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Title: Szczegółowe mapowanie genu związanego z pierwszym etapem rozwoju włośników u jęczmienia (Hordeum vulgare L.)
Authors: Gajewska, Patrycja
Advisor: Szarejko, Iwona
Janiak, Agnieszka
Keywords: genetyka molekularna; geny markerowe; jęczmień zwyczajny; mutacje roślin; włośniki korzeniowe; mapy genetyczne
Issue Date: 2018
Publisher: Katowice : Uniwersytet Śląski
Abstract: Root hairs are the part of root architecture contributing significantly to the root surface area. In many species including barley (Hordeum vulgare L), the patterning of rhizodermis is the first stage of root hairs development. It results in the formation of two types of cells: trichoblasts, which became the root hair cells and atrichoblasts, which are not capable of producing the root hairs. In H. vulgare trichoblasts and atrichoblasts differentiation depends on the asymmetrical elongation of daughter cells that are produced after symmetrical division of their mother cell. As a result, the shorter cells develop into the trichoblasts. The aim of this study was to identify a gene responsible for the first stage of barley root hairs morphogenesis, i.e. the rhizodermis differentiation in barley. A root hairless mutant rhl1.b (root hairless1) obtained after chemical mutagenesis of variety ‘Karat’ was used in the study. Additionally, a spontaneous root hairless mutant brb (bald root barley) from cv. ‘Pallas' was analyzed. In previous studies of the Department of Genetics, University of Silesia, the rhl1 gene was mapped on chromosomes 7HS. The presented thesis demonstrates a map-based gene isolation approach, comprising of: development of mapping population and construction of high resolution genetic map of the region of interest, transition from genetic map to the physical map of barley, selection and sequencing candidate genes, and the identification of a mutation, which may be responsible for the mutant phenotype. At first, mapping population consisting of 4952 F₂ individuals was derived from the cross between rhl1.b mutant and ‘Morex’ variety. Based on this population, the genetic map was developed using SSR, SNP and InDel markers. The markers were selected from literature data on barley genetic maps and the internet databases (Genome Zipper, Ensembl Plants). Genotyping of the polymorphic SNP/InDel markers was performed using a sub-population of 1472 F₂ plants and for 228 recombinants between the SSRs flanking rhl1 locus. Finally, following several rounds of map enrichment, the markers flanking the rhl1 gene region were identified: MLOC_4840 (2.2 cM distal from the gene position) and MLOC_35776 (1.5 cM at the proximal site of the gene). They established an interval of a total length of 3.7 cM, which corresponds to 577 kbp region on the physical map of barley genome, according to Ensembl Plant database. The utilization of published physical barley genome map allowed to skip the “chromosome walking” phase of candidate gene search. Five candidate genes were identified within the physical interval on the 7HS chromosome, and all of them were sequenced in rhl1.b mutant and ‘Karat’ variety. Only in 140 one candidate gene, HORVU7Hr1G030250 (MLOC_38567) the A→T mutation was identified, differing the mutant from its parent variety. The mutation was located in the 3’ splice-junction site, between sixth exon and sixth intron and it lead to the loss of splicing acceptor site. The co-segregation of the candidate gene with the root hair phenotype was confirmed, based on the analysis of F₂ rhl1.b x ‘Morex’mapping population. The in silico analysis of the mutation site and its potential influence on the splicing process and the structure of the synthesized protein showed that the mutation causes the retention of the of last intron, the frameshift, the synthesis of 71 abnormal amino acids and the introduction of premature STOP codon in mRNA of rhl1 mutant. The intron retention in the mRNA of the gene in the mutant rhl1.b roots was confirmed experimentally. The analysis of protein sequence encoded by HORVU7Hr1G030250 gene resulted in the identification of two conserved domains: bHLH and LRL, which are the key elements for the regulatory properties of bHLH transcription factor protein. In rhl1.b mutant, the synthesis of mRNA with a premature STOP codon can lead to transcript degradation, as the reduced expression of the HORVU7Hr1G030250 gene in all root zones of the mutant, compared to the 'Karat' variety, was observed. The mutation may also result in the synthesis of the bHLH protein with the abnormal sequence of the LRL domain, which in Arabidopsis is involved in the regulation of root hair development. The above mentioned findings suggest that the HORVU7Hr1G030250 gene is the most promising candidate responsible for the rhizodermis patterning in barley and that the identified mutation causes the lack of root epidermal cells differentiation. We did not find any changes between brb mutant, allelic to rhl1.b, and its parent variety ‘Pallas’ in the sequence of HORVU7Hr1G030250 candidate gene, both in the gene body and a substantial region of upstream and downstream sequences, neither in any other candidate genes. In the presented thesis, this result was discussed in the light of the literature data and the analysis of the expression profiles of selected genes that may be the possible target genes for HORVU7Hr1G030250. Such analysis was performed for both hairless mutants and their parent varieties.
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