Authors: Areeqa Shamshad, Muhammad Rashid, Ljupcho Jankuloski...

BACKGROUND

Chemical mutagenesis has been successfully used for increasing genetic diversity in crop plants. More than 800 novel mutant types of rice ( L.) have been developed through the successful application of numerous mutagenic agents. Among a wide variety of chemical mutagens, ethyl-methane-sulfonate (EMS) is the alkylating agent that is most commonly employed in crop plants because it frequently induces nucleotide substitutions as detected in numerous genomes.

METHODS

In this study, seeds of the widely consumed Basmati rice variety (Super Basmati, L.) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1.0%, and 1.25% to broaden its narrow genetic base.

RESULTS

Sensitivity to a chemical mutagen such as ethyl methanesulfonate (EMS) was determined in the M1 generation. Results in M1 generation revealed that as the levels of applied EMS increased, there was a significant reduction in the germination percent, root length, shoot length, plant height, productive tillers, panicle length, sterile spikelet, total spikelet, and fertility percent as compared to the control under field conditions. All the aforementioned parameters decreased but there was an increase in EMS mutagens in an approximately linear fashion. Furthermore, there was no germination at 1.25% of EMS treatment for seed germination. A 50% germination was recorded between 0.50% and 0.75% EMS treatments. After germination, the subsequent parameters, viz. root length and shoot length had between 05.0% and 0.75% EMS dose levels. Significant variation was noticed in the photosynthetic and water related attributes of fragrant rice. The linear increase in the enzymatic attributes was noticed by the EMS mediated treatments. After the establishment of the plants in the M1 generation in the field, it was observed that for fertility percentage was at EMS 1.0% level, for the rice variety.

CONCLUSION

Hence, it is concluded that for creating genetic variability in the rice variety (Super Basmati), EMS doses from 0.5% to 0.75% are the most efficient, and effective.

Topics: Ethyl Methanesulfonate; Oryza; Mutation; Mutagens; Mutagenesis

PubMed: 37780391
DOI: 10.7717/peerj.15821

Authors: Xin Lian, Yan Liu, Huihui Guo...

As the gene pool is exposed to both strain on land resources and a lack of diversity in elite allotetraploid cotton, the acquisition and identification of novel alleles has taken on epic importance in facilitating cotton genetic improvement and functional genomics research. Ethyl methanesulfonate (EMS) is an excellent mutagen that induces genome-wide efficient mutations to activate the mutagenic potential of plants with many advantages. The present study established, determined and verified the experimental procedure suitable for EMS-based mutant library construction as the general reference guide in allotetraploid upland cotton. This optimized method and procedure are efficient, and abundant EMS mutant libraries (approximately 12 000) in allotetraploid cotton were successfully obtained. More than 20 mutant phenotypes were observed and screened, including phenotypes of the leaf, flower, fruit, fiber and plant architecture. Through the plants mutant library, high-throughput and high-resolution melting technology-based variation evaluation detected the EMS-induced site mutation. Additionally, based on overall genome-wide mutation analyses by re-sequencing and mutant library assessment, the examination results demonstrated the ideal quality of the cotton EMS-treated mutant library constructed in this study with appropriate high mutation density and saturated genome. What is more, the collection is composed of a broad repertoire of mutants, which is the valuable resource for basic genetic research and functional genomics underlying complex allotetraploid traits, as well as cotton breeding.

Topics: Ethyl Methanesulfonate; Fertility; Gene Library; Genetic Association Studies; Genome, Plant; Genome-Wide Association Study; Germ Cells, Plant; Germination; Gossypium; Mutagens; Mutation; Polymorphism, Single Nucleotide; Quantitative Trait, Heritable; Tetraploidy

PubMed: 32239588
DOI: 10.1111/tpj.14755

Authors: Xuejiao Zhu, Jiao Huo, Zhu Zeng...

The main goal of the study was to investigate the genotoxic response of N-ethyl-N-nitrosourea (ENU) and ethyl methanesulfonate (EMS) at low doses in a multi-endpoint genotoxicity assessment platform in rats and to derive potential thresholds and related metrics. Male Sprague-Dawley rats were treated by daily oral gavage for 28 consecutive days with ENU (0.25 ~ 8 mg/kg bw) and EMS (5 ~ 160 mg/kg bw), both with six closely spaced dose levels. Pig-a gene mutation assay, micronucleus test, and comet assay were performed in several timepoints. Then, the dose-response relationships were analyzed for possible points of departure (PoD) using the no observed genotoxic effect level and benchmark dose (BMD) protocols with different critical effect sizes (CES, 0.05, 0.1, 0.5, and 1SD). Overall, dose-dependent increases in all investigated endpoints were found for ENU and EMS. PoDs varied across genetic endpoints, timepoints, and statistical methods, and selecting an appropriate lower 95% confidence limit of BMD needs a comprehensive consideration of the mode of action of chemicals, the characteristics of tests, and the model fitting methods. Under the experimental conditions, the PoDs of ENU and EMS were 0.0036 mg/kg bw and 1.7 mg/kg bw, respectively.

Topics: Rats; Animals; Male; Ethyl Methanesulfonate; Ethylnitrosourea; Dose-Response Relationship, Drug; Rats, Sprague-Dawley; Micronucleus Tests; DNA Damage; Mutagens; Mutagenicity Tests

PubMed: 35793016
DOI: 10.1007/s11356-022-21605-z

Authors: Kenta Shirasawa, Hideki Hirakawa, Tsukasa Nunome...

Genome-wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro-Tom genome were identified by a whole-genome shotgun sequencing analysis to estimate the spectrum and distribution of whole-genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired-end reads for four EMS-induced mutants and three gamma-ray-irradiated lines as well as a wild-type line were obtained by next-generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma-ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1,140,687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild-type Micro-Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild-type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse-genetic approaches.

Topics: Base Sequence; DNA Copy Number Variations; Ethyl Methanesulfonate; Gamma Rays; Genes, Plant; Genome, Plant; INDEL Mutation; Solanum lycopersicum; Mutation; Plants, Genetically Modified; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Surveys and Questionnaires

PubMed: 25689669
DOI: 10.1111/pbi.12348

Authors:

Topics: Animals; Antineoplastic Agents, Alkylating; Carcinogens; Ethyl Methanesulfonate; Government Regulation; Humans; Mutagens; Occupational Exposure; United States

PubMed: 21852839
DOI: No ID Found

Authors: Hongchun Xiong, Huijun Guo, Meiyu Fu...

Hexaploid wheat (Triticum aestivum), a major staple crop, has a remarkably large genome of ~14.4 Gb (containing 106 913 high-confidence [HC] and 159 840 low-confidence [LC] genes in the Chinese Spring v2.1 reference genome), which poses a major challenge for functional genomics studies. To overcome this hurdle, we performed whole-exome sequencing to generate a nearly saturated wheat mutant database containing 18 025 209 mutations induced by ethyl methanesulfonate (EMS), carbon (C)-ion beams, or γ-ray mutagenesis. This database contains an average of 47.1 mutations per kb in each gene-coding sequence: the potential functional mutations were predicted to cover 96.7% of HC genes and 70.5% of LC genes. Comparative analysis of mutations induced by EMS, γ-rays, or C-ion beam irradiation revealed that γ-ray and C-ion beam mutagenesis induced a more diverse array of variations than EMS, including large-fragment deletions, small insertions/deletions, and various non-synonymous single nucleotide polymorphisms. As a test case, we combined mutation analysis with phenotypic screening and rapidly mapped the candidate gene responsible for the phenotype of a yellow-green leaf mutant to a 2.8-Mb chromosomal region. Furthermore, a proof-of-concept reverse genetics study revealed that mutations in gibberellic acid biosynthesis and signalling genes could be associated with negative impacts on plant height. Finally, we built a publically available database of these mutations with the corresponding germplasm (seed stock) repository to facilitate advanced functional genomics studies in wheat for the broad plant research community.

Topics: Triticum; Exome Sequencing; Mutation; Mutagenesis; Genomics; Ethyl Methanesulfonate; Genome, Plant

PubMed: 37401008
DOI: 10.1111/pbi.14111

Authors: Mark W Dodson, Lok K Leung, Mohiddin Lone...

Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

Topics: Alleles; Animals; Animals, Genetically Modified; Autophagy; Codon, Nonsense; Cytosol; Drosophila Proteins; Drosophila melanogaster; Endosomes; Ethyl Methanesulfonate; Female; Humans; In Situ Nick-End Labeling; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Lysosomes; Microscopy, Confocal; Microscopy, Fluorescence; Mutation; Phenotype; Protein Serine-Threonine Kinases

PubMed: 25288684
DOI: 10.1242/dmm.017020

Authors: Kanive Parashiva Guruprasad, Advait Subramanian, Vikram Jeet Singh...

BACKGROUND

Ayurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Rasayana is one of the eight branches of Ayurveda which refers to rejuvenant therapy. It has been reported that rasayanas have immuno-modulatory, antioxidant and antitumor functions, however, the genotoxic potential and modulation of DNA repair of many rasayanas have not been evaluated.

METHODS

The present study assessed the role of Brahmarasayana (BR) on Ethyl methanesulfonate (EMS)-and Methyl methanesulfonate (MMS)-induced genotoxicity and DNA repair in in vivo mouse test system. The mice were orally fed with BR (5 g or 8 mg / day) for two months and 24 h later EMS or MMS was given intraperitoneally. The genotoxicity was analyzed by chromosomal aberrations, sperm count, and sperm abnormalities.

RESULTS

The results have revealed that BR did not induce significant chromosomal aberrations when compared to that of the control animals (p >0.05). On the other hand, the frequencies of chromosomal aberrations induced by EMS (240 mg / kg body weight) or MMS (125 mg / kg body weight) were significantly higher (p<0.05) to that of the control group. The treatment of BR for 60 days and single dose of EMS or MMS on day 61, resulted in significant (p <0.05) reduction in the frequency of chromosomal aberrations in comparison to EMS or MMS treatment alone, indicating a protective effect of BR. Constitutive base excision repair capacity was also increased in BR treated animals.

CONCLUSION

The effect of BR, as it relates to antioxidant activity was not evident in liver tissue however rasayana treatment was observed to increase constitutive DNA base excision repair and reduce clastogenicity. Whilst, the molecular mechanisms of such repair need further exploration, this is the first report to demonstrate these effects and provides further evidence for the role of brahmarasayana in the possible improvement of quality of life.

Topics: Animals; Bone Marrow Cells; Cells, Cultured; Chromosome Aberrations; DNA Damage; DNA Repair; Ethyl Methanesulfonate; Humans; Male; Methyl Methanesulfonate; Mice; Plant Preparations; Plants, Medicinal

PubMed: 22853637
DOI: 10.1186/1472-6882-12-113

Authors: Haoqi Shi, Peiwen Xu, Wen Yu...

Ralstonia solanacearum severely damages the growth of tobacco (Nicotiana tabacum L.) and causes great economic losses in tobacco production. To investigate the root metabolism and transcriptional characteristics of tobacco bacterial wilt susceptible variety Cuibi-1 (CB-1) and resistant new line KCB-1 (derived from an ethyl methanesulfonate (EMS) mutant of CB-1) after infestation with R. solanacearum, root metabolism and transcriptional characteristics were investigated using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Differences in resistance between KCB-1 and CB-1 were observed in several aspects: (1) The phenylpropanoid pathway was the main pathway of resistance to bacterial wilt in KCB-1 compared with CB-1. (2) KCB-1 had more differential metabolic markers of disease resistance than CB-1 after infection with R. solanacearum. Among them, the differential coumarin-like metabolites that affect quorum sensing (QS) and biofilm formation of R. solanacearum differ in KCB-1 and CB-1. (3) KCB-1 inhibited production of the R. solanacearum metabolite putrescine, and the level of putrescine in tobacco was positively correlated with susceptibility. (4) Compared with CB-1, the metabolites of KCB-1 had less differential nitrogen sources during the infestation of R. solanacearum, which was detrimental to the growth and reproduction of R. solanacearum. (5) Both indole-3-acetic acid (IAA) and abscisic acid (ABA) in CB-1 and KCB-1 were involved in the response to R. solanacearum infestation, but the levels of IAA and ABA in KCB-1 were greater than in CB-1 at 24 h post inoculation (hpi). In conclusion, R. solanacearum caused reprogramming of both root metabolism and transcription in KCB-1 and CB-1, and the transcriptional and metabolic characteristics of resistant tobacco were more unfavorable to R. solanacearum.

Topics: Abscisic Acid; Coumarins; Ethyl Methanesulfonate; Nitrogen; Plant Diseases; Putrescine; Nicotiana; Transcriptome

PubMed: 36055574
DOI: 10.1016/j.ygeno.2022.110471

Authors: Fanfan Li, Stephen Bordeleau, Kyung Hee Kim...

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

Topics: Alkaloids; Analgesics, Opioid; Catharanthus; Chlorophyll; Ethyl Methanesulfonate; Gene Expression Regulation, Plant; Indoles; Plant Proteins; Receptors, Opioid; Secologanin Tryptamine Alkaloids; Transcription Factors; Vinblastine; Vincristine

PubMed: 36055422
DOI: 10.1016/j.phytochem.2022.113422