合成甘蓝型油菜中双隐性雄性核不育材料的发现及遗传规律分析

从甘蓝型油菜与白菜型油菜的种间杂交获得的甘蓝型油菜(Brassica napus)中发现了雄性不育单株,兄妹交株系和不育株与甘蓝型油菜常规杂交F1和F2株系的育性分离分析表明,该不育材料属于双隐性雄性核不育类型.利用育性分离株系的可育株自交和可育株与不育株间兄妹交等方法筛选出7个纯合可育株系,等位测验表明这7个纯合可育株系(B1～B7)中存在两种基因型:Ms1Ms1ms2ms2和ms1ms1Ms2MS2.该材料对油菜核不育基因定位和杂种优势利用研究有重要意义.     

温室冬繁水稻低温敏核不育系原种方法的初步研究

１９９５年１—５月,在广州利用玻璃温室加温冬繁籼型水稻低温敏核不育系培矮６４ｓ和ＧＤ－２ｓ原种．当不育系进入育性转换敏感期时,温室室温调控至日均温约２３℃,检查花粉育性和自然结实率,结果表明,培矮６４ｓ的结实率为５０．７％,单株产量平均为３．５ｇ．培矮６４ｓ原种温室冬繁的成功,为现有种量少、繁种难、提纯复壮株严格的低温敏核不育系加速世代扩大繁殖系数提供了一种可靠的途径．     

水稻离体育性变异研究

对野败型不育系珍汕97A、保持系珍汕97B、恢复系IR24、IR26、泰引1号、明恢63、红莲型不育系红源A、包台型不育系包源A、光敏核不育系农垦58s、温敏核不育系W6154s等10个水稻材料的幼穗在不同的培养基上培养、再生植株及对其后代进行育性鉴定,探讨了体细胞无性系变异中雄性不育突变发生的机率以及影响离体筛选雄性不育变异体的因素, 结果表明：在5个材料 (珍汕97B、红源A、包源A、W6154s和IR26)中共获得了29例雄性不育变异株, 其中R1代有24株, R2代有5株。在R1代, 共获得2*!368株再生植株, 雄性不育变异的频率为1.02%(0.96%～1.08％)。在珍汕97B和泰引1号R2代各发现一个株系分离出雄性不育和育性正常植株。出现不育株系的频率分别为2.22％和1.89％。水稻花粉败育类型可分为无花粉、典败、圆败和染败4种类型。同时, 还发现了不育花粉败育类型之间可以相互转换这一现象,在IR26和明恢63 R1代再生植株中, 各发现一株嵌合体。在泰引1号和珍汕97B R2代再生植株中分离出不育株。在影响离体筛选雄性不育变异体的因素中, 基因型的差异是主要的,在所试10个材料中,除农垦58s、IR24、泰引1号和珍汕97A,都有雄性不育变异株产生。外植体的脱分化对产生雄性不育变异是必需的, 在这一过程中,2,4-D起决定性的作用。随着继代时间的延长,发生雄性不育变异的频率也随之提高。雄性不育变异频率在R2代高于R1代。     

萍乡显性核不育水稻育性相关基因的定位和遗传分析

萍乡显性核不育水稻(Pingxiang Dominant Genic Male Sterile Rice,PDGMSR)是在水稻中首次发现的显性核不育材料,其育性由两对显性基因互作控制,一对是萍乡显性核不育基因Ms-p,另一对是显性上位恢复基因(dominant epistatic fertility restorer gene,Rfe)。两者共同存在时显性上位恢复基因能抑制不育基因的表达,从而使育性表现可育。本实验用一个对萍乡显性核不育水稻有恢复能力的水稻品种E823与萍乡显性核不育水稻配制杂交组合,将(萍乡核不育水稻/E823)F2作为定位群体,根据F3株系的育性分离,选择育性分离株系对应F2单株(基因型为Ms-pMs-pRefrfe和Ms-pms-pRferfe)构建可育池,用对应F2株系中的不育单株(基因型为Ms-pMs-prferfe或Ms-pms-prferfe)构建不育池,将显性上位恢复基因Rfe定位在水稻10染色体RM311和RM3152一侧,遗传距离分别为7.9cM和3.6cM。根据已有的Ms-p的定位结果,合成10染色体部分微卫星引物,对不育单株进行分析,发现RM171和RM6745位于Ms-p的两侧,距离分别为0.3cM和3.0cM。根据10染色体的测序结果,将Ms-p界定在约730kb的范围内,并构建了Ms-p的电子重叠群。植物显性核不育的育性恢复机理存在“复等位基因”和“显性上位互作”两种假说,贺浩华等用经典的遗传学方法证明了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。理论上认为,确定其遗传机理最为有效的方法是基因定位,如果不育基因和恢复基因位于同一位点,则其遗传机理属于“复等位基因”,否则为“显性上位互作”。本实验将不育基因和恢复基因定位在水稻10染色体不同的位点,用基因定位的方法证实了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。     

水稻光敏核不育系的选育

湖北光敏感核不育水稻的发现,为两系杂交稻奠定了基础。农垦58S为晚梗型,经济性状不理想,直接利用于生产较困难,必须将光敏核不育特性转育到优良的籼、粳品种中,选育出符合生产要求的光敏核不育系,才有生产利用价值。光周期诱导水稻雄性育性转换特性是由1—2对主效基因控制的,可以通过杂交转育。自八十年代以来,湖北和全国的水稻遗传育种学家开展了水稻光敏核不育系的选育,到1991年止,由农垦58S为源的通过省级以上鉴定的水稻光(温)敏核不育系18个,其中籼稻10个粳稻8个,由光、温敏核不育系配制的两系杂交稻组合开始用于生产。     

玉米CMS-C同质异核不育系育性恢复的遗传研究

玉米是最早利用细胞质雄性不育系生产杂交种的作物之一,C型细胞质雄性不育系（C-type cytoplasmic male sterile, CMS-C）在杂交种生产中具有重要的作用,育性恢复的稳定性直接影响其应用价值。然而,玉米CMS-C的育性恢复机理复杂,且至今仍不明确。为进一步探究玉米CMS-C育性恢复的影响因素,本研究以玉米CMS-C同质异核不育系C48-2、C黄早四和C478为母本,分别与测验系18白、自330、5022以及恢复系A619组配杂交获得F₁。其中育性恢复F₁通过自交获得F₂,并以育性恢复F₁为父本分别给育性保持F₁授粉,组配双交群体,共获得4个F₂群体,6个双交群体。同时以不育系C48-2、C黄早四和C478为母本,各自的保持系48-2、黄早四和478为父本杂交组配不完全双列杂交F₁。将所有杂交组合的F₁、F₂以及双交组合群体分别在不同年份不同地点种植观察,通过植株田间育性调查并结合室内花粉镜检鉴定育性表现。结果表明：1) 同一测验系对玉米CMS-C同质异核不育系的恢保关系不同,暗示不育系的核背景参与调控育性恢复表现;2) 在不同年份不同地点对(C48-2×A619) F₂群体进行种植观察,发现不同环境下F₂群体可育株与不育株的分离比均符合15∶1,但在云南种植的可育株的育性级别主要为Ⅲ和Ⅳ级,而在四川种植的可育株的育性级别主要为Ⅴ级,表明环境对恢复系A619恢复后代的育性表现有影响;3) 通过恢保关系测定发现18白不能恢复C478,48-2也不能恢复C478,但双交群体[(C478×18白) F_1S×(C48-2×18白) F_1F]后代却出现了可育株与不育株的分离;同理,双交群体[(C48-2×自330) F_1S×(C478×自330) F_1F]的后代也出现了可育株与不育株的分离。因此,本文推测C48-2、C478核背景中存在微效恢复基因,这些微效基因与18白、自330中的微效恢复基因通过杂交聚合后能使C478、C48-2的育性恢复,暗示玉米CMS-C的育性恢复呈现一定的剂量效应。这些结果为进一步认识玉米CMS-C育性恢复的复杂性和多样性奠定了基础,为深入研究玉米CMS-C育性恢复机理以及加快CMS-C在不育化制种中的应用提供重要参考。     

二角型非1B/1R 小麦CMS不育恢复体系的建立

通过对具有二角山羊草细胞质的1B/1R型小麦雄性不育系m s(A e.bicorn is)-5-1回交置换,获得了新型二角山羊草细胞质小麦雄性不育系m s(A e.bicor)-V 9125和m s(A e.bicor)-M 853.利用染色体制片、分子标记、原位杂交和酸性聚丙烯酰胺凝胶电泳对其保持系V 9125和M 853进行分子细胞遗传学检测,并对其育性恢复性机理进行了初步研究.结果表明:(1)V 9125和M 853均为非1B/1R易位系.V 9125为小麦-簇毛麦易位系,m s(A e.bicor)-V 9125是二角山羊草细胞质与普通小麦核背景中簇毛麦外源染色体互作产生的一类新的核质互作不育系.(2)M 853为小麦-滨麦易位系,m s(A e.bicor)-M 853是二角山羊草细胞质与普通小麦核背景中滨麦外源染色体互作产生的一类新的核质互作不育系.(3)利用一些普通小麦品种(系)与这两个不育系杂交,m s(A e.bicor)-V 9125和m s(A e.bicor)-M 853仅与T 6-3杂交育性得到恢复,且恢复度较高,变异小,而与其它大部分普通小麦杂交F1表现雄性不育,且扬花期花药不外露.而m s(A e.bicor)-5-1与包括T 6-3在内的普通小麦品种(系)杂交F1育性得到不同程度的恢复.从而得出结论,二角山羊草细胞质与小麦细胞核的互作存在两个不同的不育-恢复系统.     

温敏核不育系株1S育性感光感温特性及繁殖条件研究

在育性敏感期,株1S经22℃处理5d、23℃处理10d仍稳定不育,是目前温敏核不育系中临界不育温度最低、对短期低温最钝感的不育系.对冷水灌溉繁殖该不育系的适宜条件研究表明,适宜的水温为20.5℃,开始冷水灌溉的时期为雌雄蕊形成期,历期为18d,冷水深度变幅为3-18cm,随着幼穗的发育和伸长需逐渐增加其冷水深度.     

水稻光(温)敏核不育系与核质互作不育系的遗传关系剖析

1991─1992年,分析了7个光(温)敏核不育系与15 个核质互作不育系杂交F1及部分F2植株在长日照和短日照条件下的育性表现,结果清楚地表明,有些光敏核不育系能够保持核质互作不育系的雄性不育性,有些光(温)敏核不育系则能够恢复或部分恢复; 有些光敏核不育系对某一核质互作不育系具有保持能力,对另一核质互作不育系则具有恢复能力; 并初步推测光(温)敏核不育基因与核质互作不育基因是独立发生的,当核质互作不育系中细胞质和细胞核的隐性不育基因一起作用时,能够掩盖光(温)敏不育基因及其育性恢复基因的表达。     

籼型光敏核不育水稻育性可转换性的基因定位和基因互作研究

本研究以来源于农垦58S的籼型光敏核不育系培矮64S(短日条件下育性难转换)和8902S(短日条件下育性易转换)及其F１、F２群体为材料,通过短日不同光温和不同生态条件4种处理,利用RFLP分子标记研究了影响光敏核不育水稻在短日条件下的育性可转换性的遗传、基因定位和基因互作,主要结果表明：影响光敏核不育水稻的育性可转换性表现为微效基因的作用,定位了7个控制光敏核不育水稻的育性可转换性QTL,即S2、S3a、S3b、S5、S8和S10。揭示了基因互作真实存在于光敏核不育水稻中,基因互作形式和互作类型对光敏核不育水稻的育性可转换性的影响表现多种多样,不同类型的基因互作所解释的遗传变异处于2.15%～10.07%之间。     

温度对双低两用核不育水稻96-5-2S与培矮64S育性的影响

在自然变温、人工控温及冷水灌溉条件下,比较研究了温度对双低两用核不育水稻96－5－2S与两用核不育水稻培矮64S育性影响的差异。结果表明：（1）当它们在雄性育性转换温敏感期1－12d平均自然日均温23.0-23.8℃的低温时,96－5－2S表现不良,套袋自交结实率为0,而培矮46S可育,套袋自交结实率为0．1％－4．5％;（2）在它们雄性育性转换温敏感期用22℃恒温处理5d,96－5－2S败育彻底,套袋自交结实率为0,而培矮64S可育,套袋自交结实率为10．7％;用17℃恒温处理6d,96－5－2S与培矮64S均可育,但96－5－2S套袋自交结实率（6．8％）显著高于培矮64S（2．5％）;（3）在它们雄性育性转换温和不同温度的冷水串灌15d,水深维持在20cm左右,当水温为22－22．5℃时,96－5－2S不育,结实率为0,而培矮64S可育,结实率为18．5％;当水温为19．5－21．5℃时,96－5－2S与培矮64S均可育,但96－5－2S结实率（2．5％－45．1％）显著或极显著低于培矮64S（50．4％－56．9％）以上结果说明：导致双低两用核不育水稻96－5－2S雄性不育的起点温度与导致其生理不育的下限温度均低,其不育性比培矮64S更稳定,耐寒性比培矮64S更强,即可确保制种安全,又可确保自身繁殖,对加快两系法杂交水稻的发展步伐将起到重要的促进作用。     

Comparative proteomics analysis reveals the mechanism of fertility alternation of thermosensitive genic male sterile rice lines under low temperature inducement

Thermosensitive genic male sterile (TGMS) rice line has made great economical contributions in rice production. However, the fertility of TGMS rice line during hybrid seed production is frequently influenced by low temperature, thus leading to its fertility/sterility alteration and hybrid seed production failure. To understand the mechanism of fertility alternation under low temperature inducement, the extracted proteins from young panicles of two TGMS rice lines at the fertility alternation sensitivity stage were analyzed by 2DE. Eighty‐three protein spots were found to be significantly changed in abundance, and identified by MALDI‐TOF‐TOF MS. The identified proteins were involved in 16 metabolic pathways and cellular processes. The young panicles of TGMS rice line Zhu 1S possessed the lower ROS‐scavenging, indole‐3‐acetic acid level, soluble protein, and sugar contents as well as the faster anther wall disintegration than those of TGMS rice line Zhun S. All these major differences might result in that the former is more stable in fertility than the latter. Based on the majority of the 83 identified proteins, together with microstructural, physiological, and biochemical results, a possible fertile alteration mechanism in the young panicles of TGMS rice line under low temperature inducement was proposed. Such a result will help us in breeding TGMS rice lines and production of hybrid seed.     

双低两用温敏核不育水稻低温条件下花粉发育的细胞学观察

双低两用温敏核不育水稻96－5－2S在11.31－20.19℃低温条件下,花粉母细胞形成、减数分裂和花粉后期发育均正常,最后形成充满淀粉的成熟花粉,而作对照的两用温敏不育水稻陆18S在相同低温条件下,除花粉母细胞形成早期发育正常外,花粉母细胞晚期、减数分裂期均有异常,败育主要发生在单核小孢子靠边期,没有形成成熟花粉,结果表明,96－5－2S桫胜殖期抗寒性强,低温生理障碍雄性不育临界温度低。     

Effect of Growth Regulators and Chemicals on Pollen Sterility in TGMS Lines of Rice

Thermosensitive genic male sterility (TGMS) in rice is a widely adopted technique for successful hybrid rice production in Asia. TGMS lines remain male sterile when daily mean temperature is above the critical sterility temperature and are therefore used as female parents. The same line will remain fertile when mean temperature is below the critical sterility temperature. Achievement of 100% male sterility in TGMS lines is important for the successful utilization of TGMS lines as female parents in hybrid rice production. This study examined the external application of some growth regulators and chemicals and their effect on pollen sterility. Among the various treatments, ethrel (800 ppm), salicylic acid (600 ppm) and maleic hydrazide (0.2%) induced a significantly higher percentage of male sterility in the TGMS lines. The sprayed plants also showed higher total phenol accumulation in their flag leaves. The results suggest that it is possible to achieve 100% male sterility in TGMS lines with the external application of growth regulators and chemicals.     

Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryza sativa L.) with molecular markers

The thermo-sensititve genic male-sterile (TGMS) gene in rice can alter fertility in response to temperature and is useful in the two-line system of hybrid rice production. However, little is known about the TGMS gene at the molecular level. The objective of this study was to identify molecular markers tightly linked with the TGMS gene and to map the gene onto a specific rice chromosome. Bulked segregant analysis of an F₂ population from 5460s (a TGMS mutant line) x Hong Wan 52 was used to identify RAPD markers linked to the rice TGMS gene. Four hundred RAPD primers were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants; of these, 4 primers produced polymorphic products. Most of the polymorphic fragments contained repetitive sequences. Only one singlecopy sequence fragment was found, a 1.2-kb fragment amplified by primer OPB-19 and subsequently named TGMS1.2. TGMS1.2 was mapped on chromosome 8 with a RIL population and confirmed by remapping with a DHL population. Segregation analysis using TGMS1.2 as a probe indicated that TGMS1.2 both consegregated and was lined with the TGMS gene in this population. It is located about 6.7 cM from the TGMS gene. As TGMS1.2 is linked to the TGMS gene, the TGMS gene must be located on chromosome 8.This research was supported by the Rockefeller Foundation and China National High-Tech Research and Development Program. The first author is a Rockefeller Career Fellow at Texas Tech University     

A bentazon and sulfonylurea sensitive mutant: breeding,genetics and potential application in seed production of hybrid rice

The use of a thermosensitive genic male sterility (TGMS) system in two-line hybrid rice breeding is affected greatly by the sterility instability of TGMS lines caused by temperature fluctuation beyond their critical temperatures for fertility reversion. To prevent seed production from self contamination, we have developed a system to secure seed purity using a herbicide-sensitive TGMS mutant, M8077S, obtained by radiation. Genetic analysis, using the F₁, F₂ and F₃ populations derived from this mutant and other normal varieties, revealed that bentazon lethality/sensitivity was controlled by a single recessive gene, which was named bel. The mutant can be killed at the seedling stage by bentazon at 300 mg/l or higher, a dosage that is safe for its F₁ hybrids and all other normal varieties. This mutant is also sensitive to all the tested sulfonylurea herbicides. Response of segregating plants to these two types of herbicide indicated that sulfonylurea sensitivity was also controlled by bel. By crossing this mutant with Pei-Ai 64S, an F₂ population was developed for genetic mapping. Surveying the two DNA pools from sensitive and non-sensitive F₂ plants identified four markers that were polymorphic between the pools. The putative linked markers were then confirmed with the F₂ population. The bel locus was located on chromosome 3, 7.1 cM from the closest microsatellite marker RM168. Phenotypic analysis indicated that the bel gene had no negative effect on agronomic traits in either a homozygous or heterozygous status. The mutant M8077S is valuable in the development of a TGMS breeding system for preventing impurity resulting from temperature fluctuation of the TGMS. Several two-line hybrid rice crosses using this system are under development.     

温度对温敏核不育水稻eui突变体最上节间伸长的影响

以培矮64S为对照, 采用田间调查和人工温度处理方法研究了温度对温敏核不育水稻（Oryza sativa）eui突变体（双低培eS）最上节间伸长的影响。结果表明, 双低培eS穗颈伸出度与抽穗前12–17天（花粉母细胞形成期至减数分裂期）的日均温度呈显著负相关。在温度敏感期分别进行人工温度处理, 在18–26℃条件下穗颈伸出度为正值且不包颈; 在28℃条件下出现包颈现象。在可育温度（20℃）和不育温度（24℃）条件下, 双低培eS最上节间中GA1、IAA和ZR含量极显著地高于培矮64S, 而ABA含量则显著低于培矮64S, 最上节间中最内层薄壁细胞数目分别比培矮64S多1 177和823个, 细胞平均长度分别比培矮64S长23.2和16.7 μm。温敏核不育水稻eui突变体最上节间伸长是由于节间最内层薄壁细胞数目增多和细胞长度增加双重作用所致, 其中以细胞伸长为主, 且随着处理温度的升高, 最上节间最内层薄壁细胞数目减少, 细胞平均长度变短。eui基因还可能通过调节激素间的平衡来控制温敏核不育水稻eui突变体最上节间的伸长生长。     

Identification of a deletion in <Emphasis Type="Italic">tms2</Emphasis> and development of gene-based markers for selection

Rice is one of the most important food crops. The temperature-sensitive genic male sterility (TGMS) system provides a great potential for improving food production by hybrids. The use of TGMS system is simple, inexpensive, effective, and eliminates the limitations of the conventional three-line system. A rice gene, tms2, generated by irradiation of a japonica variety has been reported to control TGMS in several rice lines. Previous studies reported genetic markers linked to this gene, and the gene was transferred to an aromatic Thai cultivar. Using information obtained from published databases, we located positions of the reported genetic markers flanking the gene in rice genomic sequences, and developed gene-based markers located inside the flanking markers for polymorphism detection. We found that inbred indica tms2 mutant plants contain about 1 Mb of japonica DNA, in which at least 70 kb was deleted. Using RT-PCR for expression analysis, four genes out of seven genes annotated as expressed proteins located inside the deletion showed expression in panicles. These genes could be responsible for TGMS phenotypes of tms2. In addition, we developed gene-based markers flanking and inside the deletion for selecting the tms2 gene in breeding populations. By genotyping 102 diverse rice lines including 38 Thai rice lines, 5 species of wild rice, and 59 exotic rice lines including TGMS lines and cultivars with desirable traits, a gene-based marker located inside the deletion and one flanking marker were shown to be highly specific for the tms2 mutant.