Jorge Cunha

Characterization of Portuguese populations of Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi

Wild populations of Vitis vinifera L.␣have been located in Portugal. Morphological characterization was carried out in three populations located in Alcácer do Sal, Castelo Branco, and Montemor-o-Novo, and then compared using multivariate discriminant analysis. These populations were from three different hydrological basins, therefore cross-pollination was not possible. It was verified that in each population all plants were different. The data suggest that the frequency of female and male plants is rather variable in wild populations. The morphology of the adult leaf, from the Alcácer do Sal population, had particular features when compared with Castelo Branco and Montemor-o-Novo populations, which were more homogeneous. The length of teeth compared with width at the end of the base, and the density of prostrate hairs between and on main veins (lower side) were the variables which allowed the best discrimination among populations.

Flower development and sex specification in wild grapevine

BackgroundWild plants of Vitis closely related to the cultivated grapevine (V. v. vinifera) are believed to have been first domesticated 10,000 years BC around the Caspian Sea. V. v. vinifera is hermaphrodite whereas V. v. sylvestris is a dioecious species. Male flowers show a reduced pistil without style or stigma and female flowers present reflexed stamens with infertile pollen. V. vinifera produce perfect flowers with all functional structures. The mechanism for flower sex determination and specification in grapevine is still unknown.ResultsTo understand which genes are involved during the establishment of male, female and complete flowers, we analysed and compared the transcription profiles of four developmental stages of the three genders. We showed that sex determination is a late event during flower development and that the expression of genes from the ABCDE model is not directly correlated with the establishment of sexual dimorphism. We propose a temporal comprehensive model in which two mutations in two linked genes could be players in sex determination and indirectly establish the Vitis domestication process. Additionally, we also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role involved in sex differentiation. Also, the detection of differentially transcribed regions that extended existing gene models (intergenic regions) between sexes suggests that they may account for some of the variation between the subspecies.ConclusionsThere is no evidence of differences of expression levels in genes from the ABCDE model that could explain the shift from hermaphroditism to dioecy. We propose that sex specification occurs after floral organ identity has been established and therefore, sex determination genes might be having an effect downstream of the ABCDE model genes.For the first time a full transcriptomic analysis was performed in different flower developmental stages in the same individual. Our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across developmental stages and genders that will contribute for future work in sex determination in seed plants.

Portuguese Vitis vinifera L. Germplasm: Accessing Its Diversity and Strategies for Conservation

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world. In the different Portuguese agro-ecosystems, grapevine plays an impor‐ tant role either as a border culture or as an extensive crop. The surface area used by vine‐ yards amounts to 4.9 % of the arable land [1], representing 240,000 ha, being the 7th largest area in the world and the 4th in the European Union [2]. In 2011 Portugal produced 5.9 mil‐ lion hectoliters of which 2.9 million hectoliters were exported, making the country the 12th world wine producer [2]. There are fourteen wine regions with Protected Geographical Indi‐ cation (Figure 1) and 31 wine areas with Designation of Origin status including Porto, estab‐ lished since 1756, the oldest legally established wine production region in the world. Each one of the wine regions has a particular set of grapevine cultivars adapted to its specific ter‐ roirs. Officially there are 343 cultivars allowed to be use in wine production in Portugal [3].

Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification

Key messageRNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment.AbstractThe molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.

VviAPRT3 and VviFSEX: Two Genes Involved in Sex Specification Able to Distinguish Different Flower Types in Vitis

Vitis vinifera vinifera is a hermaphrodite subspecies, while its ancestor, Vitis vinifera sylvestris, is dioecious. We have identified two genes that together allow the discrimination between male, female and hermaphrodite Vitis plants. The sex locus region on chromosome 2 was screened resulting in the discovery of a new gene, VviFSEX. The same screening revealed another gene, VviAPRT3, located in the sex region, that be used as a sex marker. Both genes are good candidates to be involved in flower sex differentiation in grapevine. To assess their role in sex specification, spatial and temporal expression analysis was performed. The expression of VviFSEX is detected in petals, stamens and carpel primordia of all flower types, making its putative function unclear; however, female plants display a single allele for this gene, while male and hermaphrodites display two alleles. On the other hand, the specific expression of VviAPRT3 in the carpel primordial of male plants suggests a possible role in the abortion of pistil structures. We propose a model to explain the carpel abortion in male flowers and the absence of stamen viability in female flowers. In addition, this work reinforces the presence of a sex locus on Vitis chromosome 2.

Vitis Flower Sex Specification Acts Downstream and Independently of the ABCDE Model Genes

The most discriminating characteristic between the cultivated Vitis vinifera subsp. vinifera and the wild-form Vitis vinifera subsp. sylvestris is their sexual system. Flowers of cultivars are mainly hermaphroditic, whereas wild plants have female and male individuals whose flowers follow a hermaphroditic pattern during early stages of development and later develop non-functional reproductive organs. In angiosperms, the basic developmental system for floral organ identity is explained by the ABCDE model. This model postulates that regulatory gene functions work in a combinatorial way to confer organ identity in each whorl. In wild Vitis nothing is known about the function and expression profile of these genes. Here we show an overall view of the temporal and spatial expression pattern of the ABCDE genes as well as the pattern of VviSUPERMAN that establishes a boundary between the stamen and the carpel whorls, in the male, female and complete flower types. The results show a similar pattern in Vitis species suggesting that the pathway leading to unisexuality acts independently and/or downstream of B- and C- function genes.