Ethyl Methanesulfonate Mutagenesis Research Articles

De novo assembly and transcriptome analysis of two contrary tillering mutants to learn the mechanisms of tillers outgrowth in switchgrass (Panicum virgatum L.).

Tillering is an important trait in monocotyledon plants. The switchgrass (Panicum virgatum), studied usually as a source of biomass for energy production, can produce hundreds of tillers in its lifetime. Studying the tillering of switchgrass also provides information for other monocot crops. High-tillering and low-tillering mutants were produced by ethyl methanesulfonate mutagenesis. Alteration of tillering ability resulted from different tiller buds outgrowth in the two mutants. We sequenced the tiller buds transcriptomes of high-tillering and low-tillering plants using next-generation sequencing technology, and generated 34 G data in total. In the de novo assembly results, 133,828 unigenes were detected with an average length of 1,238 bp, and 5,290 unigenes were differentially expressed between the two mutants, including 3,225 up-regulated genes and 2,065 down-regulated genes. Differentially expressed gene analysis with functional annotations was performed to identify candidate genes involved in tiller bud outgrowth processes using Gene Ontology classification, Cluster of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. This is the first study to explore the tillering transcriptome in two types of tillering mutants by de novo sequencing.

Constitutive Expresser of Pathogenesis Related Genes 1 Is Required for Pavement Cell Morphogenesis in Arabidopsis.

For over 50 years, researchers have focused on the mechanisms underlying the important roles of the cytoskeleton in controlling the cell growth direction and cell expansion. In our study, we performed ethyl methane sulfonate mutagenesis on Col-0 background and identified two new CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED GENES 1 (CPR1) alleles with pavement cell (PC) morphogenetic defects. Morphological characterizations showed that polar growth initiation and expansion of PCs are seriously suppressed in cpr1. Closer cytoskeleton investigation showed that the directional arrangement of microtubules (MTs) during PC development is defective and the cortical fine actin filaments cannot be aggregated effectively to form actin cable networks in cpr1 mutants. These results suggest that the abnormal PC morphogenesis in cpr1 is accompanying with the aberrant arrangement of cytoskeleton. Site-directed mutagenesis and knockout within the F-box-associated (FBA) domain, which is reported to be a motif for recognizing particular substrates of CPR1, proved that the FBA domain is indispensable for normal CPR1 regulation of the PC morphogenesis. Further genetic analysis indicated that the defects on PC morphogenesis of cpr1 depend on two lipase-like proteins, ENHANCED DISEASE SUSCEPTIBILITY 1 and PHYTOALEXIN DEFICIENT 4. Our results provide further insights into the relationship between the cytoskeleton and PC morphogenesis, and suggest that the cytoskeleton-mediated PC morphogenesis control might be tightly linked to plant defense responses.

A new method for generation and screening of Chinese cabbage mutants using isolated microspore culturing and EMS mutagenesis

A new method for generation and screening Chinese cabbage mutants was employed in this study. In order to quickly obtain homozygous mutants in Chinese cabbage, isolated microspore culturing and ethyl methanesulfonate mutagenesis were performed on a doubled haploid line. Microspores were treated with different concentrations (0.04, 0.08, 0.12 %, v/v) of ethyl methanesulfonate solution for 10 min, and mutagenic microspores were cultured in NLN-13 medium. A total of six stable inheriting mutants were selected, which accounted for 0.46 % of 1304 regenerated plants. The mutants exhibited variation in traits including overall plant shape, floral organs and leaves. These results could be applied in future functional genomics studies on Chinese cabbage.

Cloning and functional analysis of pale-green leaf (PGL10) in rice (Oryza sativa L.)

Leaf color mutants commonly found in rice have important implications in basic research and breeding science. In this study, we isolated a pale-green leaf mutant (pgl10) from the offspring of the rice cultivar Nipponbare (Oryza sativa L. spp. japonica) through ethyl methanesulfonate mutagenesis. Compared with the wild-type Nipponbare, the pgl10 mutant had phenotypically pale-green leaves with significantly decreased chlorophyll (a and b) and carotenoid contents. Transmission electron micrographs showed that pgl10 had less grana lamellae of chloroplasts than Nipponbare. The results of tissue-specific gene expression analysis revealed that pgl10 was expressed in various rice organs, including roots, stems, leaves, sheaths, and spikes. The expression of Chl synthesis-associated gene in pgl10 was decreased. Genetic analysis suggested that PGL10 was controlled by a recessive gene. Map-based cloning and genome sequencing data showed that pgl10 was a frameshift mutation caused by a single base insertion on chromosome 10. Bioinformation analysis indicated that PGL10 encoded protochlorophyllide oxidoreductase B. Therefore, pgl10 can be a genetic material for further studies on PGL10.

Improvement of Xylanase Production by Bacillus subtilis in Submerged Fermentation after UV and Chemicals Mutagenesis

Aims: Bacillus subtilis is one of the most utilized industrial microorganisms for its high capability of producing large amount of extracellular enzymes including xylanase. This extracellular enzyme has been used extensively for many years in food and detergents industry. In recent years, many attempts have been made for the improvement of microbial xylanase production by inducing mutagenesis. Thus, the objectives of this study are to produce mutants of B. subtilis using UV and chemicals mutagenesis, subsequently to elucidate the xylanase activity and finally to identify the most effective mutagenic agent for overproduction of this enzyme. Methodology: In this study, ultraviolet (UV) irradiation and chemical mutagens of ethyl methanesulfonate (EMS) and acridine orange (AO) have been selected because of their simplicity of handling and cost-effectiveness as compared to recombinant DNA technology. The efficiency of these mutagenesis was further assessed in submerged fermentation in shake flask culture compared with the non-treated wild type control strain. Results: Based on our results, B. subtilis that had been induced with 50 μg/mL of AO for 30 min exhibited the overproduction of xylanase with 1.580±0.027 U/mL in submerged fermentation. Indeed, the highest xylanase overproduction reached the maximum peak of 1.638±0.027 U/mL after Original Research Article Ho and Chor; JABB, 3(2): 42-57, 2015; Article no.JABB.2015.031 43 30 min of induction using 100 ug/mL of EMS. Apparently, when compared to the non-treated control, xylanase activity experienced the highest percentage of overproduction as much as 15.68% from EMS mutagenesis induced for 30 min in contrast to only 11.58% from B. subtilis that had been induced with 50 μg/mL of AO for 30 min. Conclusion: In a nutshell, based on our results, EMS evidenced among the most promising mutagenic agent of inducing B. subtilis that capable of overproduction of xylanase to the greater extend as compared to UV irradiation and mutagenic agent of AO.

Optimization of EMS Mutagenesis on Petunia for TILLING

The development of a chemically-mutagenized population of Petunia hybrida could enable the identification of novel alleles for crop improvement. Conditions were determined for mutagenizing petunia with ethyl methanesulfonate (EMS), while minimizing deleterious effects on viability and fertility. A mutagenized population of the doubled haploid P. hybrida line ‘Mitchell Diploid’ was developed as a resource for TILLING. Three mutagenesis parameters were investigated in this study: the imbibition of seeds prior to EMS treatment, the EMS concentration, and EMS exposure time. Based on these results, the mutagenesis of 2000 petunia seeds for TILLING was conducted with a 12 hour imbibition followed by exposure to 0.1% EMS for 12 hours.

Studying circadian rhythm and sleep using genetic screens in Drosophila.

The power of Drosophila melanogaster as a model organism lies in its ability to be used for large-scale genetic screens with the capacity to uncover the genetic basis of biological processes. In particular, genetic screens for circadian behavior, which have been performed since 1971, allowed researchers to make groundbreaking discoveries on multiple levels: they discovered that there is a genetic basis for circadian behavior, they identified the so-called core clock genes that govern this process, and they started to paint a detailed picture of the molecular functions of these clock genes and their encoded proteins. Since the discovery that fruit flies sleep in 2000, researchers have successfully been using genetic screening to elucidate the many questions surrounding this basic animal behavior. In this chapter, we briefly recall the history of circadian rhythm and sleep screens and then move on to describe techniques currently employed for mutagenesis and genetic screening in the field. The emphasis lies on comparing the newer approaches of transgenic RNA interference (RNAi) to classical forms of mutagenesis, in particular in their application to circadian behavior and sleep. We discuss the different screening approaches in light of the literature and published and unpublished sleep and rhythm screens utilizing ethyl methanesulfonate mutagenesis and transgenic RNAi from our lab.

Strategies for gene disruption in Drosophila.

Drosophila melanogaster has been a classic model organism for the studies of genetics. More than 15,000 Drosophila genes have been annotated since the entire genome was sequenced; however, many of them still lack functional characterization. Various gene-manipulating approaches in Drosophila have been developed for the function analysis of genes. Here, we summarize some representative strategies utilized for Drosophila gene targeting, from the unbiased ethyl methanesulfonate (EMS) mutagenesis and transposable element insertion, to insertional/replacement homologous recombination and site-specific nucleases such as the zinc-finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. Specifically, we evaluate the pros and cons of each technique in a historical perspective. This review discuss important factors that should be taken into consideration for the selection of a strategy that best fits the specific needs of a gene knockout project.

Next-Gen Sequencing-Based Mapping and Identification of Ethyl Methanesulfonate-Induced Mutations in Arabidopsis thaliana.

Forward genetic analysis using ethyl methanesulfonate (EMS) mutagenesis has proven to be a powerful tool in biological research, but identification and cloning of causal mutations by conventional genetic mapping approaches is a painstaking process. Recent advances in next-gen sequencing have greatly invigorated the process of identifying EMS-induced mutations corresponding to a specific phenotype in model genetic hosts, including the plant Arabidopsis thaliana and the nematode Caenorhabditis elegans. Next-gen sequencing of bulked F2 mutant recombinants produces a wealth of high-resolution genetic data, provides enhanced delimitation of the genomic location of mutations, and greatly reduces hands-on time while maintaining high accuracy and reproducibility. In this unit, a detailed procedure to simultaneously map and identify EMS mutations in Arabidopsis is described.

Ethyl methanesulfonate induces mutations in Caenorhabditis elegans embryos at a high frequency

Mutagenesis protocols typically call for exposure of late-stage larvae or adults to a mutagen with the intention of inducing mutations in a robust germ line. Instead, ca. 16,000 CB665 [unc-58(e665)] one- to four-cell embryos of the nematode Caenorhabditis elegans were hand selected and exposed to ethyl methanesulfonate (EMS) for 50min. Twenty-one reversion mutants were recovered, of which 17 were intragenic suppressors of the e665 mutation. The mutation frequency was 6.5-fold higher than when CB665 adults were similarly mutagenized, which was predicted given that cell-cycle checkpoints are muted in C. elegans embryos. The mutation spectrum was similar to that obtained after standard EMS mutagenesis.

Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase.

BackgroundYeasts tolerant to toxic inhibitors from steam-pretreated lignocellulose with xylose co-fermentation capability represent an appealing approach for 2nd generation ethanol production. Whereas rational engineering, mutagenesis and evolutionary engineering are established techniques for either improved xylose utilisation or enhancing yeast tolerance, this report focuses on the simultaneous enhancement of these attributes through mutagenesis and evolutionary engineering of Saccharomyces cerevisiae harbouring xylose isomerase in anoxic chemostat culture using non-detoxified pretreatment liquor from triticale straw.ResultsFollowing ethyl methanesulfonate (EMS) mutagenesis, Saccharomyces cerevisiae strain D5A+ (ATCC 200062 strain platform), harbouring the xylose isomerase (XI) gene for pentose co-fermentation was grown in anoxic chemostat culture for 100 generations at a dilution rate of 0.10 h-1 in a medium consisting of 60% (v/v) non-detoxified hydrolysate liquor from steam-pretreated triticale straw, supplemented with 20 g/L xylose as carbon source. In semi-aerobic batch cultures in the same medium, the isolated strain D5A+H exhibited a slightly lower maximum specific growth rate (μmax = 0.12 ± 0.01 h-1) than strain TMB3400, with no ethanol production observed by the latter strain. Strain D5A+H also exhibited a shorter lag phase (4 h vs. 30 h) and complete removal of HMF, furfural and acetic acid from the fermentation broth within 24 h, reaching an ethanol concentration of 1.54 g/L at a yield (Yp/s) of 0.06 g/g xylose and a specific productivity of 2.08 g/gh. Evolutionary engineering profoundly affected the yeast metabolism, given that parental strain D5A+ exhibited an oxidative metabolism on xylose prior to strain development.ConclusionsPhysiological adaptations confirm improvements in the resistance to and conversion of inhibitors from pretreatment liquor with simultaneous enhancement of xylose to ethanol fermentation. These data support the sequential application of random mutagenesis followed by continuous culture under simultaneous selective pressure from inhibitors and xylose as primary carbon source.

Cloning of a rice male sterility gene by a modified MutMap method

A pollen defective male sterile rice mutant, osms55, was isolated from an elite indica cultivar HHZ using EMS (ethyl methanesulfonate) mutagenesis strategy. Genetic analysis showed that osms55 was controlled by a single recessive gene. Genome-wide SNP analysis using the high-throughput Illumina Infinium iSelect SNP (50 K) microarray technology indicated that the genetic makeup of osms55 is the same as wild type (WT) HHZ. Using a modified MutMap method, we successfully identified a mutation in the LOC_Os02g40450 (MER3) gene that is co-segregated with the male sterility phenotype. The mutation is located at the intron splice-recognition site, leading to a 15 nucleotide deletion in the fifth exon. Different from the published MutMap method that aligns the mutant pool DNA sequence with the assembled WT genome, the method used in this study was to align the re-sequencing data of the mutant pool DNA and WT HHZ with the Nipponbare reference genome. The resulting SNPs of mutant/Nipponbare and WT HHZ/Nipponbare were further compared to determine the candidate mutant gene. This modified method does not need an assembled WT genome as reference and thus is more cost-effective and widely applicable.

Identification and utilization of cleistogamy gene cl7(t) in rice (Oryza sativa L.)

Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved economic traits were obtained, which can be used for the selection of ecologically safe GM rice varieties.

Morphological characteristics and gene mapping of a dense panicle (dp2) mutant in rice (Oryza sativa L.)

A dense panicle mutant (dp2) derived from the Oryza sativa ssp. japonica cultivar Nipponbare through ethyl methane sulfonate mutagenesis was used in present study. Compared to the wild type, the panicle of dp2 mutant exhibited more branches and denser grains. Further more, the number of spikelets per panicle, number of primary branches and secondary branches of dp2 mutant were significantly increased while the panicle length, and 1,000-grain weight were significantly decreased. The results from the genetic analysis indicated that the dense panicle phenotype was controlled by a single dominance nuclear gene. Polymorphic analysis of SSR and InDel markers demonstrated that the DP2 gene was located at the long arm of chromosome 2, which was further mapped between SSR markers RM341 and RM13356 in a physical region of 398 kb. Within this region, the RCN2 (LOC_Os02g32950) gene which was annotated relating to the development of rice panicle was found. Compared to the wild type, the sequence of RCN2 gene in the dp2 mutant showed that two SNPs replacement had taken place in the promoter region (G–A) and the intron region (A–T), respectively. The dp2 mutant could be a novel mutant of RCN2 gene and this novel mutant might be useful for further studies on this gene.

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

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.

Ethyl Methanesulfonate (EMS)-Mediated Mutagenesis of Cucumber (<i>Cucumis sativus</i> L.)

Ethyl methanesulfonate (EMS) is a stable and effective chemical mutagen. In this study, cucumber (Cucumis sativus L. cv. “Shannong No. 5”) seeds were treated by 1% EMS for 12 h, 24 h and 48 h to optimize EMS mutagenesis and determined median lethal dose of EMS (1% EMS and 24 h) for “Shannong No. 5”. After treated by 1% EMS for 24 h, 541 M1 plants were grown in greenhouse for phenotype investigation. The fertility of M1 cucumbers was very low, and only 79 lines produced seeds after self crossing. 60 independent M2 families comprising 600 M2 plants were investigated for phenotypic alteration, and 11 individual mutant lines were isolated into six groups: short-fruit mutants, long-fruit mutants, small-flower mutants, big-flower mutants, opposite-tendril mutants and clustered-leaf mutants. The mutation frequency was 18.3%. Two selected representatives, short-fruit mutants and clustered-leaf mutants, showed 1:3 of segregation ratio in M2 populations. This ratio is consistent with classic Mendelian model, indicating that the two kinds of mutants may be controlled by a single recessive gene, respectively. Long-fruit phenotype was stably inherited and no segregation was observed in M3 generation, indicating that this mutant line may be homozygous.

Optimization of EMS Mutagenesis on Petunia for TILLING

The development of a chemically-mutagenized population of Petunia hybrida could enable the identification of novel alleles for crop improvement. Conditions were determined for mutagenizing petunia with ethyl methanesulfonate (EMS), while minimizing deleterious effects on viability and fertility. A mutagenized population of the doubled haploid P. hybrida line ‘Mitchell Diploid’ was developed as a resource for TILLING. Three mutagenesis parameters were investigated in this study: the imbibition of seeds prior to EMS treatment, the EMS concentration, and EMS exposure time. Based on these results, the mutagenesis of 2000 petunia seeds for TILLING was conducted with a 12 hour imbibition followed by exposure to 0.1% EMS for 12 hours.

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

Genetic improvements for many fiber traits are obtained by mutagenesis of elite cottons, mitigating genetic uniformity in this inbred polyploid by contributing novel alleles important to ongoing crop improvement. The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate (EMS) mutagenesis of two G. hirsutum breeding lines, TAM 94L-25 and Acala 1517-99, were characterized for basic components of fiber quality and selected yield components. Across all measured traits, the ranges of phenotypic values among the mutant lines were consistently larger than could be explained by chance (5.27-10.1 for TAM 94 L-25 and 5.29-7.94 standard deviations for Acala 1517-99-derived lines). Multi-year replicated studies confirmed a genetic basis for these differences, showing significant correlations between lines across years and environments. A subset of 157 lines selected for superior fiber qualities, including fiber elongation (22 lines), length (22), lint percent (17), fineness (23), Rd value (21), strength (19), uniformity (21) and multiple attributes in a selection index (26) were compared to 55 control lines in replicated trials in both Texas and Georgia. For all traits, mutant lines showing substantial and statistically significant improvements over control lines were found, in most cases from each of the two genetic backgrounds. This indicates that genetic improvements for a wide range of fiber traits may be obtained from mutagenesis of elite cottons. Indeed, lines selected for one fiber trait sometimes conferred additional attributes, suggesting pleiotropic effects of some mutations and offering multiple benefits for the incorporation of some alleles into mainstream breeding programs.

EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration. Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.

Improvement of antagonism and carbendazim tolerance in native Trichoderma isolates through ethyl methane sulfonate (EMS) mutagenesis

Genetic enhancement of TCT4 and TCT10 was aimed in the present paper. Trichoderma reesei (TCT10/M18) mutant isolate evolved by ethyl methane sulfonate mutations was found to exhibit altered properties compared to its parent isolates. This mutant grew well in the potato dextrose agar (PDA) medium containing carbendazim (50 ppm). RAPD-PCR results suggested the uniqueness of mutants, which was useful in differentiating mutant and wild Trichoderma isolates. These mutants established well in the rhizosphere of rough lemon seedlings. The seedlings treated with carbendazim followed by an application of carbendazim-resistant mutant (TCT10/M18) resulted in a better seedling emergence and a less dry root rot disease caused by Fusarium solani in nursery conditions.