Earliest evidence of a primitive cultivar of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. during the Bronze Age in Sardinia (Italy)

The discovery of the Nuragic culture settlement of Sa Osa, Cabras-Oristano, Sardinia, has made it possible to investigate the domestication status of waterlogged uncharred grape pips that were recovered from three wells dating from the Middle and Late Bronze Age (ca. 1350–1150 bc). Applying the stepwise linear discriminant analysis method, a morphological comparison of archaeological seeds and modern wild and cultivated Sardinian grapes pips was performed to determine the similarities between them. The results showed that the archaeological seeds from the Middle Bronze Age have intermediate morphological traits between modern wild and cultivated grape pips from Sardinia. In contrast, the analyses performed on the archaeological seeds from the Late Bronze Age showed a high degree of similarity with the modern cultivars in Sardinia. These results provide the first evidence of primitive cultivated Vitis vinifera in Sardinia during the Late Bronze Age (1286–1115 cal bc, 2σ). This evidence may support the hypothesis that Sardinia could have been a secondary domestication centre of the grapevine, due to the presence of ancient cultivars that still exhibit the phenotypic characteristics of wild grapes.     

Multiple loss-of-function 5-O-glucosyltransferase alleles revealed in Vitis vinifera,but not in other Vitis species

Key message

Wild and loss-of-function alleles of the 5 - O - glucosyltransferase gene responsible for synthesis of diglucoside anthocyanins in Vitis were characterized. The information aids marker development for tracking this gene in grape breeding.

Abstract

Anthocyanins in red grapes are present in two glycosylation states: monoglucoside (3-O-glucoside) and diglucoside (3, 5-di-O-glucoside). While monoglucoside anthocyanins are present in all pigmented grapes, diglucoside anthocyanins are rarely found in the cultivated grape species Vitis vinifera. Biochemically 3-O-glucoside anthocyanins can be converted into 3,5-di-O-glucoside anthocyanins by a 5-O-glucosyltransferase. In this study, we surveyed allelic variation of the 5-O-glucosyltransferase gene (5GT) in 70 V. vinifera ssp. vinifera cultivars, 52 V. vinifera ssp. sylvestris accessions, 23 Vitis hybrid grapes, and 22 accessions of seven other Vitis species. Eighteen 5GT alleles with apparent loss-of-function mutations, including seven premature stop codon mutations and six frameshift indel mutations, were discovered in V. vinifera, but not in the other Vitis species. A total of 36 5GT alleles without apparent loss-of-function mutations (W-type) were identified. These W-type alleles were predominantly present in wild Vitis species, although a few of them were also found in some V. vinifera accessions. We further evaluated some of these 5GT alleles in producing diglucoside anthocyanins by analyzing the content of diglucoside anthocyanins in a set of representative V. vinifera cultivars. Through haplotype network analysis we revealed that V. vinifera ssp. vinifera and its wild progenitor V. vinifera ssp. sylvestris shared many loss-of-function 5GT alleles and extensive divergence of the 5GT alleles was evident within V. vinifera. This work advances our understanding of the genetic diversity of 5GT and provides a molecular basis for future marker-assisted selection for improving this important wine quality trait.     

Vitis vinifera L.: Wild or cultivated? Study of the grape pips found at Petra, Jordan; 150 B.C. – A.D. 40

The identification of carbonised grape pips (Vitis vinifera ssp.) is problematic, and the morphological features generally used to distinguish the wild subspeciesV. vinifera ssp.sylvestris from the cultivated subspeciesV. vinifera ssp.vinifera are not satisfactory. Different biometric studies were carried out on Nabataean and Roman seeds found at Petra, Jordan, dated to 150 B.C. – A.D. 400, and the results were compared to known phytogeographical and climatic data. Depending on the identification method selected, the seeds were attributed either to wild grapevines (based on the ratio of breadth over length, and on discriminant analyses of size variables such as pip length, stalk length, and chalaza position), or to an archaic variety of vine with seeds morphologically close to those of wild grapevines (ratio of stalk length over total pip length). The methods used here were applied to European grape pips; they should be tested on Near Eastern material. Archaeological data did not clarify the situation, and the importance of viticulture, which was prohibited during the Nabataean period, remains difficult to evaluate at Petra.     

Study of the relationship between the cultivars of Vitis vinifera and the white-fruited and hermaphrodite Chinese wild grapes

Chinese wild grapes are almost exclusively dioecious and black-fruited, with rare reports of white and hermaphrodite types in V. davidii. To reveal the molecular mechanisms of these phenotypic variations, specific primers were designed to detect the genotypes of mybA-related genes in Vitis species, including the Chinese wild Vitis species, V. riparia, V. rupestris, cultivars of Vitis vinifera and its hybrids. We report here that three mybA-related genes, VvmybA1a, VvmybA2 and VvmybA3, were only detected in cultivars of V. vinifera and its hybrids, but not in V. riparia, V. rupestris or Chinese wild Vitis species, indicating that these genes could be used to test the genetic relationship to V. vinifera. On the other hand, the genes were not detected in the dioecious varieties of V. davidii, but were in the hermaphrodites. In particular, the white-fruited varieties were homozygous for VvmybA1a and showed a low expression of mybA-related genes and UFGT during the entire maturation period. Simple sequence repeat analysis showed that the hermaphrodite varieties of V. davidii, including the white-fruited varieties, were more closely related to V. vinifera cv. Pinot Noir and V. labruscana cv. Kyoho. These results suggested that the white-fruited and hermaphrodite varieties of V. davidii could be the result of its crossing with V. vinifera. It provides a new approach to identify truly Chinese wild varieties and to search for possible hybridization events.     

Genomics Assisted Ancestry Deconvolution in Grape

The genus Vitis (the grapevine) is a group of highly diverse, diploid woody perennial vines consisting of approximately 60 species from across the northern hemisphere. It is the world’s most valuable horticultural crop with ~8 million hectares planted, most of which is processed into wine. To gain insights into the use of wild Vitis species during the past century of interspecific grape breeding and to provide a foundation for marker-assisted breeding programmes, we present a principal components analysis (PCA) based ancestry estimation method to calculate admixture proportions of hybrid grapes in the United States Department of Agriculture grape germplasm collection using genome-wide polymorphism data. We find that grape breeders have backcrossed to both the domesticated V. vinifera and wild Vitis species and that reasonably accurate genome-wide ancestry estimation can be performed on interspecific Vitis hybrids using a panel of fewer than 50 ancestry informative markers (AIMs). We compare measures of ancestry informativeness used in selecting SNP panels for two-way admixture estimation, and verify the accuracy of our method on simulated populations of admixed offspring. Our method of ancestry deconvolution provides a first step towards selection at the seed or seedling stage for desirable admixture profiles, which will facilitate marker-assisted breeding that aims to introgress traits from wild Vitis species while retaining the desirable characteristics of elite V. vinifera cultivars.     

An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes × wild Chinese Vitis species

A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3–28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4–55.4%) and plantlet development (11.15–44.6%) were all highest when embryos were cultured on Woody Plant Medium?+?5.7 μM indole-3-acetic acid?+?4.4 μM 6-benzylaminopurine?+?1.4 μM gibberellic acid?+?2% sucrose?+?0.05% casein hydrolysate?+?0.3% activated charcoal?+?0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes?×?wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes.     

Genetic relationship between Chinese wild Vitis species and American and European Cultivars based on ISSR markers

Inter-simple sequence repeat (ISSR) markers were employed to detect the genetic diversity among 70 grape accessions including 52 clones of 17 Chinese wild grape species, seven interspecific hybrids, 10 Vitis vinifera L. cultivars, and one strain of Vitis riparia L. A total of 119 polymorphic bands with an average of 11.9 per primer were observed. The unweighted pair-group method (UPGMA) analysis indicated that the 70 clones or accessions had a similarity range from 0.08 to 0.93, indicating that abundant diversities exist among these accessions. Based on cluster analysis and principal coordinate analysis, all accessions could be divided into two major groups, the Chinese wild grape group, and the American and European cultivar group. The largest distance was found among V. riparia MichX, Vitis piasezkii, V. vinifera L. interspecific hybrid (Vitis binifera × V. labrusca) and the wild grapes native to China.     

High throughput analysis of grape genetic diversity as a tool for germplasm collection management

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI?=?0.839 and 0.865, respectively) than V. vinifera cultivars (GDI?=?0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI?=?0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.     

Genetic variability and relationships between and within grape cultivated varieties and wild species based on SRAP markers

Sequence-related amplified polymorphism (SRAP) markers were used to assess genetic relationships among 76 grape genotypes including Chinese indigenous and newly bred varieties, representatives of foreign grape varieties, and wild Vitis species. Nineteen informative primers were selected from 100 SRAP primer pairs due to their ability to produce clearly and repeatedly polymorphic and unambiguous bands among the varieties. A total of 228 bands were produced; 78.63% of them were polymorphic; the average polymorphism information content (PIC) is 0.76. Genetic relationships were obtained using Nei and Li similarity coefficients. Cluster analysis of SRAP markers through the unweighted pair-group method of arithmetic averages (UPGMA) analysis and principal coordinate analysis (PCoA) were largely consistent. The definition of clusters in the dendrogram and PCoA plot is the same and some degree of grouping by types of grape, ecogeographical origin, and taxonomic status of the varieties was revealed. Three main groups were found after cluster analysis, i.e., table grape of Vitis vinifera; table grape of Euro-America hybrid and wine grape of V. vinifera; wild Vitis species. Groupings indicated a divergence between the table and wine-type varieties of V. vinifera. The results showed that the wild Vitis species that originated from America and China could be clearly differentiated and Vitis hancockii is the most distant from the others of Asian Vitis species. The results also indicated that SRAP markers are informative and could distinguish bud sports of grape. The present analysis revealed that Chinese cultivated and wild grape germplasm are highly variable and have abundant genetic diversity.     

Dimorphism in inflorescence scent of dioecious wild grapevine

Wild grapevine (Vitis vinifera subsp. sylvestris) is the dioecious ancestral form of grapevine, from which the domesticated cultivars have derived (V. vinifera subsp. vinifera). Little is known about the floral scent compounds of wild grapevine that is considered as being partly insect pollinated. The knowledge of volatiles released by male and female inflorescence may contribute to the understanding of the pollination biology of this endangered taxon. Inflorescence scents of male and female individuals were collected by dynamic headspace and analysed by thermal desorption-GC/MS. A total of 17 compounds of C5-branched chain alcohols, aliphatics, aromatics, and terpenoids were identified with benzyl alcohol being most abundant in both sexes. Eight of the compounds were sex-specific and differences in 1,2-dimethoxybenzene were most obvious. This aromatic compound was a main constituent in the scent of females (30%), but it did not occur in males. Some of the main compounds of the scent samples are known to be detected by beetles (Cerambycidae) or attract sweat bees (Halictidae) and honey bees (A. mellifera), all well-known inflorescence visitors in wild grapevine. The data presented here are an important step in understanding the chemical communication between wild grapevine and its inflorescence visitors/potential pollinators.     

Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model‐based approach implemented in the software structure , we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars.     

The effects of artificial selection on sugar metabolism and transporter genes in grape

Sugar content is a key feature of grape quality. The sugar content of grapes has been significantly improved after nearly a thousand years of artificial selection. However, the mechanism underlying the changes in the grape sugar content during the process of artificial selection remains largely unknown although several genes involved in sugar metabolism and transportation in grape have been identified. In this study, the genomes of 13 wild Vitis species and 14 cultivated Vitis vinifera accessions were resequenced to 2–5 X depth using the Illumina Hiseq2000 platform. Genetic variation of 138 genes involved in sugar biosynthesis and transport was investigated, and 7,690 and 12,717 single nucleotide polymorphisms/insertions and deletions (SNPs/InDel) were identified within the cultivated V. vinifera and wild Vitis species, respectively. The percentages of SNPs/InDels were 0.93 and 1.54 % in cultivated and wild species, respectively, and the wild Vitis species had 1.65-fold more SNPs/InDels than the cultivated V. vinifera. Moreover, the distribution of SNPs/InDels in gene regions was also investigated. Eight genes (HT4, PPFTK4, PPFTK6, PMT3, SPS1, HT8, HT15, SUSy1) showed low level of allelic diversity in cultivated species, suggesting they might have undergone purifying selection during the domestication process of grapes. Our genome DNA resequencing data provided a valuable resource for analyzing the effects of artificial selection on trait-related pathways in grape. The result that eight genes showed lower level of DNA variation in cultivated species than in wild species will be very helpful in understanding sugar accumulation in grapes.     

Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs)

Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%–88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.     

Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.     

Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes

Skin color is one of the most important fruit traits in grape, and has become greatly diversified due to hybridization and human selection. Many studies concerning the genetic control of grape color in European species (Vitis vinifera L.), especially the role of MYB-related genes, have been reported. On the other hand, there have been few studies of the MYB-related genes in grapes belonging to V.?×labruscana L.H. Bailey, a subgroup of grapes that originated from the hybridization of V. labrusca with V. vinifera. In the present study, we found a novel functional haplotype, HapE2 (consisting of the genes VlMYBA2 and VlMYBA1?C3), in diploid V.?×labruscana. Moreover, we developed a method to determine the haplotype compositions of tetraploid grapes by means of quantitative real-time PCR, and investigated the relationship between haplotype composition and skin color. The color locus in V.?×labruscana grapes usually consists of functional haplotypes (HapE1 and/or HapE2), and non-functional haplotype HapA. The number of functional haplotypes in the genome was found to be correlated with the level of anthocyanin in the skin. Anthocyanin contents of grapes that contained HapE2 were significantly higher than those containing HapE1. These results suggest that the number and kind of functional haplotypes at the color locus are the major genetic factors that determine skin color variation. These findings provide new knowledge about the unique genetic control of color in V.?×labruscana grapes, and should contribute to development of new cultivars that have the desired color and anthocyanin content.     

The timing and the mode of evolution of wild grapes (Vitis)

Wild grapes are woody climbers, found mostly in temperate regions of the northern hemisphere, comprising the genus Vitis. Despite its importance, the evolutionary history of Vitis is still contentious. Past studies have led to conflicting hypotheses about the phylogeny, speciation events, and biogeographic history of the genus. Here we investigate the evolutionary history of Vitis using data from four chloroplast spacers (trnH-psbA, trnK-rps16, trnF-nahJ, and rpl32-trnL) and the nuclear gene RPB2-I, and we explore mechanisms that could have shaped the observed distribution of current species. Maximum likelihood and Bayesian analyses provided similar results, strongly supporting the presence of two subgenera and suggesting a species clustering within subgenus Vitis that mainly mirrors the disjunction between the Old and New World. Vitis vinifera subsp. sylvestris was found to be sister to the Asian species while three major clades were found in the American species. A network approach confirmed the main geographic groups and highlighted different chloroplast haplotype patterns between Asian and American species. Molecular dating analysis provided the time boundaries to discuss our results. Our study shows wild grape diversification to be a continuous and complex process that concerned the Tertiary as well as the Quaternary, most likely involving both geographical and climatic forces. Local variations in extent and timing of these forces were discussed based on observed differences between groups. In the context of the Tertiary–Quaternary debate, we provide evidence in favor of the “continuous hypothesis” to explain present diversity. Finally, two directions for future research are highlighted: (i) was the earliest grape American or Asian? and (ii) are all modern grape species real?