QTL detection and candidate gene analysis of grape white rot resistance by interspecific grape (Vitis vinifera L. × Vitis davidii Foex.) crossing

Grape white rot,a devastating disease of grapevines caused by Coniella diplodiella(Speg.)Sacc.,leads to significant yield losses in grape.Breeding grape cultivars resistant to white rot is essential to reduce the regular use of chemical treatments.In recent years,Chinese grape species have gained more attention for grape breeding due to their high tolerance to various biotic and abiotic factors along with changing climatic conditions.In this study,we employed whole-genome resequencing(WGR)to genotype the parents of‘Manicure Finger’(Vitis vinifera,female)and‘0940’(Vitis davidii,male),along with 101 F1 mapping population individuals,thereby constructing a linkage genetic map.The linkage map contained 9337 single-nucleotide polymorphism(SNP)markers with an average marker distance of 0.3 cM.After 3 years of phenotypic evaluation of the progeny for white rot resistance,we confirmed one stable quantitative trait locus(QTL)for white rot resistance on chromosome 3,explaining up to 17.9%of the phenotypic variation.For this locus,we used RNA-seq to detect candidate gene expression and identified PR1 as a candidate gene involved in white rot resistance.Finally,we demonstrated that recombinant PR1 protein could inhibit the growth of C.diplodiella and that overexpression of PR1 in susceptible V.vinifera increased grape resistance to the pathogen.

Discovery and genome-guided mapping of REN12 from Vitis amurensis, conferring strong, rapid resistance to grapevine powdery mildew

Powdery mildew resistance genes restrict infection attempts at different stages of pathogenesis.Here,a strong and rapid powdery mildew resistance phenotype was discovered from Vitis amurensis‘PI 588631’that rapidly stopped over 97%of Erysiphe necator conidia,before or immediately after emergence of a secondary hypha from appressoria.This resistance was effective across multiple years of vineyard evaluation on leaves,stems,rachises,and fruit and against a diverse array of E.necator laboratory isolates.Using core genome rhAmpSeq markers,resistance mapped to a single dominant locus(here named REN12)on chromosome 13 near 22.8–27.0 Mb,irrespective of tissue type,explaining up to 86.9%of the phenotypic variation observed on leaves.Shotgun sequencing of recombinant vines using skim-seq technology enabled the locus to be further resolved to a 780 kb region,from 25.15 to 25.93 Mb.RNASeq analysis indicated the allele-specific expression of four resistance genes(NLRs)from the resistant parent.REN12 is one of the strongest powdery mildew resistance loci in grapevine yet documented,and the rhAmpSeq sequences presented here can be directly used for marker-assisted selection or converted to other genotyping platforms.While no virulent isolates were identified among the genetically diverse isolates and wild populations of E.necator tested here,NLR loci like REN12 are often race-specific.Thus,stacking of multiple resistance genes and minimal use of fungicides should enhance the durability of resistance and could enable a 90%reduction in fungicides in low-rainfall climates where few other pathogens attack the foliage or fruit.

Control of ovule development in Vitis vinifera by VvMADS28 and interacting genes

Seedless grapes are increasingly popular throughout the world,and the development of seedless varieties is a major breeding goal.In this study,we demonstrate an essential role for the grapevine MADS-box gene VvMADS28 in morphogenesis of the ovule.We found that VvMADS28 mRNA accumulated in the ovules of a seeded cultivar,‘Red Globe’,throughout the course of ovule and seed development,especially within the integument/seed coat.In contrast,in the seedless cultivar‘Thompson Seedless’,VvMADS28 was expressed only weakly in ovules,and this was associated with increased levels of histone H3 lysine 27 trimethylation(H3K27me3)within the VvMADS28 promoter region.RNAi-mediated transient suppression of VvMADS28 expression in‘Red Globe’led to reduced seed size associated with inhibition of episperm and endosperm cell development.Heterologous overexpression of VvMADS28 in transgenic tomatoes interfered with sepal development and resulted in smaller fruit but did not obviously affect seed size.Assays in yeast cells showed that VvMADS28 is subject to regulation by the transcription factor VvERF98,and that VvMADS28 could interact with the Type I/MβMADS-domain protein VvMADS5.Moreover,through DNA-affinity purification-sequencing(DAP-seq),we found that VvMADS28 protein specifically binds to the promoter of the grapevine WUSCHEL(VvWUS)gene,suggesting that maintenance of the VvMADS28–VvMADS5 dimer and VvWUS expression homeostasis influences seed development.Taken together,our results provide insight into regulatory mechanisms of ovule and seed development associated with VvMADS28.

葡萄(Vitis)不同种叶片中的过氧化物酶同工酶谱及其与抗病性关系的初步研究

本试验测定不同抗黑痘病葡萄种与品种间健康叶片 Px 酶谱变化,未发现以往有人在农作物上报道过的 Px 酶带有规律的增或减的变化。但试验发现除个别种类外,普遍测得感病叶较健叶 Px 同工酶带色泽加深或酶带增多。分析 Px 同工酶对感病有敏感反应的8个不同抗病性葡萄品种健病叶 Px 同工酶谱变化,发现叶片感病后与抗病性有关的变化酶带有 P_(XA)、P_(XB)、P_(XD)带。本文对3种不同抗病性类型(轻感病型、中感病型、高感病型) Px 同工酶的这些酶带变化规律作了初步分析与讨论。

Polyphenols from Vitis thunbergii Sieb. et Zucc.

Aim Isolation and structural elucidation of the constituents from the aerial part of Vitis thunbergii . Methods To isolate chemical constituents, column chromatography and HPLC were used. Physico chemical characterization and spectroscopic analysis were employed for structural identification. Results Eleven polyphenols were isolated and identified. Conclusion Compound 1 is a new compound and its structure was characterized to be 3,5 dimethoxyl 4 hydroxyl phenylpropanol 9 O β D glycopyranoside.

Genome and transcriptome analysis of the grapevine(Vitis vinifera L.) WRKY gene family

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors(TFs)that are involved in the regulation of various physiological processes,such as development and senescence,and in plant response to many biotic and abiotic stresses.Despite the growing number of studies on the genomic organisation of WRKY gene family in different species,little information is available about this family in grapevine(Vitis vinifera L.).In the present study,a total number of 59 putative grapevine WRKY transcription factors(VvWRKYs)were identified based on the analysis of various genomic and proteomic grapevine databases.According to their structural and phylogentic features,the identified grapevine WRKY transcription factors were classified into three main groups.In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine,the VvWRKYs expression profiles were examined in publicly available microarray data.Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues,organs and developmental stages,as well as in response to biotic and abiotic stresses.To also extend our analysis to situations not covered by the arrays and to validate our results,the expression profiles of selected VvWRKYs in response to drought stress,Erysiphe necator(powdery mildew)infection,and hormone treatments(salicilic acid and ethylene),were investigated by quantitative real-time reverse transcription PCR(qRT-PCR).The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine(Vitis vinifera L.)

Downy mildew of grapevine(Vitis vinifera L.),caused by the oomycete pathogen Plasmopara viticola,is one of the most serious concerns for grape production worldwide.It has been widely reported that the pathogenesis-related 4(PR4)protein plays important roles in plant resistance to diseases.However,little is known about the role of PR4 in the defense of grapevine against P.viticola.In this study,we engineered loss-of-function mutations in the VvPR4b gene from the cultivar“Thompson Seedless”using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance.Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred.Infection assays using leaf discs showed that,compared to wild-type plants,the VvPR4b knockout lines had increased susceptibility to P.viticola.This was accompanied by reduced accumulation of reactive oxygen species around stomata.Measurement of the relative genomic abundance of P.viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen.Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine(Vitis vinifera)

Grapevine(Vitis vinifera),one of the most economically important fruit crops in the world,suffers significant yield losses from powdery mildew,a major fungal disease caused by Erysiphe necator.In addition to suppressing host immunity,phytopathogens modulate host proteins termed susceptibility(S)factors to promote their proliferation in plants.In this study,CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)technology was used to enable the targeted mutagenesis of MLO(mildew resistance Locus O)family genes that are thought to serve as S factors for powdery mildew fungi.Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes,VvMLO3 and VvMLO4,in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless.The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%.Among the 20 VvMLO3/4-edited lines obtained,one was homozygous for a single mutation,three harbored biallelic mutations,seven were heterozygous for the mutations,and nine were chimeric,as indicated by the presence of more than two mutated alleles in each line.Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth,while the remaining lines exhibited senescence-like chlorosis and necrosis.Importantly,four VvMLO3-edited lines showed enhanced resistance to powdery mildew,which was associated with host cell death,cell wall apposition(CWA)and H2O2 accumulation.Taken together,our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance,such as disease resistance in grapevines.

Anthocyanin profi les and color properties of red wines made from Vitis davidii and Vitis vinifera grapes

Spine grape(Vitis davidii Foex.)is an important wild grape species native to China.Fifteen red spine grape clones and three red Vitis vinifera grape varieties were used to evaluate the differences in the anthocyanin profiles and color properties of wines made from V.davidii and V.vinifera grapes.Among spine wines,‘Junzi#2’wine had the highest total phenolic and total anthocyanin,‘Xiangzhenzhu’wine had the highest total flavonoids,and‘Junzi#1’wine had the highest total tannin.The anthocyanin compositions of all of the spine wines were dominated by Mv-3,5-diglucoside.The total individual anthocyanin contents in spine wines,except‘Gaoshan#5’,‘Junzi#5055’,‘Junzi#5061’,and‘Junzi#5044’,were signifi cantly higher than in V.vinifera wines.Most of the spine wines had a stronger red intensity and a brighter chroma with a bluer hue than V.vinifera wines.Correlation analysis revealed that the color properties were closely related to the anthocyanin composition.These results suggest that wines made from spine grapes may be useful for the wine industry for their color properties and high individual anthocyanin contents.

Cgr1,a ripe rot resistance QTL in Vitis amurensis‘Shuang Hong’grapevine

Ripe rot is a serious grapevine disease in Vitis L.and Muscadinia(Planch.)Small.However,resistance to this disease has been reported in some oriental Vitis species.To identify resistance-related Quantitative Trait Loci(QTLs)from the Chinese grape species V.amurensis,an F1 population of V.vinifera‘Cabernet Sauvignon’×V.amurensis‘Shuang Hong’was used to map the ripe rot resistance loci expected in‘Shuang Hong’grape.A total of 7598 single nucleotide polymorphisms(SNPs)between the parents were identified in our previous study,and 934 SNPs were selected for genetic map construction.These SNPs are distributed across the 19 chromosomes covering a total of 1665.31 cM in length,with an average of 1.81 cM between markers.Ripe rot resistance phenotypes among the hybrids were evaluated in vitro using excised leaves for three consecutive years from 2016 to 2018;a continuous variation was found among the F1 hybrids,and the Pearson correlation coefficients of the phenotypes scored in all three years were significant at the 0.01 level.Notably,the first QTL reported for resistance to grape ripe rot disease,named Cgr1,was identified on chromosome 14 of‘Shuang Hong’grapevine.Cgr1 could explain up to 19.90%of the phenotypic variance.In addition,a SNP named‘np19345’was identified as a molecular marker closely linked to the peak of Cgr1 and has the potential to be developed as a marker for the Cgr1 resistance haplotype.

The potential role of grape (Vitis vinifera L.) in prevention of threatened abortion via immunomodulatory and anti-inflammatory abilities: a hypothesis

Threatened abortion is a common problem in early pregnancy. This early vaginal bleeding happens in about 25% of pregnant women. The medications including progesterone, uterine muscle relaxant or human chorionic gonadotropin have essential effects in developing pregnancy, but the clinical data are insufficient to prescribe them. In recent decades, medicinal herbs can help us to present new treatments. Grape (Vitis vinifera L.) can protect the fetus from the perspective of Persian medicine. So, we hypothesize about the salutary effects of grape in miscarriage prevention. We found five standard expected mechanisms of grape to prevent threatened abortion: immunomodulatory and anti-inflammatory, anti-oxidant, anti-contraction, hormonal and anti-stress activities. Grape reduces nitric oxide, prostaglandin E2, expression of nuclear factor κB and other pro-inflammatory cytokines like IL (Interleukin)-1β, IL-6, IL-8, and tumor necrosis factor-α. It also elevates anti-inflammatory mediators and expression of peroxisome proliferator-activated receptor-γ. Grape polyphenols have a crucial role in fetus protection with high antioxidant power and other functions such as prevention of stress-triggered abortion with proanthocyanidins, or hormonal effects and inhibition of uterine contractions with resveratrol. So according to these studies, grape probably has effects on the immune and endocrine factors involved in threatened miscarriage.

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures.However,the accumulation of metabolites during cold acclimation(CA)in V.amurensis remains largely unknown.In this study,plantlets of V.amurensis and V.vinifera cv.Muscat of Hamburg were treated at 4℃ for 24 and 72 h,and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry.Most of the identified metabolites,including carbohydrates,amino acids,and organic acids,accumulated in the two types of grape after CA.Galactinol,raffinose,fructose,mannose,glycine,and ascorbate were continuously induced by cold in V.amurensis,but not in Muscat of Hamburg.Twelve metabolites,including isoleucine,valine,proline,2-oxoglutarate,and putrescine,increased in V.amurensis during CA.More galactinol,ascorbate,2-oxoglutarate,and putrescine,accumulated in V.amurensis,but not in Muscat of Hamburg,during CA,which may be responsible for the excellent cold tolerance in V.amurensis.The expression levels of the genes encodingβ-amylase(BAMY),galactinol synthase(GolS),and raffinose synthase(RafS)were evaluated by quantitative reverse transcription-PCR.The expression BAMY(VIT_02s0012 g00170)and RafS(VIT_05s0077 g00840)were primarily responsible for the accumulation of maltose and raffinose,respectively.The accumulation of galactinol was attributed to different members of GolS in the two grapes.In conclusion,these results show the inherent differences in metabolites between V.amurensis and V.vinifera under CA.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

Synthesis of Amurensin H, a New Resveratrol Dimer from the Roots of Vitis Amurensis

Amurensin H(1) is a new resveratrol dimer isolated from the roots of Vitis amurensis Rupr. Its structure was determined by spectroscopic methods. II was synthesized from resveratrol with an oxidative coupling reaction as a key step.

The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.

A novel glycoside hydrolase 74 xyloglucanase CvGH74A is a virulence factor in Coniella vitis

Grape white rot is a destructive fungal disease occurring worldwide.Recently,Coniella vitis was identified as the predominant pathogen causing this disease in China.As the periderms of grape shoots are severely degraded by C.vitis,it was speculated that cell wall-degrading enzymes(CWDEs)might play a key role in the pathogenesis of this disease.Therefore,this study aimed to examine the hydrolytic activity of the CWDEs of C.vitis.The results showed that xylanase(Xy)and xyloglucanase(XEG)had high levels of hydrolytic activity both in vitro and in vivo.Furthermore,a high-virulence fungal strain exhibited higher levels of Xy and XEG activities compared with a low-virulence strain.The genome of the fungus was found to harbor two XEG-coding genes CvGH74A and CvGH74B,which belonged to the glycoside hydrolase(GH)74 family.The expression level of CvGH74A was found to be high during pathogen infection.CvGH74A gene deletion mutants were generated using the split-marker method.The deletion of CvGH74A decreased both the hydrolytic activities of XEG and Xy and also the ability of the fungus to infect the grape leaves.No differences in the hyphal growth,morphology of colonies,or conidiation were found between theΔCvGH74A mutant strains and the wild-type strain.Together,these results suggested that CvGH74A acted as an important virulence factor,and its enzymatic activity might regulate the virulence of the pathogen.This study was novel in reporting that GH74 XEG acted as a virulence factor in C.vitis.

The EST Analysis of A Suppressive Subtraction cDNA Library of Chinese Wild Vitis pseudoreticulata Inoculated with Uncinula necator

Uncinula necator is a worldwide serious fungus disease causing annual heavy lost on grapevine production. In order to get more informations on defense related EST sequences and help breeding program, we constructed and characterized a suppression subtractive hybridization cDNA library with artificially inoculated leaves and control Chinese wild Vitis pseudoreticulata clone Baihe-35-1, which is highly resistant to powdery mildew. In the library, the length of 58 EST fragments known as putative functions varied from 130 to 800 bp, and 60% of the ESTs exhibited high similarity to known sequences in database of GenBank with BLASTX analysis. These genes were involved in stress/defense response, detoxification, signal transduction, disease defense, and etc., and 14 ESTs remained unknown or hypothetical proteins, which may be new genes. The experiment provided an important basis for studying the disease-resistance mechanism and obtaining the genes for the aim of improving grapevine powdery mildew resistance.

Relative quantification of phosphoproteomic changes in grapevine (Vitis vinifera L.) leaves in response to abscisic acid

In a previous transcriptomic analysis,abscisic acid(ABA)was found to affect the abundance of a number of transcripts in leaves of Cabernet Sauvignon grapevines with roots that had been exposed to 10μM ABA for 2 h.Other work has indicated that ABA affects protein abundance and protein phosphorylation as well.In this study we investigated changes in protein abundance and phosphorylation of Cabernet Sauvignon grapevine leaves.Protein abundance was assessed by both label-free and isobaric-label quantitive proteomic methods.Each identified common proteins,but also additional proteins not found with the other method.Overall,several thousand proteins were identified and several hundred were quantified.In addition,hundreds of phosphoproteins were identified.Tens of proteins were found to be affected in the leaf after the roots had been exposed to ABA for 2 h,more than half of them were phosphorylated proteins.Many phosphosites were confirmed and several new ones were identified.ABA increased the abundance of some proteins,but the majority of the proteins had their protein abundance decreased.Many of these proteins were involved in growth and plant organ development,including proteins involved in protein synthesis,photosynthesis,sugar and amino-acid metabolism.This study provides new insights into how ABA regulates plant responses and acclimation to water deficits.

Transcriptome of Erysiphe necator-infected Vitispseudoreticulata leaves provides insight into grapevineresistance to powdery mildew

Powdery mildew(PM),which is caused by the pathogen Erysiphe necator(Schw.)Burr.,is the single most damaging disease of cultivated grapes(Vitis vinifera)worldwide.However,little is known about the transcriptional response of grapes to infection with PM.RNA-seq analysis was used for deep sequencing of the leaf transcriptome to study PM resistance in Chinese wild grapes(V.pseudoreticulata Baihe 35-1)to better understand the interaction between host and pathogen.Greater than 100 million(M)90-nt cDNA reads were sequenced from a cDNA library derived from PM-infected leaves.Among the sequences obtained,6541 genes were differentially expressed(DEG)and were annotated with Gene Ontology terms and by pathway enrichment.The significant categories that were identified included the following:defense,salicylic acid(SA)and jasmonic acid(JA)responses;systemic acquired resistance(SAR);hypersensitive response;plant–pathogen interaction;flavonoid biosynthesis;and plant hormone signal transduction.Various putative secretory proteins were identified,indicating potential defense responses to PMinfection.In all,318 putative R-genes and 183 putative secreted proteins were identified,including the defense-related R-genes BAK1,MRH1 and MLO3 and the defense-related secreted proteins GLP and PR5.The expression patterns of 16 genes were further illuminated by RT-qPCR.The present study identified several candidate genes and pathways that may contribute to PM resistance in grapes and illustrated that RNA-seq is a powerful tool for studying gene expression.The RT-qPCR results reveal that effective resistance responses of grapes to PM include enhancement of JA and SAR responses and accumulation of phytoalexins.

Identification and Characterization of MicroRNAs and Their Targets in Grapevine(Vitis vinifera)

MicroRNAs （miRNAs） are a class of newly identified, small, non-coding RNAs that play vital roles in regulation. Based on miRNAs unique features of expression pattern, evolutionary conservation, secondary structure and genetic requirements for biogenesis, computational predication strategy is adopted to predicate the novel miRNAs. In this research, potential miRNAs and their targets in grapevine （Vitis vinifera） were predicted. We used previously known plant miRNAs against grapevine genome sequence databases to search for potential miRNAs. A total of 81 potential miRNAs were detected following a range of strict filtering criteria. Using these potential miRNA sequences, we could further blast the mRNA database to find the potential targets in this species. Comparative analysis of miRNAs in grapevine and other species reveals that miRNAs exhibit an evolutional conservation, the number and function of miRNAs must have significantly expanded during the evolution of land plants. Furthermore divergence made versatile functions of miRNAs feasible. Cluster of miRNAs likely represents an ancient expression mechanism. Predicted target genes include not only transcription factors but also genes implicated in floral development, signal transduction, diseases and stress response. Till now, little is known about experimental or computational identification of miRNA in grapevine species. Increased knowledge of the biological mechanisms of the grapevine will allow targeted approaches to increase the quality of fruit and reduce the impact of parasites together with stress, which could enable a sustainable, environmentally-sound, farming policv.