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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 (<0.025). The haplophase alignment identified > 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.

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Grapevine, as other woody perennials, has been considered a recalcitrant crop to produce transgenic plants. Since the production of transgenic and/or edited plants requires the ability to regenerate plants from transformed tissues, this step is often the biggest bottleneck in the process. The objective of this work is to review the state of the art technologies and strategies for the improvement of grapevine transformation and regeneration, focusing on three aspects: (i) problems associated with grapevine transformation; (ii) genes that promote grapevine regeneration; and (iii) vehicles for gene delivery. Concerning the first aspect, it is well documented that one of the main factors explaining the low success rate in obtaining transgenic plants is the regeneration process. After transgenic integration into receptor cells, tissue culture is required to regenerate transgenic seedlings from transformed cells. This process is time consuming and often requires the addition of environmentally damaging reagents (antibiotics and herbicides) to the culture medium to select transgenic plants. On the other hand, the expression of genes such as the so-called developmental regulators (DR), which induce specific development programs, can be used to avoid traditional tissue culture methods. The ectopic expression of specific combinations of DR in somatic cells has the potential to induce de novo meristems in diverse crops, including grapevine. Successful genome editing by de novo reprogramming of plant meristems in somatic tissues has been reported. Moreover, it has been shown that the expression of certain transcription factors can increase the regeneration efficiency in wheat, citrus, and rice. Finally, recent reports showed the use of nanoparticles, such as carbon dots (CDs), as an attractive alternative to Agrobacterium- and biolistic-mediated plant genetic transformation. In this way, the use of antibiotics in culture media is avoided, overcoming the loss of viability of plant tissues and accelerating the regeneration processes. It has been shown that CDs can act as a vehicle to transport plasmids to plant cells in transient transformation in several crops without negative impacts on photosynthesis or growth. Based on these advances, it is possible to combine these new available strategies and technologies to overcome the regeneration problems of species such as grapevine and other crops considered as recalcitrant.

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Grapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.

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Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.

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Fruit size is a highly important trait for most fruit and vegetable crops. This trait has been a main selection target and could be involved in divergent selection processes leading to the differentiation between modern table and wine cultivars. Even though its determination is highly influenced by cultural practices, several regions within the grapevine genome have been identified affecting berry size, either directly or indirectly through their effect on seed content. Using grapevine seeded cultivars, we have analyzed the relationship between ovary cell number and the final size of ovaries and berry fruits. We also performed the characterization of the grapevine AINTEGUMENTA-LIKE family, since it is well reported in Arabidopsis that AINTEGUMENTA (ANT) regulates cell proliferation and organ growth in flower organ primordia by maintaining the meristematic competence of cells during organogenesis. Here we show that orthologous grapevine gene expression associate with flower developmental stages suggesting a similar biological role for this gene family in this species. Moreover, we detected a correlation between those organs size and the level of expression of VviANT1 the grapevine homolog of AtANT. This grapevine gene also co-localizes in linkage group 18 with the confidence interval of a previously detected QTL for berry size. Thus our results suggest the involvement of ANT in the regulation of berry size in grapevine.

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Sunlight exposure has multiple effect on fruits, as it affects the light climate perceived by fruit photoreceptors and fruit tissue temperature. In grapes (Vitis vinifera L.), light exposure can have a strong effect on fruit quality and commercial value; however, the mechanisms of light action are not well understood. The role of fruit-localized photoreceptors in the control of berry quality traits was evaluated under field conditions in a commercial vineyard in Mendoza (Argentina). Characterization of the diurnal dynamics of the fruit light environment in a vertical trellis system indicated that clusters were shaded by leaves during most of the photoperiod. Supplementation of the fruit light environment from 20 days before veraison until technological harvest showed that red (R, 660 nm) and blue (B, 470 nm) light strongly increased total phenolic compound levels at harvest in the berry skins without affecting sugar content, acidity or berry size. Far-red (FR, 730 nm) and green (G, 560 nm) light supplementation had relatively small effects. The stimulation of berry phytochromes and cryptochromes favored accumulation of flavonoid and non-flavonoid compounds, including anthocyanins, flavonols, flavanols, phenolic acids and stilbenes. These results demonstrate that the chemical composition of grape berries is modulated by the light quality received by the clusters under field conditions, and that fruit photoreceptors are not saturated even in areas of high insolation and under management systems that are considered to result in a relatively high exposure of fruits to solar radiation. Therefore, manipulation of the light environment or the light sensitivity of fruits could have significant effects on critical grape quality traits.

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Anthocyanin profiles are commonly used for grapevine cultivar identification because it is currently accepted that this trait is closely related to their genetic characteristics. Nevertheless, the extent of the variation for the anthocyanin profiles among clones of the same cultivar has not yet been studied in depth. The relative concentration of anthocyanins of 131 Malbec clones grown in the same vineyard was investigated by HPLC-DAD and the use of comprehensive statistic procedures. Complementarily, the expression level of structural and regulatory genes was studied via real time polymerase chain reaction. Significant variation was identified among the profiles of the clones, mainly due to variations in the amounts of malvidin derivatives. Finally, the differential expression in F3'5'H, OMT1 and AM2 genes seems to be related to the malvidin content variation. This work shows the existence of variation for the anthocyanin profiles among clones from the same grapevine cultivar and the putative involvement of genes related to hydroxylation, methylation, and transport of anthocyanins on the basis of such variation.

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BACKGROUND: Ultraviolet-B radiation (UV-B, 280-315 nm) is a natural component of sunlight, which has numerous regulatory effects on plant physiology. The nature of the response to UV-B is dependent on fluence rate, dose, duration and wavelength of the UV-B treatment. Some reports have analyzed the changes in gene expression caused by UV-B light on several plant species using microarray technology. However, there is no information on the transcriptome response triggered by UV-B in grapevine. In this paper we investigate the gene expression responses of leaves from in vitro cultured Vitis vinifera cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m-2 d-1) administered at two different fluence rates (16 h at ≅ 8.25 µW cm-2, 4 h at ≅ 33 µW cm-2) using a new custom made GrapeGen Affymetrix GeneChip®. RESULTS: The number of genes modulated by high fluence rate UV-B doubled the number of genes modulated by low fluence UV-B. Their functional analyses revealed several functional categories commonly regulated by both UV-B treatments as well as categories more specifically modulated depending on UV-B fluence rate. General protective responses, namely the induction of pathways regulating synthesis of UV-B absorbing compounds such as the Phenylpropanoid pathway, the induction of different antioxidant defense systems and the activation of pathways commonly associated with pathogen defense and abiotic stress responses seem to play critical roles in grapevine responses against UV-B radiation. Furthermore, high fluence rate UV-B seemed to specifically modulate additional pathways and processes in order to protect grapevine plantlets against UV-B-induced oxidative stress, stop the cell cycle progression, and control protein degradation. On the other hand, low fluence rate UV-B regulated the expression of specific responses in the metabolism of auxin and abscisic acid as well as in the modification of cell walls that could be involved in UV-B acclimation-like processes. CONCLUSION: Our results show the UV-B radiation effects on the leaf transcriptome of grapevine (Vitis vinifera cv. Malbec) plantlets. Functional categories commonly modulated under both UV-B treatments as well as transcripts specifically regulated in an UV-B-intensity dependent way were identified. While high fluence rate UV-B had regulatory effects mainly on defense or general multiple-stress responses pathways, low fluence rate UV-B promoted the expression of genes that could be involved in UV-B protection or the amelioration of the UV-B-induced damage. This study also provides an extensive list of genes regulating multiple metabolic pathways involved in the response of grapevine to UV-B that can be used for future researches.

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