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.     

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.     

Genetic Analysis of East Asian Grape Cultivars Suggests Hybridization with Wild Vitis

Koshu is a grape cultivar native to Japan and is one of the country’s most important cultivars for wine making. Koshu and other oriental grape cultivars are widely believed to belong to the European domesticated grape species Vitis vinifera. To verify the domesticated origin of Koshu and four other cultivars widely grown in China and Japan, we genotyped 48 ancestry informative single nucleotide polymorphisms (SNPs) and estimated wild and domesticated ancestry proportions. Our principal components analysis (PCA) based ancestry estimation revealed that Koshu is 70% V. vinifera, and that the remaining 30% of its ancestry is most likely derived from wild East Asian Vitis species. Partial sequencing of chloroplast DNA suggests that Koshu’s maternal line is derived from the Chinese wild species V. davidii or a closely related species. Our results suggest that many traditional East Asian grape cultivars such as Koshu were generated from hybridization events with wild grape species.     

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.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

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.     

Genomic amplification of the <Emphasis Type="Italic">Gret1</Emphasis> retroelement in white-fruited accessions of wild <Emphasis Type="Italic">Vitis</Emphasis> and interspecific hybrids

Retrotransposons are retrovirus-related mobile sequences that have the potential to replicate via RNA intermediates and increase the genome size by insertion into new sites. The retroelement, Gret1, has been identified as playing a key role in generating fruit color variation in cultivated grape (Vitis vinifera L.) due to its insertion into the promoter of VvMybA1. Fruit color variation is an important distinguishing feature of cultivated grapes and virtually no fruit color variation is observed in wild grape species. The presence and relative copy number of Gret1 was assessed using quantitative PCR on 22 different Vitis species, only four of which (plus interspecific hybrids) are known to contain white accessions. Gret1 copy number was observed to vary by species as well as by color within species and was significantly higher in white-fruited accessions across all taxa tested. Additionally, genomic regions surrounding Gret1 insertion were sequenced in white V. vinifera, hybrid, V. labrusca, V. aestivalis, and V. riparia accessions.     

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.     

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.     

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?     

Identification and characterisation of differentially expressed leaf proteins among Vitis species

Grapes are commercially grown worldwide for fresh fruit and wine. They are mainly classified into bunch and muscadine grapes. These species differ in their sugar content and composition, photosynthetic efficiency and tolerance to abiotic and biotic stresses. Grape berry relies on carbohydrates produced during photosynthesis to support its development and composition. In view of the unique physiology and genetic make‐up of muscadine grape, a proteomics study was performed to increase our knowledge of Vitis leaf proteome in order to improve enological and disease tolerance characteristics of grape species. A high throughput two‐dimensional gel electrophoresis (2‐DE) was conducted on muscadine, bunch and hybrid bunch leaf proteins. The differentially expressed proteins were excised from 2‐DE gels, subjected to in‐gel trypsin digestion, and analysed in MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed by searching against Viridiplantae database using Matrix Science algorithm. Proteins were mapped to universal protein resource to study gene ontology. We have discovered >255 proteins with pIs between 3.5 and 8.0 and molecular weight between 12 and 100 kDa among the Vitis species. Comparative analysis of leaf proteome showed that 54 polypeptides varied qualitatively and quantitatively among the three Vitis species studied. Of these, seven proteins were unique to muscadine, two proteins were present in both muscadine and bunch, while 28 proteins were common to all the three species. Bioinformatic analysis of these proteins showed that they are involved in signal transduction pathway, transport of metabolites, energy metabolism, protein trafficking, photosynthesis and defence. We have also identified proteins unique to muscadine grape that are involved in defence and stress tolerance. In addition, photosynthesis‐related proteins were found to be more abundant in Vitis vinifera grape compared to other Vitis species.     

Genetic diversity in some grape varieties revealed by SCoT analyses

Start codon targeted (SCoT) polymorphic markers were used to assess genetic relationships among 64 grape varieties. Seventeen informative primers were selected from 36 SCoT primers based on their ability to produce clear and repeatable polymorphic and unambiguous bands among the varieties. A total of 131 bands were produced; 93.1% of them were polymorphic; the average polymorphism information content was 0.82. Cluster analysis of SCoT markers through the unweighted pair-group method of arithmetic averages analysis and principal coordinate analysis were largely consistent. The partition of clusters in the dendrogram and PCoA plot was similar and some degree of grouping by types of grape and taxonomic status of the varieties was revealed. Four main groups were found after cluster analysis, i.e. table grape of Vitis vinifera; table grape of Euro-America hybrid; wine grape of V. vinifera and wild Vitis species. The results showed that the wild Vitis species originated from America and China could be clearly differentiated. The results also indicated that SCoT markers are informative and could be used to detect polymorphism for grape varieties.     

Genetic Divergence between Camellia sinensis and Its Wild Relatives Revealed via Genome-Wide SNPs from RAD Sequencing

Tea is one of the most popular beverages across the world and is made exclusively from cultivars of Camellia sinensis. Many wild relatives of the genus Camellia that are closely related to C. sinensis are native to Southwest China. In this study, we first identified the distinct genetic divergence between C. sinensis and its wild relatives and provided a glimpse into the artificial selection of tea plants at a genome-wide level by analyzing 15,444 genomic SNPs that were identified from 18 cultivated and wild tea accessions using a high-throughput genome-wide restriction site-associated DNA sequencing (RAD-Seq) approach. Six distinct clusters were detected by phylogeny inferrence and principal component and genetic structural analyses, and these clusters corresponded to six Camellia species/varieties. Genetic divergence apparently indicated that C. taliensis var. bangwei is a semi-wild or transient landrace occupying a phylogenetic position between those wild and cultivated tea plants. Cultivated accessions exhibited greater heterozygosity than wild accessions, with the exception of C. taliensis var. bangwei. Thirteen genes with non-synonymous SNPs exhibited strong selective signals that were suggestive of putative artificial selective footprints for tea plants during domestication. The genome-wide SNPs provide a fundamental data resource for assessing genetic relationships, characterizing complex traits, comparing heterozygosity and analyzing putatitve artificial selection in tea plants.     

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.     

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.     

Vitis Phylogenomics: Hybridization Intensities from a SNP Array Outperform Genotype Calls

Understanding relationships among species is a fundamental goal of evolutionary biology. Single nucleotide polymorphisms (SNPs) identified through next generation sequencing and related technologies enable phylogeny reconstruction by providing unprecedented numbers of characters for analysis. One approach to SNP-based phylogeny reconstruction is to identify SNPs in a subset of individuals, and then to compile SNPs on an array that can be used to genotype additional samples at hundreds or thousands of sites simultaneously. Although powerful and efficient, this method is subject to ascertainment bias because applying variation discovered in a representative subset to a larger sample favors identification of SNPs with high minor allele frequencies and introduces bias against rare alleles. Here, we demonstrate that the use of hybridization intensity data, rather than genotype calls, reduces the effects of ascertainment bias. Whereas traditional SNP calls assess known variants based on diversity housed in the discovery panel, hybridization intensity data survey variation in the broader sample pool, regardless of whether those variants are present in the initial SNP discovery process. We apply SNP genotype and hybridization intensity data derived from the Vitis9kSNP array developed for grape to show the effects of ascertainment bias and to reconstruct evolutionary relationships among Vitis species. We demonstrate that phylogenies constructed using hybridization intensities suffer less from the distorting effects of ascertainment bias, and are thus more accurate than phylogenies based on genotype calls. Moreover, we reconstruct the phylogeny of the genus Vitis using hybridization data, show that North American subgenus Vitis species are monophyletic, and resolve several previously poorly known relationships among North American species. This study builds on earlier work that applied the Vitis9kSNP array to evolutionary questions within Vitis vinifera and has general implications for addressing ascertainment bias in array-enabled phylogeny reconstruction.