José Miguel Martínez-Zapater

Plants go through two distinct stages in their vegetative phase, with the juvenile stage being characterized by a lack of maturity to respond to flowering induction stimuli and the adult stage marked by the presence of this capacity. Phase transition has been extensively analysed in herbaceous species such as Arabidopsis and maize, where the sequential activity of miR156 and miR172 in the control of the juvenile to adult phase transition has been determined. Contrarily, little is known about most woody perennial crops, where phase transition appears to be dissociated, with a first transition from juvenile to adult vegetative state in the first year and a subsequent induction to flower in later years under flowering-inductive environmental conditions. This significantly extended vegetative phase makes fruit production depend on the grafting of adult vegetative materials. A particular aspect of grapevine vegetative phase transition is that it is marked by the differentiation of tendrils, a modified sterile reproductive organ adapted to climbing, which is continuously generated with different patterns in different Vitis species. When the grapevine plant reaches flowering inductive condition in later years, it produces inflorescences in place of some tendrils. As a first step to understand the regulation of phase change in grapevine, we have performed a detailed gene expression analysis of the juvenile-to-adult phase transition during the development of grapevine plantlets grown from seeds. The RNA-seq analysis demonstrated that miR156 was significantly repressed in the grapevine’s adult phase, where the appearance of tendrils acts as a marker of the transition. Consistent with the results reported in other species, we observed the activation of several SPL genes, known to be targets of miR156, providing evidence for the conservation of the miR156-SPLs regulatory module in grapevine. However, no variation was detected in the expression of miR172 and TPS genes were found downregulated, two key determinants in the transition to flowering in other species. This could be explained considering that grapevines do not flower during the first years of growth. Interestingly, we were able to observe the overexpression of several genes known to be involved in the establishment of flower meristem identity, which in the case of grape had also been detected along tendril development, consistent with the proposed common ontogenetic origin of tendrils and inflorescences in the Vitaceae family.

Societal Impact Statement: Table grape production is a traditional practice in southeastern Spain, where locals have produced fresh grapes and raisins for centuries. Many of these vines are now centenary, and they represent a useful source of diversity for developing future table grape varieties with improved traits. Genetic analysis showed that many of the local varieties identified in this study were traditional varieties from Western Mediterranean countries. Others were not identified, and they might be old grape varieties of previous importance in the region. The conservation and characterization of these varieties could be key to ensuring current and future vineyard sustainability.Summary: Current worldwide table grape production focuses on a reduced number of Vitis vinifera L. varieties. However, traditional farmers have grown many table grape varieties for centuries, as they provided a steady source of fresh fruit and raisins. These ancient living genotypes potentially store a genetic diversity that can be used now to ensure future grape production. Here we focused on the study of grapevines found across Almería, one of the Spanish regions with longer tradition in table grape production. After an exhaustive inspection, we located 220 old (some centenary) vines producing grapes consumed by owners or in abandoned areas no longer devoted to agriculture. Some of these vines were identified by comparing their simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) genetic profiles with available data from international databases. We found that, while grape growers' efforts focused on the cultivation of traditional grape varieties from Western Mediterranean regions, they also cultivated few exogenous varieties if they provided additional fruit features. Other vines were found to have genetic profiles that did not match reference datasets. Interestingly, some of them were found in multiple locations, suggesting they are endangered varieties with some previous relevance in the region. Besides, first‐degree relationships support the autochthonous origin of many of these unidentified genotypes. Locals kept a high number of different grapevine varieties, now considered reservoirs of genetic diversity. Traditional farming practices have been useful to prevent the loss of this diversity, which now needs to be preserved and further studied to contribute to the sustainability of viticultural systems.

To preserve their varietal attributes, established grapevine cultivars (Vitis vinifera L. ssp. vinifera) must be clonally propagated, due to their highly heterozygous genomes. Malbec is a France-originated cultivar appreciated for producing high-quality wines and is the offspring of cultivars Prunelard and Magdeleine Noire des Charentes. Here, we have built a diploid genome assembly of Malbec, after trio binning of PacBio long reads into the two haploid complements inherited from either parent. After haplotype-aware deduplication and corrections, complete assemblies for the two haplophases were obtained with a very low haplotype switch-error rate ( 25% of polymorphic regions. Gene annotation including RNA-seq transcriptome assembly and ab initio prediction evidence resulted in similar gene model numbers for both haplophases. The annotated diploid assembly was exploited in the transcriptomic comparison of four clonal accessions of Malbec that exhibited variation in berry composition traits. Analysis of the ripening pericarp transcriptome using either haplophases as a reference yielded similar results, although some differences were observed. Particularly, among the differentially expressed genes identified only with the Magdeleine-inherited haplotype as reference, we observed an over-representation of hypothetically hemizygous genes. The higher berry anthocyanin content of clonal accession 595 was associated with increased abscisic acid responses, possibly leading to the observed overexpression of phenylpropanoid metabolism genes and deregulation of genes associated with abiotic stress response. Overall, the results highlight the importance of producing diploid assemblies to fully represent the genomic diversity of highly heterozygous woody crop cultivars and unveil the molecular bases of clonal phenotypic variation.

More than 100 grapevine varieties are registered as suitable for wine production in “Douro” and “Trás-os-Montes” Protected Designations of Origin regions; however, only a few are actually used for winemaking. The identification of varieties cultivated in past times can be an important step to take advantage of all the potential of these regions grape biodiversity. The conservation of the vanishing genetic resources boosts greater product diversification, and it can be considered strategic in the valorisation of these wine regions. Hence, one goal of the present study was to prospect and characterise, through molecular markers, 310 plants of 11 old vineyards that constitute a broad representation of the grape genetic patrimony of “Douro” and “Trás-os-Montes” wine regions; 280 samples, grouped into 52 distinct known varieties, were identified through comparison of their genetic profiles generated via 6 nuclear SSR and 43 informative SNP loci amplification; the remaining 30 samples, accounting for 13 different genotypes, did not match with any profile in the consulted databases and were considered as new genotypes. This study also aimed at evaluating the population structure among the 65 non-redundant genotypes identified, which were grouped into two ancestral genetic groups. The mean probability of identity values of 0.072 and 0.510 (for the 6 SSR and 226 SNP sets, respectively) were determined. Minor differences were observed between frequencies of chlorotypes A and D within the non-redundant genotypes studied. Twenty-seven pedigrees were confirmed and nine new trios were established. Ancestors of eight genotypes remain unknown.

The South Caucasus is recognised as the primary Vitis vinifera L. (grapevine) domestication centre and has a high diversity of wild and cultivated grapevines. Archaeological findings indicate that winemaking activities have existed in Armenia for more than 6,000 years, viticulture being one of the most important activities of the modern Armenian agricultural sector. Despite this relevance, some grapevines in local collections have not yet been properly identified, thus hindering the efficient conservation, characterisation and eventual use of autochthonous genetic resources. In the present study, a combined SNP and SSR profiling strategy was used for the genetic identification of a series of grapevine accessions from the Grape Collection of the International Academy of Viticulture and Winemaking in Nalbandyan, presumed to be autochthonous Armenian varieties. The results provided useful information for the correct identification of these genetic resources, revealing multiple cases of synonyms, homonyms and misnames. The genetic data made it possible to confirm the pedigree proposed for some of the cultivars identified in this study and to clarify the origin of others. In addition, we propose, for the first time, a series of new trios and duos involving autochthonous Armenian grapevines. The singularity of this genetic pool compared to other Western and Central European varieties, as well as the potential novel sources of variability in traits of interest (e.g., seedlessness) that were found, highlight the importance of improving knowledge of the Armenian grapevine genetic pool.

Grapevine ( Vitis vinifera L.) cultivars are clonally propagated to preserve their varietal attributes. However, novel genetic variation still accumulates due to somatic mutations. Aiming to study the potential impact of clonal propagation history on grapevines intra-cultivar genetic diversity, we have focused on ‘Malbec’. This cultivar is appreciated for red wines elaboration, it was originated in Southwestern France and introduced into Argentina during the 1850s. Here, we generated whole-genome resequencing data for four ‘Malbec’ clones with different historical backgrounds. A stringent variant calling procedure was established to identify reliable clonal polymorphisms, additionally corroborated by Sanger sequencing. This analysis retrieved 941 single nucleotide variants (SNVs), occurring among the analyzed clones. Based on a set of validated SNVs, a genotyping experiment was custom-designed to survey ‘Malbec’ genetic diversity. We successfully genotyped 214 samples and identified 14 different clonal genotypes, that clustered into two genetically divergent groups. Group-Ar was driven by clones with a long history of clonal propagation in Argentina, while Group-Fr was driven by clones that have longer remained in Europe. Findings show the ability of such approaches for clonal genotypes identification in grapevines. In particular, we provide evidence on how human actions may have shaped ‘Malbec’ extant genetic diversity pattern.

Paradoxically, continuous vegetative multiplication of traditional grapevine cultivars aimed to maintain cultivar attributes in this highly heterozygous species ends in the accumulation of considerable somatic variation. This variation has long contributed to cultivar adaptation and evolution under changing environmental and cultivation conditions and has also been a source of novel traits. Understanding how this somatic variation originates provides tools for genetics-assisted tracking of selected variants and breeding. Potentially, the identification of the mutations causing the observed phenotypic variation can now help to direct genome editing approaches to improve the genotype of elite traditional cultivars. Molecular characterization of somatic variants can also generate basic information helping to understand gene biological function. In this chapter, we review the state of the art on somatic variation in grapevine at phenotypic and genome sequence levels, present possible strategies for the study of this variation, and describe a few examples in which the genetic and molecular basis or very relevant grapevine traits were successfully identified.

Seedlessness is greatly prized by consumers of fresh grapes. While stenospermocarpic seed abortion determined by the SEED DEVELOPMENT INHIBITOR (SDI) locus is the usual source of seedlessness in commercial grapevine (Vitis vinifera) cultivars, the underlying sdi mutation remains unknown. Here, we undertook an integrative approach to identify the causal mutation. Quantitative genetics and fine mapping in two 9Crimson Seedless9 (CS)-derived F1 mapping populations confirmed the major effect of the SDI locus and delimited the sdi mutation to a 323-kb region on chromosome 18. RNA-seq comparing seed traces of seedless and seeds of seeded F1 individuals identified processes triggered during sdi-determined seed abortion, including activation of salicylic acid-dependent defenses. The RNA-seq dataset was investigated for candidate genes and, while no evidence for causal cis-acting regulatory mutations was detected, deleterious nucleotide changes in coding sequences of the seedless haplotype were predicted in two genes within the sdi fine mapping interval. Targeted re-sequencing of the two genes in a collection of 124 grapevine cultivars showed that only the point variation causing the Arg197Leu substitution in the seed morphogenesis regulator gene AGAMOUS-LIKE 11 (VviAGL11) was fully linked with stenospermocarpy. The concurrent post-zygotic variation identified for this missense polymorphism and seedlessness phenotype in seeded somatic variants of the original stenospermocarpic cultivar supports a causal effect. We postulate that seed abortion caused by this amino acid substitution in VviAGL11 is the major cause of seedlessness in cultivated grapevine. This information can be exploited to boost seedless grape breeding.