Mutation tests in Neurospora crassa

Brockman, Herman E.; Serres, Frederick J. de; Ong, Tong-man; DeMarini, David M.; Katz, Alan J.; Griffiths, Anthony John; Stafford, Robert

doi:10.1016/0165-1110(84)90004-6

Cited by 39 publications

(4 citation statements)

References 74 publications

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“…Among these compounds, EMS has become one of the most effective, reliable, powerful and frequently used chemical mutagens in plants [8]. EMS mainly induces C–to-T substitutions resulting in C/G to T/A transitions [9,10] and at a low frequency, EMS generates G/C to C/G or G/C to T/A transversions through 7-ethylguanine hydrolysis or A/T to G/C transitions through 3-ethyladenine pairing errors [9-12].…”

Section: Introductionmentioning

confidence: 99%

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EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

et al. 2014

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BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin.ResultsHere, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified.ConclusionsThis new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

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Section: Introductionmentioning

confidence: 99%

“…Molecular screening of mutations was developed much later after the efficiency of chemical mutagens producing small deletions and point mutations had been improved [8,9,15,16] and DNA sequencing allowed for the identification of such point mutations. Nevertheless, direct sequencing in large populations is a slow and expensive process.…”

Section: Introductionmentioning

confidence: 99%

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

et al. 2014

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“…EMS mutagenesis has multiple advantages, such as high mutation frequency, easy screening, and stable inheritance, which is why EMS is the most widely used chemical mutagen in plants [ 47 ]. The combination of high-throughput sequencing with bulk segregant analysis (BSA) has laid the foundation for rapid mining of new genes using mutants, which has greatly facilitated functional genome studies.…”

Section: Discussionmentioning

confidence: 99%

A single amino acid residue substitution in BraA04g017190.3C, a histone methyltransferase, results in premature bolting in Chinese cabbage (Brassica rapa L. ssp. Pekinensis)

et al. 2021

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Background Flowering is an important inflection point in the transformation from vegetative to reproductive growth, and premature bolting severely decreases crop yield and quality. Results In this study, a stable early-bolting mutant, ebm3, was identified in an ethyl methanesulfonate (EMS)-mutagenized population of a Chinese cabbage doubled haploid (DH) line ‘FT’. Compared with ‘FT’, ebm3 showed early bolting under natural cultivation in autumn, and curled leaves. Genetic analysis showed that the early-bolting phenotype was controlled by a single recessive nuclear gene. Modified MutMap sequencing, genotyping analyses and allelism test provide strong evidence that BrEBM3 (BraA04g017190.3 C), encoding the histone methyltransferase CURLY LEAF (CLF), was the strongly candidate gene of the emb3. A C to T base substitution in the 14th exon of BrEBM3 resulted in an amino acid change (S to F) and the early-bolting phenotype of emb3. The mutation occurred in the SET domain (Suppressor of protein-effect variegation 3–9, Enhancer-of-zeste, Trithorax), which catalyzes site- and state-specific lysine methylation in histones. Tissue-specific expression analysis showed that BrEBM3 was highly expressed in the flower and bud. Promoter activity assay confirmed that BrEBM3 promoter was active in inflorescences. Subcellular localization analysis revealed that BrEBM3 localized in the nucleus. Transcriptomic studies supported that BrEBM3 mutation might repress H3K27me3 deposition and activate expression of the AGAMOUS (AG) and AGAMOUS-like (AGL) loci, resulting in early flowering. Conclusions Our study revealed that an EMS-induced early-bolting mutant ebm3 in Chinese cabbage was caused by a nonsynonymous mutation in BraA04g017190.3 C, encoding the histone methyltransferase CLF. These results improve our knowledge of the genetic and genomic resources of bolting and flowering, and may be beneficial to the genetic improvement of Chinese cabbage.

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“…The induction of genetic diversity is followed by screening or selection steps to deplete the unfit mutants while preserving the more fit mutants that will be the target for the next round of mutagenesis [4]. Genomic diversity may occur naturally [5] or can be induced randomly using radiation, mutagenic chemicals, or engineered mutator strains [6][7][8][9]. More focused random mutagenesis methods include error-prone PCR and DNA shuffling (Figure 2).…”

Section: Directed Evolutionmentioning

confidence: 99%

Predicting Drug Resistance Using Deep Mutational Scanning

2020

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Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.

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Mutation tests in Neurospora crassa

Cited by 39 publications

References 74 publications

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

A single amino acid residue substitution in BraA04g017190.3C, a histone methyltransferase, results in premature bolting in Chinese cabbage (Brassica rapa L. ssp. Pekinensis)

Predicting Drug Resistance Using Deep Mutational Scanning

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