葡萄初级核心种质资源MybA基因型分析

探究MybA类基因在不同类型葡萄品种中的分布,可为葡萄品种鉴定,以及有色葡萄育种的亲本选择提供依据。本研究以欧亚种、欧美杂种、法美杂种、山欧杂种以及美洲种在内的118个葡萄初级核心种质为材料,对其MybA基因型进行分析。结果表明:欧亚种及其杂种普遍具有VvmybA1基因的等位基因VvmybA1a,仅10个欧亚种及其杂种品种中没有检测到VvmybA1a基因;欧亚种、欧美杂种以及法美杂种中普遍同时具有VvmybA1、VvmybA2和VvmybA3基因,仅少数品种未检测到VvmybA2或VvmybA3基因;山欧杂种中北玫、公酿1号和熊岳白葡萄同时具有VvmybA1、VvmybA2和VvmybA3基因,北醇和北红中仅检测到VvmybA1和VvmybA3基因;仅在具有美洲种血缘的葡萄品种中检测到VlmybA2基因,而5个认为是美洲种的品种未检测到VlmybA2基因,且检测到了欧亚种特有的VvmybA1a等位基因,推测它们为含美洲种血缘较多的欧美杂种,而非纯美洲种。     

A Basic Peroxidase Isoenzyme, Marker of Resistance Against Plasmopara viticola in Grapevines, is Induced by an Elicitor from Trichoderma viride in Susceptible Grapevines

The constitutive expression of basic peroxidase isoenzymes in the Plasmopara viticola -resistant ( Vitis vinifera × Vitis rupestris) × Vitis riparia crossing and in the P. viticola -susceptible V. vinifera parent species was studied. The results illustrate that both leaves and stems of the ( V. vinifera × V. rupestris) × V. riparia crossing showed the differential expression of a basic peroxidase isoenzyme B₃ (pl = 8.9), this being almost completely absent from the P. viticola -susceptible V. vinifera parent species. To test whether the basic peroxidase isoenzyme B₃ may be considered as a molecular marker of disease resistance in Vitis spp., suspension cell cultures derived from the P. viticola -susceptible V. vinifera parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus Trichoderma viride , a specific and well-known elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B₃ in the cell cultures. These results suggest that the basic peroxidase isoenzyme B₃ may be considered as a marker of disease resistance in Vitis species since it is present in the P. viticola -resistant ( V. vinifera × V. rupestris) × V. riparia hybrid and is induced by the elicitor Onozuka R-10 in cell cultures of the P. viticola -susceptible Vitis vinifera parent species. This conclusion is supported by the presence of this isoenzyme in other resistant and its absence in other susceptible Vitis spp.     

Genetic Diversity in Wild Grapes Native to China Based on Randomly Amplified Polymorphic DNA (RAPD) Analysis

The use of the RAPD technique was investigated on a set of 73 genotypes of 18 wild grape species native to China, and one interspecific hybrid, seven Vitis vinifera L. cultivars, one rootstock cultivar and one strain of V. riparia L. Genetic diversity among these grapes was investigated based on RAPD analysis. The screening of 280 decamer oligonucleotides allowed the selection of 20 primers used for the analysis. A total of 191 RAPD markers were produced from the 20 selected primers. Relationships among the 83 clones or accessions based on their genetic distances were clustered using unweighted pair-group method arithmetic average (UPGMA) analysis in a dendrogram. Twenty-two clusters which fortunately adapted to 22 grape species level were clearly resolved on the dendrogram. The 18 wild grape species native to China were grouped into ten subclusters. The largest distance was found between V. riparia L., V. vinifera L., interspecific hybrid ( V. vinifera L.× V. larbrusca L.) and the wild grapes native to China. Among the wild grapes native to China, the largest distance was found between V. hancockii Hance and the other wild species. V. qinlingensis P.C.He was the second. Large genetic variation occurred among the different flower-type clones in one species.     

Chloroplast microsatellite polymorphisms in Vitis species.

The use of consensus chloroplast microsatellites primers for dicotyledonous chloroplast genomes revealed the existence of intra and interspecific length variation within the genus Vitis. Three chloroplast microsatellite loci were found to be polymorphic in samples of Vitis vinifera, Vitis berlandieri, Vitis riparia, and Vitis rupestris out of a total of 10 consensus primer pairs tested. These polymorphisms were always due to a variable number of mononucleotide residues within A and (or) T stretches in the amplified regions. Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplast in V. vinifera and to characterise the chloroplast haplotypes present in wine grape cultivars of this species grown in Spain and Greece. The different distribution of haplotype frequencies in the two ends of the Mediterranean growth area suggests the existence of independent domestication events for grapevine.     

葡萄Golden2-like转录因子家族的鉴定及其表达分析

本研究对葡萄（Vitis vinifera L.）的Golden2-like （GLK）转录因子家族进行了全基因组鉴定和表达模式分析,并利用品种‘玫瑰香’（V.vinifera cv.Muscat Hamburg）进一步验证其在低温胁迫下的响应。结果显示,葡萄Golden2-like家族共46个成员,分为5个亚族,同一亚族的保守结构域相似。46个VvGLK分别定位于细胞核、叶绿体、细胞质和过氧化物酶体中,其启动子区域含多种逆境应答顺式作用元件。基因芯片分析结果表明,22个Golden2-like基因在果实发育过程中变化显著。同时,有15、15和9个基因分别响应盐、干旱和低温胁迫。qRT-PCR分析发现26个基因参与低温应答。VvGLK41在所有胁迫处理中均下调表达。     

葡萄Actin基因家族的鉴定及进化和表达分析

采用生物信息学方法从葡萄(Vitis vinifera Linn.)全基因组中鉴定Actin基因家族,并对各基因的染色体定位和结构特征,编码蛋白质的理化性质、亚细胞定位、二级结构、三级结构和系统进化,以及不同组织的基因表达进行研究.结果表明:葡萄Actin基因家族16个基因分布在12条染色体上.16个基因的结构特征及其编码蛋白质的理化性质差异较大.16个基因的长度及其内含子总长度的变化范围较大,编码序列(CDS)和外显子总长度的变化范围较小.除登录号GSVIVG01008254001和GSVIVG01014035001的基因外,其他14个基因的GC含量均低于其CDS的GC含量.除登录号GSVIVG01008254001的基因外,其他15个基因编码的蛋白质的理论相对分子质量为12534.54~82612.33,理论等电点为pI 4.92~pI 9.13.16个基因编码蛋白质的消光系数为14105~73645,脂肪族氨基酸指数为65.54~92.06,其中9个为稳定蛋白,7个为不稳定蛋白.除登录号GSVIVG01014035001的基因外,其他15个基因编码的蛋白质均为亲水性蛋白.登录号GSVIVG01016517001的基因编码的蛋白质定位于细胞质和细胞核,其他15个基因编码的蛋白质定位于细胞质.二级结构和三级结构显示:葡萄Actin基因家族16个基因编码的蛋白质均由α螺旋、无规则卷曲和延伸链构成,且总体以无规则卷曲为主.系统进化分析和不同组织的基因表达分析结果显示:与拟南芥〔Arabidopsis thaliana(Linn.)Heynh.〕相似,葡萄Actin基因家族16个基因编码的蛋白质分为3个亚家族,ClassⅡ亚家族(营养型)包括登录号GSVIVG01003099001和GSVIVG01026580001的基因编码的蛋白质,这2个基因在所有组织中的表达均较高;ClassⅢ亚家族(生殖型)包括登录号GSVIVG01033494001、GSVIVG01024980001和GSVIVG01016550001的基因编码的蛋白质,这3个基因在花粉、雄蕊和花中的表达均较高;ClassⅠ亚家族包括其他11个基因编码的蛋白质,这11个基因在各组织中的表达总体上较低.研究结果显示:葡萄Actin基因家族的表达具有组织特异性.     

Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx

Two linkage maps for grape (Vitis spp.) have been developed based on 81 F(1) plants derived from an interspecific cross between the wine cultivar Moscato bianco (Vitis vinifera L.) and a Vitis riparia Mchx. accession, a donor of pathogen resistance traits. The double pseudotest-cross mapping strategy was applied using three types of molecular markers. The efficiency of SSRs to anchor homologous linkage groups from different Vitis maps and the usefulness of AFLPs in saturating molecular linkage maps were evaluated. Moreover, the SSCP technique was developed based on sequence information in public databases concerning genes involved in flavonoid and stilbene biosynthesis. For the maternal genetic map a total of 338 markers were assembled in 20 linkage groups covering 1,639 cM, whereas 429 loci defined the 19 linkage groups of the paternal map which covers 1,518 cM. The identification of 14 linkage groups common to both maps was possible based on 21 SSR and 19 AFLP loci. The position of SSR loci in the maps presented here was consistent with other published mapping experiments in Vitis.     

Resistance gene analogs are candidate markers for disease-resistance genes in grape ( Vitis spp.)

A set of NBS-containing sequences was isolated from genomic DNA of two grape species ( Vitis amurensis and Vitis riparia) and characterised in a panel of Vitis genotypes carrying different levels of resistance against downy mildew and other diseases. A PCR-mediated approach made use of degenerate primers designed on conserved regions encoding known R-genes, and provided the source for cloning grape analogous sequences. Cloned sequences were digested with ten endonucleases and 29 out of 71 putative recombinant clones, which showed unique restriction patterns, were sequenced. Using a threshold value of 40% identity, at least 12 grape NBS-sequences had a high overall similarity with known R-genes, such as the Arabidopsis gene RPS5 and the tobacco gene N. The presence of internal conserved motifs provided evidence that sequences isolated from grape may belong to the NBS-LRR gene family. A cluster analysis based on the deduced amino acid sequence and carried out on grape NBS-sequences, together with several analogous domains of known R-genes, classified grape sequences into three major groups. A grape sequence of each group was used as a probe on Southern blots with digested genomic DNA from resistant and susceptible grapes. One of the NBS-containing probes showed a clear-cut separation between resistant species and susceptible varieties. This evidence makes the probe a candidate marker for disease resistance genes in Vitis germplasm.     

Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress

ABSTRACT: BACKGROUND: Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew. RESULTS: Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles. CONCLUSIONS: The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.     

Identification of Vitis vinifera (-)-alpha-terpineol synthase by in silico screening of full-length cDNA ESTs and functional characterization of recombinant terpene synthase

The flavour and aroma of certain Vitis vinifera grape varieties is dominated by volatile terpenes and small volatile aldehydes. Monoterpenes contribute to the final grape and wine aroma and flavour in form of free volatiles and as glycoside conjugates of monoterpene alcohols. Typical monoterpenol components of the cultivar Gewürztraminer and other aroma-rich grape varieties are linalool, geraniol, nerol, citronellol, and alpha-terpineol. In a functional genomics effort to identify genes for the formation of monoterpene alcohols in V. vinifera, a database of full-length cDNA sequences was screened in silico and yielded two clones for putative monoterpene synthases. The gene products were functionally characterized by expression in Escherichia coli, in vitro enzyme assay and gas chromatography-mass spectrometry (GC-MS) product identification as multi-product (-)-alpha-terpineol synthases.