滇型水稻细胞质雄性不育恢复基因Rf-D1(t)克隆与遗传特性分析

CMS/Rf测交F1结实率与花粉可育率检测表明,Ansanbyeo和南34对三种不同细胞质来源的粳稻不育系(CMS-DT1,CMS-BT和CMS-HL)都有较强的恢复力.根据已公布的水稻基因组序列,在水稻恢复基因定位区段内设计引物,确定出一对与两个滇型CMS的恢复系Ansanbyeo和南34恢复基因紧密连锁的分子标记引物M43804和M43558,首次克隆了滇型恢复基因Rf-D1(t),编码16个PPR蛋白.序列比对表明,该基因与BT型恢复基因Rf-1相似度达99%,两个恢复基因之间在ORF区仅存在一个碱基差异,即Rf-D1(t)第1 149 bp位的A变成Rf-1的C,并产生一个氨基酸的改编即K(赖氨酸)变成N(天冬酰胺).该差异处于一个PPR结构域中,暗示该氨基酸是恢复基因的关键位点之一,它的改变可能影响对水稻CMS育性恢复力的变化.     

滇型水稻细胞质雄性不育恢复基因Rf-D1（t）的克隆与遗传特性分析

CMS/Rf测交F1结实率与花粉可育率检测表明,Ansanbyeo和南34对三种不同细胞质来源的粳稻不育系(CMS-DT1,CMS-BT和CMS-HL)都有较强的恢复力。根据已公布的水稻基因组序列,在水稻恢复基因定位区段内设计引物,确定出一对与两个滇型CMS的恢复系Ansanbyeo和南34恢复基因紧密连锁的分子标记引物M43804和M43558,首次克隆了滇型恢复基因Rf-D1(t),编码16个PPR蛋白。序列比对表明,该基因与BT型恢复基因Rf-1相似度达99%,两个恢复基因之间在ORF区仅存在一个碱基差异,即Rf-D1(t)第1149bp位的A变成Rf-1的C,并产生一个氨基酸的改编即K(赖氨酸)变成N(天冬酰胺)。该差异处于一个PPR结构域中,暗示该氨基酸是恢复基因的关键位点之一,它的改变可能影响对水稻CMS育性恢复力的变化。     

海拔及细胞质背景对水稻分离群体Rf-1位点基因型频率的影响

水稻的演化及水稻细胞质雄性不育育性恢复基因的种质分布与地理分布关系密切。Rf-1是水稻克隆唯一的育性恢复基因。为研究海拔和籼粳细胞质因素对水稻分离群体的Rf-1位点基因型频率的效应,本研究用两个云南高海拔地方粳稻品种与具籼稻细胞质和恢复基因的偏粳品系南34正反交,在4个海拔点种植F1与育性稳定的不育系测交获得测交群体,并自交产生F2群体;同时在室内将经17℃和14℃温度处理正处于开花期的F1稻穗与不育系授粉获得测交群体。然后对分离群体用位于Rf-1位点的PCR标记M45461进行基因型频率分析。结果显示:在总的25分离群体中,有11个具有由南34提供籼稻细胞质,14具有由云南高海拔粳稻提供的粳稻细胞质;在所有分离群体中,14个发生遗传偏分离。在14个以粳稻细胞质为背景分离群体中仅有4个发生遗传偏分离,但在11个具有由南34提供的籼稻细胞质背景的分离群体中有10个发生遗传偏离,而且都偏向南34,偏向南34基因型的频率基本是随海拔的升高而增加。结果说明海拔、温度和细胞质背景均对分离群体Rf-1位点的基因型频率具有影响,而且达到显著或极显著水平;细胞质背景对雄配子在Rf-1位点的基因型具极大的选择效应。本研究可为探讨水稻恢复基因与水稻生态适应性的关系及恢复基因与水稻的分化关系提供理论参考。     

滇1型杂交水稻质核雄性不育恢复基因的研究

用混合品系分析方法(BLA)，在滇1型杂交水稻的保持系和恢复系中的DNA各取20个样混合，对OSR—33引物进行筛选，结果发现OSR—33引物在保持系和恢复系中有遗传差异。然后又用这对引物分别对保持系和恢复系的单株DNA样品进行扩增。另外，用滇1型杂交粳稻的保持系和恢复系与不育系进行测交，杂种F1花粉的育性经1％的I—KI染色。用扩增出的带型与F1花粉的育性进行相关分析，结果表明扩增出带型与花粉的育性呈显著正相关，两种带型对应的花粉可育率均值经t测验差异极显著，表明OSR—33标记与滇1型细胞质雄性不育育性恢复基因连锁，由于OSR—33引物是设计在第10染色体长臂的中部，所以，滇1型细胞质雄性不育育性恢复的一个基因可初步定位于第10染色体长臂的中部。     

几种小麦雄性不育系及其保持系、恢复系的种子高分子量谷蛋白亚基比较研究

为了阐明同核不同细胞质的小麦雄性不育系及其保持系、恢复系之间在种子高分子量谷蛋白亚基异同,通过SDS-聚丙烯酰胺凝胶(SDS-PAGE)对T型、Q型、AL型3种小麦雄性不育系及其保持系、恢复系共42个小麦种质系的高分子量谷蛋白亚基(HMW-GS)进行分析.结果表明,保持系绵阳92-8与其3个不育系(T A92-8、Q A92-8、AL92-8)在Glu-A 1和Glu-B 1位点上没有明显差异,但在Glu-D 1位点彼此有显著差异.T型、Q型、AL型不育胞质彼此有一定的差异,不育系与其核供体保持系之间是否有差异,主要取决于特定核质互作关系.恢复系和保持系在谷蛋白亚基组成上也存在一定的差异,主要表现在Glu-A 1位点恢复系多数无带,保持系一般有谱带2*；在Glu-B 1位点,恢复系谱带6、8频率高,谱带7频率低,而保持系相反,即谱带6、8频率低,谱带7频率高.此外,在本研究供试材料中保持系有较多的优质亚基组合,而恢复系有较少的优质亚基组合,这说明恢复系品质不够优良,在以后的改良中需要加强品质选育工作.本研究为T型、Q型、AL型不育系的区分提供了一个有效的方法,对小麦胞质雄性不育性的遗传与生理机制研究有一定的意义.     

云南地方老品种水稻中恢复基因位点遗传差异分析

目前生产上使用的水稻恢复系之间的遗传基础过于狭窄,应积极开发水稻新恢复系的选育工作。云南地理气候条件特殊,地方老品种资源丰富,是筛选新恢复系的理想材料。以61份云南地方老品种水稻资源为父本,通过测交筛选到6份可以恢复野败型不育系的品种。再利用5对特异引物,对这6份恢复材料和7份恢复系的恢复基因位点遗传差异进行分析。结果表明：5对特异引物对6份恢复材料的扩增结果各不一样,而与对照恢复系仅有2.8 kb的一条扩增带型一致。说明云南地方老品种水稻中蕴藏的恢复基因与目前生产上应用的恢复基因的亲缘关系较远。从云南地方老品种水稻中发掘利用新型恢复基因和资源,是突破水稻恢复基因遗传基础狭窄、筛选新质源恢复系的一种有效策略。     

中国西双版纳水稻老品种在育性恢复基因Rf-1位点的遗传多样性

云南西双版纳素有"物种基因库"的美称,是稻种资源的多样性中心区和优异种质富集区之一.本研究利用8个位于Rf-1位点的PCR分子标记对53份西双版纳地方籼稻、粳稻和光壳稻种质资源的遗传多样性和遗传分化进行了分析,共检测到15种多态性扩增片段.Shannon's信息指数和Nei'S遗传多样性指数分析表明西双版纳水稻地方品种在Rf-1位点具有较高的遗传多样性,这些遗传变异主要来自于不同品种之间的遗传差异.分析表明西双版纳籼稻的遗传多样性要显著高于粳稻和光壳稻.结果还表明籼稻与粳稻和光壳稻之间的分化要显著大于粳稻与光壳稻之间的分化,光壳稻与粳稻的亲缘关系更近.这一结果可能表明水稻Rf-1位点的遗传分化与水稻的籼粳分化紧密相关,还暗示水稻Rf-1位点遗传分化可能是与籼粳分化同时进行的.     

三种水稻胞质雄性不育恢复基因的比较

用籼型强恢复系密阳46、H804,含广亲和基因S5n的粳型恢复系02428、T984及H921分别与三种水稻雄性不育胞质野败(CMS-WA),矮败(CMS-DA),印尼水田谷败育(暂名CMS-IA)不育系测交,分析各组合F1、F2及BCF1代的育性表现以研究雄性不育胞质恢复性的遗传。密阳46和H804对CMS-WA、CMS-DA、CMS-IA的恢复性均由两对主效恢复基因控制。T984及H921对野败、矮败和印尼水田谷败育均含有一对主效恢复基因。02428对野败和印尼水田谷不育胞质不含恢复基因,对矮败可能含一对弱恢复基因。对矮败胞质雄性不育的恢复基因作了讨论,推测存在强弱差异的可能。籼粳杂种后代育性表现较品种间组合育性表现更易受环境影响。     

利用PCR标记对恢复水稻BT型胞质雄不育性的核基因Rf-1进行遗传制图

Rf-1基因能恢复盯型雄性不育细胞质引起的花粉不育，Rf-1基因所在的第10染色体上的9个限制性片段长度多态性(RFLP)标记被转换成PCR标记。利用9个标记和1042株组成的分离群体进行遗传分析。结果发现，Rf-1基因位于S12564 Tsp5091和C1361 MwoI之间。两个标记位点的遗传距离估计为0．3cM。     

滇-型杂交水稻质核雄性不育恢复基因的SSR分子标记

用混合品集分析方法(BLA),在保持系和恢复系中的DNA各取20个样混合,对PMRF、OSR-33、RM294、RG304、RG257和RM228六对引物进行筛选,结果发现PMRF、OSR-33和RM228三对SSR引物在保持系和恢复系的混合之间有遗传差异.用这三对引物分别对不同的40个保持系和60个恢复系DNA样品进行扩增.另外,用滇-型杂交粳稻的保持系40份和恢复系60份与不育系进行测交,杂种F1花粉的育性经1%的I-KI染色,其花粉的可育率有高有低.用扩增出的带型与F1花粉的育性进行相关分析,结果表明扩增出带型与花粉的育性呈显著正相关,两种带型对应的花粉可育率均值经t测验差异极显著,表明PMRF、OSR-33和RM228标记与滇-型细胞质雄性不育育性恢复基因连锁,并且PMRF、OSR-33sSR和RM228引物是设计在第10染色体长臂的中部,所以,滇-型细胞质雄性不育育性恢复的一个基因可初步定位于第10染色体长臂的中部.     

Identification of restorers and maintainers for developing basmati and non-basmati hybrids in rice, Oryza sativa

Twenty‐seven improved aromatic lines of germplasm and 18 non‐aromatic disease‐resistant genotypes of rice were test‐crossed with four cytoplasmic male‐sterile lines (IR 58025A, IR 62829A, PMS 3A and PMS 10A). Thirteen aromatic and 10 non‐aromatic genotypes were selected based on pollen fertility, and crosses were repeated to confirm sterility‐maintaining and fertility‐restoring ability. Genotypes were categorized as effective restorers (> 80% spikelet fertility), partial restorers (21‐79% spikelet fertility) and maintainers (< 1% spikelet fertility). The effective basmati restorers identified were Basmati 385, Chandan, P1031‐8‐5‐1, HKR 241‐IET‐12020, SAF Khalsa 7 and Karnal Local. The basmati maintainers identified were Basmati 370, Pusa basmati 1, P615‐K‐167‐13 and P1173‐4‐1. The frequency of restorers obtained was higher for the non‐aromatic than the aromatic basmati type. The performance of restorers varied with cytoplasmic male‐sterile (CMS) line, location and season of testing. The differential ability to restore fertility in the CMS lines that have the wild abortive (WA) cytosterile system could result from different nuclear backgrounds of the CMS lines. These restorers and maintainers possess acceptable grain dimensions, a desirable degree of aroma, volume expansion through linear kernel elongation and cooking quality characteristics of basmati rice. These genotypes will contribute to developing basmati hybrids and provide restorers and maintainers with acceptable key basmati quality characteristics.     

几个水稻不育系对白叶枯病的抗性

测定了８对不育系（Ａ）和保持系（Ｂ）以及一些杂交稻组合对６个小种的４０株白叶枯病菌的抗性，比较了不同类型不育系对６个小种群的抗性差异。结果表明，冈型、印尼水田谷型和大多数野败型不育系的抗性很差，ＢＴ型的抗性较高。菌株Ａｈ２８对冈４６Ａ和野败珍汕９７Ａ的致病力强。比较了６个恢复系（Ｃ）和８个杂交组合对不同小种的抗性程度。结果显示，杂交稻的抗病性主要受父本（恢复系）核基因的控制，但同时又受不育胞质的影     

甘蓝型油菜双低萝卜质不育恢复系快速改良技术研究

通过采取不同的常规育种策略和小孢子培养、分子标记辅助选择等现代生物技术,研究了提高甘蓝型油菜萝卜质不育恢复系改良效率的方法。结果表明,在甘蓝型油菜萝卜质不育恢复系杂交转育过程中,采用杂交后代与萝卜质不育系杂交,可使杂交后代分离群体中不具备恢复基因的单株在花期表现出来,无须测交即可进行选择;杂交后代通过小孢子培养,可将稳定纯合时间由传统自交8个世代缩短到3个世代;通过萝卜特异分子标记辅助选择可大大提高选择的精度,减少筛选所需的人力、土地和时间,选择效率提高90%。试验筛选出19个在萝卜质不育恢复系中能够扩增出单一、易于辨别的谱带的SCAR标记引物,其中10个引物在现有的甘蓝型油菜萝卜质不育恢复系中均能检测出特异标记。筛选出的甘蓝型油菜萝卜质不育恢复系的特异分子标记与前人研究存在差异,标记相似系数为48.28%~93.10%。通过分子标记无法区分高硫甙和低硫甙恢复系,选育低硫甙萝卜质不育恢复系还需要借助品质分析仪。成功筛选得到6个甘蓝型油菜低芥酸、低硫甙、抗寒、结角正常、农艺性状优良的萝卜质不育恢复系,为将甘蓝型油菜萝卜质不育杂种优势应用于我国油菜生产奠定了基础。     

滇Ⅰ型粳稻不育系花粉败育状况分类研究

对供试的114个滇I型粳稻不育系花粉镜检，结果表明：滇I型不育系的大部分材料中均有染败花粉粒，但染败花粉粒所占比率因保持系来源的不同而有差异。其中有23份材料的花粉粒全部为圆败和碘败，这些材料以圈败花粉粒所占的比率较大，圆败花粉率占50％以上的有16份，占无染败花粉材料的69．56％，滇I型不育系中未发现全部为碘败花粉的材料。用败育程度较高(花粉粒全部为圆败和碘败)的不育系材料分别与南29和南34测交，杂交组合育性均可恢复，南29和南34对败育程度较高的滇I型不育系仍具有恢复力。     

Use of androgenesis in intersubspecific hybrid rice breeding

Significant differences of callus induction, green plant regeneration and culture efficiency were observed among restorer lines and their hybrids in rice. Average callus induction percentage of F₁, hybrids was about twice that of their parents. Twenty doubled haploid (DH) lines that showed normal fertility with both indica and japonica CMS lines, were selected as widely compatible restorers (WCRs). TG8 derived from CPSLO17/Minghui 63 not only showed normal fertility to both indica and japonica test varieties and CMS lines, but also exhibited superior agronomic traits, in particular short plant, desired plant type, high tillerability, large panicles, good grain quality and resistance to rice blast disease. Strong heterosis for yield was observed in crosses between indica or japonica CMS lines and TG8. TG8 possessed a dominant, widely compatible gene (WCG) that was inherited from the variety CPSLO17. Results confirmed that the anther culture technique is a quick and effective way of developing widely compatible restorers in rice and that it is applicable to the direct use of intersubspecific heterosis with the three-line method.     

红色稻米亚种间杂种优势利用途径的探讨

有色稻米优良的营养品质和特异性状与低产的突出矛盾一直制约着其生产的发展，杂种优质利用是解决这一问题的首要措施。无论“三系法”还是“两系法”，有色稻米亚种间杂种优势利用的前提必须保证亲本之一携有广亲和基因有色基因，以利用有色基因的显性遗传本质；同时又要克服双亲间的杂交不亲和性和目标性状的负向优势。     

Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer

Cytoplasmic male sterility of Dian‐type 1 (CMS‐D1) was developed 30 years ago in Yunnan. A major gene conferring fertility restoration for the CMS‐D1 system was detected by microsatellite markers in advanced inbred lines consisting of 196 maintainers and 62 restorers developed in breeding programmes of hybrid rice involving the CMS‐D1 system. The gene was mapped between two simple sequence repeat markers, OSR33 and RM228, on chromosome 10, and was temporarily designated as Rf‐D1(t). The genetic distances of the gene to the two microsatellite markers were 3.4 and 5.0 cM, respectively. This linkage was confirmed by using an F2 population derived from a cross between a CMS‐D1 line and a restorer. This study also demonstrated that using OSR33 was reliable and efficient for identification of restoring lines in hybrid rice breeding with the CMS‐D1 system.     

Identification and transfer of a new cytoplasmic male sterility source from Oryza perennis into indica rice (O. sativa)

Summary Most of the commercial hybrids of indica rice are based on wild abortive (WA) source of cytoplasmic-genetic male sterility (CMS). Such cytoplasmic uniformity may lead to genetic vulnerability to disease and insect pests. To overcome this problem, diversification of CMS sources is essential. Crosses of 46 accessions of O. perennis and two accessions of O. rufipogon as female parents were made with two restorers (IR54, IR64) of WA cytosterility. Sterile hybrids were backcrossed with the respective recurrent parents. Of all the backcross derivatives, one line having the cytoplasm of O. perennis Acc 104823 and the nuclear background of IR64 was found to be stable for male sterility. The newly developed CMS line has been designated as IR66707A. This line is completely sterile (0% seed set) under selfed conditions. Crosses of IR66707A with 10 restorers of WA cytoplasm showed almost complete (93–100%) pollen sterility, indicating that the male sterility source of IR66707A is different from WA sterility. Southern hybridization of IR66707A, O. perennis (cytoplasmic donor), IR66707B (maintainer) and V20A (WA cytoplasm) using mitochondrial DNA specific probes (5 endonucleases × 8 probes) showed identical banding patterns between IR66707A and O. perennis. However, in more than half of the combinations, different banding patterns were observed between IR66707A and IR66707B and between IR66707A and V20A. The results suggest that IR66707A has the same cytoplasm as the donor (O. perennis), and CMS may not be caused by any major rearrangement or modification of mtDNA. The new CMS source identified will be useful in cytoplasmic diversification in hybrid rice breeding.     

Establishment of a random-mating population of 'Polima' CMS restorers in Brassica napus by use of dominant genie male sterility

Five restorers of ‘polima’ cytoplasmic male sterility (pol CMS) cannot restore the fertility in dominant genie male sterility (DGMS). A dominant male sterility gene from both, a DGMS line Rs l046AB and DGMS hybrid ‘Zhongza No. 3’, was successfully introduced into Polima cytoplasm. A random-mating population of pol CMS restorers was established by using many double-low pol CMS restorers as pollinators to cross continuously to the DGMS plants which had Polima cytoplasm.     

Identification and characterization of new sources of cytoplasmic male sterility in rice

Search for male sterility-inducing cytoplasm in wild species of the genus Oryza was attempted with a view to diversify the base of the cytoplasmic genetic male sterility system currently used in the development of commercial rice hybrids. A total of 132 interspecific crosses were made involving accessions of four wild and two cultivated species, all belonging to the A genome. Wild accessions possessing sterility-inducing cytoplasms were identified following reciprocal and sterile F₂ backcross methods. Sterile segregants were pursued through substitution backcrosses to develop cytoplasmic male-sterile (CMS) lines. CMS lines were developed with the cytoplasm of either O. rufipogon (VNI) or O. nivara (DRW 21018, DRW 21001, DRW 21039, DRW 21030 and RPW 21111). Based on shape, staining, and abortive pattern of pollen and also on type of interaction with a set of restorers and maintainers for known cytoplasmic male steriles of WA source (V 20A), the newly-developed CMS lines were grouped into four classes. Of these, RPMS 1 and RPMS 2 showed gametophytic male sterility with a restorer reaction different from WA cytoplasmic male-sterile stocks.