甘蓝型油菜EMS诱变及多分枝和长角突变体分析

油菜是世界上最重要的油料作物之一,同时也是我国食用油和饲用蛋白质的主要来源。采用0.8%甲基磺酸乙酯(ethyl methane sulfonate,EMS)溶液处理甘蓝型油菜玻里马胞质雄性不育系(pol cms)的保持系2B种子,从田间M_2诱变群体中筛选获得1 495个变异株,包括叶色、叶型、早花、株高和株型、雄性不育、裂蕾、花瓣形状和颜色、多分枝和长角等变异性状,占M_2诱变群体的7.4%。对多分枝和长角突变材料的农艺性状进行观察和统计分析,结果表明多分枝突变材料平均一次有效分枝比野生型多1.03倍,单株产量增加41.48%;长角突变材料角果长度为野生型的1.69倍,但果粒数无差异,单株产量提高49.66%。研究结果为今后进一步发掘这些突变性状相关基因,并应用于油菜品种改良奠定了基础。     

甘蓝型油菜理化诱变和突变体库的构建

利用射线与甲基磺酸乙酯（EMS）溶液诱变技术复合处理甘蓝型油菜高油605的成熟种子。经田间M2筛选和M3验证, 共筛选到152个叶色、叶形、株高、分枝数、分枝角度、茎径、茎色、花色、花瓣数、花瓣大小、花蕊形态、雄性不育、死蕾及开花期等性状发生变异的突变体, 占M2诱变群体总数的12.67%。利用水培法进行M3和M4株系的子叶和根系变异性状筛选和验证, 发现子叶和根系变异频率分别为12.78%和7.07%。现已构建了包括叶片、株型、花器、子叶、根系及部分生理性状变异类型的突变体库, 可为今后油菜遗传改良和功能基因组学研究提供更多的种质资源。     

甘蓝型油菜花瓣缺失基因的图谱定位

在无花瓣品系APT02和正常有花瓣品种中双4号构建的的F2分离群体中,运用AFLP和SRAP两种标记技术对甘蓝型油菜花瓣缺失基因进行分子标记和图谱定位。在两亲本间筛选20对AFLP引物和170对SRAP 引物,进一步通过BSA法筛选,获得了与甘蓝型油菜花瓣缺失基因WHB连锁的1个SRAP标记e8m3_4（600bp）和1个AFLP标记E3247_15（150bp）,标记与基因WHB之间的遗传距离分别为5 cM和13.5cM;构建了一个甘蓝型油菜(Brassica napus.L )的分子标记遗传连锁图谱,该图谱共包含213个AFLP标记、56个SRAP标记和1个形态标记,分布于17个主要连锁群、两个三联体和4个连锁对中,遗传图距总长2487.1cM,标记间平均距离为10.09 cM。通过图谱定位,控制花瓣缺失性状的基因WHB被定位到第4连锁群(LG4)上。     

栽培萝卜植株地上部表型多样性分析

萝卜是我国重要的蔬菜作物,为了充分了解和有效利用国家蔬菜种质资源库栽培萝卜种质资源遗传多样性,以中期库经过初步鉴定的635份栽培萝卜种质为材料,对其植株地上部分39个表型性状进行了系统调查和分析。结果表明:(1)在19个质量性状中,下胚轴颜色的Shannon-Weiner多样性指数最高,为1.72,长角果着生姿势最低,为0.47,平均多样性指数为1.11。(2)20个数量性状的变异系数分布范围是13.33%~93.97%,平均变异系数为37.96%,其中小裂片间距的变异系数最大,花萼长度的变异系数最小;这20个数量性状的Shannon-Weiner多样性指数的变化范围为1.20~2.15,多样性指数平均值是1.94;其中花瓣宽和千粒重的多样性指数最高(2.15),其次分别是花瓣开展度(2.14)、子叶长(2.13)、花瓣长(2.13)、花萼长(2.12)和子叶凹槽深(2.11);基生叶叶片宽度的多样性指数最低(1.20)。(3)采用SAS 9.2对参试材料20个数量性状的主成分分析结果表明,7个主成分因子的累计贡献率达72.87%。(4)根据非加权组平均法(UPGMA),将供试材料分为5大类群,其中第Ⅰ类群包含1份种质,该种质为德国引进,角果长度最长为204.70 mm,角果喙长最长为122.90 mm,且长角果颜色为紫色;第Ⅱ类群包含4份种质,角果平均长度、喙长、平均单角果粒数、花瓣平均长度、花瓣平均宽度和花瓣开展度6个数量性状数值在5个类群中最小;第Ⅲ类群包含1份种质,其子叶宽度、子叶叶柄长度、花瓣长度、花瓣宽度、花萼长度、花瓣开展度在5个类群中均最大,且叶脉色和叶柄色均为紫色;第Ⅳ类群包含428份材料,其又可分为两个亚群,第Ⅳ-1亚群包含50份材料,主要是长荚萝卜和油萝卜,第Ⅳ-2亚群包含378份材料,花叶类型占98.94%;第Ⅴ类群包含201份材料,全部为板叶类型材料。研究结果为辨识和利用栽培萝卜种质资源提供了有效信息。     

γ射线诱变籼稻保持系T98B突变体的鉴定与农艺性状分析

辐射诱变是农作物种质创新的重要手段。本研究以杂交稻骨干亲本T98B为研究对象,以300Gy剂量的60Co-γ射线对其成熟种子进行处理,从辐射后代中鉴定出农艺性状变异材料,划分了变异类群,分析了各类型突变频率,为遗传改良奠定了材料基础。结果显示,经γ射线处理后的M1和M2种子的发芽率受到了显著抑制,分别比对照T98B降低了22.35%和14.67%;从M2群体中初步筛选出了207个变异单株;经M3~M5的遗传稳定性测试后最终获得了168份可稳定遗传的突变体,总突变频率达到了0.673%。按照变异部位将突变体划分成叶变异、茎/蘖变异和穗粒/花变异等3种类群,各类群分别含有34份、37份和97份突变体,占比20.24%、20.02%和57.74%;育性、叶色和株高等性状的突变频率较高,分别达到了0.212%,0.092%和0.088%。此外,本文简要描述了各类群的表型变异特点。结果表明,利用γ射线诱变能产生丰富的农艺性状突变体,本研究为水稻遗传改良提供新的种质资源奠定了基础。     

甘蓝型油菜无花瓣矮秆新种质APL03的选育

以正常四瓣双低甘蓝型油菜品系ZH004为母本、无花瓣种质APL01为父本杂交,对无花瓣及矮秆等性状进行多代定向选择,育成APL03新种质。该种质群体无花瓣株率迭100％,单株无花瓣度〉90％,植株高度130—140cm,单株有效角果数〉400个,种子含油率高达43％-47％,兼有抗倒伏、抗耐菌核病等特性,成为培育适于全程机械化作业栽培油菜新品种的基础材料。     

人工合成甘蓝型油菜特长角性状的遗传分析

以人工合成甘蓝型油菜特长角品系H218普通甘蓝型油菜中油821和MSP334为材料, 配制成两个杂交组合的各6个世代群体, 即P1、P2、F1、F2、B1、B2, 研究了全果长、果身长、果喙长3个角果性状的遗传特点。结果表明, 两个组合的全果长、果身长、果喙长在F2群体中近乎呈现连续正态分布, 显示为多基因控制的数量性状。两个组合的全果长广义遗传力为65.89%~70.77%、狭义遗传力为44.01%~46.78%, 基因数目均为5对; 果身长广义遗传力为60.14% ~ 63.38%、狭义遗传力为46.89%~47.38%, 基因数目均为6对; 果喙长广义遗传力为26.36% ~ 46.44%, 狭义遗传力为18.08%~37.87%, 基因数目均为2对。基因效应初步分析表明, 两个组合的3个性状基因效应均符合加性-显性二基因互作模型, 存在显著的上位性效应。两个组合的全果长、果身长、果喙长加性效应、显性效应都很重要, 全果长、果身长的加性效应大于显性效应, 果喙长显性效应大于加性效应。     

一种花色突变雄性不育油菜的发现

在甘蓝型油菜杂交种C022(其母本为由3对基因控制育性的隐性细胞核雄性不育系9012A)不育株开放受粉的后代中,发现一种稀有的黄白双色嵌合花瓣的甘蓝型油菜突变体991S。其具有3个形态特征:(1)4片花瓣每片中央均为条带状黄色色斑,而两侧为白色,为嵌合双色花瓣;(2)4片花萼也可发生中间条带状白化;(3)目前只在不同群体的雄性不育株中出现,与同源的黄色花不育株形态相似,植株纤细矮小,花器也较小,花瓣较为平整,雌蕊弯曲,雄蕊退化,花药干缩。通过对其材料来源及后代花色表型分析,初步认为黄白双色性状由可局部表达的隐性白化基因控制。     

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。     

甘蓝型油菜-白芥渐渗系材料农艺性状和种子品质分析

远缘杂交能够有效拓宽物种的遗传背景,产生丰富的遗传变异。前期研究中利用体细胞融合技术获得了甘蓝型油菜扬油6号(Y6)与白芥的属间杂种,将杂种与Y6连续回交和自交得到了一系列稳定遗传的渐渗系株系。本研究以62个渐渗系株系为材料,对这些株系进行14个农艺性状的考察,并通过相关性分析、主成分分析、回归分析和聚类分析等方法,探究甘蓝型油菜单株产量与农艺性状之间的关系及影响单株产量的主要因素。结果显示,甘蓝型油菜-白芥渐渗系株系的农艺性状存在较大差异;单株产量与全株角果数、有效分枝数和每角粒数等性状呈极显著正相关,而与分枝点高度呈极显著负相关。主成分分析的结果表明,4个主成分可以概括14个农艺性状的大部分信息,这些主成分主要与角果数目、株高、角果长度和千粒重等性状指标相关。回归分析结果表明,千粒重、每角粒数、二次有效分枝数、株高和全株角果数对单株产量具有重要影响。综合各项指标,筛选出一些具有优异农艺性状的材料,如L8等株系角果数目明显增多;L3、L7等株系分枝数目明显增多;L42、L49等株系千粒重明显增加;L3、L8等株系单株产量明显提高。利用近红外光谱仪分析渐渗系材料种子品质性状,发现硫代葡萄糖甘、芥酸、亚麻酸等品质性状存在较大变异,可用于选育具有优良品质性状的材料。本研究表明远缘杂交对于甘蓝型油菜种质资源创新、提高油菜产量提供了一条有效途径,研究结果为利用这些种质资源进行育种实践提供了理论依据。     

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.     

裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

EMS-induced polygenic mutation rates for nine quantitative characters in Drosophila melanogaster.

Polygenic mutations were induced by treating Drosophila melanogaster adult males with 2.5 mM EMS. The treated second chromosomes, along with untreated controls, were then made homozygous, and five life history, two behavioral, and two morphological traits were measured. EMS mutagenesis led to reduced performance for life history traits. Changes in means and increments in genetic variance were relatively much higher for life history than for morphological traits, implying large differences in mutational target size. Maximum likelihood was used to estimate mutation rates and parameters of distributions of mutation effects, but parameters were strongly confounded with one another. Several traits showed evidence of leptokurtic distributions of effects and mean effects smaller than a few percent of trait means. Distributions of effects for all traits were strongly asymmetrical, and most mutations were deleterious. Correlations between life history mutation effects were positive. Mutation parameters for one generation of spontaneous mutation were predicted by scaling parameter estimates from the EMS experiment, extrapolated to the whole genome. Predicted mutational coefficients of variation were in good agreement with published estimates. Predicted changes in means were up to 0.14% or 0.6% for life history traits, depending on the model of scaling assumed.     

Whole-genome effects of ethyl methanesulfonate-induced mutation on nine quantitative traits in outbred Drosophila melanogaster

We induced mutations in Drosophila melanogaster males by treating them with 21.2 mm ethyl methanesulfonate (EMS). Nine quantitative traits (developmental time, viability, fecundity, longevity, metabolic rate, motility, body weight, and abdominal and sternopleural bristle numbers) were measured in outbred heterozygous F3 (viability) or F2 (all other traits) offspring from the treated males. The mean values of the first four traits, which are all directly related to the life history, were substantially affected by EMS mutagenesis: the developmental time increased while viability, fecundity, and longevity declined. In contrast, the mean values of the other five traits were not significantly affected. Rates of recessive X-linked lethals and of recessive mutations at several loci affecting eye color imply that our EMS treatment was equivalent to approximately 100 generations of spontaneous mutation. If so, our data imply that one generation of spontaneous mutation increases the developmental time by 0.09% at 20 degrees and by 0.04% at 25 degrees, and reduces viability under harsh conditions, fecundity, and longevity by 1.35, 0.21, and 0.08%, respectively. Comparison of flies with none, one, and two grandfathers (or greatgrandfathers, in the case of viability) treated with EMS did not reveal any significant epistasis among the induced mutations.     

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.     

Ethyl methanesulfonate mutant library construction in Gossypium hirsutum L. for allotetraploid functional genomics and germplasm innovation

As the gene pool is exposed to both strain on land resources and a lack of diversity in elite allotetraploid cotton, the acquisition and identification of novel alleles has taken on epic importance in facilitating cotton genetic improvement and functional genomics research. Ethyl methanesulfonate (EMS) is an excellent mutagen that induces genome‐wide efficient mutations to activate the mutagenic potential of plants with many advantages. The present study established, determined and verified the experimental procedure suitable for EMS‐based mutant library construction as the general reference guide in allotetraploid upland cotton. This optimized method and procedure are efficient, and abundant EMS mutant libraries (approximately 12 000) in allotetraploid cotton were successfully obtained. More than 20 mutant phenotypes were observed and screened, including phenotypes of the leaf, flower, fruit, fiber and plant architecture. Through the plants mutant library, high‐throughput and high‐resolution melting technology‐based variation evaluation detected the EMS‐induced site mutation. Additionally, based on overall genome‐wide mutation analyses by re‐sequencing and mutant library assessment, the examination results demonstrated the ideal quality of the cotton EMS‐treated mutant library constructed in this study with appropriate high mutation density and saturated genome. What is more, the collection is composed of a broad repertoire of mutants, which is the valuable resource for basic genetic research and functional genomics underlying complex allotetraploid traits, as well as cotton breeding.     

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。     

Molecular dosimetry studies of forward mutation induced at the yg2 locus in maize by ethyl methanesulfonate

The yg2 assay in Zea mays detects forward mutation in somatic cells within leaf primordia of embryos and it was used in an analysis of the molecular dosimetry of ethyl methanesulfonate (EMS). Parallel genetic and molecular dosimetry experiments were conducted in which the frequency of forward mutation and the level of covalently bound ethyl DNA adducts were determined. Prepared kernels were treated for 8 h at 20 degrees C with 1-10 mM EMS. EMS induced a direct concentration-dependent increase in mutation induction proportional to the exposure concentration (slope = 0.93). The kinetics of mutation induction demonstrated in the intact maize system were consistent with the kinetics observed earlier in in vitro model systems using cultured mammalian cells, and contrasted with the exponential increase in mutation induction characteristic of microbial species. Parallel molecular dosimetry experiments were conducted using [3H]EMS. DNA was extracted and purified from embryonic tissues containing the leaf primordia, the target tissue of the yg2 assay. A linear increase in the molecular dose was observed as a function of EMS concentration. Using concentration as a common parameter between the parallel genetic and dosimetry studies, mutation induction appeared to increase nearly in a direct proportion to the molecular dose. However, studies in other genetic systems indicate that the levels of specific DNA adducts, such as O6-ethylguanine (O6-EtGua) show a better correlation with mutation induction kinetics than molecular dose. Neither molecular dose, nor O6-EtGua levels account for differences in the absolute frequencies of mutation induction observed in different genetic systems. Therefore, reliable assessment of health risks posed to humans by chemical mutagens appears to require consideration of other factors in addition to DNA dose or adduct formation, including differences in repair capabilities and in the size of the genetic targets in humans relative to the model genetic systems under study.