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|Autori: ||Fornasiero, Alice|
|Supervisore afferente all'Università: ||MORGANTE, MICHELE|
|Centro di ricerca: ||DIPARTIMENTO DI SCIENZE AGROALIMENTARI, AMBIENTALI E ANIMALI - DI4A|
|Titolo: ||Identification and mapping of loci controlling viability in Vitis vinifera crosses|
|Abstract (in inglese): ||The present research work is part of the NOVABREED project, funded by the European Research Council (ERC). The focus of NOVABREED is the concept of pan-genome as a comprehensive representation of a species genome: its main aim is to characterise the dispensable portion that arises as a consequence of structural variation, and its contribution to the intra-specific genetic and phenotypic variation (Morgante, De Paoli, & Radovic, 2007). The focus of the present work is on the Vitis vinifera genome. The goal of the present research is to understand the genetic bases of inbreeding depression in grapevine through the identification of loci controlling viability and survival and their relationship with structural variation.
Vitis vinifera is a very variable species with high levels of both nucleotide diversity and structural variation (Gabriele Magris, PhD thesis, 2016). Cultivated varieties are highly heterozygous (Jaillon et al., 2007) and are expected to carry a large mutational load that is reflected in rather severe inbreeding depression observed upon crossing of related individuals or selfing. We set out to identify loci that are responsible for inbreeding depression both individually as well as a consequence of epistatic interactions. Through the analysis of segregation distortion, defined as deviation of segregation ratio from the expected Mendelian ratio, we explored progenies segregation pattern with the goal of isolating causative mutations of the distortion.
In order to characterize segregation distortion in Vitis vinifera, progenies obtained from self-crosses of six varieties and from one out-cross were genotyped using a Genotyping-by-Sequencing approach. The technique used, known as double digest Restriction Site Associated DNA Sequencing (ddRAD-seq) (Peterson, Weber, Kay, Fisher, & Hoekstra, 2012), subsamples the genome at homologous locations among individuals by coupling double restriction enzyme digestion to a selection of genomic fragments by size, allowing fine-scale control of the fraction of regions represented in the final library. SNP genotyping, by means of the software Stacks (Catchen, Hohenlohe, Bassham, Amores, & Cresko, 2013), allowed the identification of several regions of single locus distortion in each progeny assessed.
Progenies deriving from self-fertilization of the six cultivars Cabernet franc, Pinot Noir, Primitivo, Rkatsiteli, Sangiovese and Schiava Grossa, showed distortion in 12 different loci. Progeny of the out-cross between Schiava Grossa and Rkatsiteli did not show any locus of segregation distortion. Overall, ten loci of distortion revealed lethal effects, whereas two loci revealed severe deleterious effects. Among loci with lethal effect, seven showed the complete absence of a homozygous genotype, corresponding to the action of recessive alleles; two showed the action of partially dominant alleles and one showed nearly complete dominance of the lethal allele. Deleterious loci exhibited recessive and overdominant allelic effect on distortion, respectively. In three different varieties, chromosome 8 was revealed to harbour non overlapping loci of distortion with different allelic effects. Analysis of selected progenies over two vegetative seasons showed that five loci caused embryo or seedling lethality in early stages of growth, while two loci exerted their lethal effects on growth only after the first wintering. Fine mapping of the distorted regions allowed to narrow down the causal locus to less than 34 Kbp, in the best case.
Genotype data of the progeny of Rkatsiteli self-cross and of the progeny of Schiava Grossa and Rkatsiteli out-cross was used to build genetic linkage maps of the parental varieties. The three genetic maps were used to analyse recombination frequency along the genome of Vitis vinifera.
To identify structural variants (SVs), single nucleotide polymorphisms (SNPs) and small insertion-deletion polymorphisms (INDELs) contributing to the genetic load in the grapevine genome and leading to segregation distortion in the progenies of selfing, candidate loci were screened for mutations affecting genes. Haplotype phasing of alleles allowed to identify mutations belonging to the haplotype that generated segregation distortion in the progenies of selfing. Furthermore, expression of genes affected by such variation was evaluated in a panel of six varieties in three different tissues (leaf, berry and tendril).
The analysis of two-loci segregation distortion was also performed in order to identify epistatic interactions. Fisher’s test of independence revealed one strong signal of interaction between loci on chromosome 1 and chromosome 11 in the variety Rkatsiteli, evidenced also by the pseudo-linkage signal in the genetic map. Further evidences showed that the interaction was actually due to a balanced translocation, which was validated through a PCR-based assay. In order to evaluate gene expression differences between the haplotype carrying the translocation and the normal haplotype in Rkatsiteli, we performed allele specific expression (ASE) analysis for each of the three tissues. Interestingly, the analysis revealed no significant difference in the allele-specific expression profile in the tissues.
In order to detect the balanced translocation in other grapevine varieties, a panel of 196 cultivars was tested, revealing that three varieties - Alexandroouli, Mtsvane Kachuri and Gorula - carried the rearrangement. All four varieties originated from Georgia and belonged to the Proles pontica, although not all the varieties of this Proles showed the translocation. This suggests that Georgian varieties are distinct and genetically variable from western one and that translocation could be an ancient event never imported to the West and Central Europe varieties (Proles occidentalis).
Our study showed that self-fertilization of Vitis vinifera cultivars lead to high levels of segregation distortion in the progenies due to the presence of unfavourable alleles in genes. Future studies will be oriented to the characterization of the identified genes.
Furthermore, we generated a fine-scale map of recombination frequency along the genome of Vitis vinifera.
Lastly, we detected and validated a balanced translocation involving chromosome 1 and chromosome 11 in the variety Rkatsiteli and we found the chromosomal rearrangement in other three grapevine varieties|
|Parole chiave: ||Vitis vinifera; Segregation distortion; Lethal and deleterious alleles|
|MIUR : ||Settore BIO/18 - Genetica|
Settore AGR/07 - Genetica Agraria
|Corso di dottorato: ||Dottorato di ricerca in Scienze e biotecnologie agrarie|
|Ciclo di dottorato: ||29|
|Università di conseguimento titolo: ||Università degli Studi di Udine|
|Luogo di discussione: ||Udine|
|Altre informazioni: ||Co-supervisore: Fabio Marroni|
|Citazione: ||Fornasiero, A. Identification and mapping of loci controlling viability in Vitis vinifera crosses. (Doctoral Thesis, Università degli Studi di Udine, 2017).|
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