Ethyl Methanesulfonate Mutagenesis Research Articles

High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis

High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis

Fine mapping and candidate gene analysis of leaf tip premature senescence and Dwarf Mutant dls-1 in Rice

Premature leaf senescence negatively affects rice yield and quality. Identifying premature senescence mutants and examining their gene functions are important for the genetic improvement of crops. In this study, a leaf senescence mutant dls-1 was obtained from japonica Yundao by ethyl methane sulfonate mutagenesis, which showed the phenotypes of premature leaf senescence, reduced plant height and decreased yield-associated traits including panicle length, seed setting rate, and 1000-grain weight. Biochemical analysis revealed the accumulation of reactive oxygen species in dls-1 leading to increased apoptosis rates, stomata length extension, higher water loss, and a decline in leaf silicification. Transcriptomic data showed that pathogen resistance-related genes were down-regulated in dls-1, causing increased sensitivity to bacterial-induced blight. Genetic analysis indicated that the mutant trait of dls-1 was controlled by a single recessive nuclear gene, and the gene was fine-mapped to a 159 kb region on rice chromosome 1 using map-based cloning strategy. A total of 23 open reading frames were found in the interval, and no similar phenotypic genes have been reported. However, all candidate coding genes and promoter regions were sequenced, and no mutation sites were found. RNA-seq analysis and qRT-PCR showed that the candidate gene LOC_Os01g71670, encodes a rice endo-1,3-β-glucanase, showed significantly different expression levels in dls-1 compared to the wild-type. LOC_Os01g71670 is highly homologous with GII in barley, and Gns4 and glu1 in rice, which participates in the hormone response pathway affecting plant growth and development, and also involve in the regulation of PR2 expression. Taken together with the phenotypes of dls-1, we conclude that LOC_Os01g71670 may be the candidate gene, causing the premature senescence of the leaf tip and loss of pathogen resistance.

Allele-specific suppression in Caenorhabditis elegans reveals details of EMS mutagenesis and a possible moonlighting interaction between the vesicular acetylcholine transporter and ERD2 receptors.

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

The Genome-Wide EMS Mutagenesis Bias Correlates With Sequence Context and Chromatin Structure in Rice.

Ethyl methanesulfonate (EMS) is a chemical mutagen believed to mainly induce G/C to A/T transitions randomly in plant genomes. However, mutant screening for phenotypes often gets multiple alleles for one gene but no mutant for other genes. We investigated the potential EMS mutagenesis bias and the possible correlations with sequence context and chromatin structure using the whole genome resequencing data collected from 52 rice EMS mutants. We defined the EMS-induced single nucleotide polymorphic sites (SNPs) and explored the genomic factors associated with EMS mutagenesis bias. Compared with natural SNPs presented in the Rice3K project, EMS showed a preference on G/C sites with flanking sequences also higher in GC contents. The composition of local dinucleotides and trinucleotides was also associated with the efficiency of EMS mutagenesis. The biased distribution of EMS-induced SNPs was positively correlated with CpG numbers, transposable element contents, and repressive epigenetic markers but negatively with gene expression, the euchromatin marker DNase I hypersensitive sites, and active epigenetic markers, suggesting that sequence context and chromatin structure might correlate with the efficiency of EMS mutagenesis. Exploring the genome-wide features of EMS mutagenesis and correlations with epigenetic modifications will help in the understanding of DNA repair mechanism.

CsUFO is involved in the formation of flowers and tendrils in cucumber.

An unusual flower and tendril (uft) mutant in cucumber was caused by a mutation in Csa1G056950 encoding an F-box protein. Flowers and tendrils are important agronomic and yield traits of cucumber (Cucumis sativus L.). In this study, we identified an unusual flower and tendril (uft) mutant from an ethyl methanesulfonate (EMS) mutagenesis population. Genetic analysis revealed that the phenotype of the uft mutant was regulated by a single recessive nuclear gene. Map-based cloning and MutMap+ results demonstrated that Csa1G056950 (CsUFO), encoding an F-box protein, was the causal gene for the uft mutant phenotype of cucumber. A single nucleotide polymorphism (SNP) mutation (C to T) in the second exon of CsUFO resulted in premature translation termination. The expression level of CsUFO was significantly decreased in apical buds of the uft mutant compared with the wild-type (WT) WD1. Transcriptome analysis indicated that many genes for organ development were down-regulated in uft plants, suggesting CsUFO-associated networks that regulate flower and tendril development. These findings provide a new insight into understanding the molecular mechanisms of flower organogenesis in cucumber.

Characterization and fine mapping of a new dwarf mutant in Brassica napus

BackgroundPlant height is an important plant characteristic closely related to yield performance of many crops. Reasonable reduction of plant height of crops is beneficial for improving yield and enhancing lodging resistance.ResultsIn the present study, we described the Brassica napus dwarf mutant bnd2 that was isolated using ethyl methanesulfonate (EMS) mutagenesis. Compared to wild type (WT), bnd2 exhibited reduced height and shorter hypocotyl and petiole leaves. By crossing the bnd2 mutant with the WT strain, we found that the ratio of the mutant to the WT in the F2 population was close to 1:3, indicating that bnd2 is a recessive mutation of a single locus. Following bulked segregant analysis (BSA) by resequencing, BND2 was found to be located in the 13.77–18.08 Mb interval of chromosome A08, with a length of 4.31 Mb. After fine mapping with single nucleotide polymorphism (SNP) and insertion/deletion (InDel) markers, the gene was narrowed to a 140-Kb interval ranging from 15.62 Mb to 15.76 Mb. According to reference genome annotation, there were 27 genes in the interval, of which BnaA08g20960D had an SNP type variation in the intron between the mutant and its parent, which may be the candidate gene corresponding to BND2. The hybrid line derived from a cross between the mutant bnd2 and the commercial cultivar L329 had similar plant height but higher grain yield compared to the commercial cultivar, suggesting that the allele bnd2 is beneficial for hybrid breeding of lodging resistant and high yield rapeseed.ConclusionIn this study, we identified a novel dwarf mutant of rapeseed with a new locus, which may be useful for functional analyses of genetic mechanisms of plant architecture and grain yield in rapeseed.

A single SNP in Brcer1 results in wax deficiency in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Cuticle wax covering the surface of plants is mainly composed of derivatives of long-chain saturated fatty acids. Chinese cabbage leaves with wax deficiency has a bright green phenotype, which makes it attractive and improves its commercial value. In this study, a wax deficiency mutant, cer1, was obtained from an ethyl methanesulfonate (EMS) mutagenesis population in Chinese cabbage. Scanning electron microscopy (SEM) revealed that cuticular wax in the leaves and stems of mutants was significantly less than that in the wild type (WT). The mutants decreased the total wax, alkanes, ketones and alcohols contents in leaves, but aldehydes content increased compared to WT. Genetic analysis showed that the mutant cer1 phenotype was controlled by a single recessive nuclear gene. MutMap and Kompetitive Allele-Specific PCR (KASP) analyses indicated that BraA09g066480.3C, which encoded aldehyde decarboxylase, might be the causal gene. A single nucleotide substitution (SNP) from C to T in the fourth exon of BraA09g066480.3C resulted in an amino acid substitution from serine to phenylalanine, which led to changes in the three-dimensional structure of proteins. SNP 45,148,866 occurred in the highly conserved fatty acid hydroxylase domain. This study revealed that SNP is effective for marker-assisted selection of wax deficient Chinese cabbage and provides convenience for breeding new varieties exhibiting a wax deficient phenotype.

Mutants of Lotus japonicus deficient in flavonoid biosynthesis

Spatiotemporal features of anthocyanin accumulation in a model legume Lotus japonicus (Regel) K.Larsen were elucidated to develop criteria for the genetic analysis of flavonoid biosynthesis. Artificial mutants and wild accessions, with lower anthocyanin accumulation in the stem than the standard wild type (B-129 ‘Gifu’), were obtained by ethyl methanesulfonate (EMS) mutagenesis and from a collection of wild-grown variants, respectively. The loci responsible for the green stem of the mutants were named as VIRIDICAULIS (VIC). Genetic and chemical analysis identified two loci, namely, VIC1 and VIC2, required for the production of both anthocyanins and proanthocyanidins (condensed tannins), and two loci, namely, VIC3 and VIC4, required for the steps specific to anthocyanin biosynthesis. A mutation in VIC5 significantly reduced the anthocyanin accumulation. These mutants will serve as a useful system for examining the effects of anthocyanins and proanthocyanidins on the interactions with herbivorous pests, pathogenic microorganisms and nitrogen-fixing symbiotic bacteria, Mesorhizobium loti.

Simultaneous saccharification and co-fermentation with a thermotolerant Saccharomyces cerevisiae to produce ethanol from sugarcane bagasse under high temperature conditions

Lignocellulosic ethanol production at high temperature offers advantages such as the decrease of contamination risk and cooling cost. Recombinant xylose-fermenting Saccharomyces cerevisiae has been considered a promising strain for ethanol production from lignocellulose for its high inhibitor tolerance and superior capability to ferment glucose and xylose into ethanol. To improve the ethanolic fermentation by xylose at high temperature, the strain YY5A was subjected to the ethyl methanesulfonate (EMS) mutagenesis. A mutant strain T5 was selected from the EMS-treated cultures to produce ethanol. However, the xylose uptake by T5 was severely inhibited by the high ethanol concentration during the co-fermentation in defined YPDX medium at 40 °C. In this study, the simultaneous saccharification and co-fermentation (SSCF) and the separate hydrolysis and co-fermentation (SHCF) processes of sugarcane bagasse were assessed to solve this problem. The xylose utilization by T5 was remarkably improved using the SSCF process compared to the SHCF process. For the SHCF and SSCF processes, 48% and 99% of the xylose in the hydrolysate was consumed at 40 °C, respectively. The ethanol yield was enhanced by the SSCF process. The ethanol production can reach to 36.0 g/L using this process under high-temperature conditions.

Mutagenesis of Rhodosporidium toruloides using ethyl methanesulfonate for enhancing astaxanthin production and evaluation scavenging activity against 2,2-diphenyl-1-picrylhydrazyl of astaxanthin

Astaxanthin is a valuable carotenoid which has been approved as a food coloring by the US Food and Drug Administration. This work aimed to attain Rhodosporidium toruloides mutants for enhanced astaxanthin accumulation using ethyl methanesulfonate mutagenesis (EMS). EMS treatment was shown efficient at different investigated concentration included 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 6.0% in affecting the yeast for enhanced astaxanthin production. Among the screened mutants, E4 (1719.9 µg/L), an EMS-induced mutant at 3.5% concentration, exhibited the highest astaxanthin production which was significantly higher than that of the wild strain (increased 4.7 times). A mutant yeast strain E4 showed a short lag phase of about 2 hours, then 32 hours of log phase, 35 hours of stationary phase and then entered to death phase after 70 hours of inoculation. The best time to collect astaxanthin was after 96 hours of inoculation in Hansen broth, the accumulated astaxanthin content was 1719 µg/L. Crude extract of astaxanthin from mutant strain E4 had the scavenging capacity of DPPH free radicals (IC50 = 9.106 µg/mL) 10.6 times higher than that of vitamin E and their’s application direction in the pharmaceutical industry, nutritional supplements, as well as bioproduct used in the aquaculture industry.

Character Identification of an Early Flowering Mutant

The concept of gene-function-genetic trait was introduced to explore the effects of early flowering on the growth and development of maize at the jointing stage and to obtain early flowering mutants using ethyl methanesulfonate mutagenesis. First, we studied gene expression, phytohormones, and lignin content to explore the physiological peculiarities of the early flowering mutant. Then we analyzed the genetic features of the mutants during the jointing stage by measuring physiological and biochemical indices of drought tolerance. The results showed that the photosynthetic rate of the mutant was significantly higher than that of the control and the rate of accumulation of dry matter was rapid. In addition, the lignin content increased while drought resistance diminished. Therefore, we concluded that early flowering leads to faster overall growth and development.

Mutation of the PHYTOENE DESATURASE 3 gene causes yellowish-white petals in Brassica napus

Oilseed rape (Brassica napus) with yellow flowers is an attractive ornamental landscape plant during the flowering period, and the development of different petal colors has become a breeding objective. Although yellowish flower color is a common variant observed in field-grown oilseed rape, the genetics behind this variation remains unclear. We obtained a yellowish-white flower (ywf) mutant from Zhongshuang 9 (ZS9) by ethyl methanesulfonate mutagenesis (EMS) treatment. Compared with ZS9, ywf exhibited a lower carotenoid content with a reduced and defective chromoplast ultrastructure in the petals. Genetic analysis revealed that the yellowish-white trait was controlled by a single recessive gene. Using bulked-segregant analysis sequencing (BSA-seq) and kompetitive allele-specific PCR (KASP), we performed map-based cloning of the ywf locus on chromosome A08 and found that ywf harbored a C-to-T substitution in the coding region, resulting in a premature translation termination. YWF, encoding phytoene desaturase 3 (PDS3), was highly expressed in oilseed rape petals and involved in carotenoid biosynthesis. Pathway enrichment analysis of the transcriptome profiles from ZS9 and ywf indicated the carotenoid biosynthesis pathway to be highly enriched. Further analyses of differentially expressed genes and carotenoid components revealed that the truncated BnaA08.PDS3 resulted in decreased carotenoid biosynthesis in the mutant. These results contribute to an understanding of the carotenoid biosynthesis pathway and manipulation of flower-color variation in B. napus.

CsKDO is a candidate gene regulating seed germination lethality in cucumber.

Seed germination plays an important role in the initial stage of plant growth. However, few related studies focused on lethality after seed germination in plants. In this study, we identified an Ethyl methanesulfonate (EMS) mutagenesis mutant Csleth with abnormal seed germination in cucumber (Cucumis sativus L.). The radicle of the Csleth mutant grew slowly and detached from the cotyledon until 14 d after sowing. Genetic analysis showed that the mutant phenotype of Csleth was controlled by a single recessive gene. MutMap+ and Kompetitive Allele Specific PCR (KASP) genotyping results demonstrated that Csa3G104930 encoding 3-deoxy-manno-octulosonate cytidylyltransferase (CsKDO) was the candidate gene of the Csleth mutant. The transition mutation of aspartate occurred in Csa3G104930 co-segregated with the phenotyping data. CsKDO was highly expressed in male flowers in wild type cucumbers. Subcellular localization results showed that CsKDO was located in the nucleus. Overall, these results suggest CsKDO regulates lethality during seed germination in cucumber.

Improvement of cadmium tolerance and accumulation of Phragmites spp. Tabarka by ethyl methane sulfonate mutagenesis

Improvement of cadmium tolerance and accumulation of <i>Phragmites</i> spp. Tabarka by ethyl methane sulfonate mutagenesis

A temperature-sensitive FERONIA mutant allele that alters root hair growth.

In plants, root hairs undergo a highly polarized form of cell expansion called tip-growth, in which cell wall deposition is restricted to the root hair apex. In order to identify essential cellular components that might have been missed in earlier genetic screens, we identified conditional temperature-sensitive (ts) root hair mutants by ethyl methanesulfonate mutagenesis in Arabidopsis thaliana. Here, we describe one of these mutants, feronia-temperature sensitive (fer-ts). Mutant fer-ts seedlings were unaffected at normal temperatures (20°C), but failed to form root hairs at elevated temperatures (30°C). Map based-cloning and whole-genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation localized to plasma membranes, but was subject to enhanced protein turnover at elevated temperatures. While tip-growth was rapidly inhibited by addition of rapid alkalinization factor 1 (RALF1) peptides in both wild-type and fer-ts mutants at normal temperatures, root elongation of fer-ts seedlings was resistant to added RALF1 peptide at elevated temperatures. Additionally, at elevated temperatures fer-ts seedlings displayed altered reactive oxygen species (ROS) accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other Catharanthus roseus receptor-like kinase 1L (CrRLK1L) receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but is not located within the recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN 2 binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.

ZmSKS13, a cupredoxin domain-containing protein, is required for maize kernel development via modulation of redox homeostasis.

The SKU5 similar (SKS) genes encode a family of multi-copper-oxidase-like proteins with cupredoxin domains similar to those in laccase and ascorbate oxidase. Although SKS proteins are known to function in root growth and cotyledon vascular patterning in Arabidopsis, their role in plant reproductive processes is poorly understood. Here, we identified a seed mutant of maize (Zea mays), generated by ethyl methane sulfonate (EMS) mutagenesis, that we designated defective kernel-zk1 (dek-zk1). The mutant produced small, shriveled kernels with an aberrant basal endosperm transfer layer (BETL) and placento-chalazal (PC) layer and irregular starch granules. Map-based cloning revealed that Dek-zk1 encodes an SKU5 similar 13 (GenBank: ONM36900.1), so it was named ZmSKS13. ZmSKS13 comprises a paralogous pair with Zm00001d012524, but the transcript abundance of ZmSKS13 in developing kernels is 15 times higher than that of Zm00001d012524, resulting in dek-zk1 mutation conveying a distinct kernel phenotype. ZmSKS13 loss of function led to overaccumulation of reactive oxygen species (ROS) and severe DNA damage in the nucellus and BETL and PC layer cells, and exogenous antioxidants significantly alleviated the defects of the mutant kernels. Our results thus demonstrate that ZmSKS13 is a novel regulator that plays a crucial role in kernel development in maize through the modulation of ROS homeostasis.

The mutation of ent-kaurene synthase, a key enzyme involved in gibberellin biosynthesis, confers a non-heading phenotype to Chinese cabbage (Brassica rapa L. ssp. pekinensis)

The presence of a leafy head is a vital agronomic trait that facilitates the evaluation of the yield and quality of Chinese cabbage. A non-heading mutant (nhm1) was identified in an ethyl methanesulfonate mutagenesis population of the heading Chinese cabbage double haploid line FT. Segregation analysis revealed that a single recessive gene, Brnhm1, controlled the mutant phenotype. Using MutMap, Kompetitive allele-specific PCR, and cloning analyses, we demonstrated that BraA07g042410.3C, which encodes an ent-kaurene synthase involved in the gibberellin biosynthesis pathway, is the nhm1 mutant candidate gene. A single-nucleotide mutation (C to T) in the fourth exon of BraA07g042410.3C caused an amino acid substitution from histidine to tyrosine. Compared to that of the wild-type FT, BraA07g042410.3C in the leaves of the nhm1 mutant had lower levels of expression. In addition, gibberellin contents were lower in the mutant than in the wild type, and the mutant plant phenotype could be restored to that of the wild type after exogenous GA3 treatment. These results indicate that BraA07g042410.3C caused the non-heading mutation. This is the first study to demonstrate a relationship between gibberellin content in the leaves and leafy head formation in Chinese cabbage. These findings facilitate the understanding of the mechanisms underlying leafy head development in Chinese cabbage.

Characterization and antioxidant activity of avenanthramides from selected oat lines developed by mutagenesis technique

From a mutagenized oat population, produced by ethyl methanesulfonate mutagenesis, hulled grains from 17 lines with elevated avenanthramide (AVN) content were selected and their AVN structures, concentrations and antioxidant potentials were determined by HPLC-MS2 and HPLC equipped with an on-line ABTS+ antioxidant detection system. The data obtained showed qualitative and quantitative differences in the synthesis of AVNs in the different lines, with a total AVN concentration up to 227.5 µg/g oat seed flour in the highest line, compared with 78.2 µg/g seed in the commercial line, SW Belinda. In total, 25 different AVNs were identified with avenanthramide B structures being among the most abundant, and AVN C structures having the highest antioxidant activity. The findings indicate the potential of oat mutagenesis in combination with a high precision biochemical selection method for the generation of stable mutagenized lines with a high concentration of total and/or individual AVNs in the oat seed grain.

Non-Recombinant Mutagenesis of Bacillus Tropicus CUIMW1718 for Hyper Production of Alginase

Microbial extracellular enzymes occupied a prominent place in industrial sector due to their multifunctional ability. Extensive application of these, gap between production and demand is widening. In order to fulfil this gap, researchers focusing on various strain improvement methods such as recombinant and non-recombinant mutagenesis. In the present research work we made an attempt to screen high yielding industrial important extracellular alginase producing Bacillus tropicus CUIMW1718 strain by treating with inexpensive mutagens such as UV, EMS and EtBr. In this indigenous strain was subjected to Ultraviolet (UV) irradiation, Ethidium bromide (EtBr), Ethyl Methane Sulfonate (EMS) mutagenesis, followed by cross mutation of Ultraviolet (UV) irradiated strain with Ethyl Methane Sulfonate (EMS) and Ethidium bromide (EtBr). High yielding mutant strains were selected based on zone of clearance.

Isolation of dominant Arabidopsis seiv mutants defective in VND7-induced xylem vessel cell differentiation.

The plant-specific NAC transcription factor VASCULAR-RELATED NAC-DOMAIN 7 (VND7) functions in xylem vessel cell differentiation in Arabidopsis thaliana. To identify novel factors regulating xylem vessel cell differentiation, we previously performed ethyl methanesulfonate mutagenesis of a transgenic 35S::VND7-VP16-GR line in which VND7 activity can be induced post-translationally by glucocorticoid treatment. We successfully isolated mutants that fail to form ectopic xylem vessel cells named seiv (suppressor of ectopic vessel cell differentiation induced by VND7) mutants. Here, we isolated eight novel dominant seiv mutants: seiv2 to seiv9. In these seiv mutants, ectopic xylem vessel cell differentiation was inhibited in aboveground but not underground tissues. Specifically, the shoot apices of the mutants, containing shoot apical meristems and leaf primordia, completely lacked ectopic xylem vessel cells. In these mutants, the VND7-induced upregulation of downstream genes was reduced, especially in shoots compared to roots. However, endogenous xylem vessel cell formation was not affected in the seiv mutants. Therefore, the seiv mutations identified in this study have repressive effects on cell differentiation in shoot meristematic regions, resulting in inhibited ectopic xylem vessel cell differentiation.