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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Identifying natural modifiers of meiotic crossover frequency in Arabidopsis thaliana

Lawrence, Emma Jane January 2019 (has links)
During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, producing crossovers. This generates genetic diversity and is required for balanced homolog segregation. Despite the critical functions of crossovers, their frequency and distribution varies extensively within and between species. This crossover variation can be caused by trans-modifiers within populations, which encode diffusible molecules that influence crossover formation elsewhere in the genome. This project utilised natural accessions of Arabidopsis thaliana to identify trans-modifying loci underlying crossover variation within the species. I performed Quantitative Trait Loci (QTL) mapping using a fluorescence-based crossover reporter system to measure recombination frequency in a genomic interval on chromosome 3, termed 420. Mapping in a Col-420 × Bur-0 F2 population revealed four major recombination QTLs (rQTLs) that influence crossover frequency. A novel recessive rQTL on chromosome 1 that reduced crossovers within the interval was fine-mapped to a premature stop codon in TATA Binding Protein (TBP)-associated factor 4b (TAF4b) in Bur-0 (taf4b-1). TAF4b is a subunit of the TFIID complex, a multi-protein general transcription factor complex comprising TBP and numerous TAFs that forms a component of the pre-initiation complex that recruits RNA polymerase II to promoters. Transformation-based complementation experiments and the isolation of several independent taf4b alleles provided genetic proof that TAF4b is essential for wild-type levels of crossover within 420. Analysis of the prevalence of the taf4b-1 mutation in the global Arabidopsis accession collection demonstrated its specificity to three accessions in the British Isles. A combination of cytology, genetic analysis using additional fluorescent reporter lines, and sequencing in F2 recombinant populations demonstrated a genome-wide reduction in crossover frequency in taf4b-1. In addition, RNA sequencing identified numerous transcriptional changes in taf4b-1. Both up- and down-regulated gene sets displayed significant enrichment for genes that are predominantly expressed in meiocytes, and several gene ontology terms pertaining to protein modification and meiotic processes. These results further demonstrate the existence of genetic modifiers of crossover frequency in natural populations of A. thaliana, and the characterisation of a novel trans-modifier of recombination, TAF4b. This signifies a novel function for TAF4b in Arabidopsis, and further enhances our understanding of the molecular factors controlling the frequency and distribution of meiotic crossovers in plants.
2

Investigation of natural genetic modifiers of meiotic crossover frequency in Arabidopsis thaliana

Griffin, Catherine Helen January 2017 (has links)
Meiotic recombination, known as crossover, is a vital mechanism for generating genetic diversity in sexually reproducing populations. Recombination events are non-uniform across the genome, due to a variety of influences including chromatin structure, DNA-sequence, epigenetic marks and interference from other recombination events. These known factors do not fully explain the distribution of recombination events, and additionally do not account for all the variability in recombination frequency observed both between and within species. Furthermore, of the mechanisms that have been identified, many are not yet fully understood. In Arabidopsis thaliana, considerable variation is observed in recombination frequency and distribution between natural accessions. By investigating recombination events in A.thaliana, this project aimed to identify trans-acting modifiers of recombination frequency that varied between natural accessions. Identification of meiotic recombination modifiers was performed through Quantitative Trait Loci (QTL) mapping in A.thaliana natural-accession cross populations. Populations were generated from crosses between two accessions which differed significantly for recombination frequency as measured across a defined region of the genome flanked by a fluorescent-reporter system. F1 plants were then self-fertilised to produce segregating mosaic F2 populations for mapping. Recombination frequency for specific genomic intervals was determined for each individual in the population through measurement of the segregation of flanking fluorescence-genes expressed in the products of meiosis - seeds or pollen. Individuals were also genotyped using accession-specific markers across the genome, at a marker density of one marker per 2-5Mb, depending on the chromosome. Association of variation in recombination frequency with specific sections of the genome differing between the parental accessions through QTL mapping revealed significant modifiers of meiotic recombination segregating within the populations. This resulted in the identification of three significant large-effect modifiers that differed between Col-0 and Cvi-0 accessions, on chromosomes 1 ,2 and 5, affecting recombination in an interval in the sub-telomere region of chromosome 3. An additional modifier on chromosome 4 affecting the same sub-telomeric interval was identified that differed between the Col-0 and Can-0 accessions. Further fine-mapping of modifiers to improve location resolution was performed by repeated backcrosses into the Col-0 genetic background to remove the influence of other large-effect QTL and possible unknown small-effect modifiers. Improving the resolution provided a number of potential candidates for genes underlying the recombination phenotype for each QTL. Candidate testing was then performed, either through transformation of different accession alleles into the fluorescent-reporter system, or through analysis of T-DNA insertion lines that interrupted candidate genes. Preliminary results from T-DNA insertion mutants crossed to the fluorescent-reporter system suggest a potential role for the AT2G31510 gene in modification of meiotic recombination frequency, though the mode of action remains unknown. These results demonstrate the presence of large-effect modifiers of meiotic recombination frequency that vary between the natural A.thaliana accessions Col-0, Cvi-0 and Can-0. Confirmation of underlying genes or sequence elements and characterisation of their mechanism of action are opportunities for exploration in future experiments.
3

Analysis of gene expression data from Massive Parallel Sequencing identifies so far uncharacterised regulators for meiosis with one candidate being fundamental for prophase I in male and female meiosis

Finsterbusch, Friederike 15 February 2016 (has links)
Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-­‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search. Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knock­out oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search.

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