Development and application of KASP marker for high throughput detection of the seedless trait in grapevine

Molecular marker-assisted selection(MAS) can significantly accelerate and improve the efficiency of the breeding process in seedless grape cultivars. In this study, we developed the KASP_VviAGL11 and VviAGL11_410 markers based on a single nucleotide polymorphism(SNP) site(Chr18: 26889437(A/C)) of the VviAGL11 gene, and compared them with previously reported SSR markers p3_VvAGL11 and 5U_VviAGL11 by testing 101 cultivars and 81 F1 hybrid progenies. The results showed that both of the proposed markers obtained 100% accuracy rates in detecting allele A, which was closely associated with the seedless trait in grapes, while p3_VvAGL11 and 5U_VviAGL11 had lower accuracy rates due to their tendency to produce false positives. After careful evaluation of the technical advantages and disadvantages associated with these markers, we concluded that KASP_VviAGL11 was superior in terms of simplicity,cost-effectiveness, efficiency, and accuracy. Thus, we optimized the process of molecular MAS for seedless grapes,focusing on the KASP_VviAGL11 marker as a central component, to provide key technical support for the development of new seedless grape cultivars.

An effector of Erysiphe necator translocates to chloroplasts and plasma membrane to suppress host immunity in grapevine

The powdery mildew(Erysiphe necator)is a prevalent pathogen hampering grapevine growth in the vineyard.An arsenal of candidate secreted effector proteins(CSEPs)was encoded in the E.necator genome,but it is largely unclear what role CSEPs plays during the E.necator infection.In the present study,we identified a secreted effector CSEP080 of E.necator,which was located in plant chloroplasts and plasma membrane.Transient expressing CSEP080 promotes plant photosynthesis and inhibits INF1-induced cell death in tobacco leaves.We found that CSEP080 was a necessary effector for the E.necator pathogenicity,which interacted with grapevine chloroplast protein VviB6f(cytochrome b6-f complex iron–sulfur subunit),affecting plant photosynthesis.Transient silencing VviB6f increased the plant hydrogen peroxide production,and the plant resistance to powdery mildew.In addition,CSEP080 manipulated the VviPE(pectinesterase)to promote pectin degradation.Our results demonstrated the molecular mechanisms that an effector of E.necator translocates to host chloroplasts and plasma membrane,which suppresses with the grapevine immunity system by targeting the chloroplast protein VviB6f to suppress hydrogen peroxide accumulation and manipulating VviPE to promote pectin degradation.

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.

Advancements in plant regeneration and genetic transformation of grapevine(Vitis spp.)

Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands.Molecular genetic techniques in particular,associated with rapid technological advancements,provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance,quality,stress tolerance and disease resistance.To date,several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation,and transgenic grape lines have been obtained using established regeneration systems.Nevertheless,a wide range of factors,including genotype,explant source and culture medium,have been shown to affect the efficiency of plant regeneration.Moreover,the selection and use of acceptor materials,bacterial strain and cell density,selectable markers and selection methods also influence transformation efficiency.This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors,and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.

Spatial Distribution of Soil Organic Matter and Nutrients in the Pear Orchard Under Clean and Sod Cultivation Models

The soil organic matter and nutrients are fundamental for the sustainability of pear production, but little is known about the spatial distribution of soil organic matter and nutrients in a pear orchard. With the soil of the pear (cv. Dangshansu on P.betulifolia Bunge. rootstock) orchard under clean and sod cultivation models as test materials, the experiment was conducted to evaluate spatial variability of soil organic matter (SOM), total nitrogen (STN), total phosphorus (STP), total potassium (STK), available nitrogen (SAN), and available potassium (SAK) in and between rows at different soil depths (0-60 cm). The SOM, STN, STP, STK, SAN and SAK of the different soil layers under the two tillage models were different in the vertical direction. The SOM, STN, STP and SAN in the 0-20 cm soil layer were higher than those in the 20-40 and 40-60 cm soil layers. The STK of 40-60 cm soil layer was higher than that in the 0-20 and 20-40 cm soil layers. The STK increased with the depth of soil in the vertical direction in the clean cultivated pear orchard. Variability of the SOM, STN, STP, STK, SAN and SAK of sample sites in between rows of the same soil layer was found in the pear orchard soil in the horizontal direction under clean and sod cultivation management systems, except that STK of all sites did not show the difference in identical soil layers in the pear orchard under clean cultivation. The sod cultivation model improved the SOM, STN, and STK in the 0-20 cm soil layer in the pear orchard, and the three components increased by 12.8, 12.7 and 7.3% compared to clean cultivation, respectively. The results can be applicable to plan collection of orchard soil samples, assess orchard soil quality, and improve orchard soil management practices.

Grapevine VlbZIP30 improves drought resistance by directly activating VvNAC17 and promoting lignin biosynthesis through the regulation of three peroxidase genes

Drought stress severely affects grapevine quality and yield,and recent reports have revealed that lignin plays an important role in protection from drought stress.Since little is known about lignin-mediated drought resistance in grapevine,we investigated its significance.Herein,we show that VlbZIP30 mediates drought resistance by activating the expression of lignin biosynthetic genes and increasing lignin deposition.Transgenic grapevine plants overexpressing VlbZIP30 exhibited lignin deposition(mainly G and S monomers)in the stem secondary xylem under control conditions,which resulted from the upregulated expression of VvPRX4 and VvPRX72.Overexpression of VlbZIP30 improves drought tolerance,characterized by a reduction in the water loss rate,maintenance of an effective photosynthesis rate,and increased lignin content(mainly G monomer)in leaves under drought conditions.Electrophoretic mobility shift assay,luciferase reporter assays,and chromatin immunoprecipitation-qPCR assays indicated that VlbZIP30 directly binds to the G-box cis-element in the promoters of lignin biosynthetic(VvPRX N1)and drought-responsive(VvNAC17)genes to regulate their expression.In summary,we report a novel VlbZIP30-mediated mechanism linking lignification and drought tolerance in grapevine.The results of this study may be of value for the development of molecular breeding strategies to produce drought-resistant fruit crops.

VqMAPK3/VqMAPK6, VqWRKY33, and VqNSTS3 constitute a regulatory node in enhancing resistance to powdery mildew in grapevine

Grapevine powdery mildew is caused by Erysiphe necator,which seriously harms grape production in the world.Stilbene synthase makes phytoalexins that contribute to the resistance of grapevine against powdery mildew.A novel VqNSTS3 was identified and cloned from Chinese wild Vitis quinquangularis accession Danfeng-2.The novel VqNSTS3 was transferred into susceptible‘Thompson Seedless’by Agrobacterium-mediated transformation.The transgenic plants showed resistance to the disease and activated other resistance-related genes.VqNSTS3 expression in grapevine is regulated by VqWRKY33,and which binds to TTGACC in the VqNSTS3 promoter.Furthermore,VqWRKY33 was phosphorylated by VqMAPK3/VqMAPK6 and thus led to enhanced signal transduction and increased VqNSTS3 expression.ProVqNSTS3::VqNSTS3-GFP of transgenic VqNSTS3 in Arabidopsis thaliana was observed to move to and wrap the pathogen’s haustoria and block invasion by Golovinomyces cichoracearum.These results demonstrate that stilbene accumulation of novel VqNSTS3 of the Chinese wild Vitis quinquangularis accession Danfeng-2 prevented pathogen invasion and enhanced resistance to powdery mildew.Therefore,VqNSTS3 can be used in generating powdery mildew-resistant grapevines.

Importin-αs are required for the nuclear localization and function of the Plasmopara viticola effector PvAVH53

Plant pathogenic oomycetes deliver a troop of effector proteins into the nucleus of host cells to manipulate plant cellular immunity and promote colonization.Recently,researchers have focused on identifying how effectors are transferred into the host cell nucleus,as well as the identity of the nuclear targets.In this study,we found that the RxLR effector PvAVH53 from the grapevine(Vitis vinifera)oomycete pathogen Plasmopara viticola physically interacts with grapevine nuclear import factor importin alphas(VvImpαand VvImpα4),localizes to the nucleus and triggers cell death when transiently expressed in tobacco(Nicotiana benthamiana)cells.Deletion of a nuclear localization signal(NLS)sequence from PvAVH53 or addition of a nuclear export signal(NES)sequence disrupted the nuclear localization of PvAVH53 and attenuated its ability to trigger cell death.Suppression of two tobacco importin-αgenes,namely,NbImp-α1 and NbImp-α2,by virus-induced gene silencing(VIGS)also disrupted the nuclear localization and ability of PvAVH53 to induce cell death.Likewise,we transiently silenced the expression of VvImpα/α4 in grape through CRISPR/Cas13a,which has been reported to target RNA in vivo.Finally,we found that attenuating the expression of the Importin-αs genes resulted in increased susceptibility to the oomycete pathogen Phytophthora capsici in N.benthamiana and P.viticola in V.vinifera.Our results demonstrate that importin-αs are required for the nuclear localization and function of PvAVH53 and are essential for host innate immunity.The findings provide insight into the functions of importin-αs in grapevine against downy mildew.

Comparative transcriptomic analysis highlights contrasting levels of resistance of Vitis vinifera and Vitis amurensis to Botrytis cinerea

Botrytis cinerea is a major grapevine(Vitis spp.)pathogen,but some genotypes differ in their degree of resistance.For example,the Vitis vinifera cultivar Red Globe(RG)is highly susceptible,but V.amurensis Rupr Shuangyou(SY)is highly resistant.Here,we used RNA sequencing analysis to characterize the transcriptome responses of these two genotypes to B.cinerea inoculation at an early infection stage.Approximately a quarter of the genes in RG presented significant changes in transcript levels during infection,the number of which was greater than that in the SY leaves.The genes differentially expressed between infected leaves of SY and RG included those associated with cell surface structure,oxidation,cell death and C/N metabolism.We found evidence that an imbalance in the levels of reactive oxygen species(ROS)and redox homeostasis probably contributed to the susceptibility of RG to B.cinerea.SY leaves had strong antioxidant capacities and improved ROS homeostasis following infection.Regulatory network prediction suggested that WRKY and MYB transcription factors are associated with the abscisic acid pathway.Weighted gene correlation network analysis highlighted preinfection features of SY that might contribute to its increased resistance.Moreover,overexpression of VaWRKY10 in Arabidopsis thaliana and V.vinifera Thompson Seedless enhanced resistance to B.cinerea.Collectively,our study provides a high-resolution view of the transcriptional changes of grapevine in response to B.cinerea infection and novel insights into the underlying resistance mechanisms.

Ectopic expression of VvFUS3,B3-domain transcription factor,in tomato influences seed development via affecting endoreduplication and hormones

FUSCA3(FUS3)is a member of B3-domain transcription factor family and master regulator of seed development.It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle,especially in seed germination,dormancy,embryo formation,seed and fruit development,and maturation.However,there is limited information about its functions in seed and fruit development of grapevine.In this study,we expressed VvFUS3 in tomato for its functional characterization.Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size.Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected.However,there were no obvious differences in pollen size,shape,and viability,suggesting that VvFUS3 affects seed development but not the pollen grains.Moreover,the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3.These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size.In conclusion,our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.

Proteomic analysis of early-stage incompatible and compatible interactions between grapevine and P.viticola

Wild grapevines can show strong resistance to the downy mildew pathogen P.viticola,but the associated mechanisms are poorly described,especially at early stages of infection.Here,we performed comparative proteomic analyses of grapevine leaves from the resistant genotype V.davidii“LiuBa-8”(LB)and susceptible V.vinifera“Pinot Noir”(PN)12h after inoculation with P.viticola.By employing the iTRAQ technique,a total of 444 and 349 differentially expressed proteins(DEPs)were identified in LB and PN,respectively.The majority of these DEPs were related to photosynthesis,respiration,cell wall modification,protein metabolism,stress,and redox homeostasis.Compared with PN,LB showed fewer downregulated proteins associated with photosynthesis and more upregulated proteins associated with metabolism.At least a subset of PR proteins(PR10.2 and PR10.3)was upregulated upon inoculation in both genotypes,whereas HSP(HSP70.2 and HSP90.6)and cell wall-related XTH and BXL1 proteins were specifically upregulated in LB and PN,respectively.In the incompatible interaction,ROS signaling was evident by the accumulation of H_(2)O_(2),and multiple APX and GST proteins were upregulated.These DEPs may play crucial roles in the grapevine response to downy mildew.Our results provide new insights into molecular events associated with downy mildew resistance in grapevine,which may be exploited to develop novel protection strategies against this disease.

Identification of potential key genes in resveratrol biosynthesis via transcriptional analyses of berry development in grapevine(Vitis spp.)genotypes varying in trans-resveratrol content

Resveratrol is an important secondary metabolite not only owing to its function as a phytoalexin,but also its potential benefits to human health.In this study,the content of trans-resveratrol was documented in seven accessions of grapevine,in the seed,pulp and skin of berries,and at three developmental stages.The highest amount(2.99μg g^(−1) FW)was found in the skin of berries at the ripe stage from V.amurensis‘Tonghua-3'.Resveratrol was not detected in several samples,including skin of berries at the green hard or véraison stage from V.davidii‘Tangwei'.We carried out transcriptional profiling of developing‘Tonghua-3'and‘Tangwei'berries to identify gene expression patterns that may be linked with the difference in resveratrol content between these accessions.The expression levels of several differentially expressed genes(DEGs)with presumed function in resveratrol biosynthesis,including STILBENE SYNTHASEs(STSs),CINNAMATE 4-HYDROXYLASEs(C4Hs)and 4-COUMARATE-COA LIGASEs(4CLs),were significantly higher in‘Tonghua-3',than in'Tangwei'during the véraison and ripe stages.Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses suggested that these DEGs were enriched for multiple biological processes at the three stages of fruit development.Additionally,we identified a total of 36 transcription factors,including MYBs,WRKYs,ERFs,bHLHs and bZIPs,that were coexpressed with 17 STSs via a weighted gene co-expression network analysis,suggesting roles as regulators of resveratrol biosynthesis.Overall,these findings provide insight into genotypic differences in resveratrol biosynthesis in grapevine,as well as the molecular genetics of its regulation.

Pathogen development and host responses to Plasmopara viticola in resistant and susceptible grapevines:an ultrastructural study

The downy mildew disease in grapevines is caused by Plasmopara viticola.This disease poses a serious threat wherever viticulture is practiced.Wild Vitis species showing resistance to P.viticola offer a promising pathway to develop new grapevine cultivars resistant to P.viticola which will allow reduced use of environmentally unfriendly fungicides.Here,transmission and scanning microscopy was used to compare the resistance responses to downy mildew of three resistant genotypes of V.davidii var.cyanocarpa,V.piasesezkii and V.pseudoreticulata and the suceptible V.vinifera cultivar‘Pinot Noir’.Following inoculation with sporangia of P.viticola isolate‘YL’on V.vinifera cv.‘Pinot Noir’,the infection was characterized by a rapid spread of intercellular hyphae,a high frequency of haustorium formation within the host’s mesophyll cells,the production of sporangia and by the absence of host-cell necrosis.In contrast zoospores were collapsed in the resistant V.pseudoreticulata‘Baihe-35-1’,or secretions appeared arround stomata at the beginning of the infection period in V.davidii var.cyanocarpa‘Langao-5’and V.piasezkii‘Liuba-8’.The main characteristics of the resistance responses were the rapid depositions of callose and the appearance of empty hyphae and the plasmolysis of penetrated tissue.Moreover,collapsed haustoria were observed in V.davidii var.cyanocarpa‘Langao-5’at 5 days post inoculation(dpi)and in V.piasezkii‘Liuba-8’at 7 dpi.Lastly,necrosis extended beyond the zone of restricted colonization in all three resistant genotypes.Sporangia were absent in V.piasezkii‘Liuba-8’and greatly decreased in V.davidii var.cyanocarpa‘Langao-5’and in V.pseudoreticulata‘Baihe-35-1’compared with in V.vinifera cv.‘Pinot Noir’.Overall,these results provide insights into the cellular biological basis of the incompatible interactions between the pathogen and the host.They indicate a number of several resistant Chinese wild species that could be used in developing new cultivars having good levels of downy mildew resistance.

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.

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.

VlbZIP30 of grapevine functions in dehydration tolerance via the abscisic acid core signaling pathway

Drought stress limits the growth and development of grapevines,thereby reducing productivity,but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized.Here,we characterized a group A bZIP gene from“Kyoho”grapevine,VlbZIP30,which was shown to be induced by abscisic acid(ABA)and dehydration stress.Overexpression of VlbZIP30 in transgenic Arabidopsis thaliana enhanced dehydration tolerance.Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage.We identified an A.thaliana G-box motif(CACGTG)and a potential grapevine G-box motif(MCACGTGK)in the promoters of the 39 selected A.thaliana genes upregulated in the transgenic plants and in the 35 grapevine homologs,respectively.Subsequently,using two grapevine-related databases,we found that 74%(23/31)and 84%(21/25)of the detected grapevine genes were significantly upregulated by ABA and drought stress,respectively,suggesting that these genes are involved in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine.We propose that VlbZIP30 functions as a positive regulator of dehydration-responsive signaling in the ABA core signaling pathway.

VqMYB154 promotes polygene expression and enhances resistance to pathogens in Chinese wild grapevine

Resveratrol plays a crucial phytoalexin role in the grapevine and is beneficial to human health.However,the molecular mechanism of resveratrol accumulation in the enhancement of disease resistance is unclear.Here,we report that the transcription factor VqMYB154 from Vitis quinquangularis accession Danfeng-2 is strongly expressed under artificial inoculation with Uncinula necator and regulates resveratrol accumulation.Unlike its homolog,VqMYB154 has a pathogen-induced promoter and responds to stimulation by U.necator,Pseudomonas syringae,and other treatments.Yeast one-hybrid and GUS activity assays confirmed that VqMYB154 can activate the stilbene synthase genes VqSTS9,VqSTS32,and VqSTS42 by directly binding to their promoters.Overexpression of VqMYB154 in grape leaves resulted in activation of the stilbene pathway,upregulation of STS genes,and accumulation of stilbenoids.In addition,heterologous overexpression of VqMYB154 in Arabidopsis activated resistance-related genes and resulted in greater programmed cell death and accumulation of reactive oxygen species,which led to resistance against P.syringae.These results suggest that the transcription factor VqMYB154 from V.quinquangularis accession Danfeng-2 participates in the regulatory mechanism that improves the biosynthesis and accumulation of stilbenes and enhances resistance to disease in grapevine.

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

The CRISPR(clustered regularly interspaced short palindromic repeats)-associated protein 9(Cas9)system is a powerful tool for targeted genome editing,with applications that include plant biotechnology and functional genomics research.However,the specificity of Cas9 targeting is poorly investigated in many plant species,including fruit trees.To assess the off-target mutation rate in grapevine(Vitis vinifera),we performed whole-genome sequencing(WGS)of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type(WT)plants.In total,we identified between 202,008 and 272,397 single nucleotide polymorphisms(SNPs)and between 26,391 and 55,414 insertions/deletions(indels)in the seven Cas9-edited grapevine plants compared with the three WT plants.Subsequently,3272 potential off-target sites were selected for further analysis.Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing.In addition,we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome(PN40024)but no true off-target mutations.In conclusion,we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

Identification of long non-coding RNAs in response to downy mildew stress in grape

The importance of long non-coding RNA in plants has been reported more frequently in recent years,but there has been few specific reports on lncRNAs in grape,especially in terms of disease resistance.We performed RNA-seq on grape leaves of two species(Vitis piasezkii accession Liuba-8,Vitis vinifera cultivar Pinot Noir)sampled at six time points after inoculation,and 4011 possible lncRNAs were identified.The characteristics of grape lncRNAs were analyzed,and it was found that lncRNAs showed relatively consistent characteristics with the reported lncRNAs in model plants.3,643 lncRNAs were predicted that have cis-regulatory effects on 6,622 protein-coding genes and 91 DElncRNAs were revealed to be coexpressed with its trans-regulated coding genes.One hundred and seventeen grape microRNAs were predicted to potentially target 184 lncRNAs and six lncRNAs were predicted to be endogenous targeting mimics of 15 microRNAs,among which some miRNAs have been reported in grape disease resistance.At six time points,LncRNAs showed different expression levels and different expression patterns in two species,suggesting that lncRNAs may have a certain regulatory effect on resistance to downy mildew in grape.Finally,a lncRNA MSTRG.12742.1 which may play a positive role in grape downy mildew resistance was verified by transient transformation.Its potential target gene,VIT_204s0008g02671.1,encodes cryptochrome DASH which may regulate stomatal opening and closing of plant leaves.In this study,we provided the systematic identification of lncRNAs in the course of downy mildew of grape,laying a foundation for further studies on downy mildew and lncRNAs of grape in the future.

QTL-seq analysis of the seed size trait in grape provides new molecular insights on seedlessness

Seedlessness in grape(Vitis vinifera)is an important commercial trait for both the fresh and drying markets.However,despite numerous studies,the mechanisms and key genes regulating grape seedlessness are mostly unknown.In this study,we sequenced the genomes of the V.vinifera seeded cultivar‘Red Globe’,the seedless cultivar‘Centennial Seedless’,and the derived hybrids.Nonsynonymous single nucleotide polymorphisms(SNPs)were identified by genome sequencing and analyzed using published transcriptome data.Nonsynonymous SNPs occurred in genes related to seed development,which were identified as protein kinases,transcription factors,and cytochrome P450 s and showed differential expression during ovule development in both seeded and seedless grapes.These nonsynonymous SNP-associated genes were mainly involved in biological processes such as hormone balance,seed coat and endosperm development,reproductive organ development,oxidation and reduction,senescence and cell death.A potential quantitative trait locus(QTL)region associated with seed size was characterized based on the SNP-index,and expression analysis of candidate genes in the QTL region during ovule development in multiple seeded and seedless grape cultivars were conducted.Three SNPs were further subjected to SNa Pshot analysis and one SNP in G8 showed 67.5%efficiency in the grape progeny validation.Overall,the data obtained in this study shed light on the differences in seed development between seeded and seedless progeny at the genomic level,which provides valuable resources for future functional studies and grape breeding.