EMS诱变西瓜突变体库的构建及表型分析

采用1.0%诱变剂甲基磺酸乙酯(EMS)处理西瓜品系W1-17种子9h,然后对M1和M2代群体单株在叶、花、茎、育性、分支习性等方面进行表型变异观察,同时选取M2代典型变异株系,利用23对西瓜SSR引物进行分析鉴定,构建西瓜突变体库。结果表明:(1)EMS诱变使M1代幼苗形态呈现出叶畸形、叶褶皱、部分黄化、花畸形、雄花不散粉、卷须畸形、矮小、生长缓慢、不育等特异性状,获得由1 252个单株组成的西瓜突变体M1群体,群体总变异频率为18.33%。(2)M2代共筛选到205个突变植株,40种表型变异,表现在子叶性状(黄化、扭曲不对称、折叠等)、叶和茎性状(叶黄化、变小、裂刻变深,茎变细,节间变短,分支少等)、花性状(花变大,花色变浅,两性花,花瓣皱缩、部分退化、数目突变,柱头畸形,雄蕊不成熟等)和其他性状(生长缓慢、不育等)等方面,总的表型突变率达到了19.59%。(3)针对M2代10个典型变异植株,通过SSR引物分析发现有9份材料在DNA水平上有变异。本研究初步构建了含有120个M1代家系及1 051株M2代植株、40种表型变异的西瓜突变体库。     

Production of doubled haploids through anther culture of M<Subscript>1</Subscript> rice plants derived from mutagenized fertilized egg cells

To produce stable mutants from Mankeumbyeo, a japonica rice (Oryza sativa L.) variety, we estimated the mutation efficiency of ethyl methane sulfonate (EMS) and N-methyl-N-nitrosourea (MNU) on fertilized egg cells using doubled haploids (DHs) derived from anther culture of M₁ plants. M₁ seed production and germination were higher in 1 mM MNU than in 94.2 mM EMS. A total of 68 DHs (35.4%) were regenerated by anther culture of M₁ plants. Twenty-one DHs (30.9%) were stable mutants, 14 DHs (20.6%) were unstable mutants, and the remainder (48.5%) were normal. The frequencies of stable mutants following EMS and MNU treatments were 20.7% (three semidwarfs, one early maturation and one glabrous line) and 38.5% (three semidwarfs, two early maturation, four glabrous and one long grain line), respectively. In a field trial of seven stable mutants for yield potential, five mutants did not show a significant difference in yield as compared with the original variety. Among these five, three glabrous mutants (MK-MAC 1, MK-MAC 4 and MK-MAC 26) with a smooth leaf and hull may be considered to be improved mutant lines because of the health benefits (reduced skin damage and generation of less dust compared to the original variety) to farmers handling the plant materials. MK-MAC 26, a glabrous mutant, had also less shattering resistance than that of the original variety. These stable mutants could be used as new breeding materials.Communicated by P.P. Kumar     

An Ethylmethane Sulfonate Mutant Resource in Pre-Green Revolution Hexaploid Wheat

Mutagenesis is a powerful tool used for studying gene function as well as for crop improvement. It is regaining popularity because of the development of effective and cost efficient methods for high-throughput mutation detection. Selection for semi-dwarf phenotype during green revolution has reduced genetic diversity including that for agronomically desirable traits. Most of the available mutant populations in wheat (Triticum aestivum L.) were developed in post-green revolution cultivars. Besides the identification and isolation of agronomically important alleles in the mutant population of pre-green revolution cultivar, this population can be a vital resource for expanding the genetic diversity for wheat breeding. Here we report an Ethylmethane Sulfonate (EMS) generated mutant population consisting of 4,180 unique mutant plants in a pre-green revolution spring wheat cultivar ‘Indian’. Released in early 1900s, ‘Indian’ is devoid of any known height-reducing mutations. Unique mutations were captured by proceeding with single M₂ seed from each of the 4,180 M₁ plants. Mutants for various phenotypic traits were identified by detailed phenotyping for altered morphological and agronomic traits on M₂ plants in the greenhouse and M₃ plants in the field. Of the 86 identified mutants, 75 (87%) were phenotypically stable at the M₄ generation. Among the observed phenotypes, variation in plant height was the most frequent followed by the leaf morphology. Several mutant phenotypes including looped peduncle, crooked plant morphology, ‘gritty’ coleoptiles, looped lower internodes, and burnt leaf tips are not reported in other plant species. Considering the extent and diversity of the observed mutant phenotypes, this population appears to be a useful resource for the forward and reverse genetic studies. This resource is available to the scientific community.     

Development and utilization of a new chemically‐induced soybean library with a high mutation density

Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate (EMS)‐induced soybean (Glycine max) population, consisting of 21,600 independent M₂ lines, was developed. Over 1,000 M_{4 (5)} families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll (Chl) content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to MinnGold, where a different single nucleotide polymorphism variation in the Mg‐chelatase subunit gene (ChlI1a) results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker‐assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996     

EMS对三个玉米自交系的诱变效应分析

EMS诱变玉米花粉是玉米化学诱变的主要技术。该研究以生产上3个常用的玉米自交系K305、21-ES、R08为材料,对其花粉用不同浓度的EMS诱变处理,探讨其EMS诱变的最佳浓度范围,明确其诱变效应。结果表明：3个自交系经过不同浓度的EMS诱变后,其结实率随着浓度的增大表现出减小的趋势,从其半致死剂量来看,EMS诱变花粉的适宜浓度范围自交系K305和R08均为0.67~1.0 mL?L-1,21-ES在1.67 mL?L-1附近。 M1代不同性状其变异幅度和变异系数与对照相比主要表现出增大的趋势,其不同性状的生物学效应在材料间表现不一致,表明性状在不同材料间对EMS的敏感性不一样,生育期表现为21-ES>K305>R08;主要株型性状表现为R08>21-ES>K305;主要雄穗性状K305和21-ES比R08敏感;主要果穗性状表现为21-ES>K305>R08。 M2代整体表现为变异谱扩大,其株高、穗位高和叶面积以及主要果穗性状的变异表现复杂,主要雄穗性状中除K305的M2株系雄穗分枝数呈双向变异外,其余M2株系整体偏向于雄穗变短,雄穗分枝数减小。该研究结果为后续研究和应用打下了基础。     

Chimeras for mutations induced in dormant tomato seeds

Genetic chimeras of the VFNT cherry tomato line (Lycopersicon esculentum) were produced by mutagenizing seeds with ethyl methane sulfonate (EMS). The chimeras thereby produced were evaluated by progeny-testing the fruits of the genetically mosaic tissue. A total of 2011 M₁ plants was grown from treated seeds and evaluated by screening their 95175 (M₂) progeny for mutations affecting seedling phenotype. Three vigorous and fertile M₁ plants bearing mutant progeny with definitive phenotypes were selected for systematic harvesting and analysis. The specific location of each fruit was noted at harvest time, enabling the mutated sporogenous tissue of the mosaic M₁ plants to be traced. Sectoring appeared in both branch and floral tissues. In several cases, mutant progenies were restricted to individual branches or parts thereof. True-breeding recessive mutants whose monogenic mode of inheritance was later established occasionally segregated within M₁ fruit progenies at frequencies that indicate a non-homogeneous floral meristem origin. The data emphasize the necessity of making a well-distributed harvest of mosaic plants in order to detect as many variants as possible, as mosaic sectors may or may not recur late in ontogeny and may not contribute to sporogenous tissue early in development.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

Inhibition of cell growth by cellular differentiation into adipocyte-like cells in dexamethasone sensitive cancer cell lines

The stress responses in human body lead to secretion of cortisol hormone. The present study investigated the cellular responses on cell growth and cellular differentiation into adipocytes by exposure of synthetic stress hormone, dexamethasone (DEX) in various human cancer and normal cells. After prolonged exposure of cells with 1?μg/ml DEX for 2 weeks, population doubling time (PDT) was significantly (P?P?P?β (GRβ) and peroxisome proliferator-activated receptor γ (PPARγ) were significantly (P?P?相似文献   

The screening of mutants and construction of mutant library for <Emphasis Type="Italic">Oryza sativa</Emphasis> cv. Nipponbare via ethyl methane sulphonate inducing

Following the sequencing of rice genome, the functional analysis of unidentified genes is gaining wide importance. Mutant isolation is one of the effective ways to isolate and clone the target genes and analyze their functions. To find the various mutants in the same genetic background, seeds of Oryza sativa cv. Nipponbare were treated with ethyl methane sulphonate (EMS). A total of 1056 mutants were screened for five categories in M₂ generation with the seedling frequency of 26.29‰ at three-leaf stage, but only 264 mutants were verified in M₃ generation with a frequency of 6.57‰. Among the mutants verified in M₃ generation, the frequency of leaf mutation was the highest (2.22‰), followed by seedling height (1.74‰) and the abiotic stress tolerance mutant (1.47‰). Nineteen characteristic mutations, including a big group of abiotic stress tolerant mutants such as herbicide resistant, salt tolerant and drought tolerant were identified at this stage. By observation of rice growth characteristics at different developmental stages, another 220 mutants have been isolated and verified in the M₃ generation with the mutant frequency of 53.9‰ covering about 28 mutant traits. Among those identified, the highest frequencies were obtained for appearance of brown rice mutant with 18.37‰, followed by panicle mutant with 13.47‰, and grain mutants with 9.06‰. All the mutants screened above were suitable for gene function analysis and for utilization in agronomy.     

Genotypes and their response to in vitro production of haploids in F1 lines of Nicotiana tabacum

Summary The present article reports on pollen embryogenesis resulting in haploid plants in 14 different F₁ lines of Nicotiana tabacum. The response for pollen embryogenesis was high in cultures where the F₁ lines were derived from mutants. Present observations are in agreement with previous reports on Secale cereale where one of the parents is from a mutant M_II. These observations indicate that plant genotype is the main factor for differences in the induction of pollen embryogenesis.     

Induction of streptomycin-resistant plantlets inCapsicum annuum L. through mutagenesisin vitro

Streptomycin resistant shoots were regenerated from mutagenised (ethylemethane sulphonate treated) cotyledon explants ofCapsicum annuum Cv G₄. Streptomycin resistant shoots developed from green (unbleached) sectors of the cotyledons. Reciprocal crosses betweeen streptomycin resistant and sensitive plants have shown a non-mendelian transmission. Such mutant plants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids ofC. annuum.ABBREVIATIONS BAP 6-Benzylaminopurine - IAA Indoleacetic acid - EMS Ethyl methane sulphonate - NMU Nitrosomethyl urea     

M1 chlorophyll chimeras induced by different mutagens and their M2 chlorophyll mutation yields in rice

Studies with gamma rays, fast neutrons, and EMS treatments in rice showed higher frequency of M₁ chlorophyll chimeras in the EMS treatments, a correlation between the frequencies of M₁ chlorophyll chimeras and M₂ chlorophyll mutants, and a higher M₂ mutation frequency in the progeny of chimerical M₁ plants compared to those of normal-looking plants.     

Effects of increasing inorganic carbon supply to roots on net nitrate uptake and assimilation in tomato seedlings

We investigated the influence of an increased inorganic carbon supply in the root medium on NO^?₃ uptake and assimilation in seedlings of Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were pre-grown for 4 to 7 days with 0 or 100 mM NaCl in hydroponic culture using 0.2 mM NO^?₃ (group A) or 0.2 mM NH⁺₄ (group B) as nitrogen source. The nutrient solution for group A plants was aerated with air or with air containing 4 800 μumol mol^?1 CO₂. Nitrate uptake rate and root and leaf malate contents in these plants were determined. The plants of group B were subdivided into two sets. Plants of one set were transferred either to N-free solution containing 0 or 5 mM NaHCO₃, or to a medium containing 2 mM NO^?₃ and 5 mM NaHCO₃. Both sets of group B plants were grown for 12 h in darkness prior to 2 h of illumination, and were assayed for malate content and NO^?₃ uptake rate (only for plants grown in N-free solution). The second set of group B plants was labeled with ¹⁴C by a 1-h pulse of H¹⁴CO^?₃ which was added to a 5 mM NaHCO₃ solution containing 0 or 100 mM NaCl and 0 or 2 mM NO^?₃, and ¹⁴C-assimilates were extracted and fractionated. The roots of group B plants growing in carbonated medium accumulated twice as much malate as did control plants. This malate was accumulated only when NO^?₃ was absent from the root medium. Both a high level of root malate and aeration with CO₂-enriched air stimulated NO^?₃ uptake. Analysis of ¹⁴C-assimilates indicated that with no NO^?₃ in the medium, the ¹⁴C was present mainly in organic acids, whereas with NO^?₃, a large proportion of ¹⁴C was incorporated into amino acids. Transport of root-incorporated ¹⁴C to the shoot was enhanced by NO^?₃, while the amino acid fraction was the major ¹⁴C-assimilates in the shoot. It is concluded that inorganic carbon fixed through phosphoenolpyruvate carboxylase (EC 4.1.1.31) in roots of tomato plants may have two fates: (a) as a carbon skeleton for amino acid synthesis; and (b) to accumulate, mainly as malate, in the roots, in the absence of a demand for the carbon skeleton. Inorganic carbon fixation in the root provides carbon skeletons for the assimilation of the NH⁺₄ resulting from NO₃ reduction, and the subsequent removal of amino acids through the xylem. This ‘removal’ of NO^?₃ from the cytoplasm of the root cells may in turn increase NO^?₃ uptake.     

Diurnal Variation in Wingate-Test Performance and Associated Electromyographic Parameters

The present study was designed to evaluate time-of-day effects on electromyographic (EMG) activity changes during a short-term intense cycling exercise. In a randomized order, 22 male subjects were asked to perform a 30-s Wingate test against a constant braking load of 0.087?kg·kg^?1 body mass during two experimental sessions, which were set up either at 07:00 or 17:00?h. During the test, peak power (P_peak), mean power (P_mean), fatigue index (FI; % of decrease in power output throughout the 30 s), and evolution of power output (5-s span) throughout the exercise were analyzed. Surface EMG activity was recorded in both the vastus lateralis and vastus medialis muscles throughout the test and analyzed over a 5-s span. The root mean square (RMS) and mean power frequency (MPF) of EMG were calculated. Neuromuscular efficiency (NME) was estimated from the ratio of power to RMS. Resting core temperature, P_peak, P_mean, and FI were significantly higher (p?<?.05) in the evening than morning test (e.g., P_peak: 11.6?±?0.8 vs. 11.9?±?1 W·kg^?1). The results showed that power output decreased following two phases. During the first phase (first 20s), power output decreased rapidly and values were higher (p?<?.05) in the evening than in the morning. During the second phase (last 10s), power decreased slightly and appeared independent of the time of day of testing. This power output decrease was paralleled by evolution of the MPF and NME. During the first phase, NME and MPF were higher (p <?.05) in the evening. During the second phase, NME and MPF were independent of time of day. In addition, no significant differences were noticed between 7:00 and 17:00?h for EMG RMS during the whole 30 s. Taken together, these results suggest that peripheral mechanisms (i.e., muscle power and fatigue) are more likely the cause of the diurnal variation of the Wingate-test performance rather than central mechanisms. (Author correspondence: n_souissi@yahoo.fr)     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G₁ to early S, followed by late G₂ to M, which were more sensitive than the other phases. In both M₁ and M₂ generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G₂ and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G₁, xantha at early S, short-culm mutant at mid G₂, heading-date mutant at M to early G₁. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination.     

POLYGENIC MUTATION IN DROSOPHILA MELANOGASTER: ESTIMATES FROM DIVERGENCE AMONG INBRED STRAINS

A highly inbred line of Drosophila melanogaster was subdivided into 25 replicate sublines, which were independently maintained for 100 generations with 10 pairs of unselected flies per generation. The polygenic mutation rate (V_M) for two quantitative traits, abdominal and sternopleural bristle number, was estimated from divergence among sublines at 10 generation intervals from generations 30-100, and from response of each line to divergent selection after more than 65 generations of mutation accumulation. Estimates of V_M averaged over males and females both from divergence among lines and from response to selection within lines were 3.3 × 10^-3 V_E for abdominal bristles and 1.5 × 10^-3 V_E for sternopleural bristles, where V_E is the environmental variance. The actual rate of production of mutations affecting these traits may be considerably higher if the traits are under stabilizing selection, and if mutations affecting bristle number have deleterious effects on fitness. There was a substantial component of variance for sex × mutant effect interaction and the sublines evolved highly significant mutational variation in sex dimorphism of abdominal bristle number. Pleiotropic effects on sex dimorphism may be a general property of mutations at loci determining bristle number.     

Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants

To increase the specific free amino acid content in the japonica rice (Oryza sativa L.) cultivar Donganbyeo, mutant cell lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from embryo-cultured callus irradiated with 50 Gy gamma-rays. Four 5MT-resistant homozygous M₄ lines, MRI-40, MRI-116, MRII-8, and MRII-12, were obtained. The mean content of nine free essential amino acids were 70.1, 72.5, 31.7, and 35.4% greater than the original variety in these four mutant lines, respectively. For AFLP analysis, 8 EcoRI (+2) and 8 MseI (+3) primers used in 45 primer combinations generated a total of 3,684 bands with a mean of 82 bands, of which 361 (9.8%) were clearly polymorphic with the control cultivar, the four 5MT-resistant mutants, and five sensitive lines. The lines were grouped into three clusters through cluster analysis using unweighted pair grouping method of averages. The 36 polymorphic PCR products present only in the four homozygous 5MT-resistant lines were cloned and sequenced, and 10 of these sequenced products were converted into sequence tagged site (STS) markers. These STS primer sets were designated OSMR1–OSMR10. Six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4, OSMR5, and OSMR6) generated a single monomorphic PCR product identical in size to the original AFLP fragments. The broad applicability of these STS markers for the screening of 5MT resistance was evaluated with seven putative 5MT-resistant M₂ plants (PM-1 to PM-7). Four STS markers (OSMR1, OSMR2, OSMR4, and OSMR5) out of six STS primer sets were revealed as polymorphic products between the control cultivar and the seven M₂ plants. These markers can be utilized for the fine selection of 5MT resistance in rice, and this PCR-screening technique is less time-consuming, less labor-intensive, and more accurate and reliable than selection based solely on phenotypic evaluation involving soaking in 5MT solutions.Abbreviations AFLP Amplified fragment length polymorphism - MAS Marker-assisted selection - STS Sequence tagged site - UPGMA Unweighted pair grouping method of averages - 5MT 5-Methyltryptophan