Dataset Information

Forward Genetics Approach Reveals a Mutation in bHLH Transcription Factor-Encoding Gene as the Best Candidate for the Root Hairless Phenotype in Barley.

ABSTRACT: Root hairs are the part of root architecture contributing significantly to the root surface area. Their role is particularly substantial in maintaining plant growth under stress conditions, however, knowledge on mechanism of root hair differentiation is still limited for majority of crop species, including barley. Here, we report the results of a map-based identification of a candidate gene responsible for the lack of root epidermal cell differentiation, which results in the lack of root hairs in barley. The analysis was based on the root hairless barley mutant rhl1.b, obtained after chemical mutagenesis of spring cultivar 'Karat'. The rhl1 gene was located in chromosome 7HS in our previous studies. Fine mapping allowed to narrow the interval encompassing rhl1 gene to 3.7 cM, which on physical barley map spans a region of 577 kb. Five high confidence genes are located within this region and their sequencing resulted in the identification of A>T mutation in one candidate, HORVU7Hr1G030250 (MLOC_38567), differing the mutant from its parent variety. The mutation, located in the 3' splice-junction site, caused the retention of the last intron, 98 bp long, in mRNA of rhl1.b allele. This resulted in the frameshift, the synthesis of 71 abnormal amino acids and introduction of premature STOP codon in mRNA. The mutation was present in the recombinants from the mapping population (F₂rhl1.b × 'Morex') that lacked root hairs. The candidate gene encodes a bHLH transcription factor with LRL domain and may be involved in early stages of root hair cell development. We discuss the possible involvement of HORVU7Hr1G030250 in this process, as the best candidate responsible for early stages of rhizodermis differentiation in barley.

SUBMITTER: Gajewska P

PROVIDER: S-EPMC6129617 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Forward Genetics Approach Reveals a Mutation in bHLH Transcription Factor-Encoding Gene as the Best Candidate for the Root Hairless Phenotype in Barley.

Gajewska Patrycja P Janiak Agnieszka A Kwasniewski Miroslaw M Kędziorski Piotr P Szarejko Iwona I

Frontiers in plant science 20180903

Root hairs are the part of root architecture contributing significantly to the root surface area. Their role is particularly substantial in maintaining plant growth under stress conditions, however, knowledge on mechanism of root hair differentiation is still limited for majority of crop species, including barley. Here, we report the results of a map-based identification of a candidate gene responsible for the lack of root epidermal cell differentiation, which results in the lack of root hairs i ...[more]

PMID: 30233607

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Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.

Dataset Information

Forward Genetics Approach Reveals a Mutation in bHLH Transcription Factor-Encoding Gene as the Best Candidate for the Root Hairless Phenotype in Barley.

Publications

Forward Genetics Approach Reveals a Mutation in bHLH Transcription Factor-Encoding Gene as the Best Candidate for the Root Hairless Phenotype in Barley.

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