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.     

诱变选育具有磺胺胍抗性的L-色氨酸产生菌的研究

采用紫外线、甲基磺酸乙酯(EMS)及半导体激光诱变的方法,处理产色氨酸谷氨酸棒状杆菌GA22(Phe-+Tyr-+5MTr)选育磺胺胍抗性菌株以提高L-色氨酸的产量。用紫外照射20S、EMS处理40 min及半导体激光辐照16 min,筛选得到抗性突变株GA507(Phe-+Tyr-+5MTr+SGr),产色氨酸的量达到5.35 g/L,较出发菌株提高50.3%,并具有良好的遗传稳定性。     

小兴安岭阔叶红松林地表甲虫Metacommunity格局

Metacommunity理论框架为理解生物群落的时空格局及其构建机制提供了一个强有力的方法。然而,少有研究揭示土壤动物metacommunity的格局及其构建机制,小尺度空间的研究更是少见。于2015年分别在凉水和丰林小兴安岭典型阔叶红松林长期动态监测样地内,通过空间直观定位调查监测的方法获得地表甲虫群落,揭示小尺度空间(300 m)地表甲虫metacommunity的格局,并进一步分析地形、土壤因子与这种metacommunity格局的相关性。结果表明:镶嵌型(nestedness)和随机型(random)是小尺度空间地表甲虫metacommunity的常见格局。地表甲虫的metacommunity格局具有类群依赖性,步行虫科和葬甲科多形成镶嵌型格局,而隐翅虫科则仅形成随机型格局。地表甲虫metacommunity格局具有季节波动性,在相对温暖的季节易形成显著的镶嵌型格局,而在相对寒冷的季节多为随机型格局。凉水和丰林地表甲虫的metacommunity格局没有明显差异。地形和土壤因子对地表甲虫metacommunity格局有重要的影响,该影响过程相对复杂,依类群和时间而异。本实验表明镶嵌型和随机型是小尺度空间小兴安岭典型阔叶红松林地表甲虫的常见格局,地形、土壤因子对这些格局的形成有重要的影响。     

Fisher's geometric model predicts the effects of random mutations when tested in the wild

Fisher's geometric model of adaptation (FGM) has been the conceptual foundation for studies investigating the genetic basis of adaptation since the onset of the neo Darwinian synthesis. FGM describes adaptation as the movement of a genotype toward a fitness optimum due to beneficial mutations. To date, one prediction of FGM, the probability of improvement is related to the distance from the optimum, has only been tested in microorganisms under laboratory conditions. There is reason to believe that results might differ under natural conditions where more mutations likely affect fitness, and where environmental variance may obscure the expected pattern. We chemically induced mutations into a set of 19 Arabidopsis thaliana accessions from across the native range of A. thaliana and planted them alongside the premutated founder lines in two habitats in the mid‐Atlantic region of the United States under field conditions. We show that FGM is able to predict the outcome of a set of random induced mutations on fitness in a set of A. thaliana accessions grown in the wild: mutations are more likely to be beneficial in relatively less fit genotypes. This finding suggests that FGM is an accurate approximation of the process of adaptation under more realistic ecological conditions.     

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.     

A comparative study of different experimental protocols for mutagenesis assys with the 9-azaguanine resistance system in cultured Chinese hamster cells

Both spontaneous and EMS-induced mutant frequencies were determined in cultured cells from V79 Chinese hamsters using three different experimental protocols. After optimal expression time was attained, mutation frequencies only remained constant when a protocol was used in which the cell density was maintained below critical values both before and during mutant selection. The identification of such a palteau allows, besides more reliable and reproducible estimated of mutation frequency, reduction in the size of experiments for quantitative evaluation of mutagenicity. Determination of mutation frequencies over a wide range of expression times becomes in fact unnecessary.     

The resistances of Micrococcus radiodurans to killing and mutation by agents which damage DNA

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N′-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r.M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism.Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.     

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.