Characterization and genetic mapping of a novel recessive genic male sterile gene in sesame (Sesamum indicum L.)

Male sterile mutants play a very important role in the utilization of crop heterosis. A recessive genic male sterile (RGMS) two-type line 95ms-5AB was derived from a male sterile mutant of common white sesame (Sesamum indicum L.) cultivar Yuzhi 4 by treatment with gamma rays from ⁶⁰Co. Male sterile 95ms-5A plants did not show any other obvious differences from the male fertile 95ms-5B plants, except for having greenish, shriveled and slim anthers with few, small and degenerative pollens. Genetic analysis indicated that the male sterility of 95ms-5A was controlled by a single RGMS gene, Sims1 (Sesamum indicum male sterility 1). An allelic test with a previously identified RGMS mutant, ms86-1, confirmed that Sims1 in 95ms-5A is different from Sims2 in ms86-1. Amplified fragment length polymorphism markers linked to SiMs1 were screened using bulked segregant analysis. A genetic linkage map of the SiMs1 gene was constructed using 237 plants derived from the sib-mating between the near-isogenic lines 95ms-5A and 95ms-5B. The SiMs1 gene was found to be located in a region of 8.0 cM, at a distance of 1.2 cM from P06MG04 and 6.8 cM from P12EA14. In this genetic region, another marker P01MC08 was identified to be co-segregated with SiMs1. The linkage map constructed in this study will be very useful for marker-assisted selection and map-based cloning of SiMs1 as well as for hybrid breeding in sesame crop.     

雄性不育与可育楸树花发育的细胞学比较研究

楸树（Catalpa bungei C.A.Meyer.）属紫葳科(Bignoniaceae)梓树属(Catalpa),落叶乔木,是我国特有的珍贵优质用材树种。本文用石蜡切片法对可育株和雄性不育株楸树的大、小孢子发生及雌、雄配子体发育过程进行了详细地比较观察。结果表明：可育株和不育株楸树雌蕊的发育基本相同,胚珠倒生,薄珠心,单珠被,胚囊发育为蓼型。可育株雄蕊花药四室,药隔薄壁组织发达;异型绒粘层,由药壁绒粘层和药隔绒粘层组成;花药壁表皮细胞在小孢子母细胞减数分裂前后开始径向伸长加厚,直到花药开裂并不降解,这可能与花药开裂有关;成熟花粉为四合花粉。雄性不育株花药的早期发育到次生造胞细胞时期与可育雄蕊的相同,小孢子母细胞减数分裂前绒毡层发育不充分;四分体时期,绒毡层细胞高度液泡化,细胞质稀薄,已提前降解,小孢子四分体因绒毡层结构和功能异常而不能正常发育,因此楸树雄性不育为结构型雄性不育。     

云南紫稻细胞质雄性不育系花药发育过程中Ca2+的分布特点

运用焦锑酸钾沉淀法研究了云南紫稻细胞质雄性不育系和保持系花药在发育过程中Ca^2 的分布特点。结果表明,保持系的花粉母细胞和小孢子的胞质内部基本无Ca^2 的沉淀,后期花粉外壁出现Ca^2 的沉淀;保持系早期的绒毡层细胞形态正常,胞内有少量Ca^2 沉淀,后期绒毡层细胞开始凋亡,胞质凝集,胞内出现大量Ca^2 的颗粒。不育系花粉母细胞在减数分裂时期败育,胞质液泡化,内部出现大量Ca^2 的沉淀;不育系绒毡层细胞形态正常,胞内无Ca^2 的沉淀。绒毡层与花粉母细胞、小孢子之间出现大量Ca^2 颗粒。探讨了不育系花药花粉母细胞中以及与绒毡层细胞之间Ca^2 的异常积累与雄性不育的关系。     

A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish

To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.     

Meiotic abnormality in dominant genic male sterile <Emphasis Type="Italic">Brassica napus</Emphasis>

Rs1046AB is a dominant genic male sterile (DGMS) Brassica napus line derived from Yi-3A. Until now the molecular mechanism of its male sterility is still unknown. In this paper, cytological observations demonstrated that all cells in sterile plants contained condensed nuclei at the beginning stage of meiosis; this implied that meiotic cells were degenerating. Although 31% (93/300) cells escaped from the state of nuclei condensation in buds about 3 mm in length (in such length, normal plants are at tetrade stage), no cells could pass the pachytene stage. Then pachytene-or zygotene-like chromatin/chromosomes sometimes congregated into two or more groups with different size, which resulted in the formation of micronuclei. A nucleoplasmic bridge could also be found in some meiotic cells. Even when the “microspore’s analogue” appeared in sterile buds about 4 mm in length (in such length, mature pollens could be detected in normal buds), the nuclei condensation and escaped cells with a pachytene-like chromosome still could be found in the sterile anthers. So it could be concluded that male sterility was caused by meiotic abnormality. According to our previous research, four genes related to cell cycle/DNA processing were identified in fertile plants. RT-PCR further confirmed that three DNA repair genes were partially or completely repressed in the sterile plants and were only expressed in the early stage fertile flower buds, i.e., the buds <3 mm in length. Therefore, DGMS of rapeseed was probably caused by the abnormality in the DNA damage repair system during meiosis. According to these results, some possible mechanisms of fertility control were discussed.     

棉花细胞核雄性不育两用系差异表达基因分析

应用cDNA-AFLP对棉花ms5ms6双隐性核雄性不育两用系的不育株和可育株花粉发育的3个时期—造孢细胞时期、花粉母细胞时期和花粉粒时期进行对比分析,共得到17个差异表达片段,它们分别属于11种表达模式,其中14个片段可以在NCBI数据库中找到同源序列,功能分析表明这些片段所编码的基因可能参与了信号转导、转录、能量代谢、细胞壁发育等相关过程。Northern杂交结果证明检测片段的表达模式与cDNA-AFLP结果吻合。同时还在可育花药中发现了与玉米T型细胞质雄性不育恢复因子RF2基因高度同源的育性恢复因子类基因。     

油菜雄性核不育系及其等位可育系小孢子发育过程的比较研究

用Olympus BH2型光学显微镜对甘蓝型油菜单显性核不育系(GMS)及其等位可育系小孢子发育过程进行解剖学观察,发现在正常小孢子发育过程中,绒毡层在小孢子发育的四分体前后开始解体,为小孢子继续发育提供营养,而不育系小孢子的败育在减数分裂前就已经发生,并且不能形成四分体,小孢子逐渐解体,且小孢子解体在绒毡层解体之前发生,最后花药成为干瘪的空壳,导致不育。     

Meiotic abnormality in dominant genic male sterile Brassica napus

Rs1046AB is a dominant genic male sterile (DGMS) Brassica napus line derived from Yi-3A. Until now the molecular mechanism of its male sterility is still unknown. In this paper, cytological observations demonstrated that all cells in sterile plants contained condensed nuclei at the beginning stage of meiosis; this implied that meiotic cells were degenerating. Although 31% (93/300) cells escaped from the state of nuclei condensation in buds about 3 mm in length (in such length, normal plants are at tetrade stage), no cells could pass the pachytene stage. Then pachytene or zygotene like chromatin/chromosomes sometimes congregated into two or more groups with different size, which resulted in the formation of micronuclei. Nucleoplasmic bridge could also be found in some meiotic cells. Even when the "microspore's analogue" appeared in sterile buds about 4 mm in length (in such length, mature pollens could be detected in normal buds), the nuclei condensation and escaped cells with pachytene like chromosome still could be found in the sterile anthers. So it could be concluded that male sterility was caused by meiotic abnormality. According to our previous research, four genes related to cell cycle/DNA processing were identified in fertile plants. RT-PCR further confirmed that three DNA repair genes were partially or completely repressed in the sterile plants, and were only expressed in the early stage fertile flower buds, i.e. the buds <3 mm in length. Therefore, DGMS of rapeseed was probably caused by the abnormality in DNA damage repair system during meiosis. According to these results, some possible mechanisms of fertility control were discussed.     

长春蒲公英雄性不育株形态学观察与败育细胞学研究

在长春蒲公英(Taraxacum junpeianum Kitam.)株群中发现雄性不育现象,为研究其败育机理及特点,探寻其不育基因,采用形态观察法、石蜡切片技术和染色体压片法,对长春蒲公英野生型及其雄性不育株的花药发育过程和花粉母细胞减数分裂过程进行了观察。结果表明:(1)长春蒲公英雄性不育株花药中部发红、干瘪、无花粉散出。与野生型比较,雄性不育株雄蕊更短,子房更窄,种子形态更加狭长;(2)长春蒲公英雄性不育株败育时期为四分体到单核小孢子前期,败育方式为小孢子自身异常发育,绒毡层异常分解,互相粘连败育;(3)长春蒲公英雄性不育株花粉母细胞减数分裂二分体时期出现落后微核,随后产生极少四分体,并且四分体产生大量染色体桥,小孢子营养物质流失,彻底败育。因此,长春蒲公英雄性不育株败育彻底、稳定,并且有种的特点。小孢子自身异常发育和绒毡层异常分解是导致败育的主要原因。     

基因工程雄性不育烟草及其温度敏感性

将含有抗溴苯腈基因bxn和雄性不育基因的重组载体pTA29-Barnase/bxn导入农杆菌(Agrobacterium tumefaciens)后转化烟草(Nicotiana tabacum L.),得到33个转基因植株。在27℃/23℃培养的16株中,有7株表现部分不育,另9株全部可育。而在20℃/15℃培养的17株中,12株表现不育,5株表现部分不育。部分不育的植株上同时开放可育花朵和不育花朵,不过其中的不育花朵中的花粉萌发活力很低。将在20℃/15℃条件下表现不育的12个不育株从20℃/15℃温室移至27℃/23℃温室后30d左右,其中9株表现程度不同的育性恢复现象:5株表现部分可育,另4株表现完全可育;但仍有3株表现雄性不育。雄性不育花朵的花丝变短,花药皱瘪,不散粉。细胞学观察证明,转基因植株的花药绒毡层降解提早于四分体时期,至单核小孢子时期降解殆尽。     

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radishand has been introduced into rapeseed (Brassica napus) by protoplastfusion. As with all CMS systems, it involves a constitutivelyexpressed mitochondrial gene which induces male sterility tootherwise hermaphroditic plants (so they become females) anda nuclear gene named restorer of fertility that restores pollenproduction in plants carrying a sterility-inducing cytoplasm.A correlative approach using light and electron microscopy wasapplied to define what stages throughout development were affectedand the subcellular events leading to the abortion of the developingpollen grains upon the expression of the mitochondrial protein.Three central stages of development (tetrad, mid-microsporeand vacuolate microspore) were compared between fertile, restored,and sterile plants. At each stage observed, the pollen in fertileand restored plants had similar cellular structures and organization.The deleterious effect of the sterility protein expression startedas early as the tetrad stage. No typical mitochondria were identifiedin the tapetum at any developmental stage and in the vacuolatemicrospores of the sterile plants. In addition, some strikingultrastructural alterations of the cell's organization werealso observed compared with the normal pattern of development.The results showed that Ogu-INRA CMS was due to premature celldeath events of the tapetal cells, presumably by an autolysisprocess rather than a normal PCD, which impairs pollen developmentat the vacuolate microspore stage, in the absence of functionalmitochondria. Key words: Brassica napus, cell death, light and electron microscopy, mitochondria, plastids, pollen development, Ogu-INRA cytoplasmic male sterility, transgenic-restored plants, tapetum Received 30 September 2007; Revised 11 December 2007 Accepted 20 December 2007