Chemical Mutagen Ethyl Methanesulfonate Research Articles

Development of wilt resistant parental lines through mutagenesis in castor(Ricinus communis L.)

The present study was taken up to diversify the pistillate and monoecious lines to be used in hybridization programme. Mutagenesis was carried out using the chemical mutagen ethyl methane sulfonate (EMS) to induce mutation in popular pistillate line DPC 9 and land race Rasipuram local. The segregating mutant population was evaluated for their wilt resistance by screening in wilt sick plot. Plant to progeny row was continued up to M5 generation by selecting superior progenies in terms of yield and wilt resistance. Skewness and kurtosis indicated the presence of gene interactions for most of the traits studied. The pistillate progeny M5 29-11-5 (YRCP 2) and the monoecious progeny M4 106-2-3(YRCS 1904) were found to be the promising one and this could be further utilized as parents in hybrid development process.

Studies on Improving the Efficiency of Somatic Embryogenesis in Grapevine (Vitis vinifera L.) and Optimising Ethyl Methanesulfonate Treatment for Mutation Induction.

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP-9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar.

Influence of ethyl methane sulfonate (EMS) on M3 generations of two genotypes of tomato (Solanum lycopersicum)

Mutation breeding is considered to be economic and efficient in plant improvement, and the use of chemical mutagens such as ethyl methane sulfonate (EMS) can potentially address plant breeding challenges. The aim of this present study is to create variations from existing two genotypes of tomato (Cobra and Roma vf) using a chemical mutagen ethyl methane sulfonate (EMS) in the M3 generation. Variations like increased yield, increase in fruit size and weight, variations in leaf shape, growth habit and fruit colour were conspicuously observed in the two genotypes studied in the M3 generation. In Cobra, variant 3 recorded the highest fruit weight per plant of 1.118kg while 0.318kg was the least and it was recorded by variant 7. Variations in the fruit size are equally observed with some variants producing large fruits while others produce small fruits. Variant 2 had a single fruit weight of about 63g while variant 8 produced small fruit with a single fruit weighing about 27g. There are equally some remarkable variants in Roma vf, variant 2, had a fruit weight of 1.145kg with 56.4g fruits harvested with a single fruit weight of 20.3g. This variant produces lots of many small fruits with a creeping growth habit that makes it behave like an indeterminate variety.

Comparing the response of callus of two chrysanthemums (Dendranthema grandiflora Tzvelev) cultivars to the chemical mutagen ethyl methane sulfonate (EMS)

ISHS II International Symposium on Tropical and Subtropical Ornamentals Comparing the response of callus of two chrysanthemums (Dendranthema grandiflora Tzvelev) cultivars to the chemical mutagen ethyl methane sulfonate (EMS)

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51–3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants’ effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5–100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5–19.3 and aliphatic index between 74.7–75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: −7.17; S score: −6.54 kcal/mol) and seven mutants of KerS26uv (affinity: −7.43; S score: −7.17 kcal/mol) compared to the wild predicted structure (affinity: −6.57; S score: −6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.

Generation of Mutants by Combined Treatment of Physical and Chemical Mutagens in Rice.

Mutants can be generated by physical or chemical mutagens. The procedure includes mutagenesis treatment, post-treatment, planting of M1, harvest of M1, planting of M2, phenotypic observation or mutation scanning in M2 plants. Here we describe the combined treatment of the rice seeds from the indica variety Tenuo with 137Cs gamma rays at 100Gy and a chemical mutagen-ethyl methanesulfonate (EMS). Theoretically, the method can be used to study various plant biological processes which include plant gametogenesis in addition to the generation of mutants by straightforward physiological screens. As an example of the procedure, we screened for mutants resistant to imazethapyr, an herbicide which can kill grass weeds.

Novel Mutation in the Acetohydroxyacid Synthase (AHAS), Gene Confers Imidazolinone Resistance in Chickpea Cicer arietinum L. Plants.

Chickpea (Cicer arietinum L.) is an important crop in crop-rotation management in Israel. Imidazolinone herbicides have a wide spectrum of weed control, but chickpea plants are sensitive to acetohydroxyacid synthase (AHAS; also known as acetolactate synthase [ALS]) inhibitors. Using the chemical mutagen ethyl methanesulfonate (EMS), we developed a chickpea line (M2033) that is resistant to imidazolinone herbicides. A point mutation was detected in one of the two genes encoding the AHAS catalytic subunit of M2033. The transition of threonine to isoleucine at position 192 (203 according to Arabidopsis) conferred resistance of M2033 to imidazolinones, but not to other groups of AHAS inhibitors. The role of this substitution in the resistance of line M2033 was proven by genetic transformation of tobacco plants. This resistance showed a single-gene semidominant inheritance pattern. Conclusion: A novel mutation, T192I (T203I according to Arabidopsis), providing resistance to IMI herbicides but not to other groups of AHAS inhibitors, is described in the AHAS1 protein of EMS-mutagenized chickpea line M2033.

From Dimness to Glossiness—Characteristics of the Spring Rapeseed Mutant Form without Glaucous Bloom (Brassica napus L.)

As a result of the treatment of “Vikros” spring canola with the chemical mutagen ethyl methanesulfonate (EMS), a high-protein mutant form without glaucous bloom (wax bloom) on leaves, shoots, and siliques was isolated. Segregation into glossy and glaucous forms was always observed in the progeny of glossy plants from self-pollination, and the proportion of glaucous plants could reach up to 25%. The progeny of glaucous plants were homogeneous and did not segregate. If during the period of seed germination and seedling development the soil did not dry out and remained moist, and the average daily temperature did not exceed 16 °C, then the amount of glossy plants could reach 99%. Glossy plants possessed qualities valuable for breeding forage varieties, such as the increased content of protein in seeds (more than 30%), and change phenol metabolism, чтo прoявляется a reduced amount of lignin and sinapine in comparison with the original cultivar. In addition, plants without wax coating showed weakened shoot growth, decreased pollen fertility and seed production, and reduced lignin content in the shoots. Glossy mutants are of interest for the obtaining of fodder low-sinapine and low-lignin varieties of spring rapeseed.

Mutagenic effects of ethyl methanesulfonate on nine protein coding genes in tomato (Solanum lycopersicum L.)

The chemical mutagen ethyl methanesulfonate (EMS) has been generally utilised for the development of novel variants of commercially cultivated crop plants. The general approach involves the application of the mutagen at sub-lethal concentrations, followed by the selection of novel phenotypes based on morphometric characterization. This study was directed towards the evaluation of the effects of EMS at a concentration of 0.5 % on Solanum lycopersicum. Mutants were selected based on phenotypic variation, following which nine specific genes such as NRP2 (RNA polymerase II), HKX (hexokinase), P2D1 (photosystem II D1), NEXP (expansin), PHYSN (phytoene synthase), PCANH (carbonic anhydrase), NAGO (argonaute), PRUB (RuBisCO) and NGAG (gag-pol) were isolated via polymerase chain reaction and sequenced to determine the presence of mutations. The results demonstrated that six genes: NRP2, HKX, P2D1, NAGO, NGAG, and NEXP contained mutations that altered the sequence of translated amino acid. In addition to this, a wide range of phenotypic variants was observed. This study concluded that the selected genes were under selection pressure and that undefined physiological processes may be involved in maintaining genetic stability during exposure to chemical mutagens.

An ethyl methanesulfonate-induced neutral mutant-bridging method efficiently identifies spontaneously mutated genes in rice.

Spontaneous mutants are mainly obtained from tissue culture or natural occurrences in plants. The traditional strategy for identifying spontaneously mutated genes is to continuously backcross these mutants to another variety and develop a near-isogenic F2 population for map-based cloning or bulked segregant analysis. However, this strategy is time-consuming. Here, we have developed a new method to efficiently accelerate the identification process. The chemical mutagen ethyl methanesulfonate was first used to treat the wild type of the spontaneous mutants to induce thousands of neutral mutations. An induced individual without any statistically significant phenotypic changes which was compared with the wild type was chosen as the neutral mutant. The spontaneous mutant was then crossed with the neutral mutant to develop a pseudo-near-isogenic F2 population in which only the induced neutral mutations and the causal mutation were segregated in the genome. This population ensures that the variation of the mutated trait is controlled only by the spontaneously mutated gene. Finally, after sequencing the neutral mutant and the mutant-type DNA pool of the F2 population the spontaneous mutation will be identified quickly by bioinformatics analysis. Using this method, two spontaneously mutated genes were identified successfully. Therefore, the neutral mutant-bridging method efficiently identifies spontaneously mutated genes in rice, and its value in other plants is discussed.

The Action of Chemical Mutagens on the Characteristics of Nigella Damascena L. Plants in M1 Generation

The effect of the chemical mutagens EMS (ethyl methanesulfonate), NMU (nitrosomethylurea), and the new mutagen DG-2 on a number of traits of Nigella damascena L. of the variety “Charivnytsya” in the M1 generation was studied. It was found that treating love-in-a-mist with these chemical mutagens changed such characteristics as plant survival, plant height, number of capsules per plant, number of side shoots and number of seeds from one plant. The dependence of the change in the studied traits on the type of chemical mutagen, its concentration and exposure duration was revealed. Nitrosomethylurea had the greatest influence on plant survival. After treatment with this mutagen at a maximum concentration of 0.05% and with a maximum exposure of 16 hours, the number of surviving plants was 4.3%. The effect of DG-2 mutagen at the concentration of 0.5% and 16 hours’ exposure on the survival of M1 plants was the least different from the control group. In this treatment 60.6% of plants survived, with a control value of 61.7%. The tested mutagens caused the appearance in plants of chlorophyll morphoses with a frequency of up to 0.93%, they were characterized by a change in the color of the leaves and capsule leaflets. Nitrosomethylurea had the greatest influence on the plant height and the number of seeds per plant at the concentrations of 0.01 and 0.05% with 6 and 16 hours’ exposure, respectively. After treatment with NMU at the concentration of 0.05% with exposure of 16 hours all plants were found to be unviable. After treatment with NMU at the concentration of 0.01% with 6 hours’ exposure, the average number of seeds per plant was minimal - 17.5 pieces, and plant height - 25.9 cm. The smallest but statistically significant effect on the studied parameters was noted after treatment with mutagen DG-2. In the case of the mutagen concentration of 0.05% and 16 hours’ exposure the average plant height was 40.7 cm, and the average number of seeds - 112.0 pieces, with control values of 47.0 cm and 237.1 pieces, respectively. The revealed changes in the M1 generation imply mutations of different directions in the next generation of love-in-a-mist.

Phenotypic characterization and genetic mapping of the dwarf mutant m34 in maize

Plant height is one of the most important agronomic traits associated with yield in maize. In this study, a gibberellins (GA)-insensitive dwarf mutant, m34, was screened from inbred line Ye478 by treatment with the chemical mutagen ethylmethanesulfonate (EMS). Compared to Ye478, m34 showed a dwarf phenotype with shorter internodes, and smaller leaf length and width, but with similar leaf number. Furthermore, m34 exhibited smaller guard cells in internodes than Ye478, suggesting that smaller cells might contribute to its dwarf phenotype. Genetic analysis indicated that the m34 dwarf phenotype was controlled by a recessive nuclear gene. An F2 population derived from a cross between m34 and B73 was used for mutational gene cloning and this gene was mapped to a chromosome region between umc2189 and umc1553 in chromosome 1 bin1.10, which harbored a previously identified dwarf gene ZmVP8. Sequencing analysis showed a nucleotide substitution (G1606 to A1606) in the sixth exon of ZmVP8, which resulted in an amino acid change (E531 to K531) from Ye478 to m34. This amino acid change resulted in an α-helix changing to a β-sheet in the secondary protein structure and the ‘SPEC’ domain changed to a ‘BOT1NT’ domain in the tertiary protein structure. Taken together, these results suggested that m34 is a novel allelic mutant originally derived from Ye478 that is useful for further ZmVP8 functional analysis in maize.

Searching for the Function of Ecm14, a Pseudopeptidase, Using a Synthetic Lethal Assay

Many enzyme families contain both catalytically active and inactive members. Although these inactive enzyme‐homologs (pseudoenzymes) share significant similarity with their active counterparts, key active site modifications seem to suggest a loss of traditional catalytic activity. The human M14 metallocarboxypeptidase (MCP) family is one such enzyme family that contains several active and inactive members. The baker's yeast Saccharomyces cerevisiae contains only one M14 MCP member, Ecm14, which is produced by a non‐essential gene in the yeast genome. We show through a phylogenetic analysis that this gene is present throughout the ascomycete lineage of fungi. Although Ecm14 is very similar to human CPA1 (a catalytically active member of the M14 MCP family), multiple key modifications in Ecm14 seem to indicate a loss of traditional peptidase‐like activity. A key technique for elucidating the function of non‐essential genes, including those producing pseudoenzyme products, is the genome‐wide synthetic lethal screen. Biochemical pathways typically involve multiple gene components and many of these gene players provide redundancy in the event that other genes experience deleterious mutations. The synthetic lethal screen is used to identify novel mutant yeast strains whose survival is dependent on a particular gene of interest. Colonies that contain a putative synthetically lethal gene pair are kept alive and identified by harboring a color‐reporting plasmid that complements the deletion of the endogenous gene of interest. Here we demonstrate the insertion of the wild‐type ECM14 gene into this integral pSLS1 plasmid and the determination of gene insert orientation using restriction endonucleases. Additionally, we demonstrate the preparation of knockout yeast strains using drug resistant deletion cassettes, the transformation of these yeast with the above plasmid, and finally, genome‐wide mutagenesis attempts using the chemical mutagen ethyl methanesulfonate. Identification of synthetic lethal genes will be presented, as a step toward a better understanding of the biological role of Ecm14.Support or Funding InformationGraduate Student Grants in Aid of Research to RCM and Faculty Research Grants to PJL from the Office of Research & Creative Scholarship, Andrews University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induced mutations alter patterns of quantitative variation, phenotypic integration, and plasticity to elevated CO2 in Arabidopsis thaliana.

A key step toward predicting responses to climate change is characterizing genetic variation in populations. While short-term responses will likely be shaped by currently available genetic variation, longer-term evolutionary responses will depend on the supply of novel variation by, ultimately, mutation. Studying mutational contributions to phenotypic variation can provide insights into the extent of potential variation on which selection may operate in future human-altered environments. Here we used the chemical mutagen ethyl methanesulfonate (EMS) to explore mutational contributions to phenotypic variation, integration, and plasticity to elevated carbon dioxide (eCO2) in three accessions of Arabidopsis thaliana. We found that (1) mutagenesis increased broad-sense heritabilities and variation in plasticity to eCO2 (genotype by environment interactions); (2) mutational effects varied among the three genetic backgrounds; (3) induced mutations had non-random (biased) effects on patterns of phenotypic integration. To our knowledge, this is the first study to address the effects of chemically induced mutations on phenotypic plasticity to eCO2 in a model plant. We discuss our results in light of emerging insights from theoretical and empirical quantitative genetics, suggest potential avenues of research, and identify approaches that may help advance our understanding of climate-driven evolution in plants.

Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum.

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Exploring ethyl methanesulfonate (EMS) treated cotton (Gossypium hirsutum L.) to improve drought tolerance

The Texas High Plains often has extended periods between rainfall events, which can lead to a reduction in the yield and fiber quality of cotton (Gossypium hirsutum L.). It is known that cultivated cotton suffers from low levels of genetic diversity due to the over-use in breeding of similar gene pools, which may hinder breeding for drought tolerance. In this study, for the first time the novel variability or genetic diversity of morphological and agronomic traits possibly created by the chemical mutagen ethyl methanesulfonate (EMS) was evaluated to improve drought tolerance in cotton by traits’ response to different irrigation regimes. EMS is a chemical mutagen that has been shown to cause point mutations in the DNA of many model plants and crop species. Three EMS treated lines were advanced from the M1 to M4 generation as bulk-harvested populations. A diverse selection scheme was applied to capture most of the genetic trait-variability or diversity and superior lines in these populations. In 2014–2016 the diversity of these populations was evaluated based on four agronomic and thirteen morphological traits to determine differences in response to multiple irrigation rates. Analyses of these traits showed statistically significant (p ≤ 0.05) differences between and within populations when compared to the original non-treated EMS source, with most of the variability being observed in the high irrigation rate. However, none of the EMS treated populations had significantly (p ≤ 0.05) better lint yield than the commercial cultivar (control) in 2016. EMS yield performance was possibly constrained by the applied diverse selection scheme of this study. Traits such as total number of bolls, bolls retained at node 7 and below, and those retained between nodes 8 and 12, and bolls retained at node of first fruiting branch may be predictors to improve cotton production (yield) in water limiting environments.

Photoelectrochemical biosensor for microRNA detection based on multiple amplification strategies.

A photoelectrochemical biosensor is described for sensitive detection of microRNA-162a. A multiple amplification strategy is employed that involves (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase. Graphite-like C3N4 (g-C3N4) nanosheets were used as photoactive material. By using these amplification strategies, the detection limit is as low as 0.18fM of microRNA, and the photocurrent increases linearly with the concentration of microRNA-162a in the range from 0.5fM to 1pM. The method was successfully applied to quantify the expression level of microRNA-162a in total RNA extracted from the leaves of maize seedlings after incubation with the chemical mutagen ethyl methanesulfonate. The results confirmed the applicability of the method to the analysis of practical biological samples. Graphical Abstract Schematic of a photoelectrochemical microRNA assay based on a multiple amplification strategy involving (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension,

Induction of mutagenized tomato populations for investigation on agronomic traits and mutant phenotyping

Global demand for tomato production increased tremendously due to its diverse utility in raw, cooked and processed form of food. This necessitates the continued supply of highly nutritious and better yielding improved cultivars to the producers, considering the rapid changing agro-climatic condition. In this study, induced mutant tomato populations of widely recommended tomato genotype Arka Vikas (Sel-22) were generated using chemical mutagen ethyl methane sulfonate (EMS), hydrazine hydrates (HZ) and their combined treatments. In the in vitro study, a gradual reduction in germination percentage and seedling height occurred with the increasing concentrations of mutagens. Combination of EMS and HZ caused maximum biological inhibition followed by EMS and HZ treatments alone in M1 generation. The rate of survival and fertility in M1 plants of tomato was found highly affected due to mutagenic treatment, in which sensitivity toward combined treatment was found highest followed by EMS and HZ. Inspection on induced phenotypic variations in individual plants of M2 population resulted in identification and isolation of wide range of mutants with altered phenotypes. Highest mutation frequency was resulted by combined mutagens followed by the EMS and HZ treatment. Agronomic trait analyses showed intra and inter treatment variations in three quantitative traits (Plant height, fertile branch per plant and fruits per plant) of M2 mutagenized population. Assessment on rate of mutant recovery in M2 population showed highest mutant recovery is possible with combination treatments and then 0.02% HZ followed by 0.02% EMS. In the present study, phenotyping of the mutants revealed that vegetative organs (‘plant size’, ‘plant habit’ and ‘leaf morphology’) was the most sensitive category (69.33%) to which most of the mutant belongs, followed by ‘fruit color and size’ (20.27%) and ‘germination’ (9.79%). Comparative investigation on number of mutants and phenotype category in M2 mutant plants confirmed that the number of pleiotropic mutants was more than the non-pleiotropic mutants in all the treatment conditions. Heritability and reproducibility of the 30 putative mutants were checked in M3 generation based on the observed agro-economical traits; results showed 17 complete and 5 partial heritable mutants.

Mutational Changes In Delphinium malabaricum (Huth.) Munz.: A Potential Ornamental Plant

Abstract Mutation breeding is an established method used for crop improvement and has played a major role in the development of many new flower color/shape mutant cultivars in ornamentals. The present study is aimed at inducing mutations in Delphinium malabaricum using chemical mutagens ethyl methane sulfonate (EMS), sodium azide (SA) and physical mutagen (gamma rays). It was observed that D. malabaricum manifested specific reactions to the treatments with EMS, SA and gamma rays. Identification and selection of mutations were carried out in the second generation (M2). A variety of chlorophyll deficient mutants and high percentage of the flower color and morphological mutants were recorded. The maximum frequency of chlorophyll and flower color and morphological mutations were recorded in EMS treated plants when compared to the other two mutagens. The frequency values for the individual mutant types were varied and randomly distributed at different mutagenic treatments. The highest percentage of color mutants arose after treatments with 0.25% of EMS and the lowest at 20 kR of gamma rays. The mutants were quite distinct, as compared to the control and often had more attractive ornamental features compared to the starting material. The major commercial benefit of the application of this technology has so far been obtaining of novel flower mutants that can be used as an initial material for further breeding of new cultivars.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BackgroundFunctions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest.ResultsTo increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions.ConclusionsA soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2079-y) contains supplementary material, which is available to authorized users.