Comparative analysis of the genetic variation of flowering time genes in Vitis vinifera

Kamal N. Comparative analysis of the genetic variation of flowering time genes in Vitis vinifera. Bielefeld: Universität Bielefeld; 2018.Grapevine is one of the economically most valuable fruit crops in the world. In recent years climate change and global warming have led to significant strains on viticulture. Higher temperatures at earlier dates within the growing season advance the date of bud burst and flowering. Consequently, the predicted shift in véraison to earlier dates causes ripening to occur under higher temperatures which impairs wine quality. It is of high interest for viticulture to investigate the timing of flowering and elucidate its genetic architecture. Genetic variation between cultivars leads to differences in the expression of phenotypic traits such as flowering time. A deeper understanding of varietal differences in the phenology of flowering time is thus critical to select varieties that are well adapted for production under current and future climatic conditions. The goal of this thesis was therefore to identify alleles of Flowering Time Control (FTC) candidate genes that might be involved in the timing of flowering. More than four hundred FTC candidate genes were identified in grapevine by using functional data from A. thaliana to exploit the grapevine genome for homologues. In addition, previously defined quantitative trait locus (QTL)-regions for FTC in grapevine were considered for the selection of candidate genes. From this selection, 72 genes were chosen for an amplicon sequencing approach with a focus on flowering time related QTL-regions. The amplicons subjected to sequencing were derived from the parental lines and 35 individuals of the F1-mapping population GF.GA-47-42 x ‘Villard Blanc’. This population segregates for the trait flowering time. In order to identify alleles of amplicon sequenced genes, a bioinformatic workflow for the phasing of the alleles of each gene was established. Alleles in genomic regions of 46 genes on 16 chromosomes with a length of up to 8.3 kb were distinguished. The inheritage of alleles of closely neighbored genes remains largely constant, which indicates the functionality of the established workflow. Moreover, analyzing inheritance patterns within an F1-population including the parental lines proved to be an effective method for the validation of allele phasing results. Functional molecular markers that are capable of distinguishing between up to four alleles and suitable for analyzing polymorphisms located in genomic regions were developed. A genetic association study between alleles of FTC candidate genes and the timing of flowering was performed. Using a Wilcoxon Rank-Sum test, correlation between specific alleles of FTC candidate genes and flowering time phenotypes was investigated. This revealed a correlation within and outside of QTL-regions for flowering time on chromosome 1, 4, 14, 17, and 18. Among others, appearance of an allele of Vitis vinifera WITH NO LYSIN KINASE 6 (VvWNK6) inherited from early flowering GF.GA-47-42 was found to highly correlate with early flowering. In addition to the above mentioned population, alleles of FTC genes were analyzed in other Vitis cultivars. They were investigated for common alleles of FTC candidate genes with a focus on alleles correlating with flowering time phenotypes. Common alleles of FTC candidate genes were found across different cultivars. However, common alleles were usually only shared between a few cultivars since grapevine exhibits a high genetic diversity. Within a diverse panel of eleven well-known grapevine cultivars, all late flowering cultivars were found to harbor an allele of Vitis vinifera TOMATO MADS BOX GENE 6 (VvTM6). In order to further analyze and confirm the role of FTC candidate genes, RNA-Seq experiments were performed. Differential gene expression was analyzed over consecutive developmental stages of buds and inflorescences of early flowering GF.GA-47-42 and late flowering ‘Villard Blanc’. Both analyzed cultivars show similar expression patterns for most genes. Many of the analyzed FTC candidate genes showed differential expression between consecutive time points, indicating functional roles of these genes in the flowering control network. Moreover, most of these genes were found to be expressed in buds and inflorescences but not in leaves. This further supports their role in flowering initiation and floral development. The findings of this study provide indications of the genetic factors controlling or influencing flowering time in grapevine. For future research the clarification of these factors can contribute to the development of molecular genetic markers that are capable of predicting flowering time phenotypes. This is of crucial importance in light of the need to breed new grapevine varieties adapted for production under changing climatic conditions