Cytological and molecular characterization of a new cytoplasmic male sterility in rapeseed

It is very important for rapeseed hybrid production to develop and utilize a novel cytoplasmic male sterility (CMS) system concerning the possible risk because of a narrow cytoplasm background. Here the anatomy of anther development in the CMS system, named NCa , was observed using light microscope and transmission electron microscope, and the restriction fragment length polymorphism (RFLP) analyses of mitochondrial DNA of NCa sterile line were also performed in comparison with the other rapeseed sterile lines, such as pol , nap , ogura , and tour . The anther abortion of this CMS line occurred at the later uninucleate microspore stage, and the anatomic aborting characteristics were obviously different from all the other rapeseed CMS lines reported before. The RFLP analyses revealed that five probe/enzyme combinations could distinguish the five CMS lines. The results of anatomic observations and mitochondrial DNA polymorphism indicated that the NCa CMS system is a novel one which differs from the pol , nap , ogura , and tour systems.     

甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究

明确甘蓝型油菜温敏核雄性不育系160S花器形态变化、花药败育的时期和细胞学特征,初步探究败育的原因,为深入研究不育系160S的内在分子调控机制提供理论基础,也对其在油菜两系杂交育种中的实际应用具有指导意义。本研究在15℃和28℃条件下培养试验材料160S,利用体式显微镜分别观察花发育形态特征;采用醋酸洋红染色方法观察各时期小孢子发育形态;通过石蜡切片和苏木精-伊红染色对可育植株(MaleFertile/160S-MF)和不育植株(MaleSterile/160S-MS)花药细胞学特征进行显微观察;TUNEL染色法检测花药发育各时期绒毡层细胞凋亡情况。160S-MF在15℃表现为可育,雄蕊正常发育,成熟的花药呈黄色,形态饱满,正常开裂,表面一层有活性的花粉附着在上面;28℃条件下, 160S-MS花朵的雌蕊、萼片与160S-MF花朵无差异,但花瓣变小,花丝变短,雄蕊明显退化,花药干瘪呈黄褐色,无花粉粒附着在花药上,表现出雄性完全不育。160S-MF的小孢子能正常发育为成熟有活力的花粉。而160S-MS由于雄蕊完全败育,未观察到小孢子和花粉粒。160S-MS花药在造孢时期和花粉母细胞时期与160S-MF无明显差异,但在减数分裂期,160S-MS花药绒毡层形态和结构出现异常,绒毡层细胞排列不整齐,细胞空泡化,伴随提前解体。同时花粉母细胞发育受阻,无四分体结构形成,最终在减数分裂期完成前形成空的花粉囊。TUNEL检测发现, 160S-MS花药绒毡层细胞在减数分裂期开始凋亡。本研究结果表明, 160S属花粉母细胞败育型不育系,败育时期发生在减数分裂期,绒毡层异常降解,绒毡层未向腺质型转化,不能提供花粉母细胞发育所需要的营养物质,致使花粉母细胞发育受阻无法形成四分体结构,从而导致小孢子无法形成,花药形成空的花粉囊,产生雄性不育。     

甘蓝型油菜白花突变体的遗传研究和白花胞质雄性不育系的选育

用白花突变体和白花不育突变体分别选育白花品系G95150和白花胞质不育系7600 A。遗传研究表明,白花对黄花由一对不完全显性基因控制。7600A在农艺性状上与其保持系G95150完全一致：叶色浓绿,植株较矮（163.5 cm）、生育期较长（243天）、抗寒能力较差（冻害率100%,冻害指数33.8）。7600A花色纯白、花瓣皱缩、花药小、低温存在微量花粉。G95150和7600 A的芥酸含量为0.8854 %,硫甙含量70.5461μmol/g。     

不结球白菜Ogura雄性不育花器官形态及败育细胞学的研究

对不结球白菜细胞质雄性不育系S2108A和Y3611A及其相应的保持系S2108B和Y3611B进行了花器官形态比较和花药发育细胞学观察,结果表明:两套不育系和保持系之间在花器官的多个性状上差异极显著,而两个不育系S2108A和Y3611A之间除花蕾大小、雌蕊长和花瓣长有差异外,其他均不显著,说明从不同不育源转育来的两个不育系在花器官外形上差异不大;两个不育系花药败育表现均起始于四分体时期,且由于绒毡层的液泡化和径向膨大,挤压四分体小孢子导致败育,表明败育与绒毡层的不正常发育有关.此外,与Y3611A不同,在不育系S2108A中还存在另一种败育形式,绒毡层细胞壁发生融合,成为紧贴药室壁的类似于变形型绒毡层的周原质团,部分细胞质流人药室腔,包裹粘连四分体小孢子,成为染色很深的不连续团块状物,花药败育;两不育系花药发育存在花粉囊数目的变异,且花粉囊发育时期不同步,个别花粉囊较正常花粉囊发育推迟,在不育系Y3611A中还有巨型花粉囊的现象.     

Tournefortii male sterility system in Brassica napus. Identification, expression and genetic characterization of male fertility restorers

A germplasm collection of 152 diverse rapeseed accessions from Canada, China, France, India, Poland and South Korea was assayed for identifying new fertility restorers and sterility maintainers for a Tournefortii (tour) cytoplasmic male sterility (CMS) system in rape‐seed. Only 16 (10.5%) genotypes showed complete fertility restoration following hybridization with tour CMS line NE 409A. Notable among these were GSL 8851, GSL 8953, Mokpo # 9, Mali, Buk‐wuk‐13, Kuju‐27 and Mokpo # 84. As many as 78 (51.3%) genotypes were perfect maintainers of sterility, the remaining 58 (38.2%) genotypes were classified as partial maintainers. To study the inheritance of fertility restoration, 20 CMS (tour) rapeseed lines were crossed with the four best fertility restorers, namely GSL 8851, GSL 8953, Kuju‐27 and Mokpo # 9, to obtain F₂ and test cross populations. Segregation data indicated that fertility restoration for tour CMS was governed by two genes, of which, one is stronger than the other (χ²_12:3:1). Differences in gene interactions were also observed (χ²_9:3:4) which could be explained on the basis of influence of female parent genotypes/or modified expression of the restorer gene(s) in different genetic backgrounds. Tests of allelism indicated that the restorer genes present in the four restorers evaluated were allelic.     

Characterization of different cytoplasmic male sterility systems in Indian mustard (Brassica juncea L. Czern & Coss)

Six cytoplasmic male sterility (CMS) systems, viz. moricandia, ogura, oxyrrhina, siifolia, tournefortii and trachystoma of Indian mustard (Brassica juncea L.) were characterized for agronomic and floral characteristics. Introgression of alien cytoplasm caused alterations in different floral traits in ogura, siifolia, tournefortii and trachystoma CMS systems. Varied response to different genetic backgrounds of CMS lines indicated the presence of cytoplasmic–nuclear interaction in alteration of floral traits. On the basis of floral characteristics, CMS systems could be grouped into distinct classes. Siifolia, tournefortii and trachystoma CMS lines had narrow petals, while moricandia, ogura and oxyrrhina had wider petals, which were distinguishable on the basis of visual observations. The ratio between length and width of petals were >2.0 in wide petal group but <2.0 in narrow petal group. Further, the relative position of anther and stigma, which was estimated as the ratio between stamen and style length could differentiate the CMS systems. Stamens were longer than styles in oxyrrhina, equal in moricandia and shorter in ogura, siifolia, tournefortii and trachystoma male sterile lines. Non‐viable pollen grains were present in moricandia and oxyrrhina systems, but absent in other systems. In tournefortii and trachystoma, few flowers showed petaloid corolla. All male sterile lines, except trachystoma, which showed crooked siliqua formation were at par with their respective maintainers for flower initiation, plant height, primary branches, seeds per siliqua, seed yield, harvest index, oil and protein content. In general, flower senescence and maturity occurred earlier in male sterile lines than in their respective maintainer lines.     

甘蓝型油菜细胞质雄性不育系105A花药败育的细胞学观察

旨在了解并揭示不育系105A的花粉败育时期和细胞学特征,为进一步正确认识细胞质雄性不育系分子机制提供必要参考依据。采用石蜡制片法,对甘蓝型油菜杂交种‘青杂5号’的雄性不育系105A及其恢复系1831R的小孢子发生和花药发育过程进行观察,以确定其花粉败育的时期和细胞学特点。研究结果表明,在不育系105A花药败育过程中,一部分发生于造孢细胞时期,属于无花粉囊败育型;另一部分发生于单核晚期,属于单核败育型,其特点为单核晚期绒毡层细胞膨大向小孢子靠近,并逐步降解,其破裂胞质退化残余物侵入药室,与小孢子混合粘连在一起,甚至有些绒毡层细胞整块脱落,成为染色很深的团块状物质,占据药室一部分空间,最终小孢子降解,花药败育。     

Characterization of tribenuron-methyl-induced male sterility in Brassica juncea L.

Significant heterosis has been documented in Brassica juncea L. that are grown as agriculturally important oilseeds, vegetables and condiments crops. Male sterility induced by chemical hybridizing agents is an important pollination control system in hybrid crop breeding. Herein, we show that tribenuron-methyl (TBM), a sulfonylurea herbicide, is an effective male gametocide in B. juncea when used at a very low dosage. In the present study, foliar application of various rates of TBM induced a significant increase in pollen sterility in B. juncea (90.57–100%). TBM-treated plants exhibited reductions in size of floral organ and yield components; however, lower dose of TBM (0.075 g a.i. ha^–1) did not cause a significant reduction in seed yield per plant. Tapetum cells of TBM-treated plants were hypertrophied and degenerated earlier, and abnormal meiosis was observed at the meiotic stage. A significant decrease of acetohydroxyacid synthase (AHAS) activities was detected in buds of plants treated with 0.10 g a.i. ha^–1 TBM, and RT-qPCR analysis showed that TBM exposure perturbed AHAS expression in small buds, which support that TBM induces male sterility in B. juncea by targeting AHAS expression. Our results suggest that TBM could be used as an efficient chemical hybridization agent in B. juncea, which has practical implications for the application of hybrid breeding in B. juncea.     

Development and primary genetic analysis of a fertility temperature-sensitive polima cytoplasmic male sterility restorer in Brassica napus

Over the past decade, the polima cytoplasmic male sterility ( pol CMS) three-line and two-line systems have been developed for the production of hybrid seed in Brassica napus oilseed rape in China. The discovery of the novel pol CMS restorer line FL-204 is described here. It restores male fertility of hybrid plants in the pol CMS system, but hybrid seed production can only be carried out under autumn sowing in Wuhan in south China under moderate temperatures at flowering. The restorer cannot be used as a male for hybrid seed production in northwestern China (Gansu) under spring sowing conditions, because there it is more or less male sterile due to high temperatures at flowering. Because of this behaviour, it is referred to as a fertility temperature-sensitive restorer (FTSR) in this paper. F₂, BC₁ as well as double haploid populations were constructed to determine the inheritance of fertility restoration of FL-204 in the autumn at Wuhan and under spring sowing conditions at Gansu, respectively. Deviations from Mendelian genetics were observed. It was hypothesized that the change of fertility was the result of the interaction between nuclear genes [restoring gene ( Rf ) and temperature-sensitive genes ( ts )] and the cytoplasm. The Rf gene in FL-204 was incapable of restoring male fertility of pol CMS lines under spring sowing conditions at Gansu where it is inactivated by the recessive ts gene present in FL-204. However, the ts gene(s) could be non-functional under moderate temperature conditions at flowering at Wuhan which allows full expression of male fertility in FL-204. The recessive ts gene(s) can only be expressed in plants containing the pol sterile cytoplasm. A method for the utilization of the FTSR pol CMS restorer FL-204 for the production of hybrid seed in B. napus oilseed rape is proposed.     

中国甘蓝型油菜细胞质雄性不育的主要类型及在育种实践中的应用

我国甘蓝型油菜细胞质雄性不育的类型主要有：ogura CMS、nap CMS、pol CMS、Shan 2A CMS、Hau CMS、NCa CMS、Nsa CMS和NEA CMS。pol CMS和Shan 2A CMS在我国油菜杂种优势利用中发挥着重要的作用。新型不育胞质的育成以及在育种上的应用,不仅丰富了细胞质雄性不育种质资源,而且为我国油菜产业可持续发展提供了保证。     

特早熟甘蓝型春油菜恢复系核心种质构建

通过对特早熟甘蓝型春油菜恢复系种质资源进行评价和利用,降低育种工作中恢复系与不育系选配杂交组合的工作量,从而更好地为杂交种选育服务。通过对97份波里马雄性不育系的恢复系进行简化基因组（SLAF-seq）测序,开发SNP标记,进而分析群体遗传关系,构建核心种质群体。结果显示,共获得527 872个SLAF标签,842 248个有效SNP;利用有效SNP对97份种质资源进行聚类分析,可将其分为5类;利用Core Hunter构建核心种质,构建了C30核心种质29份,C40核心种质38份,C30和C40核心种质平均等位基因覆盖度分别为99.89%和99.96%,这些材料能够代表整个资源的变异程度,达到了构建核心种质的要求。     

油菜温敏雄性不育系373S的选育

发现和选育新型光温敏雄性不育材料,有利于深入揭示植物雄性不育的发生机制,丰富油菜杂种优势利用的方法。笔者报道一种新发现的环境敏感雄性不育系373S的选育过程及其育性随温度、日长的变化基本规律。2002年在一个天然雄性不育群体后代中发现1株嵌合不育株,经过连续自交,育成环境敏感不育系373S。2004—2006年在陕西杨凌进行分期播种试验,并进行了温度、光照脉冲处理试验。结果表明：373S是主要对温度敏感的雄性不育系,低温可育而高温不育,开花前2~9d的平均温度对育性影响显著,而日长对育性影响不明显,花蕾长度约2~3mm是育性转换的敏感时期。用一系列品种与373S测交,F1均为100%可育,在F2代温敏不育株出现几率很高,可以达到7.1%~14.6%。进一步精确分析373S光温反应特征、改良其农艺及品质性状、寻找适宜制种生态区的工作正在进行当中。     

抑制消减杂交法研究复等位基因遗传的

AB01是本课题组培育的复等位基因遗传的核雄性不育大白菜甲型“两用系”,目前已建立了一套该材料的应用技术体系,但其不育分子机制尚不明确。本研究以AB01的不育株和可育株为材料,利用抑制差减杂交技术构建了正反抑制差减cDNA文库,并通过测序及生物信息学手段寻找育性相关基因,以此来推断该材料的不育分子机制。研究中共找到27个差异表达基因,其中25个基因在NCBI数据库中均有同源序列,这些基因中7个与花发育相关,5个与脂类代谢相关,3个与活性氧及能量代谢相关,3个与光合作用及叶绿体合成相关,其余7个为功能未知基因。由此推测复等位基因遗传的核雄性不育大白菜不育的发生与脂类、能量代谢及光合作用有关。     

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.     

甘蓝型油菜NCa CMS育性相关候选线粒体基因及其恢复基因的转录调控

用10个线粒体基因为探针，对NCa不育系、保持系和可育F1幼蕾的线粒体RNA进行了Northern检测。结果表明，atp6、atp1、cox1、cox2、cob、rrn5S和rnn26S等7个线粒体基因探针在不育系、保持系、可育F1中的转录没有差异，只有orf222、orf139和atp9等3个探针检测到转录本的差异。orf222和orf139分别在不育系和可育F1中产生相同大小和丰度的转录本，但是在保持系中没有检测到转录本；orf222检测到的3条转录本分别为1．1、0．9和0．6kb，orf139检测到0．8和0．6kb2条带。atp9探针在不育系和保持系中都检测到1条0．6kb转录本，而在可育F1中检测到0．6和1．2kb的转录本。NCaCMS不育的形成可能与orf222、orf39和atp9基因的表达有关。讨论了恢复基因在F1育性恢复过程中对育性相关候选基因的可能调控方式。     

Production of a desirable Brassica oleracea CMS line using an alloplasmic B. rapa CMS line carrying Diplotaxis erucoides cytoplasm as a bridge plant

In Brassica oleracea, production of F₁ hybrid seeds mainly makes use of the improved Ogura cytoplasmic male sterile (CMS) line. However, reliance on one particular line is a risk, and it would be advantageous to develop other CMS lines. In this study, we transferred Diplotaxis erucoides cytoplasm to B. oleracea cultivars using an alloplasmic B. rapaCMS line as a bridge plant to avoid incompatibility between donor and recipient plants. The new B. oleraceaCMS lines, which were derived by four generations of backcrossing, had small rudimentary anthers with no pollen grain and showed complete male sterility. There was no functional defect in other floral organs, and the ability to receive normal pollen did not appear to be impaired. Moreover, the B. oleraceaCMS lines carrying D. erucoides cytoplasm had larger leaf areas and a normal plastochron. As a consequence, the B. oleraceaCMS lines carrying D. erucoides cytoplasm have the potential to be valuable alternatives for use in commercial B. oleracea hybrid seed production.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

Evaluation of cytoplasmic effects on agronomic and seed quality traits in two doubled haploid populations of Brassica napus L.

Cytoplasmic effects have been occasionally implicated in the inheritance of several traits in oilseed rape (Brassica napus L.), including linolenic acid concentration (18:3) in the oil. It is important that these be considered when choosing the direction of cross for producing new breeding populations. To study this phenomenon, a reciprocal cross was made between two genotypes of oilseed rape, Reston and LL09, which differed for their erucic and linolenic acid concentrations in the seed oil. Two DH populations, which were produced by microspore culture from reciprocal F₁ plants, were evaluated in the growth room for one generation and in the field at two locations in Southern Ontario in 1993and 1994. Field notes were taken on days to flower, days to maturity,plant lodging, plant height and, seed quality traits. In the growth room study, the phenotypic distribution of 18:3 differed significantly between the two reciprocal DH populations. In the field, significant reciprocal differences between the population means were detected for 18:3,flowering date and protein content in both years and for days to maturity and oil content in 1993 only. To further study the parental lines,chloroplast (cp) and mitochondrial (mt) DNA from parental lines were isolated and subjected to RFLP and RAPD analysis. Several random primers revealed reproducible DNA polymorphism (RAPD) between the parental mt DNA. It is concluded that the direction of cross should be taken into consideration by oilseed rape breeders relying solely on doubled haploids for developing genotypes with modified seed quality traits in Brassica napus L. This revised version was published online in July 2006 with corrections to the Cover Date.     

Directional transfer of a multiple-allele male sterile line in Brassica campestris L. ssp. chinensis (L.) Makino var. rosularis Tsen et Lee

To produce hybrid seeds of Wutacai (Brassica campestris L. ssp. chinensis (L.) Makino var. rosularis Tsen et Lee), a “directional transfer program” was designed to breed the multiple-allele male sterile line of Wutacai. A multiple-allele male sterile line of Naibaicai (Brassica campestris L. ssp. chinensis L., S01) was used as the male sterile resource, and an inbred line of Wutacai (WT01) was used as the target line. Recurrent backcrossing was employed to transfer the male sterility and other botanical traits simultaneously, while the genotype was identified through test crossing. The male sterility was successfully transferred from S01 to WT01. A new male sterile line, GMS-3, with similar botanical traits to WT01, was bred. Four hybrid combinations were generated with GMS-3 as the female parent. One hybrid (C1) that contained the most desirable traits was developed from the new male sterile line.