey of the grapevine VQ gene family and its transcriptional correlation with WRKY proteins 
WRKY proteins are a class of transcription factors (TFs) involved in the regulation of various physiological processes, including the plant response to biotic and abiotic stresses. Recent studies in Arabidopsis have revealed that some WRKY TFs interact with a class of proteins designed as VQ proteins because of their typical conserved motif (FxxxVQxLTG). So far, no information is available about the genomic organization and the function of VQ motif-containing protein in grapevine (Vitis vinifera L). In the current study, we analyzed the 12X V1 prediction of the nearly homozygous PN40024 genotype identifying up to 18 predicted VQ genes (VvVQ). VvVQs phylogenetic and bioinformatic analyses indicated that the intron-exon structures and motif distribution are highly divergent between different members of the grapevine VQ family. Moreover, the analysis of the V. vinifera cv. Corvina expression atlas revealed a tissue- and stage-specific expression of several members of the family which also showed a significant correlation with WRKY TFs. Grapevine VQ genes also exhibited altered expression in response to drought, powdery mildew infection, salicylic acid (SA) and ethylene (ETH) treatments. The present study represents the first characterization of VQ genes in a grapevine genotype and it is a pivotal foundation for further studies aimed at functionally characterizing this mostly unknown grapevine multigenic family.
doi:10.3389/fpls.2015.00417
PMCID: PMC4464145  PMID: 26124765
grapevine; VQ gene family; gene expression; drought treatment; biotic stress; WRKY protein; co-expression network
19.  QTL analysis of flowering time and ripening traits suggests an impact of a genomic region on linkage group 1 in Vitis 
In the recent past, genetic analyses of grapevine focused mainly on the identification of resistance loci for major diseases such as powdery and downy mildew. Currently, breeding programs make intensive use of these results by applying molecular markers linked to the resistance traits. However, modern genetics also allows to address additional agronomic traits that have considerable impact on the selection of grapevine cultivars. In this study, we have used linkage mapping for the identification and characterization of flowering time and ripening traits in a mapping population from a cross of V3125 (‘Schiava Grossa’ × ‘Riesling’) and the interspecific rootstock cultivar ‘Börner’ (Vitis riparia × Vitis cinerea). Comparison of the flowering time QTL mapping with data derived from a second independent segregating population identified several common QTLs. Especially a large region on linkage group 1 proved to be of special interest given the genetic divergence of the parents of the two populations. The proximity of the QTL region contains two CONSTANS-like genes. In accordance with data from other plants such as Arabidopsis thaliana and Oryza sativa, we hypothesize that these genes are major contributors to control the time of flowering in Vitis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-014-2310-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-014-2310-2
PMCID: PMC4145202  PMID: 25112201
20.  VitisCyc: a metabolic pathway knowledgebase for grapevine (Vitis vinifera) 
We have developed VitisCyc, a grapevine-specific metabolic pathway database that allows researchers to (i) search and browse the database for its various components such as metabolic pathways, reactions, compounds, genes and proteins, (ii) compare grapevine metabolic networks with other publicly available plant metabolic networks, and (iii) upload, visualize and analyze high-throughput data such as transcriptomes, proteomes, metabolomes etc. using OMICs-Viewer tool. VitisCyc is based on the genome sequence of the nearly homozygous genotype PN40024 of Vitis vinifera “Pinot Noir” cultivar with 12X v1 annotations and was built on BioCyc platform using Pathway Tools software and MetaCyc reference database. Furthermore, VitisCyc was enriched for plant-specific pathways and grape-specific metabolites, reactions and pathways. Currently VitisCyc harbors 68 super pathways, 362 biosynthesis pathways, 118 catabolic pathways, 5 detoxification pathways, 36 energy related pathways and 6 transport pathways, 10,908 enzymes, 2912 enzymatic reactions, 31 transport reactions and 2024 compounds. VitisCyc, as a community resource, can aid in the discovery of candidate genes and pathways that are regulated during plant growth and development, and in response to biotic and abiotic stress signals generated from a plant's immediate environment. VitisCyc version 3.18 is available online at http://pathways.cgrb.oregonstate.edu.
doi:10.3389/fpls.2014.00644
PMCID: PMC4260676  PMID: 25538713
grape; VitisCyc; grapevine pathway database; Vitis vinifera; microarray
21.  VpRFP1, a novel C4C4-type RING finger protein gene from Chinese wild Vitis pseudoreticulata, functions as a transcriptional activator in defence response of grapevine 
Journal of Experimental Botany  2011;62(15):5671-5682.
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.
doi:10.1093/jxb/err253
PMCID: PMC3223060  PMID: 21862480
C4C4-type RING finger; Chinese wild Vitis pseudoreticulata; disease resistance; powdery mildew; VpRFP1
22.  Berry skin development in Norton grape: Distinct patterns of transcriptional regulation and flavonoid biosynthesis 
BMC Plant Biology  2011;11:7.
Background
The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways.
Results
A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin.
Conclusions
This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.
doi:10.1186/1471-2229-11-7
PMCID: PMC3025947  PMID: 21219654
23.  Sugars, organic acids, and phenolic compounds of ancient grape cultivars (Vitis vinifera L.) from Igdir province of Eastern Turkey 
Biological Research  2015;48(1):2.
Background
The Eurasian grapevine (Vitis vinifera L.) is the most widely cultivated and economically important horticultural crop in the world. As a one of the origin area, Anatolia played an important role in the diversification and spread of the cultivated form V. vinifera ssp. vinifera cultivars and also the wild form V. vinifera ssp. sylvestris ecotypes. Although several biodiversity studies have been conducted with local cultivars in different regions of Anatolia, no information has been reported so far on the biochemical (organic acids, sugars, phenolic acids, vitamin C) and antioxidant diversity of local historical table V. vinifera cultivars grown in Igdir province. In this work, we studied these traits in nine local table grape cultivars viz. ‘Beyaz Kismis’ (synonym name of Sultanina or Thompson seedless), ‘Askeri’, ‘El Hakki’, ‘Kirmizi Kismis’, ‘Inek Emcegi’, ‘Hacabas’, ‘Kerim Gandi’, ‘Yazen Dayi’, and ‘Miskali’ spread in the Igdir province of Eastern part of Turkey.
Results
Variability of all studied parameters is strongly influenced by cultivars (P < 0.01). Among the cultivars investigated, ‘Miskali’ showed the highest citric acid content (0.959 g/l) while ‘Kirmizi Kismis’ produced predominant contents in tartaric acid (12.71 g/l). The highest glucose (16.47 g/100 g) and fructose (15.55 g/100 g) contents were provided with ‘Beyaz Kismis’. ‘Kirmizi Kismis’ cultivar had also the highest quercetin (0.55 mg/l), o-coumaric acid (1.90 mg/l), and caffeic acid (2.73 mg/l) content. The highest ferulic acid (0.94 mg/l), and syringic acid (2.00 mg/l) contents were observed with ‘Beyaz Kismis’ cultivar. The highest antioxidant capacity was obtained as 9.09 μmol TE g-1 from ‘Inek Emcegi’ in TEAC (Trolox equivalent Antioxidant Capacity) assay. ‘Hacabas’ cultivar had the highest vitamin C content of 35.74 mg/100 g.
Conclusions
Present results illustrated that the historical table grape cultivars grown in Igdir province of Eastern part of Turkey contained diverse and valuable sugars, organic acids, phenolic acids, Vitamin C values and demonstrated important antioxidant capacity for human health benefits. Further preservation and use of this gene pool will be helpful to avoid genetic erosion and to promote continued agriculture in the region.
doi:10.1186/0717-6287-48-2
PMCID: PMC4362841  PMID: 25654659
Table grape; Biochemical composition; HPLC; Spectrophotometer; Germplasm characterization
24.  Genetic diversity and population structure assessed by SSR and SNP markers in a large germplasm collection of grape 
BMC Plant Biology  2013;13:39.
Background
The economic importance of grapevine has driven significant efforts in genomics to accelerate the exploitation of Vitis resources for development of new cultivars. However, although a large number of clonally propagated accessions are maintained in grape germplasm collections worldwide, their use for crop improvement is limited by the scarcity of information on genetic diversity, population structure and proper phenotypic assessment. The identification of representative and manageable subset of accessions would facilitate access to the diversity available in large collections. A genome-wide germplasm characterization using molecular markers can offer reliable tools for adjusting the quality and representativeness of such core samples.
Results
We investigated patterns of molecular diversity at 22 common microsatellite loci and 384 single nucleotide polymorphisms (SNPs) in 2273 accessions of domesticated grapevine V. vinifera ssp. sativa, its wild relative V. vinifera ssp. sylvestris, interspecific hybrid cultivars and rootstocks. Despite the large number of putative duplicates and extensive clonal relationships among the accessions, we observed high level of genetic variation. In the total germplasm collection the average genetic diversity, as quantified by the expected heterozygosity, was higher for SSR loci (0.81) than for SNPs (0.34). The analysis of the genetic structure in the grape germplasm collection revealed several levels of stratification. The primary division was between accessions of V. vinifera and non-vinifera, followed by the distinction between wild and domesticated grapevine. Intra-specific subgroups were detected within cultivated grapevine representing different eco-geographic groups. The comparison of a phenological core collection and genetic core collections showed that the latter retained more genetic diversity, while maintaining a similar phenotypic variability.
Conclusions
The comprehensive molecular characterization of our grape germplasm collection contributes to the knowledge about levels and distribution of genetic diversity in the existing resources of Vitis and provides insights into genetic subdivision within the European germplasm. Genotypic and phenotypic information compared in this study may efficiently guide further exploration of this diversity for facilitating its practical use.
doi:10.1186/1471-2229-13-39
PMCID: PMC3610244  PMID: 23497049
Grapevine; Diversity pattern; Population structure; Phenotypic variation; Core collections; Vitis spp
25.  The grapevine gene nomenclature system 
BMC Genomics  2014;15(1):1077.
Background
Grapevine (Vitis vinifera L.) is one of the most important fruit crops in the world and serves as a valuable model for fruit development in woody species. A major breakthrough in grapevine genomics was achieved in 2007 with the sequencing of the Vitis vinifera cv. PN40024 genome. Subsequently, data on structural and functional characterization of grape genes accumulated exponentially. To better exploit the results obtained by the international community, we think that a coordinated nomenclature for gene naming in species with sequenced genomes is essential. It will pave the way for the accumulation of functional data that will enable effective scientific discussion and discovery. The exploitation of data that were generated independently of the genome release is hampered by their heterogeneous nature and by often incompatible and decentralized storage. Classically, large amounts of data describing gene functions are only available in printed articles and therefore remain hardly accessible for automatic text mining. On the other hand, high throughput “Omics” data are typically stored in public repositories, but should be arranged in compendia to better contribute to the annotation and functional characterization of the genes.
Results
With the objective of providing a high quality and highly accessible annotation of grapevine genes, the International Grapevine Genome Project (IGGP) commissioned an international Super-Nomenclature Committee for Grape Gene Annotation (sNCGGa) to coordinate the effort of experts to annotate the grapevine genes. The goal of the committee is to provide a standard nomenclature for locus identifiers and to define conventions for a gene naming system in this paper.
Conclusions
Learning from similar initiatives in other plant species such as Arabidopsis, rice and tomato, a versatile nomenclature system has been developed in anticipation of future genomic developments and annotation issues. The sNCGGa’s first outreach to the grape community has been focused on implementing recommended guidelines for the expert annotators by: (i) providing a common annotation platform that enables community-based gene curation, (ii) developing a gene nomenclature scheme reflecting the biological features of gene products that is consistent with that used in other organisms in order to facilitate comparative analyses.
doi:10.1186/1471-2164-15-1077
PMCID: PMC4299395  PMID: 25481684

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