Identification and map-based cloning of an EMS-induced mutation in wheat gene TaSP1 related to spike architecture.

KEY MESSAGE: A candidate gene TaSP1 related to spike shape was cloned, and the gene-specific marker was developed to efficiently track the superior haplotype in common wheat. Spike shape, an important factor that affects wheat grain yield, is mainly defined by spike length (SPL), spikelet number (SPN), and compactness. Zhoumai32 mutant 1160 (ZM1160), a mutant obtained from ethyl methane sulfonate (EMS) treatment of hexaploid wheat variety Zhoumai32, was used to identify and clone the candidate gene that conditioned the spike shape. Genetic analysis of an F2 population derived from a cross of ZM1160 and Bainong207 suggested that the compact spike shape in ZM1160 was controlled by a single recessive gene, and therefore, the mutated gene was designated as Tasp1. With polymorphic markers identified through bulked segregant analysis (BSA), the gene was mapped to a 2.65-cM interval flanked by markers YZU0852 and MIS46239 on chromosome 7D, corresponding to a 0.42-Mb physical interval of Chinese spring (CS) reference sequences (RefSeq v1.0). To fine map TaSP1, 15 and seven recombinants were, respectively, screened from 1599 and 1903 F3 plants derived from the heterozygous F2 plants. Finally, TaSP1 was delimited to a 21.9 Kb (4,870,562 to 4,892,493 bp) Xmis48123-Xmis48104 interval. Only one high-confidence gene TraesCS7D02G010200 was annotated in this region, which encodes an unknown protein with a putative vWA domain. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that TraesCS7D02G010200 was mainly expressed in the spike. Haplotype analysis of 655 wheat cultivars using the candidate gene-specific marker Xg010200p2 identified a superior haplotype TaSP1b with longer spike and more spikelet number. TaSP1 is beneficial to the improvement in wheat spike shape.

Dauer juvenile recovery transcriptome of two contrasting EMS mutants of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode Heterorhabditis bacteriophora, symbiotically associated with enterobacteria of the genus Photorhabdus, is a biological control agent against many insect pests. Dauer Juveniles (DJ) of this nematode are produced in industrial-scale bioreactors up to 100 m3 in liquid culture processes lasting approximately 11 days. A high DJ yield (> 200,000 DJ·mL-1) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24 h) Photorhabdus bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in H. bacteriophora ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with Photorhabdus supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6 h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified Caenorhabditis elegans homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3 h after Photorhabdus supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine H. bacteriophora gene models with differential expression are provided as candidate genes for further studies.

Deciphering induced variability, character association and multivariate analysis utilizing gamma rays and ethyl methanesulfonate in bread wheat (Triticum aestivum L.) genotypes with differential grain texture.

PURPOSE: Sustainable wheat production and higher genetic gains can be realized by broadening the genetic base and improving the well adapted varieties. In the present study, a multi-year experiment involving induced mutagenesis was conducted to create genetic variation, assess trait associations and genetic divergence in four wheat varieties with differential grain texture treated with six doses of gamma rays and ethyl methane sulfonate using ten agro-morphological traits. MATERIALS AND METHODS: Healthy selfed seeds of four bread wheat varieties with differential texture were irradiated using six doses ranging from 175 Gy-300 Gy of gamma rays (Co60: BARC, Mumbai) and six concentrations of ethyl methanesulfonate (0.3-1.3%) (Sigma-Aldrich, Bangalore, India) to evaluate variability, character association and degree of genetic diversity induced among the mutagenic treatments of wheat varieties with differential grain texture. RESULTS: Significant inter-population differences were observed for almost all the traits. The sample mean of twelve mutant populations in each of the cultivar exhibited superior quantitative phenotypic traits and increased values of the genetic parameters. Based on association and variability studies, plant height, spike length, grain filling period, biological yield per plant and harvest index can be used as early generation criteria for maximum genetic improvement. Multivariate studies indicated the contribution of various traits towards divergence and indicated the efficiency of mutagens in generating variability. Gamma-irradiation dosages between 200-250 Gy and 0.5-1.1% EMS for soft-textured varieties, whereas doses between 225-275 Gy and 0.5-0.9% EMS were found to be most potent for semi-hard-textured varieties. CONCLUSIONS: Assessment of mutagen sensitivity showed that semi-hard wheat varieties were responsive to both mutagens, particularly EMS and generated higher variability and divergence than the soft textured varieties. Hence, gamma rays were proved to be more effective in generating higher variability than ethyl methanesulfonate. A total of 117 putative mutants were identified with desirable agro-morphological attributes. Among these, mutants with higher inter-cluster distance can be used as parents in hybridization programs and serve as important genetic resources in future wheat improvement programs.

Enhancing cold resistance in Banana (Musa spp.) through EMS-induced mutagenesis, L-Hyp pressure selection: phenotypic alterations, biomass composition, and transcriptomic insights.

BACKGROUND: The cultivation of bananas encounters substantial obstacles, particularly due to the detrimental effects of cold stress on their growth and productivity. A potential remedy that has gained attention is the utilization of ethyl mesylate (EMS)-induced mutagenesis technology, which enables the creation of a genetically varied group of banana mutants. This complex procedure entails subjecting the mutants to further stress screening utilizing L-Hyp in order to identify those exhibiting improved resistance to cold. This study conducted a comprehensive optimization of the screening conditions for EMS mutagenesis and L-Hyp, resulting in the identification of the mutant cm784, which exhibited remarkable cold resistance. Subsequent investigations further elucidated the physiological and transcriptomic responses of cm784 to low-temperature stress. RESULTS: EMS mutagenesis had a substantial effect on banana seedlings, resulting in modifications in shoot and root traits, wherein a majority of seedlings exhibited delayed differentiation and limited elongation. Notably, mutant leaves displayed altered biomass composition, with starch content exhibiting the most pronounced variation. The application of L-Hyp pressure selection aided in the identification of cold-resistant mutants among seedling-lethal phenotypes. The mutant cm784 demonstrated enhanced cold resistance, as evidenced by improved survival rates and reduced symptoms of chilling injury. Physiological analyses demonstrated heightened activities of antioxidant enzymes and increased proline production in cm784 when subjected to cold stress. Transcriptome analysis unveiled 946 genes that were differentially expressed in cm784, with a notable enrichment in categories related to 'Carbohydrate transport and metabolism' and 'Secondary metabolites biosynthesis, transport, and catabolism'. CONCLUSION: The present findings provide insights into the molecular mechanisms that contribute to the heightened cold resistance observed in banana mutants. These mechanisms encompass enhanced carbohydrate metabolism and secondary metabolite biosynthesis, thereby emphasizing the adaptive strategies employed to mitigate the detrimental effects induced by cold stress.

Mutagenic sensitivity, effectiveness and efficiency of gamma rays and ethyl methane sulfonate on soft and semi-hard bread wheat (Triticum aestivum L.) varieties in the north-western Himalayan climate.

PURPOSE: The North-western Himalayan region requires unique varietal traits for the cultivation and quality of grain produced. Wheat varieties released for this zone in the past remained very popular among the farmers. However, with the passage of time certain traits such as the appearance of pathogenic rust races and grain softness have become threat to the fecundity of these genotypes and needs immediate improvement in this region. Mutation breeding facilitates improving one or two traits of a popular cultivar and to generate variability for most of plant traits upon which selection can be imposed. The purpose of this study is to evaluate the mutagenic sensitivity, effectiveness and efficiency of physical and chemical mutagens in four bread wheat varieties with differential grain texture. MATERIALS AND METHODS: Four bread wheat varieties; HS 490, HPW 89, HPW 360 and HPW 251 were irradiated using six doses of gamma rays (γ-rays) ranging from 175 to 300 Gy; Co60 source (BARC, Mumbai, India) and six doses of ethyl methane sulfonate (EMS) ranging from 0.3 to 1.3%; EMS (Sigma-Aldrich, Bangalore, India) to assess their mutation sensitivity, effectiveness, efficiency and spectrum of induced macro-mutations in M1 and M2 generation. RESULTS: Based on mutagen sensitivity tests, both gamma rays and ethyl methane sulfonate had similar effects as the doses/concentrations increased in all four varieties. Ethyl methane sulfonate had a discernible effect on seed germination and growth parameters as compared to gamma irradiated treatments. Pollens viability studies confirmed the differential effects of both mutagens on germination and plant survivability. The LD50 and LC50 values varied between 290-315 Gy for gamma rays and 0.90-1.35% for EMS under controlled laboratory conditions, however, the range substantially differs for gamma rays (240-290 Gy) and for EMS (0.50-1.1%) under field conditions, irrespective of the variety treated. The frequency of chlorophyll mutations was low and showed a linear correlation with the doses/concentrations of the mutagen. A total of 117 putative mutants with desirable agro-morphological characteristics were also isolated. Mutagenic effectiveness and efficiency results showed that gamma irradiation doses of 250-300 Gy and ethyl methane sulfonate of 0.7-1.3% were most potent for an effective mutation breeding programme in wheat crop. CONCLUSIONS: It was found that semi-hard textured varieties showed higher sensitivity to chemical mutagens as compared to soft-textured varieties. Gamma irradiation dose of 250-300 Gy and ethyl methane sulfonate concentration of 0.7-1.3% were found to be most effective and efficient across four bread wheat varieties and can be used in large scale mutagenesis programmes.

A large sequenced mutant library - valuable reverse genetic resource that covers 98% of sorghum genes.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

EMS-induced mutagenesis in Choy sum (Brassica chinensis var. parachinensis) and selection for low light tolerance using abiotic stress indices.

BACKGROUND: Choy Sum (Brassica rapa ssp. chinensis var. parachinensis), grown in a controlled environment, is vulnerable to changes in indoor light quality and displays distinct photo-morphogenesis responses. The scarcity of Choy Sum germplasm for indoor cultivation necessitates the development of new cultivars. Hence, this study attempted to develop mutants through chemical mutagenesis and select low-light-tolerant mutants by using abiotic stress tolerance indices. RESULTS: A mutant population of Choy Sum created using 1.5% ethyl methane sulfonate (EMS) at 4 h was manually pollinated to obtain the M2 generation. 154 mutants with reduced hypocotyl length were initially isolated from 3600 M2 seedlings screened under low light (R: FR = 0.5). Five mutants that showed reduced plant height at mature stages were selected and screened directly for shade tolerance in the M3 generation. Principal component analysis based on phenotypic data distinguished the M3 mutants from the wild type. Abiotic stress tolerance indices such as relative stress index (RSI), stress tolerance index (STI), geometric mean productivity (GMP), yield stability index (YSI), and stress resistance index (SRI) showed significant (P < 0.05), and positive associations with leaf yield under shade. M3-12-2 was selected as a shade-tolerant mutant based on high values of STI, YSI, and SRI with low values for tolerance (TOL) and stress susceptibility index (SSI). CONCLUSIONS: The results demonstrate that mutation breeding can be used to create dominant mutants in Choy Sum. Furthermore, we show that screening for low light and selection based on abiotic tolerance indices allowed the identification of mutants with high resilience under shade. This method should apply to developing new cultivars in other crop plants that can be suitable for controlled environments with stable yield performance.

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

Background: 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. Methods: 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. 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 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. 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.

Improving desirable agronomic traits of M2 lines on fourteen Ethiopian Sesame (Sesamum indicum L.) genotypes using Ethyl Methane Sulphonate (EMS).

Sesame is an important oilseed crop cultivated in Ethiopia as a cash crop for small holder farmers. However, low yield is one of the main constraints of its cultivation. Boosting and sustaining production of sesame is thus timely to achieve the global oil demand. This study was, therefore, aimed at identifying mutant genotypes targeted to produce better agronomic traits of M2 lines on fourteen Ethiopian sesame genotypes through seed treatment with chemical mutagens. EMS was used as a chemical mutagen to treat the fourteen sesame genotypes. Quantitative and qualitative data were recorded and analyzed using analysis of variance with GenStat 16 software. Post-ANOVA mean comparisons were made using Duncan's Multiple Range Test (p≤ 0.01). Statistically significant phenotypic changes were observed in both quantitative and qualitative agronomic traits of the M2 lines. All mutant genotypes generated by EMS treatment showed a highly significant variation for the measured quantitative traits, except for the traits LBL and LTL. On the other hand, EMS-treated genotypes showed a significant change for the qualitative traits, except for PGT, BP, SSCS, LC, LH and LA traits. Mutated Baha Necho, Setit 3, and Zeri Tesfay showed the most promising changes in desirable agronomic traits. To the best of our knowledge, this study represents the first report on the treatment of sesame seeds with EMS to generate desirable agronomic traits in Ethiopian sesame genotypes. These findings would deliver an insight into the genetic characteristics and variability of important sesame agronomic traits. Besides, the findings set up a foundation for future genomic studies in sesame agronomic traits, which would serve as genetic resources for sesame improvement.

Detection of genetic divergence among putative ethyl methane sulfonate mutants of super basmati using microsatellite markers.

BACKGROUND: Seeds of super basmati were mutagenized with different ethyl methane sulphonate (EMS) doses for creating genetic variability. METHODS AND RESULTS: A total of 48 randomly selected putative EMS mutants of super basmati were analyzed to dissect the genetic diversity by using 25 SSR primers located on twelve chromosomes of rice. SSRs analysis revealed that wide-range of genetic diversity is present among mutants of super basmati. A sum of 91 alleles were identified, out of these, 82 alleles were polymorphic and the rest of nine alleles were monomorphic in nature. The range of allele number was 2-10 with mean of 3.64 alleles/locus. The value of polymorphic information content was range between 0.039 (RM5) and 0.878 (RM44) with mean of 0.439 for each locus. A number of polymorphic markers showed unique bands of various sizes ranges from 75 to 1000 bp, during genetic dissection of mutant population. Dendrogram divided whole mutant population into four major groups. Phylogenic analyses revealed that 40-96%genetic similarity is present among individuals of mutant population. CONCLUSION: It is concluded that EMS induced genetic variability and SSRs markers (RM44, RM154, RM1, RM252, RM334, RM487, RM110 and RM257) could be handy for the selection of rice mutants as parents for functional genomic and molecular breeding program.

A large-scale whole-exome sequencing mutant resource for functional genomics in wheat.

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.

Screening and Identification of Garlic Leaf Blight (Pleospora herbarum)-Resistant Mutants Induced by Ethyl Methane Sulphonate.

Garlic (Allium sativum L.) is a popular condiment used as both medicine and food. Garlic production in China is severely affected by continuous cropping and is especially affected by leaf blight disease. Garlic is sterile, so it is very important to develop specialized genotypes, such as those for disease resistance, nutritional quality, and plant architecture, through genetic modification and innovation. In this experiment, we applied the induction method using EMS to mutate garlic cloves of cultivar G024. From the mutations, 5000 M0 mutants were generated and planted in the field. Then, 199 M1 mutant lines were screened according to growth potential and resistance to leaf blight. From M2 to M3, 169 generational lines were selected that grew well and were resistant to leaf blight in the field. Thereafter, their resistance to leaf blight was further analyzed in the lab; 21 lines resistant to leaf blight that had good growth potential were identified, among which 3 mutants were significantly different, and these were further screened. Also, transcriptome analysis of two mutants infected with Pleospora herbarum, A150 and G024, was performed, and the results revealed 2026 and 4678 differentially expressed genes (DEGs), respectively. These DEGs were highly enriched in hormone signaling pathway, plant-pathogen interaction, and MAPK signaling pathway. Therefore, the results provide a theoretical and technical basis for the creation of garlic germplasm resistant to leaf blight.

M2 plants derived from different tillers of a chemically mutagenized rice M1 plant carry independent sets of mutations.

Generation of mutant populations with high genetic diversity is key for mutant screening and crop breeding. For this purpose, the single-seed descent method, in which one mutant line is established from a single mutagenized seed, is commonly used. This method ensures the independence of the mutant lines, but the size of the mutant population is limited because it is no greater than the number of fertile M1 plants. The rice mutant population size can be increased if a single mutagenized plant produces genetically independent siblings. Here, we used whole-genome resequencing to examine the inheritance of mutations from a single ethyl methanesulfonate (EMS)-mutagenized seed (M1 ) of Oryza sativa in its progeny (M2 ). We selected five tillers from each of three M1 plants. A single M2 seed was selected from each tiller, and the distributions of mutations induced by EMS were compared. Surprisingly, in most pairwise combinations of M2 siblings from the same parent, ≥85.2-97.9% of all mutations detected were not shared between the siblings. This high percentage suggests that the M2 siblings were derived from different cells of the M1 embryo and indicates that several genetically independent lines can be obtained from a single M1 plant. This approach should allow a large reduction in the number of M0 seeds needed to obtain a mutant population of a certain size in rice. Our study also suggests that multiple tillers of a rice plant originate from different cells of the embryo.

Molecular rearrangements in S6 during slow inactivation in Shaker-IR potassium channels.

Voltage-gated K+ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker KV channels.

Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment.

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.

Development and characterization of gamma ray and EMS induced mutants for powdery mildew resistance in blackgram.

PURPOSE: During post-rainy and rice fallow cropping seasons, popular blackgram varieties are severely affected by powdery mildew leading to severe yield loss. The lack of natural genetic variability for powdery mildew resistance in blackgram germplasm warrants mutation breeding. Hence, in this study, blackgram cultivar CO6 was mutagenized with gamma ray and ethyl methanesulphonate (EMS) to create variability for powdery mildew resistance. MATERIALS AND METHODS: Seeds of blackgram CO6 were irradiated with three doses of gamma ray (200 Gy, 300 Gy and 400 Gy) followed by two doses of ethyl methanesulphonate (EMS; 20 and 30 mM) to achieve six combination treatments. Selected resistant mutants of M2 generation were characterized for agronomic, histological, enzyme and biochemical traits along with powdery mildew resistant LBG 17 and susceptible CO6 checks. Molecular variability was studied using 72 simple sequence repeat (SSR) markers. RESULTS: In the M2 generation, 60 powdery mildew resistant mutants were identified and a total of 25 high yielding mutants were evaluated further to confirm powdery mildew resistance and yield. Nine resistant mutants (PM 13, PM 20, PM 21, PM 42, PM 53, PM 54, PM 56, PM 57 and PM 60) and the resistant check (LBG17) showed significantly higher values for leaf density, trichome density, SOD, CAT, POX, PPO, total phenols, phytic acid and silica content. SSR markers viz., CEDG154, CEDG290, CEDG139, CEDG259, CEDG191, CEDG024, CEDG 282, CEDG 166, CEDG 232 and CEDG 088 were found polymorphic between resistant mutants and the parent CO6. CONCLUSION: The study has demonstrated that sufficient variability was induced in the blackgram for powdery mildew resistance. The elevated levels of SOD, CAT, POX, PPO, total phenols, phytic acid, and silica content observed in selected mutants may be attributed to powdery mildew resistance. The superior mutants identified in this study may be used as donors for the development of powdery mildew resistant lines or released as a new variety.

Direct access to millions of mutations by whole genome sequencing of an oilseed rape mutant population.

Induced mutations are an essential source of genetic variation in plant breeding. Ethyl methanesulfonate (EMS) mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In the present study, a rapeseed M2 population was derived from M1 parent cultivar 'Express' treated with EMS. Whole genomes were sequenced from fourfold (4×) pools of 1988 M2 plants representing 497 M2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284 442 single nucleotide polymorphisms (SNPs) per M2 DNA pool were found resulting from EMS mutagenesis. 55% of the SNPs were C → T and G → A transitions, characteristic for EMS induced ('canonical') mutations, whereas the remaining SNPs were 'non-canonical' transitions (15%) or transversions (30%). Additionally, we detected 88 725 high confidence insertions and deletions per pool. On average, each M2 plant carried 39 120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Approximately 82% of such mutations were located either 5 kb upstream or downstream (56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool 'EMSBrassica' (http://www.emsbrassica.plantbreeding.uni-kiel.de), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

Map-based cloning and transcriptome analysis of the more-tiller and small-grain mutant in rice.

MAIN CONCLUSION: A G to T nucleotide substitution of OsTSG2 led to more tillers and smaller grains in rice by participating in phytohormone signal transduction and starch and sucrose metabolism. Rice is one of the most important food crops worldwide. Grain size and tiller number are the most important factors determining rice yield. The more-tiller and small-grain 2 (tsg2) mutant in rice, developed by ethyl methanesulfonate (EMS) mutagenesis, has smaller grains, more tillers, and a higher yield per plant relative to the wild-type (WT). Based on the genetic analysis, the tsg2 traits were conferred by a single recessive nuclear gene located on the long arm of chromosome 2. After fine-mapping the OsTSG2 locus, a G to T nucleotide substitution was identified, which resulted in an A to S mutation in a highly conserved domain of the growth-regulation factor protein. The single-strand conformation polymorphism (SSCP) marker was developed based on the SNP associated with the phenotypic segregation of traits. The functional complementation of OsTSG2 from the tsg2 mutant to the WT led to an increase in grain size and weight. The differentially expressed genes (DEGs) identified by RNA sequencing were involved in phytohormone signal transduction and starch and sucrose metabolism. Enzyme-linked immunosorbent assay (ELISA) analysis detected variation in the indole acetic acid (IAA) and jasmonic acid (JA) content in the tsg2 inflorescence, while the cellular organization, degree of chalkiness, gel consistency, amylose content, and alkaline spreading value were affected in the tsg2 grains. The findings elucidated the regulatory mechanisms of the tsg2 traits. This mutant could be used in marker-assisted breeding for high-yield and good-quality rice.

Metabolomic and transcriptomic analysis of roots of tobacco varieties resistant and susceptible to bacterial wilt.

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.

Increasing the Grain Yield and Grain Protein Content of Common Wheat (Triticum aestivum) by Introducing Missense Mutations in the Q Gene.

Grain yield (GY) and grain protein content (GPC) are important traits for wheat breeding and production; however, they are usually negatively correlated. The Q gene is the most important domestication gene in cultivated wheat because it influences many traits, including GY and GPC. Allelic variations in the Q gene may positively affect both GY and GPC. Accordingly, we characterized two new Q alleles (Qs1 and Qc1-N8) obtained through ethyl methanesulfonate-induced mutagenesis. Compared with the wild-type Q allele, Qs1 contains a missense mutation in the sequence encoding the first AP2 domain, whereas Qc1-N8 has two missense mutations: one in the sequence encoding the second AP2 domain and the other in the microRNA172-binding site. The Qs1 allele did not significantly affect GPC or other processing quality parameters, but it adversely affected GY by decreasing the thousand kernel weight and grain number per spike. In contrast, Qc1-N8 positively affected GPC and GY by increasing the thousand kernel weight and grain number per spike. Thus, we generated novel germplasm relevant for wheat breeding. A specific molecular marker was developed to facilitate the use of the Qc1-N8 allele in breeding. Furthermore, our findings provide useful new information for enhancing cereal crops via non-transgenic approaches.