Linchuan Fang

Comprehensive analysis of NAC domain transcription factor gene family in Vitis vinifera

Key messageGenome-wide identification of grapevineNACdomain genes and investigation of their chromosome locations, gene structures, duplication, evolution, phylogeny and expression profiles.AbstractGrapevine is a widely used fruit crop. NAC (NAM, ATAF1/2 and CUC2) domain genes are plant-specific transcription factors (TFs) that comprise a conserved NAM domain in the N-terminus. Members of this gene family have been reported to contribute to plant development. During this study, 74 NAC genes were identified from 12× assembled grapevine genomic sequences. The duplication patterns, genomic structures and phylogeny of these 74 grapevine NAC genes were investigated. To understand the roles of VvNAC during grapevine development, their expression profiles in different tissues including leaf, tendril, inflorescence, stem, root and veraison berry skin were tested using quantitative real-time PCR. Analysis revealed expression diversity of various VvNAC genes among different grapevine tissues. To identify candidate grapevine NAC genes with a role in response to stress, publicly available microarray data were obtained to calculate their expression change under abiotic and biotic treatments, with a number of VvNAC genes displaying up-regulation after stress induction. Therefore, this study has uncovered more knowledge relating to the gene structures, chromosome organizations, evolution, expression profiles and functions of VvNAC genes.

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera

BackgroundWRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown.ResultsWe identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress.ConclusionsWe identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.

Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

BackgroundGenetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS) technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD) might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP) marker development.ResultsAn F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations.ConclusionsThe applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.

Genome Wide Transcriptional Profile Analysis of Vitis amurensis and Vitis vinifera in Response to Cold Stress

Grape is one of the most important fruit crops worldwide. The suitable geographical locations and productivity of grapes are largely limited by temperature. Vitis amurensis is a wild grapevine species with remarkable cold-tolerance, exceeding that of Vitis vinifera, the dominant cultivated species of grapevine. However, the molecular mechanisms that contribute to the enhanced freezing tolerance of V. amurensis remain unknown. Here we used deep sequencing data from restriction endonuclease-generated cDNA fragments to evaluate the whole genome wide modification of transcriptome of V. amurensis under cold treatment. Vitis vinifera cv. Muscat of Hamburg was used as control to help investigate the distinctive features of V. amruensis in responding to cold stress. Approximately 9 million tags were sequenced from non-cold treatment (NCT) and cold treatment (CT) cDNA libraries in each species of grapevine sampled from shoot apices. Alignment of tags into V. vinifera cv. Pinot noir (PN40024) annotated genome identified over 15,000 transcripts in each library in V. amruensis and more than 16,000 in Muscat of Hamburg. Comparative analysis between NCT and CT libraries indicate that V. amurensis has fewer differential expressed genes (DEGs, 1314 transcripts) than Muscat of Hamburg (2307 transcripts) when exposed to cold stress. Common DEGs (408 transcripts) suggest that some genes provide fundamental roles during cold stress in grapes. The most robust DEGs (more than 20-fold change) also demonstrated significant differences between two kinds of grapevine, indicating that cold stress may trigger species specific pathways in V. amurensis. Functional categories of DEGs indicated that the proportion of up-regulated transcripts related to metabolism, transport, signal transduction and transcription were more abundant in V. amurensis. Several highly expressed transcripts that were found uniquely accumulated in V. amurensis are discussed in detail. This subset of unique candidate transcripts may contribute to the excellent cold-hardiness of V. amurensis.

Construction of a high-density genetic map and QTLs mapping for sugars and acids in grape berries

BackgroundQTLs controlling individual sugars and acids (fructose, glucose, malic acid and tartaric acid) in grape berries have not yet been identified. The present study aimed to construct a high-density, high-quality genetic map of a winemaking grape cross with a complex parentage (V. vinifera × V. amurensis) × ((V. labrusca × V. riparia) × V. vinifera), using next-generation restriction site-associated DNA sequencing, and then to identify loci related to phenotypic variability over three years.ResultsIn total, 1 826 SNP-based markers were developed. Of these, 621 markers were assembled into 19 linkage groups (LGs) for the maternal map, 696 for the paternal map, and 1 254 for the integrated map. Markers showed good linear agreement on most chromosomes between our genetic maps and the previously published V. vinifera reference sequence. However marker order was different in some chromosome regions, indicating both conservation and variation within the genome. Despite the identification of a range of QTLs controlling the traits of interest, these QTLs explained a relatively small percentage of the observed phenotypic variance. Although they exhibited a large degree of instability from year to year, QTLs were identified for all traits but tartaric acid and titratable acidity in the three years of the study; however only the QTLs for malic acid and β ratio (tartaric acid-to-malic acid ratio) were stable in two years. QTLs related to sugars were located within ten LGs (01, 02, 03, 04, 07, 09, 11, 14, 17, 18), and those related to acids within three LGs (06, 13, 18). Overlapping QTLs in LG14 were observed for fructose, glucose and total sugar. Malic acid, total acid and β ratio each had several QTLs in LG18, and malic acid also had a QTL in LG06. A set of 10 genes underlying these QTLs may be involved in determining the malic acid content of berries.ConclusionThe genetic map constructed in this study is potentially a high-density, high-quality map, which could be used for QTL detection, genome comparison, and sequence assembly. It may also serve to broaden our understanding of the grape genome.

Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ETHYLENE RESPONSE FACTOR 057

Ethylene (ET) is a gaseous plant hormone that plays essential roles in biotic and abiotic stress responses in plants. However, the role of ET in cold tolerance varies in different species. This study revealed that low temperature promotes the release of ET in grapevine. The treatment of exogenous 1-aminocyclopropane-1-carboxylate increased the cold tolerance of grapevine. By contrast, the application of the ET biosynthesis inhibitor aminoethoxyvinylglycine reduced the cold tolerance of grapevine. This finding suggested that ET positively affected cold stress responses in grapevine. The expression of VaERF057, an ET signaling downstream gene, was strongly induced by low temperature. The overexpression of VaERF057 also enhanced the cold tolerance of Arabidopsis. Under cold treatment, malondialdehyde content was lower and superoxide dismutase, peroxidase, and catalase activities were higher in transgenic lines than in wild-type plants. RNA-Seq results showed that 32 stress-related genes, such as CBF1-3, were upregulated in VaERF057-overexpressing transgenic line. Yeast one-hybrid results further demonstrated that VaERF057 specifically binds to GCC-box and DRE motifs. Thus, VaERF057 may directly regulate the expression of its target stress-responsive genes by interacting with a GCC-box or a DRE element. Our work confirmed that ET positively regulates cold tolerance in grapevine by modulating the expression of VaERF057.

Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis

Highlight VaNAC26, a member of the NAC gene family from the wild species Vitis amurensis, plays an important role in drought tolerance by positively modulating jasmonic acid synthesis and enhancing the antioxidant system.

Overexpression of VaPAT1 , a GRAS transcription factor from Vitis amurensis , confers abiotic stress tolerance in Arabidopsis

Key messageVaPAT1functions as a stress-inducible GRAS gene and enhanced cold, drought and salt tolerance in transgenic Arabidopsis via modulation of the expression of a series of stress-related genes.AbstractThe plant-specific GRAS transcription factor family regulates diverse processes involved in plant growth, development and stress responses. In this study, VaPAT1, a GRAS gene from Vitis amurensis was isolated and functionally characterized. Sequence alignment and phylogenetic analysis showed that VaPAT1 has a high sequence identity to CmsGRAS and OsCIGR1, which belong to PAT1 branch of GRAS family and function in stress resistance. The transcription of VaPAT1 was markedly induced by stress-related phytohormone abscisic acid (ABA) and various abiotic stress treatments such as cold, drought and high salinity, however, it was repressed by exogenous gibberellic acid (GA) application. Overexpression of VaPAT1 increased the cold, drought and high salinity tolerance in transgenic Arabidopsis. When compared with wild type (WT) seedlings, the VaPAT1-overexpression lines accumulated higher levels of proline and soluble sugar under these stress treatments. Moreover, stress-related genes such as AtSIZ1, AtCBF1, AtATR1/MYB34, AtMYC2, AtCOR15A, AtRD29A and AtRD29B showed higher expression levels in VaPAT1 transgenic lines than in WT Arabidopsis under normal growth conditions. Together, our results indicated that VaPAT1 functions as a positive transcriptional regulator involved in grapevine abiotic stress responses.

Patterns of Gene Duplication and Their Contribution to Expansion of Gene Families in Grapevine

Grapevine is an important fruit crop that has undergone a long history of evolution. Analysis of the whole genome sequence of grapevine has revealed presence of an early palaeo-hexaploid along with three complements. Thus, gene duplication and genome expansion are common in this genome. In this study, we identified 17,922 duplicated genes in the whole grapevine genome. Among these, 2,039; 628; 1,428; 722; and 2,942 were identified respectively as produced by genome-wide, tandem, proximal, retrotransposed, and DNA-based transposed duplications. Analyses of the evolutionary patterns for different types of duplication using non-synonymous and synonymous substitution rates uncovered a series of underlying rules. Thereafter, all the grapevine genes were classified into families, and the contributions of different types of duplication to the expansion of large families were revealed. No duplication type was solely responsible for the formation of any large gene family, but some families showed enrichment of a special type of duplication. On the basis of this study, we believe that uncovering the underlying rules for gene duplications, expansions of gene families, and their evolutionary styles will contribute significantly to a comprehensive understanding of the features of the grapevine genome.