DNA methylation affected by male sterile cytoplasm in rice (Oryza sativa L.)

Male sterile cytoplasm plays an important role in hybrid rice, and cytoplasmic effects are sufficiently documented. However, no reports are available on DNA methylation affected by male sterile cytoplasm in hybrid rice. We used a methylation-sensitive amplified polymorphism technique to characterize DNA methylation in four male sterile cytoplasms that are widely commercialized in China. In total, 12 pairs of selective primers in combinations of EcoRI and MspI/HpaII amplified 350 bands among four male sterile (A) lines and the corresponding maintainer (B) lines. Sites b1 and b3 were fully methylated only in all the B lines, while b2 was fully methylated only in all the A lines. These results implied a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A and B lines was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. WA-type and Yinshui-type cytoplasms affected the methylation to a much greater degree than G-type and D-type cytoplasms, as indicated by the number and degree of methylated sites, ratio of methylated sites, number of fully methylated sites, ratio of fully methylated sites, and polymorphism between A and B lines for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the WA-type is much greater than for G- and D-types because the former is between wild and cultivated species and the latter is within indica subspecies between African and Asian cultivars. This difference in genetic distance may be responsible for the variation in methylation which we observed.     

小麦Ven型胞质雄性不育植株与可育植株花药蛋白质组差异研究

普通小麦具有偏凸山羊草（Ae. ventricosa）细胞质的不育系为Ven型胞质雄性不育系（Ven cytoplasmic male sterility, Ven CMS）,是粘类小麦CMS的一种类型。该研究对小麦Ven型雄性不育系冀5418A及其同型保持系冀5419B的单核期和二核期的花药进行差异蛋白质组学分析,探讨小麦质核互作雄性不育的分子机制。通过双向电泳分离花药蛋白,基质辅助激光解析飞行时间串联质谱（MALDI TOF TOF）对差异表达蛋白进行质谱鉴定,利用生物信息学进行差异表达蛋白鉴定和功能注释分析。结果表明,在分子量19.0～100.0 kD、等电点4～7线性范围内,共检测到约2 000个蛋白点。2个时期共检测到差异蛋白98个,其中两个时期差异表达变化一致的蛋白点56个;数据库搜索获得鉴定的蛋白点41个,其中18个蛋白的表达量在冀5418A 中显著下调,23个在冀5418B 中明显下调。在不育系和可育系中均有参与能量代谢、活性氧代谢、核糖体合成、花粉物质合成的差异蛋白。GO分析预测差异蛋白生物学过程多涉及电子传递和能量代谢、核糖体代谢、活性氧代谢等,细胞组成主要是在膜区域和线粒体,分子功能主要是DNA和RNA结合功能和水解酶等。KEGG分析表明,较多蛋白分布于碳水化合物代谢、活性氧代谢和蛋白组装和折叠途径。推测不育系冀5418A 的雄性不育性除了涉及能量代谢、活性氧清除过程,核糖体蛋白、伴侣蛋白等也有重要作用,雄性不育性可能还与蛋白质加工、物质合成过程的紊乱有关。     

DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions

Summary Mitochondrial and chloroplast DNA was isolated from fertile and cytoplasmic male sterile cultivars of cultivated onions. Restriction fragment length polymorphism led to the distinction between cytoplasms S and M. Mitochondrial DNA patterns from S cytoplasms appeared dentical and characterized mostly male sterile lines. An open-pollinated variety was found to bear this cytoplasm and thought to be the origin of S types. Mitochondrial DNA patterns from M cytoplasms were subdivided into four types, M₁ and M₂ corresponding to normal N cytoplasm, M₃ and M₄ probably corresponding to T cytoplasms. S and M cytoplasms were also distinguished by chloroplast DNA restriction patterns. Our results confirm previous genetic distinction between S, N and T cytoplasms.     

Basic studies on hybrid wheat breeding

Summary The nuclei of 12 common wheats (genome constitution AABBDD) were placed into the cytoplasms of Aegilops kotschyi and Ae. variabilis (both C^uC^uS^vS^v) by repeated backcrosses. Using these nucleus-cytoplasm hybrids, male sterility-fertility restoration relationship was investigated. Male sterility was expressed by these cytoplasms only in Slm, Splt and Mch. The other nine common wheat nuclei gave normal fertility against these cytoplasms. These cytoplasms were compared with the Triticum timopheevi cytoplasm that is now widely used in the hybrid wheat breeding program in order to investigate their effects on important agronomic traits of the 12 common wheats: The kotschyi and variabilis cytoplasms were as good as the timopheevi cytoplasm in this respect.The F₁ hybrid between (kotschyi)- or (variabilis)-Splt and CS showed normal fertility. Segregation of the fertiles and steriles in their F₂ generations followed the simple Mendelian fashion, i.e., 3 fertile1 sterile. Thus, the fertility restoration in this case is mainly controlled by a single dominant gene which will be designated as Rfv1. To determine its location, ditelo-lBS and -lBL of CS were crossed as male parents to male sterile (kotschyi)- and (variabilis)-Splt. The F₁ hybrids between the male sterile Spit's and CS ditelo-lBS became male fertile, while those between the male sterile Spit's and CS ditelo-lBL became completely male sterile. Thus, the location of the gene Rfv1 has been determined to be on the short arm of chromosome lB of CS. Furthermore, a close relationship between the fertility-restoring genes and the nucleolus organizer region was pointed out.Finally, the schemes of breeding the male sterile lines of a cultivar with these cytoplasms, and its maintainer line were formulated. The following two points were considered as the advantages of the present male sterility-fertility restoration system over that using the timopheevi cytoplasm in breeding hybrid wheat: (1) easier fertility restoration in F₁ hybrids, and (2) no need of breeding the restorer line.This work was supported by a Grand-in-Aid from the Ministry of Education, No. 386002. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 420.     

几类异质易恢复小麦雄性不育系杂种籽粒品质研究初报

利用具有粘果出羊草（Ae.kotschyi),易变山羊草（Ae.variabilis),偏凸山羊草（Ae.ventricosa)和二角山羊草（Ae.bicornis)异源细胞质小麦雄性不育系（以下简称粘,易,偏和二角型）组配成不同组合的杂种小麦,研究这4种不育系对杂种小麦籽粒品质的影响。结果表明,由它们所配制的杂种小麦在蛋白质含量,湿面筋含量,沉淀值等性状上均高于具有普通小麦细胞质的相同核型杂种小麦,尤其粘,易型某些组合在湿面筋含量,沉淀值两性状上差异达到显著水平。因此,利用这4种不育系很有希望培育出优势,高产的杂种小麦。     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

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.     

小麦D^2型与K型,V型,T型细胞质雄性不育系线粒体DNA的RAPD比较分析

采用ＲＡＰＤ方法比较新育成的小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）Ｄ２型不育系ｍｓＤ２ＣＡ８０５７与Ｋ型、Ｖ型和Ｔ型细胞质雄性不育系的线粒体ＤＮＡ（ｍｔＤＮＡ）。结果表明,ｍｓＤ２ＣＡ８０５７的ｍｔＤＮＡ不同于其它３种类型不育系,而其它类型不育系的ｍｔＤＮＡ彼此也各不相同。这一结果从分子水平上为鉴定该Ｄ２不育系的不育类型提供了证据。实验还发现在胞质来源相同而核背景不同的Ｔ型不育系间存在线粒体基因组多态性,这是关于小麦中Ｔ型不育系ｍｔＤＮＡ在常规回交转育过程中发生变异的直接证据。这种变异在很大程度上可能是受不同核背景影响的结果。     

RAPD Comparison Study on Mitochondrial DNA of the D2 Cytoplasmic Male Sterile (CMS) Line with K-, V-and T-type CMS Lines in Wheat

A new line of cytoplasmic male sterile (CMS) wheat ( Triticum aestivum L. ) named msD2-CA8057 (briefly as D:) was compared with K-, V- and T-type CMS lines by mitochondrial DNA (mtDNA) RAPD analysis. It was revealed that the mtDNA of this D2 line was different from that of the other three types although differences were also found between the K-, V- and T-type lines themselves. This result provided some evidences at the molecular level for the identification of the cytoplasm of this D2 line as a new type of CMS line. In the experiment the authors also found polymorphism between two CMS lines, which had the same source of T-type cytoplasm but had different nuclear background. This was the first time to provide direct evidence about the mtDNA mutation in T-type CMS wheat lines during the routine backcross process. Such changes are most likely resulted from the influence of different nuclear background.     

重庆温光型雄性不育小麦的染色体分析

本研究采用种子醇溶蛋白酸性聚丙烯酰胺凝胶电泳（APAGE）和染色体C分带技术对重庆温光型雄性不育小麦的染色体组成进行分析,结果表明：不育系染色体1B短臂（1BS）已被黑麦1R短臂（1RS）取代,具有1RS/1BL易位染色体及普通小麦细胞质。 Abstract:Seed gliadin acid-polyacrlamide-gel-electrophoresis(APAGE) and chromosome C-banding techniques were used to identify chromosome constitution of Chongqing thermo-photo sensitive male sterile wheat.Results showed that 1BS of the male sterile lines was substituted by 1RS of rye.They were 1B/1R wheat with Triticum aestivum cytoplasm.     

水稻7种不同细胞质源滇型不育系间恢保关系研究

本研究选用7种细胞质源不同的滇型不育系为母本,10个常用品种或品系为父本,得到70个杂交组合.通过分析70个杂交后代的花粉可育度和自然结实率可知,这7个细胞质源不同的滇型不育系之间的保持关系基本一致;5个滇一型恢复系对这7个不育系的恢复能力差异较大.鉴于7种不同细胞质源不育系的保持关系相同,使转育同核异质不育系成为可能,从而为实现滇型杂交水稻细胞质多样化提供了实验依据.     

Mitochondrial DNA modifications associated with cytoplasmic male sterility in rice

Summary Mitochondrial DNA was isolated from fertile and cytoplasmic male sterile lines of rice. Restriction analysis showed specific modifications in the male sterile cytoplasm. In addition to the major mitochondrial DNA, three small plasmid-like DNA molecules were detected by agarose gel electrophoresis in both cytoplasms. An additional molecule was specifically found in the sterile cytoplasm. These mitochondrial DNA modifications support the hypothesis of the mitochondrial inheritance of the cytoplasmic male sterility in rice.     

水稻CMS不育系细胞质遗传效应的研究进展

在三系杂交水稻生产中,不育系的细胞质对杂种一代的表现具有一定的效应。同一种不育细胞质对不同性状的效应是不同的;不同的不育细胞质对同一性状的效应也存在着差异;且对大多数性状表现为负向效应。可通过扩大不育细胞质源以选用优质细胞质或选用强优恢复系来减少或消除其负效应,培育同核异质的多胞质不育系来适应杂交水稻生产的需要。     

Variable presence of the 1.94kb mitochondrial plasmid in maize S cytoplasm and its relationship to cytoplasmic male sterility

Mitochondrial DNA (mtDNA) was isolated from over 100 different maize nucleo-cytoplasmic combinations. DNA preparations were assayed for the presence of the 1.94kb mitochondrial plasmid by agarose gel electrophoresis and hybridization to a recombinant clone of the plasmid. The plasmid was present in all tested inbreds which carried N, male fertile, cytoplasm or the cytoplasmically male sterile (cms) groups,cms-T andcms-C. However, members of thecms-S group differed with respect to the presence of the plasmid. Cytoplasms I, J and S possessed the plasmid, whereas cytoplasms B, CA, D, G, H, IA, ME, ML, PS, RD and VG did not.Cms-S group lines which had spontaneously reverted to fertility (nuclear and cytoplasmic revertants) did not exhibit a concomitant change in 1.94kb plasmid levels, although all such lines showed the previously reported alteration in levels of the linear mtDNAs, S1 and S2. The presence or absence of the plasmid was not correlated with (i) frequency of reversion to fertility, (ii) the degree of male sterility expressed, (iii) the presence or absence of standard nuclear restorer to fertility genes and (iv) nuclear genotype. Latin American races carrying RU cytoplasm possessed the plasmid, as did sweet corn varieties. The relevance of the data tocms and evolution of thecms-S group is discussed.     

Efficiency of one- and two-stage selection indexes for improvement of net economic merit in White Leghorns

Summary Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet lines carrying normal and introduced sources of male sterile cytoplasms have been characterized and compared on the basis of restriction enzyme analysis. Normal cytoplasm was shown to contain mt and ctDNAs which differed from those of the male sterile cytoplasms examined in the present investigation. On the other hand, four groups of male sterile cytoplasms could be differentiated by their own characteristic mtDNA digest patterns, while two were separated by ctDNA comparisons. In addition, a greater degree of variability of the mitochondrial genome is suggested. Our results also imply strict maternal inheritance of mt and ctDNAs. Thus, the organelle DNA assay provides a positive and alternative means of identifying various male sterile cytoplasms.     

Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf₁ is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf₆, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf₆ was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf₆ was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes.     

三例小麦细胞质雄性不育系线粒体DNA的扩增片段长度多态性分析

细胞质雄性不育是小麦杂种优势利用的重要途径,为了鉴定3例小麦雄性不育系的细胞质类型,对其线粒体DNA(mtDNA)进行扩增片段长度多态性(Amplified fragment length polymorphism,AFLP)分析。文中利用差速离心法和不连续蔗糖密度梯度超速离心法提取纯化小麦线粒体。结果表明:通过该提取方法获得的mtDNA,其质量和纯度能够满足PCR反应和遗传学分析。在64对选扩引物中,筛选到了4对特异性引物,其中引物E1/M7在ms(Kots)-90-110不育系扩增出3条特异条带;引物E4/M2在ms(Ven)-90-110不育系扩增出2条特异条带;引物E7/M6在ms(S)-90-110不育系中扩增出2条特异条带;引物E6/M4在ms(Kots)-90-110不育系中扩增出2条特异条带。这些特异引物可以用来作为鉴定具有粘果山羊草Aegilops kotschyi、偏凸山羊草Ae.ventricosa、斯卑尔脱小麦Triticum spelta 3类不育细胞质型小麦雄性不育系的细胞质分子标记,为研究小麦细胞质雄性不育机理奠定了分子基础。     

矮败蓝标型小麦不育系的选育与研究

利用蓝粒太谷核不育硬粒小麦89-2343[AABB 4D(MS2)/4E]与普通小麦7739-3(2n=42)杂交、回交所产生的蓝粒可育株与白粒矮败材料杂交、回交,育成了一份矮败蓝粒小麦.选用13份遗传背景不同的白粒普通小麦与之杂交、回交,育成了13份矮败蓝粒小麦.对后代的粒色和育性分离进行分析,蓝粒矮败不育株占22.1%,白粒非矮秆可育株占77.7%,表明蓝粒基因、Ms2和Rht10均位于附加染色体上,且连锁紧密;但不同轮回亲本,矮败蓝粒的传递率有差异,477A的传递率最高,接近50%.细胞学分析表明矮败蓝粒小麦仍为单体附加系;探讨了矮败蓝粒小麦在群体改良和杂种小麦生产中的应用.