主要农作物细胞质雄性不育系育性恢复基因研究进展

提升作物产量、抗逆性和品质的主要手段之一是利用杂种优势,其中细胞质雄性不育/恢复(CMS/Rf)系统是应用最广的雄性不育系统。研究细胞质雄性不育系育性恢复机理是“三系”法选育的重要分子遗传学基础。目前,各个作物已经创制了不同类型的不育系。随着分子技术、基因组学和测序技术的深入,大量育性恢复基因已被定位,且部分已被克隆和功能鉴定。针对主要作物中恢复基因的遗传模式,分子标记定位、克隆及CMS/Rf系统在杂交育种中的应用进行了系统总结。希望本文能为今后恢复系分子标记辅助选育,或利用转基因、基因编辑手段创制新恢复系提供思路。     

青花菜雄性不育系选育研究初报

以具有细胞质雄性不育基因的青花菜不育材料CMS94008为不育源,通过杂交和连续回交的方法选育出不育性稳定、经济性状良好的一批青花菜胞质雄性不育系,并以CMS92100、CMS93-2、CMS9905、CMS95234等不育材料及其转育父本为代表,对不育系的主要特征及农艺性状进行系统的观察鉴定。     

大白菜细胞质雄性不育的分子生物学研究

细胞质雄性不育(cytoplasmic male sterility,CMS)是作物杂种优势利用的一 条重要途径。对其机制的研究不仅具有重要的理论意义,而且具有重要的应用价值。对大白菜而言,利用CMS最关键的环节是要育成不育系和其相应的保持系。利用不育系和其它材料杂交产生F1代种子;用不育系和保持系杂交来繁殖不育系;保持系自交繁殖种子。但采用常规的方法来选 育不育系和其相应的保持系周期长、见效慢,远不能满足生产发展的需要。现代生物技术为创造新的不育系和保持系提供了便利。本论文对大白菜细胞质雄性不育的分子机理和 RAPD分析的有关问题进行了研究。     

青花菜两类雄性不育系花器官形态结构的比较

青花菜是一种重要的十字花科蔬菜作物,具有良好的抗癌功效。随着青花菜栽培面积的不断扩大和抗癌药物的开发,对青花菜种子的需求量也不断增加。利用雄性不育系生产青花菜F1杂交种子是其杂种优势利用的重要途径,但常用的细胞质雄性不育系在生产中存在前期死蕾较重、花蜜量少和种子产量不高等问题。本课题组用优良的青花菜自交系为父本,以甘蓝显性细胞核雄性不育材料79-399-3和细胞质不育材料OguraCMSR3629为不育源,通过回交转育的方法获得了多个青花菜显性细胞核雄性不育系和细胞质不育系,为了进一步有效利用其青花菜雄性不育系,寻求更好的不育源。本研究以青花菜高代自交系(保持系)8554、8590、93219及由其经多代回交转育而成的两类雄性不育系为试材,对其死蕾数、花器官形态和蜜蜂访花情况进行了比较研究。结果表明,在花蕾长、花蕾直径、单枝死蕾数、花冠直径、雄蕊长、花瓣长、花药长、蜜蜂访花次数、来访蜜蜂数及访花时间等方面,经相同自交系转育而成的显性细胞核雄性不育系(DGMS)与细胞质雄性不育系(CMS)间的差异有统计学意义,总体表现为DGMS优于CMS。     

叶用芥菜细胞质雄性不育系0912A的胞质效应和杂种优势分析

通过异地多代的育性鉴定及花器形态观察来调查叶用芥菜细胞质雄性不育系0912A的不育性及结籽能力;同时分别以不育系0912A和其保持系0912B为母本,与5个典型叶用芥菜自交系配成10个同核异质的杂交种,以研究hau胞质的胞质效应并测定杂种优势.结果显示:该不育系败育彻底稳定,不育株率和不育度均为100％,有正常的结籽能力;不育系杂种一代与产最相关性状的杂种优势明显,但品质性状无优势;hau胞质在单株重上的负效应较明显,在其他性状上的负效应相对较弱,但hau胞质组合F1的单株重杂种优势依然显著,且胞质效应受杂交父本核影响,通过选择合适的杂交父本配组,细胞质的负效应可以减轻或消除.     

不同倍性不结球白菜Pol CMS及保持系生理生化特性比较

对二、四倍体不结球白菜Pol CMS及其保持系花蕾和薹叶进行生理生化特性比较分析,结果表明:不育系花蕾中可溶性蛋白、可溶性糖、脯氨酸含量均显著低于保持系,且各指标在不育系中四倍体低于二倍体,而在保持系中则四倍体高于二倍体;不育系MDA含量,POD、SOD、CAT活性均高于保持系,不育系中四倍体均高于二倍体.而保持系中四倍体均低于二倍体;薹叶中MDA含量,POD、SOD、CAT活性均为不育系高于保持系,不育系中四倍体高于二倍体;POD、EST同工酶显示不育系与保持系间均具特异酶带,但不同倍性间并无差异.     

大白菜萝卜细胞质雄性不育系RC7的选育及其特性研究

利用甘蓝型油菜萝卜细胞质雄性不育材料RC97-1为不育源,采用种间杂交,将RC97-1的不育性导入大白菜,再通过连续回交转育和严格经济性状选择育成新的大白菜胞质雄性不育系RC7.RC7表现为:蜜腺正常,雌蕊功能健全,能吸引蜜蜂等昆虫正常采蜜传粉;在不同的生态区和播期条件下不育性稳定,不育株率和不育度均达100%,且自然结实性良好,配合力高;高抗霜霉病,抗病毒病和黑斑病;生长整齐,叶色正常,生育期85 d,株高58.6 cm,株幅45.3 cm,叶球纵径和横径分别为48.5和16.3 cm,球形指数2.98,单球质量3.6 kg左右.RC7的一代杂种金秋70和金秋90抗病性强,适应性广,优质、丰产、稳产.因此,RC7是优良的大白菜萝卜细胞质雄性不育系,具有较高的利用价值.     

甘蓝型油菜新型不育系花器特征及mtDNA的RAPD分析

对新选育的甘蓝型油菜PL CMS不育系和Pol CMS及Ogu CMS不育系材料花器形态进行比较,并对3种不育系10份材料mtDNA进行RAPD分析.结果表明,新选育的PL CMS不育系与Pol CMS、Ogu CMS在花瓣长和宽、花冠直径、雄蕊大小等形态特征方面有极显著差异;mtDNA分析结果表明,新发现的细胞质雄性不育材料L04-02A、L04-05A、L04-01A为同一类型的细胞质雄性不育系,它们与Pol CMS及Ogu CMS不育材料有很大差异,由此推知PL CMS不育系是一种新型甘蓝型油菜不育系.     

油菜细胞质雄性不育与育性恢复机理的研究进展

油菜细胞质雄性不育不仅是研究核质互作的理想材料,同时也是杂种优势利用的最有效方式之一。目前对油菜细胞质雄性不育的研究主要包括不育基因的来源、不育基因的结构特征、不育基因的作用机理以及育性恢复的分子机制等。对目前国际上主要的油菜细胞质雄性不育类型(pol CMS、nap CMS、kos CMS、ogu CMS和tour CMS)在分子水平上的研究进展进行了综述。包括线粒体不育基因相关区域的确定和结构特点,不育形成的分子机理以及恢复基因的定位和作用机制等。     

水稻雄性不育系及雄性不育突变体筛选研究进展

 为了获得新细胞质源的水稻雄性不育系及雄性不育突变体,以解决目前杂交水稻生产上所面临的不育系来源单一的问题,人们采用了常规育种、诱变育种、组织培养和离体诱变等方法,其中离体筛选尤显其优势性,但也有不少问题有待解决,如能通过离体培养定向地筛选出雄性不育系及雄性不育突变体,无疑将大大提高工作效率。此外,对影响离体筛选雄性不育突变体的因素进行了探讨。     

Development and utilization of an InDel marker linked to the fertility restorer genes of CMS-D8 and CMS-D2 in cotton

Molecular Biology Reports - The cytoplasmic male sterility (CMS) system is a useful tool for commercial hybrid cotton seed production. Two main CMS systems, CMS-D8 and CMS-D2, have been recognized...     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

利用RNA指纹技术分析大白菜胞质雄性不育相关基因的差异表达

采用改进的RNA指纹技术(RAP-PCR)对两套大白菜胞质雄性不育材料及其杂种F1花蕾的总RNA进行了分析, 通过对186条随机引物的筛选, 获得4个重复性较好的差异片段S47-412, S93-622, S176-343, S199-904, 并对其克隆、测序。根据各差异条带的测序结果合成长引物进行验证, S47, S93所对应的差异消失, 而S176, S199长引物验证的结果与RAP-PCR相似, 序列分析表明: S176-343, S199-904两差异片段均和油菜Polima不育胞质线粒体基因组orf224/atp6位点有着极高的同源性, 两差异片段序列部分重叠, 这说明两差异片段可能和大白菜胞质雄性不育有很高的相关性。     

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理, 将甘蓝型油菜(Brassica napus L.)和白菜(B. campestris L. ssp. chinensis Makino)杂交并连续回交6代获得白菜OguCMS, 在与保持系花药细胞学比较的基础上, 运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片断, 利用RACE克隆得到该片断的cDNA全长, 命名为BcMYBogu(GenBank 登录号: EF127861), 对其氨基酸序列和表达特征进行研究。结果表明, 白菜OguCMS绒毡层在四分体后增生, 高度液泡化, 导致小孢子花粉外壁异常, 细胞质同外壁分离并降解; 花药变白; BcMYBogu具有典型的MYB DNA结合域—W残基和SH[AL]QKY[RF]基序; 系统进化分析显示BcMYBogu与AtMYB26, AtMYB32和AtMYB4等聚类在同一分枝; RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达, 但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。     

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F₁s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Plant regeneration from protoplasts of cytoplasmic male sterile lines of rice (Oryza sativa L.)

This study compared plant regeneration from protoplasts isolated from suspension cultures of threeJaponica rice (Oryza sativa L.) lines with different male sterile cytoplasms. More than 180 green plants were regenerated from protoplasts from 5–8 month old suspensions of IR58024A, a line with the WA type of cytoplasmic male sterility (CMS). About 40% of the calli recovered from protoplasts produced green plants. ShuangbaiA (BT type of CMS) and Tai2A (Dian I type of CMS), both from Zhejiang province of China, responded less well in culture. ShuangbaiA produced green plants from 6.6% of calli, although initial protoplast yield per gram fresh weight was higher than for IR58024A. Tai2A showed lower protoplast yield, and only 1.1% of the calli produced green plants. Flow cytometric analyses of nuclear DNA content indicated that many of the regenerated plants were tetraploid. The percentage of tetraploids varied in the different lines. The male sterile characteristics of the original lines were maintained in the regenerated plants. Pollen abortion occured earliest in IR58024A and latest in Tai2A. IR58024A is a promising rice genotype for use as a recipient in direct gene transfer experiments.Abbreviations BAP 6-benzylaminopurine - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxyacetic acid - IRRI International Rice Research Institute - LS Linsmaier and Skoog's (1965) medium - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - WA wild abortive