Agronomic evaluation of eight 41 B×110 richter grapevine genotypes as rootstock candidates for mediterranean viticulture

Choosing the most appropriate rootstock(s)is a key decision for the profitability of vineyards;therefore,there must be a sufficient range of rootstocks in the market adapted to different environmental conditions and production objectives.However,rootstock-breeding programs have been scarce in recent decades,and most of the rootstocks used today were bred a century ago,when the needs of the sector were very different from today.In this work,we aimed to evaluate new rootstock candidates before their introduction in the market.An agronomic evaluation was conducted on eight novel rootstock genotypes obtained from the first generation of the cross-pollination of 41 B Millardet et de Grasset(41 B)and 110 Richter(110 R)grafted with‘Syrah’and‘Tempranillo’and planted in a typical vineyard of the Ebro Valley in Spain.During the four consecutive growing seasons(2016–2019),growth,yield and berry composition parameters at harvest were collected.A linear mixed-effects model was constructed,considering year and block as random effects.Multiple factor analysis and hierarchical clustering on principal components were performed to establish clusters of genotypes with similar behaviour.The rootstock candidates showed a very wide performance range compared to their parents.The trial allowed us to identify two very promising candidates(RG8 and RG10),whose registration as commercial rootstocks is already in progress.

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.

VitisMod:一个葡萄基因共表达数据库

葡萄转录组已在各种组织、发育阶段、生物胁迫、非生物胁迫和其他条件下被测定。目前,仍没有简单实用的网络工具来探索这些宝贵的数据。本文从美国国立生物技术信息中心的基因表达数据库(NCBI GEO)下载1019个基因表达芯片数据,进行权重基因共表达网络分析(WGCNA)。鉴定到41个基因共表达模块。功能富集分析发现这些模块具有不同的功能,并与实验/表型相关。通过模块内连接度筛选枢纽基因,这些基因可能具有重要功能。通过关联推定(Guilt-by-association)原理对模块内功能未知的基因进行功能预测。最后,构建了免费的网络工具VitisMod,为葡萄的基因功能研究提供新资源,网址为:http://bioinformatics.fafu.edu.cn/grape。

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.

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。

葡萄(Vitis spp.)果皮颜色及果实着色性状分析

为了明确葡萄(Vitis spp.)果实的着色规律,对中国农业科学院郑州葡萄种质资源圃保存的302个葡萄品种(种)的果皮颜色及果实着色状况进行了调查分析,并对喷水处理后10个葡萄品种的果实着色状况以及75个葡萄品种果实转色期与成熟期的关系进行了研究。结果表明:在果实成熟期,葡萄的果皮颜色共有绿黄色、黄绿色、粉红色、紫红色、紫黑色和蓝紫色6种,其中,绿黄色和黄绿色为无色葡萄的果皮颜色,其余4种颜色为有色葡萄的果皮颜色;紫红色葡萄品种(种)数量最多,蓝紫色葡萄品种(种)数量最少。供试202个有色葡萄品种(种)果实的平均着色时间为18.7 d,其中,欧亚种(Vitis vinifera Linn.)果实的平均着色时间长于欧美杂种(V.vinifera×V.labrusca Linn.);56.9%的有色葡萄品种(种)果实在7月上旬开始着色,转色期果穗上的着色果粒数量变化呈"S"形曲线;早熟葡萄果实的平均着色时间最短(11.5 d),而中熟和晚熟葡萄果实的平均着色时间则相对较长(分别为18.7和21.0 d)。160个有色葡萄品种(种)的果穗着色方式包括穗肩先着色、穗尖先着色和穗中先着色3种,以穗肩先着色的品种(种)数量最多(72个);175个有色葡萄品种(种)的果粒着色方式包括粒尖先着色、中部先着色、顶部先着色和均匀着色4种,以粒尖先着色的品种(种)数量最多(116个)。喷水处理可以促进多数葡萄品种果实着色,但对少数葡萄品种果实着色无明显影响。91.1%的葡萄品种果实符合"早着色,早成熟;晚着色,晚成熟"的规律,但少数中熟和晚熟葡萄品种从果实开始着色到完全成熟经历的时间相对较长。分析结果显示:光照和温度是影响葡萄果实着色的主要环境因子。

196份葡萄属(Vitis L.)种质资源耐热性评价

以国家果树种质郑州葡萄圃保存的196份代表性葡萄属种质为试材,利用叶绿素荧光参数法对其耐热性进行了鉴定评价。结果表明:196份葡萄属植物的耐热性叶绿素荧光参数Fv/Fm数值在0.0792~0.6836之间,品种间抗性存在差异,耐热性服从正态分布,表现为多基因控制的数量性状。利用有序样品最优分割聚类法,将其耐热性分为弱、中、强3种类型,叶绿素荧光参数分级阈值分别对应≤0.3、(0.3,0.5]、>0.5,利用该分级标准筛选出了腺枝葡萄双溪03、刺葡萄梅岭山1301、菱叶葡萄0945和和田绿葡萄等48份耐热性强的葡萄种质,起源于我国的野生葡萄耐热性整体上高于其他类群或品种。研究结果对于利用抗性亲本选育耐热葡萄新品种和葡萄抗热机理研究具有参考意义。

中国葡萄属（VitisL．）分类研究

报导了中国葡萄属植物现存全部种类的分类处理结果，这些结果是在对植物野外和栽培观察及标本室研究基础上获得的；标本室的研究除了中国之外还包括参阅欧洲的一些著名标本馆内早期植物学家鉴定名称所依据的标本，因而所有种类的确定较之过去更为可靠．文中发表了一些新分类群和新组合，给出了鉴定种类的检索表．

葡萄属植物(Vitis L.)再生系统的研究进展

阐述了葡萄属植物再生系统的研究进展,论述和分析了影响葡萄属植物离体再生的主要因素,包括葡萄基因型、外植体类型、外植体生理状态、培养基类型、植物激素种类及浓度、培养方式、培养条件等,并对其研究现状、存在问题及应用前景作了简要概述。

山葡萄(Vitis amurensis)资源核心种质的初步构建

采用省份分组-花型分组-聚类-分组法构建了山葡萄核心种质,在此基础上,考虑到山葡萄品种在生产上和雄株山葡萄在科学研究上的作用,把未进入核心种质的山葡萄品种和雄株山葡萄也并入核心种质。该核心种质包括48份资源(占资源总数的31%),其中来自吉林省的资源18份(占37.5%),黑龙江省的资源29份(占60.4%),辽宁省的资源1份(占2.1%);两性花资源22份(占45.8%),雌能花资源21份(占43.8%),雄株5份(占10.4%)。除出汁率外,其他性状的符合度达72%以上;除果粒质量外,其他性状的变幅吻合度达79%以上。以上结果表明,所构建的核心种质较好地代表了评价的山葡萄资源。

中国野生刺葡萄(Vitis davidii Foex)及其单品种葡萄酒中花旗松素含量分析

利用高效液相色谱-串联质谱联用技术对原产中国的野生刺葡萄果实的果皮、果肉、种子及单品种葡萄酒中的花旗松素含量(以鲜质量计算)进行测定与分析。结果表明:刺葡萄果实果皮中花旗松素含量最高,其含量在刺葡萄黑、白果实中分别为(2.44±0.18)、(2.03±0.14)mg/kg,在种子中分别为(1.66±0.13)、(1.38±0.12) mg/kg,果肉中含量最低,分别为(0.36±0.02)、(0.25±0.02)mg/kg。刺葡萄黑、白果实中各个部位花旗松素含量均无明显差异,但对照品种黑色果实果皮中的花旗松素含量显著高于白色果实,这说明花旗松素含量的高低与葡萄果皮颜色并无直接关系。在单品种酒中,白葡萄酒由于酿造工艺不同,果皮和种子中的花旗松素无法进入酒中,花旗松素含量仅为红葡萄酒的20%;葡萄酒中的花旗松素含量略低于果实是因为提取果实中花旗松素的溶剂是甲醇,而葡萄酒中只是水和乙醇的自然浸渍。

葡萄(vitis)植株不同部位与不同节位叶片中过氧化物酶同工酶的变化

试验发现,葡萄叶片过氧化物酶同工酶存在明显的组织部位特异性,因叶片着生的枝条不同部位、同一枝条不同节位等发生变化.两个品种测定结果,在上、中、下部枝条中,均以上部枝一次梢4～5节位叶片酶带数最多、最稳定.在生长期,上部枝一次梢不同节位叶片中,过氧化物酶同工酶酶带数与酶活性均随叶片节位升高而递减;至果实成熟期,酶相对活性随叶片节位上升显现低一高一低的变化趋势.本文对葡萄叶片中过氧化物酶同工酶的这些变化规津以及不同生长期的适宜采样部位作了分析与讨论.

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.

浙江葡萄属(Vitis L.)植物新资料

该文报道了在浙江葡萄属(Vitis L.)分类研究中的新发现:(1)描述了开化葡萄(V.kaihuaica Z.H.Chen,F.Chen et W.Y.Xie)、秀丽葡萄(V.amoena Z.H.Chen,F.Chen et W.Y.Xie)2新种和腺枝龙泉葡萄(V.longquanensis var.glandulosa Z.H.Chen,F.Chen et W.Y.Xie)1新变种;(2)将V.adenoclada Hand.-Mazz.作为毛葡萄(V.heyneana Roem.et Schult.)的变种处理,即腺枝毛葡萄[V.heyneana Roem.et Schult.var.adenoclada(Hand.-Mazz.)Z.H.Chen,F.Chen et W.Y.Xie];(3)报道了蓝果刺葡萄[V.davidii(Roman.Du Caill.)Foex var.cyanocarpa(Gagnep.)Sarg.]在浙江的分布新记录。

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.

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.