Ethyl Methanesulfonate Treatment Research Articles

Genome-Wide Identification of Height-Related Genes Using Three Maize Dwarfs and RNA-Seq

Plant height is an important grain yield-associated trait in maize. To date, few genes related to plant height have been characterized in maize. To better understand the genetic mechanisms of plant height in maize, we revealed the transcriptional changes of three dwarf mutants compared to the wild type. By ethyl methane sulfonate treatment of the wild-type maize cultivar PH6WC, we obtained three dwarfs—PH6WCdwarf1 (pd1), PH6WCdwarf2 (pd2), and PH6WCdwarf3 (pd3)—and their plant heights were reduced by 42%, 38%, and 24%, respectively. RNA-Seq data suggested that 1641 differentially expressed genes (DEGs) overlapped with each other among the three dwarfs at the seedling stage. Further analysis showed that the DEGs were divided into four groups with different expression patterns. Functional analysis revealed that these DEGs were commonly enriched in 47 GO terms mainly involved in cytokinesis, hormone, and energy metabolism pathways. Among them, An1, involved in the GA biosynthesis pathway, and mutations in An1 result in reduced plant height. EREB182 encodes ethylene-responsive element binding protein 2, which is critical for internode elongation. Microtubule-related genes Zmtub2, Zmtub3, Zmtub5, Zmtub6, and TUBG2 were commonly enriched among the three comparisons. Previous studies have shown that mutations in microtubule-associated genes cause the dwarf phenotype. However, nearly half of the common DEGs had no functional information, such as Zm00001d000107, Zm00001d000279, etc., implying their novel and specific functions in maize. Overall, this study identifies several potential plant height-related genes and contributes to linking genetic resources with maize breeding.

Genomic variation induced by a low concentration of ethyl methanesulfonate (EMS) in quinoa ‘Longli-4’ variety

Quinoa (Chenopodium quinoa, 2n = 4x = 36), a super pseudocereal crop, has been introduced into China nearly 60 years. Many excellent varieties have been developed through massive selection; however, few are developed through mutagenesis breeding. In this study, the ‘Longli-4’ variety, locally cultivated in Gansu province, Northwest China, was selected for experimentation. The grains of ‘Longli-4’ were treated with ethyl methanesulfonate (EMS) at a concentration of 0.8% for 8 h. Nine plants from independent M2 families were randomly selected to investigate the mutagenesis effect of EMS on the quinoa genome. The results indicated that the single nucleotide polymorphisms (SNPs) induced by EMS were unevenly distributed across all 18 chromosomes, with an average mutation frequency of 91.2 SNPs/Mb, ranging from 4.5 to 203.5 SNPs/Mb. A significant positive correlation between the number of SNPs and chromosome length was identified through linear model analysis. Transitions from G/C to A/T were the most predominated in all variant categories, accounting for 34.4–67.2% of the mutations, and SNPs were significantly enriched in intergenic regions, representing 69.2–75.1% of the total mutations. This study provides empirical support for the application of low concentration EMS treatment in quinoa breeding.

Repeat treatment of organotypic airway cultures with ethyl methanesulfonate causes accumulation of somatic cell mutations without expansion of bronchial-carcinoma-specific cancer driver mutations

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25–100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DisclaimerThis article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

Induction of genetic diversity in gepak kuning soybean cultivar and M.1.1.3 line using ethyl methane sulfonate in M1 generation

Abstract Genetic diversity is the main determining factor in plant breeding activities. Various breeding methods can be used to induce plant variability, including mutation breeding. The chemical mutagen commonly used for mutagenesis is Ethyl Methane Sulfonate (EMS). This study explores the effects of EMS mutagen on the morphological and agronomic diversity of gepak kuning cultivar and M.1.1.3 soybean line. The research was conducted in Tambon Tunong Village, North Aceh District, Indonesia, using a single-factor Randomized Block Completely Design (RCBD). Concentrations of 0%, 0.05%, 0.075%, and 0.1% EMS were tested on the gepak kuning cultivar and M.1.1.3 line. Treating gepak kuning soybeans with EMS significantly impacted various variables, including harvesting age, pod number, seed weight, leaf appearance, and production. Additionally, the treatment resulted in changes in the morphology of the plant, particularly in the shape of its branches. The EMS treatment on M.1.1.3 line also significantly affected various variables like plant height, flowering age, harvesting age, and yield components. However, it led to sterile plants in the line.

Phenotypic characterization of ethyl methanesulfonate (EMS) induced bigger pod (bp) with multiple seed mutant in lentil (Lens culinaris Medik.)

Lentils (Lens culinaris Medik.) is a protein- and nutrient-rich crop with limited genetic diversity and climate adaptability, making it ideal for mutation breeding research. Hence, in this study, we aimed to select and characterize distinct mutations in the flower and pod traits of lentil crops for enhanced yield and yield stability. In this study, healthy and viable lentil seeds were mutagenized using different concentrations of ethyl methanesulfonate (EMS) (0.10%, 0.25%, 0.50%, 0.75%, and 1.0%). The mutagenized lentil populations were cultivated up to the third generation (M3) to identify stable mutations in the flower and pod structures with increasing plant height. The notably larger 'bigger pod’ (bp) mutant, characterized by its unusually large pods containing 6–7 seeds per pod, was meticulously studied and quantified for its morphological traits in subsequent generations. Morphological observations of the tall bp mutant revealed significant mutations induced by EMS (0.10%) treatment. The quantitative trait means and confidence intervals showed that the mutant and parent cultivar L-4076 exhibited considerable variation. Seed yield (g) and yield-related components, including pods per plant, pod length (cm), and seeds per pod, were significantly higher in the mutant. Seed yield exhibited a significant positive phenotypic correlation with pod length, followed by branches per plant, seeds per pod, plant height, and pods per plant. The novel bp mutant induced in the present study produced increased number of seeds per plant and ensured optimal resource utilization, thus possibly contributing to yield improvement and stability. Further, it also aided in understanding the genetic networks that control legume flower and pod architecture and in genomics-assisted breeding for the development of superior lentil cultivars.

Effect of chemical mutagen (Ethyl methane sulfonate) on Money plant (Epipremnum aureum)

Epipremnum aureum, commonly known as pothos, is a hardy indoor foliage ornamental crop. Mutation breeding in Golden pothos is needed to meet the demand for more valuable and highly sought-after cultivars. The present study aimed to exploit variability on various parameters viz. new leaf emergence, survival percentage, plant height, length and width of leaves. The experiment was set up using a Complete Randomized Design (CRD) with 8 treatments and 3 replications. Rooted cuttings of Golden pothos were treated with varying concentrations of Ethyl Methane Sulfonate (EMS) for 4 hours, and a control treatment was included. The results showed that all the treated cuttings with EMS delays the new leaf emergence period compared to the control. Compared to the control, the minimum number of new leaf emergences was found in EMS-treated plants. The maximum survival percentage (100%) was recorded in 0.1%, 0.5% and control treatment. The lowest plant height (15.3 cm ) was observed in treatment T4 (1.5 % EMS) treated plants. However, maximum plant height was observed in the control treatment at 150 days of interval. In comparison to the control, EMS decreased the length and width of leaves at higher dosages. The minimum leaf length and width, i.e. 2.40 cm and 1.20 cm, were recorded in plants treated with EMS at 2%, while maximum length and width were observed in control and low EMS-treated plants. The study showed that ethyl methane sulfonate affected plant development characteristics and showed some dwarfness of money plants. This appears to be the first report of EMS treatment on pothos.

Improving French bean yield potential through induced mutagenesis using EMS and SA

IntroductionFrench bean (Phaseolus vulgaris L.) holds global significance as one of the most consumed legumes, with commercial value surpassing that of all other legume crops combined. In India, the consumption of French beans has grown steadily, especially in the North Eastern region, driven by heightened consumer interest in its nutritional benefits. Considering these factors, we initiated an induced mutagenesis program to enhance the genetic diversity of locally grown French bean genotypes, traditionally cultivated for their superior adaptability.MethodsTo achieve this, we initiated an induced mutagenesis program. Seeds from the village seed stock were subjected to treatments with varying doses of ethyl methane sulfonate (EMS) ranging from 0.1% to 0.4% and sodium azide (SA) from 0.1% to 0.4%. The objective was to increase yield potential and enhance genetic diversity.ResultsThe treatment with EMS and SA led to a non-specific, dosage-independent reduction in biophysiological characteristics in French bean mutants. Notably, the 0.4% SA treatment significantly inhibited germination and fertility, causing a decrease in chlorophyll (10.02 mg. g-1 FW) and carotenoid (1.57 mg. g-1 FW) levels. This suggests a disruption in genes associated with chlorophyll and carotenoid synthesis. However, in the M2 generation, the mutagenic treatments substantially improved yield and associated traits. The highest pod yield per plant was recorded at 79.50 gm for the 0.2% EMS treatment. A character association study revealed strong correlations (0.217 to 0.995) between pod yield and other agronomic traits.DiscussionThe results indicate that selecting mutants based on these traits in populations treated with EMS and SA can significantly increase crop yield. The 0.2% SA and 0.2% EMS M2 mutant populations exhibited the highest induced variability, making them ideal for selecting higher-yielding mutant lines for further breeding generations. The increased yields in these mutant lines, derived from a local cultivar, show promise for meeting the growing demand for French bean production through their widespread cultivation.

Study the influence of Mutagenic Treatment on Cell Suspension Culture of Elephantopus scaber L. for the Quantitative enhancement of Bioactive constituents and their Antioxidant potentials

In the present study, the impact of the chemical mutagen, Ethyl methanesulfonate (EMS) was evaluated on the quantitative enhancement of sesquiterpene, flavonoids (Quercetin and Rutin) and Stigmasterol compound Elephantopus scaber. The leaves of E. scaber L. were used for the callus induction. MS basal medium supplemented with different hormone combinations of 2,4-D and BAP (1:1 ratio) was used. The cell suspension culture was treated with three concentrations of EMS (0.1%, 0.2% and 0.5%) and treatment durations (1h, 2h and 3h). Mutagenesis is an important strategy to improve production of secondary metabolites using cell suspension culture. The results obtained revealed that the concentrations and treatment durations of EMS were most effective in enhancement in the content of studied metabolites. EMS treatment of 0.1% concentration was effective. The analysis was done using HPTLC technique. The highest content of Deoxyelephantopin recorded in 1h EMS treatment and 0.1% concentration compared to control sample. Quercetin, Rutin content was also high in 1h EMS treatment and 0.1% concentration compared to control sample. The Stigmasterol content was more in 2h EMS treatment and 0.1% concentration compared to control sample. The EMS treated samples showed potential scavenging activity compared to control samples IC50 value. The method was simple, precise, eco-friendly.

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.

New mutations of flower shape in Nigella damascena L., its pleiotropic effects and patterns of inheritance

Two mutants with short sepals were identified after ethyl methanesulfonate treatment of Nigella damascena seeds. In one of them (“shs1” gene = short sepal 1), isolated from the line with double flowers, the sepals, in addition to reduced size, were divided into several rounded lobes, which granted the flower an original rose-like appearance of ornamental value. Another mutant with reduced sepals (“shs2” gene = short sepal 2) was isolated from the line with simple flowers. The allelism test showed that these two genes were non-allelic. Both mutants as pollen parents were crossed with the same line with single flowers. In a dihybrid cross, simple flower, non-reduced sepals (wild type) × double flower, reduced sepals (“shs1” gene) F1 hybrids demonstrated a wild phenotype. F2 progeny, in addition to two parental classes, showed two recombinant classes in a 9:3:3:1 ratio, indicating that flower shape and sepal size were inherited monogenously and independently, and the plant with rose-like flowers was a double recessive homozygote. Reduced sepals (“shs2” gene) in crosses with the single flower line of wild type were inherited as a monogenic recessive trait, showing a 3:1 segregation ratio in F2. Both mutant genes had a number of similar pleiotropic effects, which, however, were different in strength. Thus, both mutant genes shortened leaf segments, divided the cotyledon leaves into several lobes, and caused disturbances in the female generative sphere, leading to a lack of seed setting. At the same time, the identification of mutants as early as at the cotyledon stage, due to the pleiotropic effect, makes it possible to select and maintain them, especially with regard to the mutant with rose-like flowers, which is highly decorative.

Chemosensitivity analysis of tiger nuts (Cyperus esculentus L.) using ethyl methanesulfonate (EMS) and colchicine mutagens

Four hundred tubers of four genotypes, two brown and two black tiger nuts were subjected to Ethyl Methanesulfonate (EMS) and Colchicine treatments at concentrations of 0 %, 0.1 %, 0.25 %, 0.5 % and 1.0 % for 24 h. Each genotype had twenty tubers treated with each of the five different concentrations and were planted using Complete Randomized Design (CRD) in a greenhouse. Quantitative data was collected and LD50 and RD50 were analysed using Excell 2016 and Genstat 11.2. A general decreasing trend in percentage germination and plant height was observed with increasing concentrations of mutagens applied. An EMS treatment had LD50 and RD50 values of 0.97 % and 1.49 % for black and 0.63 % and 1.63 % for brown genotypes.Similarly, the percentage colchicine treatment had LD50 and RD50 values of 1.65 % and 19.51 % concentrations for black and 0.91 % and 1.71 % concentrations for brown genotypes.

Effect of ethyl methanesulfonate mediated mutation for enhancing morpho-physio-biochemical and yield contributing traits of fragrant rice.

Chemical mutagenesis has been successfully used for increasing genetic diversity in crop plants. More than 800 novel mutant types of rice (Oryza sativa 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. In this study, seeds of the widely consumed Basmati rice variety (Super Basmati, Oryza sativa 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. 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 LD50 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 LD50 for fertility percentage was at EMS 1.0% level, for the rice variety. 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.

Ethyl Methanesulfonate (EMS) Mutagen Toxicity-Induced DNA Damage, Cytosine Methylation Alteration, and iPBS-Retrotransposon Polymorphisms in Wheat (Triticum aestivum L.)

The use of mutagens in plant breeding is used to create new germplasm, increase agricultural yield, quality, and resistance to diseases and pests. Mutagens are physical or chemical factors that can alter the DNA or RNA structure of an organism, causing mutations above the expected level. One of the most common and potent chemical mutagens is EMS (ethyl-methane sulfonate), which produces point mutations in plants, but to a lesser degree can also cause the loss or deletion of a chromosomal region. This study used inter-primer binding site (iPBS) and coupled restriction enzyme digestion inter-primer binding site (CRED-iPBS) technique analysis to determine the effect of EMS mutagens on methylation rates in wheat genotypes at seedling growth stage. Treatments with five different EMS concentrations (0%; control, 0.1%, 0.2%, 0.3%, and 0.4%) at four different times (0; control, 3, 6, and 9 h) were used. Inter-primer binding site (iPBS) markers were employed to assess genomic instability and cytosine methylation in treated wheat. In seeds treated with EMS at different concentrations and times, the disappearance of regular bands and the formation of new bands due to the effects of the EMS mutagen revealed that genetic diversity exists. The CRED-iPBS analysis revealed that the 3 h + 0.1% EMS treatment produced the highest MspI polymorphism value (19.60%), while the 9 h + 0.1% EMS treatment produced the lowest value (10.90%). The mutagenic effects of EMS treatments had considerable polymorphism on a variety of impacts on the cytosine methylation and genomic instability of wheat. According to the current research, EMS mutagenesis may be a practical method for accelerating breeding programs to produce enough genetic diversity in wheat populations. Mutation-assisted breeding and the subsequent selection of desirable mutants using genetic markers may also be carried out in wheat utilizing an integrated strategy.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

AbstractInduced mutations are important for genetic research and breeding. Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations. However, mutations must be fixed prior to phenotyping or field trials, which requires additional rounds of self-pollination. Microspore culture is an effective method to produce double-haploid (DH) plants that are fixed homozygotes. In this study, we conducted ethyl methanesulfonate (EMS)-induced mutagenesis of microspore cultures of barley (Hordeum vulgare) cultivar ‘Hua30’ and landrace ‘HTX’. The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration. The two genotypes showed different regeneration efficiencies. The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations, revealed by whole-genome sequencing, highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants. Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture (somaclonal variation) rather than EMS-induced mutagenesis. In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials, this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations (mainly by fine-tuning the mutagen dosage).

Influence of EMS applications on fatty acids composition in safflower

Safflower is a valuable oil plant with 13–46% seed oil content rich in fatty acids, especially linoleic acid. In latest years, the safflower varieties with excessive oleic acid content have been started to be grown due to their high storage stability and suitability for frying. The research aimed to determine the influence of Ethyl Methane Sulfonate (EMS), which is the most commonly used chemical mutagen in experimental genetics, on the fatty acids’ composition of safflower oil. In this study, seven different EMS rates were applied to 20 seeds harvested in August. As a result of the study, the primary fatty acids of the oil of the control plot plants were identified as 73% linoleic, 12% oleic, 5.8% palmitic, and 2.2% stearic acid. Compared to the control, the highest reduction among fatty acids was observed as 8.40% in oleic acid with 2 ​h of 0.64% EMS treatment. However, there were increases in oleic acid, stearic acid, and linoleic acid contents in some applications. In addition, lauric acid, which was not detected in the control treatments, was detected in most of the other applications, where the highest lauric acid ratio of 14.945% was determined under EMS treatment of 0.64% for 2 ​h. The results showed that the influence of EMS mutagenesis on safflower fatty acid composition might be affected differently by different mutation levels. Therefore, there is a need to evaluate the impact of EMS mutagenesis on fatty acids across successive generations to confirm the results of this study.

Induction of Protocorm-Like Bodies (PLBs) Phalaenopsis spp. Hybrids Mutation through Ultraviolet Irradiation (UV254) and Ethyl Methane Sulfonate (EMS)

Phalaenopsis sp. is the most-produced orchid species in Indonesia. Compared to conventional breeding, mutation induction by using mutagens, such as Ultraviolet Light-C (λ = 254 nm) (UV254) and Ethyl Methane Sulfonate (EMS), could probably result in new superior orchid variants. This research aims to get some mutants with phenotypes that have visual differences in the Phalaenopsis spp. hybrids wild type. There were 4 durations of UV254 irradiation: 5’ on, 85’ off; 10’ on, 80’ off (1 day and 7 days for each treatment); 4 concentrations of EMS used in this research: 0.05%; 0.06%; 0.07%; 0.08% for 6 hours of immersion; selected UV254 irradiation (5’ on, 85’ off (7 days)) combined with these concentrations. UV254 irradiation treatment (5’ on, 85’ off (1 day and 7 days); 10’ on, 80’ off (7 days)) resulted in some mutants with leaf phenotypes that were visually different from the wild type; 0.05% EMS treatment resulted in PLBs mutant with a visually larger size than the wild type; 0.08% EMS treatment and combination treatments (for EMS 0.05% and 0.08% for each treatment) resulted in non-growing albino PLBs. Hence, mutation induction using UV254 and EMS in this research produced several most likely mutants having visual differences that may be more desirable than the wild type.

Identification and characterization of a novel gene controlling floral organ number in rice (Oryza sativa L.).

Floral organ number is crucial for successful seed setting and mature grain development. Although some genes and signaling pathways controlling floral organ number have been studied, the underlying mechanism is complicated and requires further investigation. In this study, a floral organ number mutant was generated by the ethyl methanesulfonate treatment of the Korean japonica rice cultivar Ilpum. In the floral organ number mutant, 37% of the spikelets showed an increase in the number of floral organs, especially stamens and pistils. Histological analysis revealed that the number of ovaries was determined by the number of stigmas; spikelets with two or three stigmas contained only one ovary, whereas spikelets with four stigmas possessed two ovaries. The floral organ number mutant showed pleiotropic phenotypes including multiple grains, early flowering, short plant height, and reduced tiller number compared with the wild-type. Genetic and MutMap analyses revealed that floral organ number is controlled by a single recessive gene located between the 8.0 and 20.0 Mb region on chromosome 8. Calculation of SNP-index confirmed Os08g0299000 as the candidate gene regulating floral organ number, which was designated as FLORAL ORGAN NUMBER7 (FON7). A single nucleotide polymorphism (G to A) was discovered at the intron splicing donor site of FON7, which caused the skipping of the entire sixth exon in the mutant, resulting in the deletion of 144 bp. Furthermore, the T-DNA-tagged line displayed the same floral organ number phenotype as the fon7 mutant. These results provide valuable insight into the mechanism of floral organ differentiation and formation in rice.

Construction of EMS-Induced Peanut Mutant Libraries and Identification of Pod-Related Traits Mutant Lines

Peanut (Arachis hypogaea L.) is an oil and economic crop of vital importance, and peanut pod is the key organ influencing the yield and processing quality. Hence, the Pod-related traits (PRTs) are considered as important agronomic traits in peanut breeding. To broaden the variability of PRTs in current peanut germplasms, three elite peanut cultivars were used to construct Ethyl methane sulfonate (EMS)-induced mutant libraries in this study. The optimal EMS treatment conditions for the three peanut varieties were determined. It was found that the median lethal dose (LD50) of EMS treatment varied greatly among different genotypes. Finally, the EMS-induced peanut mutant libraries were constructed and a total of 124 mutant lines for PRTs were identified and evaluated. Furthermore, “M-8070”, one of the mutant lines for pod constriction, was re-sequenced via high-throughput sequencing technology. The genome-wide variations between “M-8070” and its wild parent “Fuhua 8” (FH 8) were detected. 2994 EMS-induced single nucleotide polymorphisms (SNPs) and 1188 insertion-deletions (InDels) between “M-8070” and its wild parent were identified. The predominant SNP mutation type was C/G to T/A transitions, while the predominant InDel mutation type was “1-bp”. We analyzed the distribution of identified mutations and annotated their functions. Most of the mutations (91.68% of the SNPs and 77.69% of the InDels) were located in the intergenic region. 72 SNPs were identified in the exonic region, leading to 27 synonymous, 43 non-synonymous and 2 stop-gain variation for gene structure. 13 Indels were identified in the exonic region, leading to 4 frame-shift, 8 non-frame-shift and 1 stop-gain variations of genes. These mutations may lead to the phenotypic variation of “M-8070”. Our study provided valuable resources for peanut improvement and functional genomic research.

Characteristics of a radish mutant with longer siliques

The silique is an important organ in plant reproduction and maintains biodiversity among species; however, little is known about the regulation of radish (Raphanus sativus L.) silique development. In this study, we conducted research on the radish long silique (ls) mutant and wild-type (WT) radish and compared their morphological and molecular markers. The results showed that the mutant obtained by ethyl methane sulfonate treatment had the following stable characteristics: the lengths of mutant and WT siliques were 20.50 and 9.10 cm, respectively; and the ovule number per silique of the mutant and WT was 9.5 and 4.5, respectively. Polymerase chain reaction (PCR) analysis revealed abundant polymorphisms between the ls mutant and WT in HZ001, SRC9-022, and OIL2F11 simple-sequence repeat molecular markers, and the expression of LS1 and LS2 (RsNAC66). Arabidopsis thaliana-transformed plants with RsNAC66 overexpression were obtained by the floral dip method. Quantitative PCR showed that LS2 (RsNAC66) was more highly expressed in transformed lines than in WT, and the expression of LS2 (RsNAC66) in the transformed lines was higher in siliques than leaves. Phenotypic analysis revealed abnormal ovule numbers and shoots, and altered plant height in the transformed plant. Phenotypic and gene expression analyses showed that LS2 (RsNAC66) plays an important role on silique length and the number of seeds per silique. Together, the results showed that the radish ls mutant is specific and stable, and thus constitutes an excellent research resource for improving the seed yield of radishes.

Short grain 5 controls grain length in rice by regulating cell expansion

Grain shape is a crucial determinant of grain weight and quality and plays a vital role in rice breeding. Although many grain shape-related genes have been reported, the regulatory relationship between them has not been well characterized in rice. In this study, we report the isolation of a short-grain-length mutant called sg5 from the heavy-panicle-type hybrid rice elite restorer line ‘ShuhuiR498′ (R498) after ethyl methanesulfonate (EMS) treatment. MutMap cloning revealed that SG5 encodes a Myb-like transcription factor. A missense mutation in the first exon of SG5 was found to cause an amino acid change from leucine to proline at position 197 in the mutant SG5 protein. Gene knockout and genetic complementation experiments confirmed that the point mutation in SG5 was responsible for the sg5 mutant phenotype. SG5 is mainly expressed in young panicles and hulls. In addition, the SG5 protein is found in the nucleus and does not affect subcellular localization. Histochemical observation and gene expression analysis indicated that SG5 regulates spikelet hull development by mediating cell expansion. Moreover, the expression levels of BG1, GS2, and DEP1 were reduced in sg5 plants, and dual-luciferase (LUC) assays showed that SG5 can bind to the BG1 gene promoter. The effect of pyramiding sg5 and GS3 suggests that sg5 and GS3 regulate grain length independently. The results of our study show that the missense mutation in sg5 is essential for the molecular function of SG5 and SG5 is involved in regulating cell expansion and expression of grain-shape-related genes to regulate grain length. This work provides new data to help study and understand the molecular function of SG5.