Genetic investigation of seed development in grapevine

In a comprehensive attempt to understand the molecular and cellular processes driving seedlessness in grapevine, a seeded variety (wild-type) and its seedless somatic variant (mutant) were characterized at the morphological, genomic and transcriptomic levels in relation to berry development and seed content. The overall importance of clonal variability and the application of Next Generation Sequencing technology in highlighting the molecular events during seed formation within a developing berry have been clearly demonstrated. In this thesis three hypothesis were formulated, tested and confirmed. First it was hypothesized that the mutant has a gross morphology identical to the wild-type except for berry size and seed content. In testing this hypothesis quantitative and qualitative traits that relate to berry development and seed content were compared in the two clones. Here traits that were significantly different in the two lines are those that relate only to berry size and seed content. This evaluation was performed both in control conditions (self-pollination) and after anther/stigma removal which allowed the investigation of a possible role for parthenocarpy, stenospermocarpy or other mechanisms in promoting the phenotype of the seedless somatic variant. The second hypothesis states that the mutant is sterile or partly sterile hence cannot produce viable seeds. In order to verify this hypothesis pollen germination and viability assays were carried out in both clones. The tests confirmed pollen germination and vitality percentage of the mutant was significantly lower than that of the wild-type. The third hypothesis concerned the existence of genomic/transcriptomic differences between the two lines and could be tested through the power of the Next generation Sequencing technology. In particular, we raised the following questions. Are there somatic mutations that can allow the wild-type and mutant to be distinguished? What are the temporal and spatial changes that could occur in their respective transcriptomes? Especially how does expression levels of key regulatory genes change before, during and after fertilization in the two clones? These key questions were addressed with the aid of Molecular marker analysis, Array based SNP genotyping method and RNA-Seq approach. Using 58 microsatellites, the analyzed loci showed identical profile in the wild-type and the mutant. The 20K grapevine Illumina Chip revealed 16333 identical SNP loci in the two clones, thus a further confirmation of the true identity of the seedless line. Conversely variant calling from RNA-Seq enabled the identification of several somatic mutations at the whole-genome level in the two lines. At the same time, RNA-Seq allowed the creation of inventories of gene expression at successive stages of seed formation. i.e. stages E-L 15 (single flowers in compact groups), E-L 27 (young berries enlarging) and E-L 38 (berries harvest-ripe). Here the transcriptomes revealed by Illumina mRNA-Seq technology had approximately 98% of grapevine annotated transcripts and about 80% of them were commonly expressed in the two lines. Differential gene expression analysis revealed a total of 1075 differentially expressed genes (DE) in the pairwise comparison of developmental stages, which included DE genes specific to the wild-type background, DE genes specific to the mutant background and DE genes commonly shared in both backgrounds. The analysis of differential expression patterns and functional category enrichment of wild-type and mutant DE genes highlighted significant coordination and enrichment of pollen and ovule developmental pathways. The expression of some selected DE genes was further confirmed by real-time RT-PCR analysis. To the best of our knowledge the work presented in this thesis represents the most comprehensive attempt to characterize the genetic bases of seed formation in grapevine. We have shown that a seeded wine grape and its seedless somatic variant are similar in several biological processes except for berry size and seed content. With a high throughput method we could identify an inventory of genes with altered expression in the mutant compared to the wild-type, which may be responsible for the seedless phenotype. The genes located within known genomic regions regulating seed content may be used for the development of molecular tools to assist table grape breeding. Therefore the data reported here have provided a rich genomic resource for practical use and functional characterization of the genes that potentially underpin seedlessness in grapevine.