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Frenkel O.,Cornell University | Frenkel O.,Israel Agricultural Research Organization | Portillo I.,University of Bologna | Brewer M.T.,Cornell University | And 4 more authors.
Plant Pathology | Year: 2012

Transcriptome sequences of the grape powdery mildew fungus Erysiphe necator were used to develop microsatellite markers (EST-SSRs) to study its relatively unexplored population structure in its centre of diversity in eastern North America. Screening the transcriptome sequences revealed 116 contigs with candidate microsatellites, from which 11 polymorphic microsatellite markers were developed from 31 markers tested. Eight of these markers were used to genotype isolates from different regions and hosts in the eastern USA and compare them to samples from southern France and Italy. Genetic diversity in the eastern USA is much greater than in Europe. Bayesian cluster analyses showed that 10 isolates from North America have high affinities with, but differ from, European group A; these are referred to as A-like isolates. No isolates with close affinity to European group B were found in the eastern USA. Bayesian analyses also detected genetic differentiation between isolates from Vitis rotundifolia and isolates from other Vitis hosts. Genetic differentiation detected between the northeastern and southeastern USA was mostly attributable to the A-like isolates in the southeast, which are significantly more aggressive than the other populations. This research demonstrates that transcriptome sequencing of fungal pathogens is useful for developing genetic markers in protein-coding regions and highlights the role of these markers in population biology studies of E. necator. © 2011 The Authors. Plant Pathology © 2011 BSPP.

News Article | October 29, 2016

Two South Dakota State University researchers are unraveling how the genetic makeup of the grapevine root and variations in climate affect the characteristics expressed in the stem, leaves and fruit. What they discover may help plants adapt to a changing climate. Professor Anne Fennell, who has been doing research on cold hardy grapes for more than 20 years, and assistant professor Qin Ma, whose expertise is in bioinformatics and computational systems biology, are part of a multi-institutional research team working on the five-year, $4.6 million National Science Foundation project. The two SDSU Department of Agronomy, Horticulture and Plant Science researchers will receive nearly $830,000 in total funding to support their work. Fennell will focus on data generation, while Ma will do data mining and modeling using computational resources available through the state's collaborative research center, Biosystems Networks and Translational Research and Extreme Science and Engineering Discovery Environment. Allison Miller, an associate professor in biology at Saint Louis University, is the lead for the NSF project, which also involves researchers from the University of Missouri, Missouri State University, Danforth Plant Center and Missouri Botanical Garden in St. Louis, as well as the Grape Genetics Research Unit of the U.S. Department of Agriculture in Geneva, New York. Grapes are commonly grafted, so the root system is genetically different from the top portion that produces the stems, leaves and fruit, referred to as the scion, explained Fennell. The practice allows producers to graft a desirable variety of grapes onto rootstock that is resistant to pests and diseases. "Though grafting is a standard way of propagating grapes worldwide, we don't have a good handle on how that rootstock affects the scion," Fennell noted. However, she pointed out that researchers know that the genotype of the rootstock impacts the characteristics expressed in the scion, known as its phenotype. "This is a very complex study; each facet of the project addresses a different type of rootstock-scion interaction," she explained. First, the Missouri researchers will find out how three different rootstocks affect variation in the grape scion and how varying amounts of water affect rootstock-scion interactions. All will be grown in the same vineyard. The second portion examines different environments in northern and southern California and how they affect two different scions grafted onto two different rootstocks. "The red grape scion are grafted onto a different rootstock than the white grape scion," Fennell pointed out. In the second year of the project, the research team will begin looking at 200 different rootstock genotypes with the same red grape scion, Marquette, grown at four climatically diverse sites -- Parlier, California; Mt. Vernon, Missouri; Geneva, New York; and Brookings, SD. The rootstocks are a population developed by Jason Londo of the USDA Grape Genetics Research Unit and derived from two native grapevine species, V. rupestris and V. riparia, which have frequently been used to produce commercial rootstocks. Fennell said, "There are a lot of characteristics you can select for in rootstocks. What we're interested in is how the rootstock affects the scion, how the communication of two genetic systems impacts the scion phenotype."

Wakefield L.,Cornell University | Gadoury D.M.,Cornell University | Seem R.C.,Cornell University | Milgroom M.G.,Cornell University | And 3 more authors.
Phytopathology | Year: 2011

Asexual sporulation (conidiation) is coordinately regulated in the grape powdery mildew pathogen Erysiphe necator but nothing is known about its genetic regulation. We hypothesized that genes required for conidiation in other fungi would be upregulated at conidiophore initiation or full conidiation (relative to preconidiation vegetative growth and development of mature ascocarps), and that the obligate biotrophic lifestyle of E. necator would necessitate some novel gene regulation. cDNA amplified fragment length polymorphism analysis with 45 selective primer combinations produced ≈1,600 transcript-derived fragments (TDFs), of which 620 (39%) showed differential expression. TDF sequences were annotated using BLAST analysis of GenBank and of a reference transcriptome for E. necator developed by 454-FLX pyrosequencing of a normalized cDNA library. One-fourth of the differentially expressed, annotated sequences had similarity to fungal genes of unknown function. The remaining genes had annotated function in metabolism, signaling, transcription, transport, and protein fate. As expected, a portion of orthologs known in other fungi to be involved in developmental regulation was upregulated immediately prior to or during conidiation; particularly noteworthy were several genes associated with the light-dependent VeA regulatory system, G-protein signaling (Pth11 and a kelch repeat), and nuclear transport (importin-β and Ran). This work represents the first investigation into differential gene expression during morphogenesis in E. necator and identifies candidate genes and hypotheses for characterization in powdery mildews. Our results indicate that, although control of conidiation in powdery mildews may share some basic elements with established systems, there are significant points of divergence as well, perhaps due, in part, to the obligate biotrophic lifestyle of powdery mildews.

Cadle-Davidson L.,Grape Genetics Research Unit | Cadle-Davidson L.,Cornell University | Wakefield L.,Cornell University | Seem R.C.,Cornell University | Gadoury D.M.,Cornell University
Journal of Phytopathology | Year: 2010

RNA expression profiling of obligately parasitic plant microbes is hampered by the requisite interaction of host and parasite. This can be especially problematic in the case of powdery mildews, such as Erysiphe necator (syn. Uncinula necator), which grow superficially but tightly adhere to the plant epidermis. We developed and refined a simple and efficient technique in which nail polish was used to remove conidia, appressoria, hyphae, conidiophores, and developing ascocarps of E. necator from grapevine (Vitis vinifera) leaves and showed that RNA isolated after removal was not contaminated with V. vinifera RNA. This approach can be applied to expression analyses throughout fungal development and could be extended to other epiphytic pathogens and saprophytes.

Yang Y.,Grape Genetics Research Unit | Jittayasothorn Y.,Grape Genetics Research Unit | Jittayasothorn Y.,U.S. National Institutes of Health | Chronis D.,Robert lley Center For Agriculture And Health | And 3 more authors.
PLoS ONE | Year: 2013

Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5′ end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.

Cousins P.,Grape Genetics Research Unit
Acta Horticulturae | Year: 2014

Grapevine seedlings initially display spiral phyllotaxy of true leaves, then undergo a shift to alternate phyllotaxy with the production of the first lateral meristems (typically tendrils). The node at which the shift from spiral to alternate phyllotaxy occurs varies from about the 4th to about the 12th node on the vine. To investigate the genetic control of the transition from spiral phyllotaxy to alternate phyllotaxy, a population segregating for this trait was developed and screened. The population derived from four female parents and six male parents crossed in a Design 2 mating array (all female parents crossed to all male parents). The female parents were the pistillate flowered rootstock varieties 1613 Couderc, 93-5 Couderc (California clone), Vitis rupestris 187G, and Fercal. The male parents were staminate flowered grape rootstock germplasm, species, and species hybrid selections with diverse backgrounds, including accessions from the USDA ARS National Clonal Germplasm Repository, Davis, California (denoted with DVIT accession numbers): IAC 572, Vitis labrusca Y137 DVIT 1392, Vitis hybrid Y93 DVIT 1519, Vitis hybrid Q126 DVIT 1456, Vitis hybrid Q130 DVIT 1466, and Vitis hybrid R127 DVIT 1490. The species background of the male parents includes V. labrusca, V. mustangensis, V. riparia, V. tiliifolia, and V. rupestris. Seedlings from controlled crosses were grown in individual pots in a greenhouse with artificial illumination to provide 24 h day length. The node number of the first observed lateral meristem was recorded; the goal was 50 seedlings per population for each of 24 populations, although some populations showed poor seed germination. Male and female parents differ from one another at P ≤0.01, with male parents falling into three groups and female parents into two groups based on the mean node of phyllotaxy shift in their seedlings. Narrow sense heritability of the first tendril bearing node is estimated at 0.23 based on breeding value of male parents and at 0.45 based on breeding value of female parents.

Londo J.P.,Grape Genetics Research Unit | Johnson L.M.,Cornell University
Environmental and Experimental Botany | Year: 2014

Cultivated grapevine (Vitis vinifera) is one of the most important agricultural fruit crops in the world and grapevines are often grown in environments very different than the Mediterranean climate from where they were domesticated. To meet the environmental demands of varied climate, grapevine breeders often utilize wild grapevine species for adaptive traits, particularly in regard to abiotic stress resistance. In this study, we characterized both the chilling requirement and days to budburst phenotypes in wild grapevine species. Using survival analysis, we examined the effect of varied lengths of low temperature on budburst in 27 different genotypes of grapevine, including seven wild grapevine species, three cultivated grape varieties, and four hybrid varieties. Results of our two-year study demonstrate a wide range in both intraspecific and interspecific variation in these traits. Trends within the data allow us to categorize low and high chill species. Correlated with these categories are fast and slow maximal budburst phenotypes. When compared with geographic distribution of species and genotypes, patterns between northern and southern populations suggest that chilling requirement and budburst rate are adaptive traits. © 2013.

Centinari M.,Pennsylvania State University | Smith M.S.,Pennsylvania State University | Londo J.P.,Grape Genetics Research Unit
HortScience | Year: 2016

Spring frosts and subsequent crop losses threaten the economic sustainability of fruit crop producers all over the world. This study used a controlled-freezing technique to impose a post-budburst freezing stress to grapevine shoots forced from one-node cuttings [’Albariño’, ‘Cabernet Franc’, ‘Cabernet Sauvignon’, and ‘Pinot Grigio’ (Vitis vinifera)] and whole plants [’Noiret’ (Vitis hybrid)]. Our goal was to investigate the incidence of freeze injury among cultivars, stage of phenological development, and a potassium salt-based fertilizer (KDL) with potential cryoprotectant activity. Among the V. vinifera cultivars, the incidence of mortality of shoots exposed to - 3.5 °C was highest for ‘Albariño’ (71%) and lowest for ‘Cabernet Sauvignon’ (51%). Cuttings sprayed withKDL 24 hours before cold temperature exposure exhibited 16% lower shoot mortality and lower osmotic potential (Ψs) (-0.92 MPa) than the unsprayed cuttings (-0.77 MPa). However, application of KDL did not impact shoot mortality for whole ‘Noiret’ vines. Mortality for ‘Noiret’ shoots greatly increased with the advancement of phenological development, ranging from 10% in wooly buds to 78% in shoots ≈ 10-cm long. The practical significance of KDL remains questionable; cultivar selection still appears to be a more reliable method for avoiding spring frost, by planting late bursting cultivars in more frost-prone areas. © 2016 American Society for Horticultural Science. All rights reserved.

Gadoury D.M.,Cornell University | Wakefield L.M.,Cornell University | Cadle-Davidson L.,Grape Genetics Research Unit | Dry I.B.,CSIRO | Seem R.C.,Cornell University
Phytopathology | Year: 2012

Initiation of asexual sporulation in powdery mildews is preceded by a period of superficial vegetative growth of mildew colonies. We found evidence of a quorum-sensing signal in Erysiphe necator that was promulgated at the colony center and stimulated conidiation throughout the colony. Removal of the colony center after putative signal promulgation had no impact upon timing of sporulation by 48-h-old hyphae at the colony margin. However, removal of the colony center before signaling nearly doubled the latent period. A relationship between inoculum density and latent period was also observed, with latent period decreasing as the number of conidia deposited per square millimeter was increased. The effect was most pronounced at the lowest inoculum densities, with little decrease of the latent period as the density of inoculation increased above 10 spores/mm. Furthermore, light was shown to be necessary to initiate conidiation of sporulation-competent colonies. When plants were inoculated and maintained in a day-and-night cycle for 36 h but subjected to darkness after 36 h, colonies kept in darkness failed to sporulate for several days after plants kept in light had sporulated. Once returned to light, the dark-suppression was immediately reversed, and sporulation commenced within 12 h. Merging of colonies of compatible mating types resulted in near-cessation of sporulation, both in the region of merging and in more distant parts of the colonies. Colonies continued to expand but stopped producing new conidiophores once pairing of compatible mating types had occurred, and extant conidiophores stopped producing new conidia. Therefore, in addition to a quorum-sensing signal to initiate conidiation, there appears to be either signal repression or another signal that causes conidiation to cease once pairing has occurred and the pathogen has initiated the ascigerous stage for overwintering. © 2012 The American Phytopathological Society.

PubMed | Grape Genetics Research Unit and China Agricultural University
Type: | Journal: BMC plant biology | Year: 2015

Terpenes are of great interest to winemakers because of their extremely low perception thresholds and pleasant floral odors. Even for the same variety, terpene profile can be substantially different for grapevine growing environments. Recently a series of genes required for terpene biosynthesis were biochemically characterized in grape berries. However, the genes that dominate the differential terpene accumulation of grape berries between regions have yet to be identified.Free and glycosidically-bound terpenes were identified and quantified using gas chromatography-mass spectrometry (GC-MS) technique. The transcription expression profiling of the genes was obtained by RNA sequencing and part of the results were verified by quantitative real time PCR (QPCR). The gene co-expression networks were constructed with the Cytoscape software v 2.8.2 ( Blanc a Petits Grains berries were collected from two wine-producing regions with strikingly different climates, Gaotai (GT) in Gansu Province and Changli (CL) in Hebei Province in China, at four developmental stages for two consecutive years. GC-MS analysis demonstrated that both free and glycosidically bound terpenes accumulated primarily after veraison and that mature grape berries from CL contained significantly higher concentrations of free and glycosidically bound terpenes than berries from GT. Transcriptome analysis revealed that some key genes involved in terpene biosynthesis were markedly up-regulated in the CL region. Particularly in the MEP pathway, the expression of VviHDR (1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase) paralleled with the accumulation of terpenes, which can promote the flow of isopentenyl diphosphate (IPP) into the terpene synthetic pathway. The glycosidically bound monoterpenes accumulated differentially along with maturation in both regions, which is synchronous with the expression of a monoterpene glucosyltransferase gene (VviUGT85A2L4 (VviGT14)). Other genes were also found to be related to the differential accumulation of terpenes and monoterpene glycosides in the grapes between regions. Transcription factors that could regulate terpene synthesis were predicted through gene co-expression network analysis. Additionally, the genes involved in abscisic acid (ABA) and ethylene signal responses were expressed at high levels earlier in GT grapes than in CL grapes.Differential production of free and glycosidically-bound terpenes in grape berries across GT and CL regions should be related at least to the expression of both VviHDR and VviUGT85A2L4 (VviGT14). Considering the expression patterns of both transcription factors and mature-related genes, we infer that less rainfall and stronger sunshine in the GT region could initiate the earlier expression of ripening-related genes and accelerate the berry maturation, eventually limiting the production of terpene volatiles.

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