Genetics of Fertility Restoration in Cytoplasmic Male Sterile Pepper

Pepper hybrid seeds production using male sterility could lower cost by reducing time and labour, and increase thegenetic purity of the F1 seeds. To investigate the genetics of fertility restoration of the Peterson cytoplasmic sterility inpepper, a doubled haploid population of 115 pepper lines obtained from anther culture of the F1 hybrid between YoloWonder (sterility maintainer line) and Perennial (fertility restorer line) and the parental lines were test-crossed by 77013A(a strict cytoplasmic-genic male sterile line). The fertility of the test-crossed lines was assessed in greenhouse and openfield with the following three criteria: pollen index (PI, visual estimation of pollen amount per flower), pollen number (PN,pollen counting under microscope), and seed number (SN, the number of seeds per fruit in open pollination). Correlationsbetween the each couple of criteria within, as well as between the cultivation methods ranged from 0.55 to 0.84. Analysisof variance showed that the genotype (DH line) and environment were the significant sources of variation of the fertility.Narrow sense of heritance of fertility restoration ranged from 0.38 to 0.92, depending on the criteria and environment. Thedistribution of the progeny was continuous between the parental genotypes indicating the quantitative inheritance offertility restoration. Inferred from segregation according to Snape et al.(1984), the number of segregating genes wasestimated to be that three to four genetic factors were involved in pollen traits (PI and PN) and five to eight genetic factorsin seed production (SN). The heredity analysis of the CMS will be helpful for understanding of the genetic mechanism ofthe fertility restoration and the exploitation of the CMS in hybrid seed production.

Inheritance of Fertility Restoration for Cytoplasmic Male Sterility in a New Gossypium barbadense Restorer

In order to clarify inheritance mechanism of fertility restoration for cytoplasmic male sterility (CMS) in a new Gossypium barbadense restorer line Hai R which was found in the fertility test crossing of G. hirsutum CMS lines with G. barbadense germplasms. 23 fertility segregation populations of F2 and backcross were used to analyze the inheritance of fertility restoring gene(s) of Hai R. The result showed that Hai R had one major dominant gene (RfB) to control the CMS fertility restoration and this fertility restoration gene functioned at the sporophytic level. The sterile cytoplasm background might not only influence the transmission rate of male gamete but also that of female gamete when the restorer gene was recessive. It could be deduced that this fertility restoration gene might come from G. harknessii cotton, Hai R is of value in the application of cotton interspecific hybrid breeding.

Mapping of Rice Fertility-Restoring Genes for ID-Type Cytoplasmic Male Sterility in a Restorer Line R68

An F2 population derived from the cross Zhong 9A/R68 was used to map the fertility-restoring (Rf) gene for ID-type cytoplasmic male sterility (CMS). Two bulks (a fertile bulk and a sterile bulk) were constructed by pooling equal amount of ten highly fertile lines and ten highly sterile lines, respectively. Four hundred and thirteen pairs of simple sequence repeat (SSR) primers, which evenly distributed on 12 chromosomes of rice, were selected for analyzing polymorphisms between the parents and between the two bulks. The primer RM283 on chromosome 1 and the primers RM5756, RM258, RM6100 and RM171 on chromosome 10 were found to be polymorphic between the parents and between the two bulks. These five SSR markers were linked to fertility-restoring genes. A total of 82 excessive sterile lines were selected from Zhong 9A/R68 F2 population to estimate the genetic distance between five SSR markers and fertility-restoring genes respectively. The results indicated that one Rf gene was linked to RM283 located on chromosome 1 at a distance of 6.7 cM, and the other Rf gene was mapped to the long arm of chromosome 10 flanked by RM258 and RM6100 at the distances of 8.0 cM and 2.4 cM, respectively.

Development of New InDel Marker to Detect Genotypes of Rf-1a Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility(CMS) in japonica rice, a functional marker InDel-Rf-1a based on the difference of nucleotide sequence in Rf-1a locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-1arf-1a showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-1a locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-1a locus could be distinguished distinctly. Therefore, the marker InDel-Rf-1a could be widely used for genetic identification and marker-assisted selection(MAS) in breeding japonica restorer lines.

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice(Oryza rufipogon)

This study was conducted to develop and characterize a novel cytoplasmic male sterile(CMS)source which was identified from Dongxiang wild rice(Oryza rufipogon)by crossing Dongxiang wild rice as female with Zhongzao 35,an indica inbred variety,as male and continuous backcrossing with Zhongzao 35.Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm(CMS-WA).Sequential planting showed that this novel CMS has complete and stable male sterility.Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm(CMS-HL)and other indica inbred varieties are the maintainers with complete maintaining ability,suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS.So far,we only discovered a unique fertility restoration source for this novel CMS.Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes.Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.

Effect of drought stress on male fertility restoration in A3 CMS-inducing cytoplasm of sorghum

Use of cytoplasmic male sterility (CMS) in hybrid breeding requires effective male fertility-restoring lines. In sorghum, very few restoring lines that can restore fertility in A_3 CMS have been reported. To identify the reasons for this deficiency, F_1 and F_2 hybrids of an A_3 CMS line crossed with the line IS1112C, a donor of fertility-restoring (Rf) genes for A_3 cytoplasm, and testcrosses of fertile plants to A_3CMS lines were grown under contrasting water availability regimes in dryland and irrigated field plots. In the irrigated plots the frequency of fertile plants in testcrosses was twice that in dryland plots (P < 0.05). Fertile plants from the F_2 family grown in the irrigated plots showed significantly higher restoration ability than fertile plants from the same family grown in dryland plots. F_3 plants from the F_2 family grown in irrigated plots yielded on average a sixfold higherfrequency of fertile plants in testcrosses than F_3 plants derived from dryland plots (P < 0.01).Fertility of testcross hybrids correlated negatively with air vapor pressure deficit (VPD) at flowering (r = - 0.96; P < 0.01) suggesting that VPD is a trigger for downregulation of Rf genes for A_3 cytoplasm.

Cytoplasmic Male Sterility in Maize

14 isoplasmic and allonuclear cytoplasmic male sterile lines were used as female parents, 8 tester lines as male parents, 101 F1 progenies were obtained. Fertility restoration response of 101 F1 progenies were investigated through field observation and pollen stainability examination under microscope. 14 isoplasmic and allonuclear cytoplasmic male sterile lines were developed by repeated backcross with recurrent male parent lines for more than 8 generations. The result shows: tester line Zifeng1 not only restored the isoplasmic and allonuclear sterile lines of group C backcrossed with Mo17, Yu30 and Heer, but also completely restored the isoplasmic and allonuclear cytoplasm male sterile lines of group T backcrossed with Mo17, HZS , 1792 ,292 and Yu30. Therefore, nuclear background limits the use of Zifeng1 as a tester for identification of cytoplasmic male sterility. Furthermore RFLPs of mitochondrial DNA of 6 isonuclear and alloplasmic cytoplasmic male sterile lines were analyzed with Bam H Ⅰ and Hind Ⅲ restriction endonuclease and mitochondrial DNA probes pBcmH3 and Cox Ⅱ. The same RFLPs were found within sterile cytoplasm of group C, including C,Chuan G, Lei 2 and Lei 3, but a different RFLP pattern was observed among sterile cytoplasm of group S, C,T and the normal cytoplasm. This result suggested that the RFLP markers tightly linked to sterile mitochondrial genes of different groups could be applied in the identifcation of cytoplasmic male sterility.

Differences in Nuclear DNA Between Male-Sterile and Male-Fertile Lines of Sorghum bicolor

Cytoplasmic male sterility (cms) is determined by nuclear-cytoplasmic interactions. Up tonow, most studies are focused on the comparison of cytoplasmic DNAs of male-sterile lines and male-fertilelines, and analysis of nuclear DNA has not been documented yet. In order to find out the possible difference innuclear genome of male-sterile line A1 Tx623 and corresponding male-fertile line Tx623 of sorghum, randomamplified polymorphic DNA (RAPD) approach was used to analyze their cytoplasmic and nuclear genomes.Total DNAs of them were amplified at first to screen primers, which were able to generate reproducible bandsspecific to male-sterile line or male-fertile line. Then the selected primers were used to amplify their mitochon-drial DNA (mtDNA) and chloroplast DNA (cpDNA). The origins of all the polymorphic fragments were ana-lyzed. After ruling out those amplified from cytoplasmic DNA, seventeen polymorphic fragments were deter-mined to be amplified from nuclear DNA. These fragments originated from nuclear DNA indicate that diffe-rences in sequence exist between the nuclear DNA of male-sterile line and male-fertile line of sorghum, whichdo not agree with the traditional standpoint that they have identical nucleus.

Characterization of Zm26Sub5 and Its Potential Roles in Regulating Pollen Fertility of S-CMS in Maize

Mitochondrial orf355-orf77 and the nuclear fertility restorer locus Rf3/rf3 mutually control the fertility of male gametes in CMS-S in maize (Zea mays L.). A fragment of gene Zm26Sub5 was identified through cDNA-AFLP from a set of Rf3/rf3 near-isogenic lines in a previous study. In the present study, real-time PCR analysis revealed that Zm26Sub5 expression levels were much higher in pollen of S-Mo17Rf3Rf3 than in pollen of S-Mo17rf3rf3, and also higher than in fresh leaves, mature leaves, and roots of both S-Mo17Rf3Rf3 and S-Mo17rf3rf3. In silico cloning of full-length cDNA was successfully implemented. Gene Zm26Sub5 was 1 451 bp in size, of which 1 329 bp encoded a protein with 443 amino acids. The structure of this gene was analyzed by comparing its full length cDNA to homologous genomic DNA sequence (GenBank accession: Ac209463.3). Subsequent sequence analysis led to sub cellular localization of protein ZM26SUB5, and construction of a phylogenetic tree. In silico mapping indicated that Zm26Sub5 was located on chromosome 5 and closed to a reported starch-filled pollen ratio QTL. ZM26SUB5, therefore might have potential roles in repressing mitochondrial PCD which is associated with sterile activity in pollen in S-type cytoplasm.

Microsatellite-Aided Screening for Fertility Restoration Genes (Rf) Facilitates Hybrid Improvement

DNA markers enabled to determine the chromosomal locations of the two Rf genes(Rf3 and Rf4) in the wild-abortive cytoplasmic male sterility(WA-CMS) system. Four simple sequence repeats(SSRs) RM171, RM258, RM315 and RM443 were used to detect the allelic status with respect to the fertility restoration genes(Rf3 and Rf4) in 300 rice cultivars or breeding lines. The results revealed that out of 300 lines, 90 lines screened had Rf3, 65 lines had Rf4, and 45 lines had Rf3 and Rf4 alleles. Furthermore, 45 lines selected using SSR markers were mated with a CMS line(IR58025A) to analyze their restoring ability. Offspring of all the test lines except HHZ8-SAL9DT1-Y1, HHZ5-SAL9-Y3-1 and IDSA77 exhibited higher pollen and spikelet fertility(> 80%), thus confirming they bear the Rf alleles. The hybrid offspring of ARH12-6-1-1-B-3-1, IR32307-10-3-2-1 and Sahel 329 had the highest pollen fertility(97.39%, 98.30% and 97.10%, respectively) and spikelet fertility(95.10%, 97.07% and 96.10%, respectively).

The genetic and molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited characteristic found in many (>150) plant species. CMS/restoration systems are useful tools for hybrid seed production, and are ideal models for study of the interactions between nuclear and mitochondrial genomes. CMS/restoration systems in rice have been widely used for hybrid seed production, greatly contributing to the food supply. This article reviews the progress of the studies on the genetic and molecular basis of cytoplasmic male sterility and fertility restoration in rice.

大豆CMS-RN型不育系育性恢复基因GmRf1的初步鉴定及其分子标记开发

在大豆杂种优势利用中,主要基于三系法进行杂交大豆品种选育。恢复系作为杂交种的父本,其所含的育性恢复(Rf,restorer-of-fertility)基因起决定作用。前期对大豆RN型细胞质雄性不育恢复系的育性恢复基因GmRf1进行了精细定位。本研究在此基础上,对GmRf1定位区间内的候选基因进行功能注释、亚细胞定位预测、序列比对和差异表达分析,明确了Glyma.16G161900基因在恢复系JLR230中所编码的1个576个氨基酸的PPR(Pentatricopeptide repeats)蛋白为GmRf1。进一步对含有GmRf1的对照材料Williams82与母本不育系JLCMS204A进行测交及F_(1)植株花粉育性鉴定,验证了GmRf1可以恢复CMS-RN型不育系育性。最后利用GmRf1在亲本间存在的单核苷酸突变位点,开发了功能性分子标记Rf1-dCAPS-2和Rf1-dCAPS-3。上述研究将为今后通过分子标记筛选或辅助选育含GmRf1基因型材料,以及通过基因工程手段创制新型恢复系奠定了理论和技术基础。

The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of the mitochondrial ATP synthase complex

Cytoplasmic male sterility(CMS)is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm.C-type CMS(CMS-C)in maize has long been used in hybrid seed production,but the underlying sterility factor and its mechanism of action remain unclear.In this study,we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize.The ATP6C protein shows stronger interactions with ATP8 and ATP9 than ATP6 during the assembly of F1F0-ATP synthase(F-type ATP synthase,ATPase),thereby reducing the quantity and activity of assem-bled F_(1)F_(o)-ATP synthase.By contrast,the quantity and activity of the F1'component are increased in CMS-C lines.Reduced F1F0-ATP synthase activity causes accumulation of excess protons in the inner membrane space of the mitochondria,triggering a burst of reactive oxygen species(ROS),premature programmed cell death of the tapetai cells,and pollen abortion.Collectively,our study identifies a chimeric mitochondrial gene(ATP6C)that causes CMS in maize and documents the contribution of ATP6C to F1F0-ATP synthase assembly,thereby providing novel insights into the molecular mechanisms of male sterility in plants.

水稻CMS-WA育性恢复基因的定位

在由227个组合组成的珍汕97A×（珍汕97B×密阳46）F6测交群体中，构建了由115个RFLP标记组成的连锁图。应用QTL分析方法，定位控制水稻CMS朩A（野败型细胞质雄性不育）育性恢复的基因。检测到1个主效基因qRf-10和3个效应较小的QTL（qRf-1、qRf-7和qRf-11），它们之间主要表现累加效应。同时，有证据表明这些基因之间存在相互作用。不仅微效基因qRf-1和qRf-11分别与主效基因qRf-10存在显著互作，qRf-10的存在与否，还影响到其他基因的相互作用，即可能存在多重互作。另外，还检测到2个控制结实率的QTL，它们不具有育性恢复作用，但在qRf-10存在的情况下，其杂合子具有提高结实率的作用。

水稻CMS-DA育性恢复基因定位及其互作分析

在由 2 10个测交组合组成的协青早A (协青早B 密阳 4 6 )F6 群体中 ,构建了由 12 9个RFLP、SSLP组成的连锁遗传图谱。应用QTL分析方法 ,对水稻矮败型胞质雄性不育育性恢复基因进行了定位。检测到一个主效基因和 3个效应较小的QTL(qRf 1、qRf 10 1、qRf 5 ) 。

玉米C型Cms育性恢复基因Rf4的SSR标记

对A6 19与Cms2 37F2 及BC1群体的育性观察结果表明 ,恢复系A6 19有一对恢复基因 ,用微卫星标记 (SSR)将A6 19的恢复基因定位在玉米第 8染色体短臂上 ,与SSR引物bnlg 2 30 7连锁 ,距bnlg2 30 712 .

玉米C型胞质雄性不育系POD、CAT、SOD活性及POD酶谱分析

以玉米C型雄性不育系C478及其保持系478、恢复系H01为材料,对叶片以及雄穗小花的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的活性和过氧化物酶同工酶谱进行比较研究。结果表明:玉米叶片在生长过程中,不育系与保持系和恢复系叶片中的POD活性有明显差异;CAT活性在抽雄期相差不大;不育系每个时期的SOD活性均显著高于保持系。玉米雄穗发育过程中,不育系雄穗小花的POD活性高于保持系和恢复系,不育系雄穗小花刚开始孕育穗长不超过5 cm(时期I)时CAT的活性显著高于保持系与恢复系,不育系雄穗小花孕育完全但没抽出(时期II)时SOD活性明显低于其保持系。玉米抽雄期和开花期的不育系与保持系、恢复系叶片中的酶谱差异明显。

大豆质核互作雄性不育系NJCMS3A双亲雄性育性基因的SSR标记

利用大豆质核互作雄性不育系NJMCS3A的质、核供体亲本N21566和N21249构建F_2和BC_1F_1育性分离群体进行雄性育性的遗传分析与基因定位。结果表明,F1正反交可育,F2和BC1F1的可育株与不育株分离比例经χ2测验分别符合3∶1和1∶1,表明NJCMS3A供体亲本雄性育性由一对基因控制,可育等位基因为显性。该基因可能是NJCMS3A的一个恢复基因。选用793对SSR引物对F_2和BC_1F_1群体分别进行育性基因定位,发现该育性基因位于O连锁群上,在Satt331和Satt477标记之间,与Satt331、CSSR133和Satt477标记距离的次序一致,分别为8.1～10.4、11.4～16.4和13.3～19.2cM。

玉米C型胞质雄性不育与可溶性糖、可溶性蛋白、游离脯氨酸含量的关系

为揭示玉米胞质雄性不育的生化机制,以玉米C型雄性不育系C478及其保持系478、恢复系H01为材料,对叶片及雄穗小花的可溶性糖、可溶性蛋白、游离脯氨酸含量进行比较研究.结果表明:玉米不育系苗期、抽雄期、开花期叶片中可溶性糖含量均显著高于保持系、恢复系;三系间可溶性蛋白含量相差小;不育系除抽雄期外叶片中的游离脯氨酸含量显著高于其保持系、恢复系.玉米雄穗发育过程中,保持系雄穗小花中的可溶性糖含量在雄穗孕育完全但没抽出期(时期Ⅱ)、散粉期(时期Ⅲ)显著高于不育系;不育系小花中各个时期的可溶性蛋白含量均低于其保持系和恢复系,特别在时期Ⅱ、时期Ⅲ显著低于保持系和恢复系;不育系每个时期小花中的游离脯氨酸含量都显著低于保持系和恢复系.

粘类小麦CMS育性基因分布区研究

为更有效地选育小麦不育系的高恢复系,以8个粘类小麦细胞质雄性不育系(Cytoplasmic malesterility,CMS)为测验种,对310份来自国内外不同地区的普通小麦品种及亲本材料对粘类小麦CMS系的育性恢保关系进行调查,并采用聚类分析法研究了粘类小麦CMS育性基因的地区分布。结果表明:(1)高恢复能力的品种在中国春麦区和南方冬麦区的分布比例均超过50%,具有保持性的品种在加拿大分布最多,我国北方冬麦区次之,春麦区分布最少。(2)不同地区品种恢复度存在差异。(3)将48个普通品种聚为3类:第1类品种对粘类CMS的平均恢复度均低于20%,属全不育和高不育类型;第2类品种的平均恢复度在20%～55%范围内,基本属于半不育类型;第3类品种的平均恢复度均大于55%,属高可育和全可育类型。(4)对不育系进行聚类:90-110核背景下,K型和V型聚为一类,Ven型和B型聚为一类;224核型下V型和B型聚为一类,再和K型聚为一类,与Ven型距离最远;相同细胞质背景下,90-110和224两种核型分别在一定水平上被分为2个类别。