Development of the BonnMu transposon resource in maize (Zea mays L.) and its application in characterizing the magenta root dwarf 1 (mrd1) mutant
Development of the BonnMu transposon resource in maize (Zea mays L.) and its application in characterizing the magenta root dwarf 1 (mrd1) mutant
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DissertationDate of Exam
02.07.2024Date of Publication
08.07.2024Advisor
Marcon, CarolineCo-Referee
Schoof, HeikoMetadata
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Abstract
Genome-wide insertional mutagenesis is a tool to generate loss-of-function mutations for virtually all genes in a genome. In this process, mutations are naturally generated by random integration of mobile DNA elements within a genome. The BonnMu resource is a European transposon-tagged mutant collection designed for functional genomics studies in maize. In this thesis the mutant repository was expanded by crossing an active Mutator (Mu) line with dent (B73, Co125) and flint (DK105, EP1 and F7) germplasms, resulting in the generation of 8,064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families identified 425,924 heritable Mu insertions affecting 36,612 (83%) of the 44,303 high-confidence gene models of maize. On average, 12 Mu insertions per gene (425,924 total insertions/ 36,612 affected genes) and 53 insertions per BonnMu F2-family (425,924 total insertions/ 8,064 families) were observed. Mu insertions and photographs depicting seedling phenotypes from segregating BonnMu F2-families are available for access through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5’ untranslated region (UTR) of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163,843 of 425,924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. In summary, the publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.
In the second project, the magenta root dwarf 1 (mrd1) mutant, identified in a forward genetic screening of BonnMu F2-families, was functionally characterized. In comparison to the wild type, mrd1 mutants accumulate anthocyanins in primary, seminal and crown roots and exhibit a limited shoot growth under light and dark conditions. The identification of a causal candidate gene was facilitated by the combination of bulked segregant RNA-seq (BSR-seq) and the availability of the sequenced Mu-tagged BonnMu F2-families, which provided a limited number of candidate mutations for the BSR-seq mapping interval. This combinatory approach identified a constitutive photomorphogenesis 9 (cop9) signalosome complex subunit 4 as the causal candidate gene that underlie the mrd1 phenotype. The COP9 signalosome (CSN) is a multiprotein complex initially identified in Arabidopsis thaliana as a repressor of photomorphogenesis. Maize mrd1 mutants show similarities to csn mutants in Arabidopsis thaliana such as the constitutive photomorphogenesis, but different patterns of anthocyanin accumulation. PCR-based co-segregation analysis identified mrd1 mutants as homozygous for a Mu insertion in the mrd1 gene. The expression of the mrd1 gene was significantly reduced in the primary roots of mrd1 mutants, compared to heterozygous and wild type plants. Microbiome profiling of the rhizosphere from crown roots of mrd1 and wild type indicated an enrichment of specific bacterial genera. Additionally, RNA-seq analysis between mrd1 and wild type crown roots highlighted a down-regulation of the mrd1 gene in the mutant. The attempt to validate the gene using an independent mutant allele generated through CRISPR/Cas9 genome editing was unsuccessful. In a future approach, the mrd1 gene could be validated either by a gene silencing strategy using RNAi or by a novel allele from publicly available mutant resources, such as the in-house BonnMu collection, which is continuously expanding.
In the second project, the magenta root dwarf 1 (mrd1) mutant, identified in a forward genetic screening of BonnMu F2-families, was functionally characterized. In comparison to the wild type, mrd1 mutants accumulate anthocyanins in primary, seminal and crown roots and exhibit a limited shoot growth under light and dark conditions. The identification of a causal candidate gene was facilitated by the combination of bulked segregant RNA-seq (BSR-seq) and the availability of the sequenced Mu-tagged BonnMu F2-families, which provided a limited number of candidate mutations for the BSR-seq mapping interval. This combinatory approach identified a constitutive photomorphogenesis 9 (cop9) signalosome complex subunit 4 as the causal candidate gene that underlie the mrd1 phenotype. The COP9 signalosome (CSN) is a multiprotein complex initially identified in Arabidopsis thaliana as a repressor of photomorphogenesis. Maize mrd1 mutants show similarities to csn mutants in Arabidopsis thaliana such as the constitutive photomorphogenesis, but different patterns of anthocyanin accumulation. PCR-based co-segregation analysis identified mrd1 mutants as homozygous for a Mu insertion in the mrd1 gene. The expression of the mrd1 gene was significantly reduced in the primary roots of mrd1 mutants, compared to heterozygous and wild type plants. Microbiome profiling of the rhizosphere from crown roots of mrd1 and wild type indicated an enrichment of specific bacterial genera. Additionally, RNA-seq analysis between mrd1 and wild type crown roots highlighted a down-regulation of the mrd1 gene in the mutant. The attempt to validate the gene using an independent mutant allele generated through CRISPR/Cas9 genome editing was unsuccessful. In a future approach, the mrd1 gene could be validated either by a gene silencing strategy using RNAi or by a novel allele from publicly available mutant resources, such as the in-house BonnMu collection, which is continuously expanding.
Classification (DDC)
630 Landwirtschaft, VeterinärmedizinRelated Publications
https://doi.org/10.1101/2024.02.24.581857Win, Yan Naing: Development of the BonnMu transposon resource in maize (Zea mays L.) and its application in characterizing the magenta root dwarf 1 (mrd1) mutant. - Bonn, 2024. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-76830
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-76830
@phdthesis{handle:20.500.11811/11646,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-76830,
author = {{Yan Naing Win}},
title = {Development of the BonnMu transposon resource in maize (Zea mays L.) and its application in characterizing the magenta root dwarf 1 (mrd1) mutant},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = jul,
note = {Genome-wide insertional mutagenesis is a tool to generate loss-of-function mutations for virtually all genes in a genome. In this process, mutations are naturally generated by random integration of mobile DNA elements within a genome. The BonnMu resource is a European transposon-tagged mutant collection designed for functional genomics studies in maize. In this thesis the mutant repository was expanded by crossing an active Mutator (Mu) line with dent (B73, Co125) and flint (DK105, EP1 and F7) germplasms, resulting in the generation of 8,064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families identified 425,924 heritable Mu insertions affecting 36,612 (83%) of the 44,303 high-confidence gene models of maize. On average, 12 Mu insertions per gene (425,924 total insertions/ 36,612 affected genes) and 53 insertions per BonnMu F2-family (425,924 total insertions/ 8,064 families) were observed. Mu insertions and photographs depicting seedling phenotypes from segregating BonnMu F2-families are available for access through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5’ untranslated region (UTR) of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163,843 of 425,924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. In summary, the publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.
In the second project, the magenta root dwarf 1 (mrd1) mutant, identified in a forward genetic screening of BonnMu F2-families, was functionally characterized. In comparison to the wild type, mrd1 mutants accumulate anthocyanins in primary, seminal and crown roots and exhibit a limited shoot growth under light and dark conditions. The identification of a causal candidate gene was facilitated by the combination of bulked segregant RNA-seq (BSR-seq) and the availability of the sequenced Mu-tagged BonnMu F2-families, which provided a limited number of candidate mutations for the BSR-seq mapping interval. This combinatory approach identified a constitutive photomorphogenesis 9 (cop9) signalosome complex subunit 4 as the causal candidate gene that underlie the mrd1 phenotype. The COP9 signalosome (CSN) is a multiprotein complex initially identified in Arabidopsis thaliana as a repressor of photomorphogenesis. Maize mrd1 mutants show similarities to csn mutants in Arabidopsis thaliana such as the constitutive photomorphogenesis, but different patterns of anthocyanin accumulation. PCR-based co-segregation analysis identified mrd1 mutants as homozygous for a Mu insertion in the mrd1 gene. The expression of the mrd1 gene was significantly reduced in the primary roots of mrd1 mutants, compared to heterozygous and wild type plants. Microbiome profiling of the rhizosphere from crown roots of mrd1 and wild type indicated an enrichment of specific bacterial genera. Additionally, RNA-seq analysis between mrd1 and wild type crown roots highlighted a down-regulation of the mrd1 gene in the mutant. The attempt to validate the gene using an independent mutant allele generated through CRISPR/Cas9 genome editing was unsuccessful. In a future approach, the mrd1 gene could be validated either by a gene silencing strategy using RNAi or by a novel allele from publicly available mutant resources, such as the in-house BonnMu collection, which is continuously expanding.},
url = {https://hdl.handle.net/20.500.11811/11646}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-76830,
author = {{Yan Naing Win}},
title = {Development of the BonnMu transposon resource in maize (Zea mays L.) and its application in characterizing the magenta root dwarf 1 (mrd1) mutant},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = jul,
note = {Genome-wide insertional mutagenesis is a tool to generate loss-of-function mutations for virtually all genes in a genome. In this process, mutations are naturally generated by random integration of mobile DNA elements within a genome. The BonnMu resource is a European transposon-tagged mutant collection designed for functional genomics studies in maize. In this thesis the mutant repository was expanded by crossing an active Mutator (Mu) line with dent (B73, Co125) and flint (DK105, EP1 and F7) germplasms, resulting in the generation of 8,064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families identified 425,924 heritable Mu insertions affecting 36,612 (83%) of the 44,303 high-confidence gene models of maize. On average, 12 Mu insertions per gene (425,924 total insertions/ 36,612 affected genes) and 53 insertions per BonnMu F2-family (425,924 total insertions/ 8,064 families) were observed. Mu insertions and photographs depicting seedling phenotypes from segregating BonnMu F2-families are available for access through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5’ untranslated region (UTR) of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163,843 of 425,924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. In summary, the publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.
In the second project, the magenta root dwarf 1 (mrd1) mutant, identified in a forward genetic screening of BonnMu F2-families, was functionally characterized. In comparison to the wild type, mrd1 mutants accumulate anthocyanins in primary, seminal and crown roots and exhibit a limited shoot growth under light and dark conditions. The identification of a causal candidate gene was facilitated by the combination of bulked segregant RNA-seq (BSR-seq) and the availability of the sequenced Mu-tagged BonnMu F2-families, which provided a limited number of candidate mutations for the BSR-seq mapping interval. This combinatory approach identified a constitutive photomorphogenesis 9 (cop9) signalosome complex subunit 4 as the causal candidate gene that underlie the mrd1 phenotype. The COP9 signalosome (CSN) is a multiprotein complex initially identified in Arabidopsis thaliana as a repressor of photomorphogenesis. Maize mrd1 mutants show similarities to csn mutants in Arabidopsis thaliana such as the constitutive photomorphogenesis, but different patterns of anthocyanin accumulation. PCR-based co-segregation analysis identified mrd1 mutants as homozygous for a Mu insertion in the mrd1 gene. The expression of the mrd1 gene was significantly reduced in the primary roots of mrd1 mutants, compared to heterozygous and wild type plants. Microbiome profiling of the rhizosphere from crown roots of mrd1 and wild type indicated an enrichment of specific bacterial genera. Additionally, RNA-seq analysis between mrd1 and wild type crown roots highlighted a down-regulation of the mrd1 gene in the mutant. The attempt to validate the gene using an independent mutant allele generated through CRISPR/Cas9 genome editing was unsuccessful. In a future approach, the mrd1 gene could be validated either by a gene silencing strategy using RNAi or by a novel allele from publicly available mutant resources, such as the in-house BonnMu collection, which is continuously expanding.},
url = {https://hdl.handle.net/20.500.11811/11646}
}
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