Application of Molecular Markers Linking to Cytoplasmic Male Sterile Loci to Assist Maintainer Line Selection and Their Selection Efficiency in Welsh Onion (Allium fistulosum L.)

Cytoplasmic male sterility exists widely in most natural populations of welsh onion （Alliumfistulosum L.）, which makes it possible to breed out many male sterile lines for heterosis utilization. Unfortunately, the breeding of cytoplasmic male sterility in welsh onion has a little progress due to the limitation of its biological characteristic and traditional selection approach. To study the feasibility and the efficiency of utilizing marker assisted selection for male sterile lines in welsh onion, one SCAR marker, SCS13, and one RAPD marker, S2002400, which could distinguish between N and S cytoplasm in several welsh onion cultivars, were identified. The two markers were then confirmed by Southern blotting, and used to screen the N or S cytoplasm of individual plants in seven welsh onion cultivars in this study. Male sterile and fertile plants were evaluated by aceto-carmine dying. The frequency of N-cytoplasmic plants and maintainer genotype was calculated in the seven open populations of welsh onion. The minimum number of plants needed to identify a maintainer was evaluated to be 95% reliable. Results showed that 20 to 80% decrease of crosses and self-crosses for identifying a maintainer genotype could be achieved by the marker-assisted selection compared with traditional selection method. It was proved that the molecular markers could precisely identify cytoplasmic types individually, performed by one generation of cross and two generations of testcrosses and self-crosses. Finally, several maintainer genotype plants were selected with the help of the two markers in the seven cultivars. The screened markers could assist and accelerate sterile and maintainer lines selection with less labor and cost.

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNAfragments about 1 600 bp were amplified using random amplified polymorphism DNA （RAPD） primer OPAl2 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region （SCAR） primers were then developed to discriminate the cytoplasmic male sterile （CMS） lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm （CMS-WA）, namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID （Indonesia paddy type） line 11-32A but not in 11-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

Influence of Soil Moisture and Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (Zea mays L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen. CMS is a useful biological tool for confinement strategies to facilitate coexistence of genetically modified (GM) and non-GM crops in case where it is required. The trait is reversible and can be restored to fertility in the presence of nuclear restorer genes (Rf genes) and by environmental impacts. The aim of this study was to investigate the influence of the level of irrigation on the stability of CMS maize hybrids under defined greenhouse conditions. Additionally the combination of irrigation and air temperature was studied. Three CMS maize hybrids were grown with different levels of irrigation and in different temperature regimes. Tassel characteristics, pollen production and fertility were assessed. The CMS stability was high in hot air temperatures and decreased in lower temperatures. The level of irrigation had no major effect on the level of sterility. The extent of these phenomena was depending on the genotype of CMS maize and should be known before using CMS for coexistence purposes.

Influence of Genetic Drift of Restoring Gene (Rf) on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

The seed samples of Yuetai A, a Honglian （HL） type cytoplasmic male sterile （CMS） line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing, Jiangsu Province and Lingshui, Hainan Province during 2004-2006. The seed impurity of Yuetai A mainly resulted from the genetic drift of restoring fertility gene （Rt） after biological contamination but not from its sterility unstability. All of the ten maintainer lines, five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants, directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating ＇iso-cytoplasm restoring-generations＇. Furthermore, high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination. After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A, the seed purity of Yuetai A and Yuetai B had been raised to 100%.

Genetic Mechanism of Dominant Earliness in Kefeng A,a New Rice Cytoplasmic Male Sterile Line

Kefeng A is an early maturing indica cytoplasmic male sterile （CMS） line of rice. Combinations derived from Kefeng A and late maturing indica restorer lines showed dominant earliness to various extents. To understand the genetic basis of dominant earliness, the genotype of photoperiod-sensitive genes in Kefeng A was analyzed using a complete set of heading time near isogenic lines （NILs） EGO to EG7, ER, LR, T65, T65E^b, T65E^bm, T65m, NIL（Hd1） and NIL（Hd4）. Results indicated that Kefeng A contained two dominant photoperiod-sensitive alleles E1 and Se-1^U on E1 and Se-1 loci, respectively, and the genotype of photoperiod-sensitivity genes for heading time in Kefeng A was E1E1e2e2E3E3Se-1^USe-1UEf-1Ef-1. Based on the detected heading time genotype, in combination with the heading time of Kefeng A and the early maturing phenomenon in its derived F1 hybrids, it is speculated that Kefeng A might carry a dominant inhibitor gene Su-E7 for the dominant photoperiod- sensitive gene E1, and a recessive inhibitor gene i-Se-1 for another dominant photoperiod-sensitive gene Se-1. The reason why F1 hybrids from Kefeng A exhibited early maturing was hereby analyzed and the breeding value of dominant earliness related genes in Kefeng A was discussed.

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility （CMS） lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-IA, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-IB, and the abortive process of SaNa-IA were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-IA was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-IA aborted in the pollen mother cell （PMC） stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-IA is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

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 the genetic purity of the F1 seeds. To investigate the genetics of fertility restoration of the Peterson cytoplasmic sterility in pepper, a doubled haploid population of 115 pepper lines obtained from anther culture of the F1 hybrid between Yolo Wonder （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 open field 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）. Correlations between the each couple of criteria within, as well as between the cultivation methods ranged from 0.55 to 0.84. Analysis of 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. The distribution of the progeny was continuous between the parental genotypes indicating the quantitative inheritance of fertility restoration. Inferred from segregation according to Snape et al.（1984）, the number of segregating genes was estimated to be that three to four genetic factors were involved in pollen traits （PI and PN） and five to eight genetic factors in seed production （SN）. The heredity analysis of the CMS will be helpful for understanding of the genetic mechanism of the fertility restoration and the exploitation of the CMS in hybrid seed production.

Mitochondrial comparative proteomic analysis of sterile line and its maintain line of purple cytoplasmic rice (Oryza sativa)

CMS/Rf systems in rice (Oryza sativa) have long been exploited for hybrid breeding to enhance productivity. Ying xiang CMS/Rf system is a new type. In this study, a mitochondrial comparative proteomic analysis of Ying xiang Sterile Line and its Maintain Line was started for a comprehensive investigation of the mitochondrial proteins’ functions in rice cytoplasmic male sterility. Mitochondria were prepared from rice shoots grown in the dark. Proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF/ MS. Using Mascot, it was found that 7 proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. 3 of them were characterized.

Studies of the Energy Release of Mitochondria from Sporophyte Cytoplasmic Male Sterile Rice by Differential Scanning Calorimetry

The differential scanning calorimetric (DSC)curves of the mitochondria isolated from two varieties of sporophyte cytoplasmic male sterile and their fertile lines of Yie Bai and Ma Xie type rice have been determined. The curves show that the energy is released continuously as temperature rise to 70℃. Some thermodynamic and kinetic parameters of the energy release of the mitochondria have been obtained. The presented results showed that the mitochondria from cytoplasmic male sterile rice released more heat and they had higher energy barrier, less rate, and more complicated mechanism than that of their fertile lines in the energy release process.

AFLP Analysis of Mitochondrial DNA from a Cytoplasmic Male Sterility Line and its Maintainer in Pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the inability of a plant to produce functional pollen, which is widespread among higher plants. CMS system is a valuable tool for plant

Maxie A- a novel cytoplasm male sterile line

The pollen abortion of Maxie A and Zhengshan97 A belong to stainless type with Ksolu-tion.The maiority of Maxie A abortive Pollenstook cireular shape and few was staining abortion

DIA-based proteome profiling with PRM verification reveals the involvement of ER-associated protein processing in pollen abortion in Ogura CMS cabbage

Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.

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 to now, most studies are focused on the comparison of cytoplasmic DNAs of male-sterile lines and male-fertile lines, and analysis of nuclear DNA has not been documented yet. In order to find out the possible difference in nuclear genome of male-sterile line A1 Tx623 and corresponding male-fertile line Tx623 of sorghum, random amplified 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 bands specific 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 analyzed. After ruling out those amplified from cytoplasmic DNA, seventeen polymorphic fragments were determined to be amplified from nuclear DNA. These fragments originated from nuclear DNA indicate that differences in sequence exist between the nuclear DNA of male-sterile line and male-fertile line of sorghum, which do not agree with the traditional standpoint that they have identical nucleus.

基于BSA重测序的辣椒CMS恢复基因连锁分子标记开发

三系配套是辣椒育种的主要研究方向,鉴定并开发辣椒恢复基因连锁标记是利用分子标记辅助选育恢复系的难点。本研究以自主选育的辣椒胞质雄性不育系014A和恢复系014C为亲本构建F2分离群体,经χ2测验可育与不育的分离比例符合3∶1,表明辣椒胞质雄性不育育性恢复性状受1对显性基因控制。采用BSA法构建可育和不育极端DNA混池,全基因组重测序并与参考基因组比对,通过SNP-index法和Fisher检验将辣椒胞质雄性不育恢复基因定位于辣椒第6号染色体顶端1.44~8.28Mb的区域内。根据亲本间SNP/InDel差异设计引物,在亲本和极端池筛选多态性引物,得到能稳定扩增出特异条带的分子标记PP5和OP59。这2个标记在极端群体验证中准确率均达到100%;标记OP59是共显性标记,位于恢复基因的候选基因T459-15819间区17232 bp,该标记在不育群体准确率为100%,在可育群体准确率为97.21%;标记PP5位于恢复基因的候选基因T459-15819下游318bp,该标记在不育群体和纯合可育群体准确率均为100%,不能区分杂合可育类型。该研究获得与恢复基因紧密连锁2个标记为加速选育辣椒CMS恢复系奠定基础。

Difference and Inheritance of the Sterility Maintenance of CMS in Indica Rice

The inheritance of the sterility maintenance ability was studied for three types of CMS lines (Zhenshan97A, XieqingzaoA and II-32A) and several lines in indica rice. The result showed that the abilities to keep the male sterility of the maintainer lines strengthened obviously with the progressive backcrossing. The critical stage to maintain the sterility of CMS lines happened usually in BC5-6 generation. The selection of the recurrent parent gave an obvious influence on the pollen fertility distribution of the later generations. The author has discussed ways of improving of the maintainer lines.

Lodging Resistance of Aromatic CMS Line Zhongzhe A and Its Derived Lines

Nineteen rice combinations derived from Zhongzhe A and its derived lines, as well as Y Liangyou 1 as the control, were used to study lodging resistance by detecting plant height and some of the traits of culm and panicle that are related to lodging. The results indicated that the lodging resistance of 16 combinations was significantly stronger than that of Y Liangyou 1. Moreover, the lodging resistances of all combinations derived from Zhongzhe 2A, Zhongzhe 3A, Huazhe A and Zhong 1A were significantly higher than that of Y Liangyou 1. Additionally, the traits related to lodging were not identical within different CMS rice lines, and the relationship between plant height and lodging resistance was only significantly (P < 0.01) detected in Zhongzhe 2A. Some traits of the second internode were closely related to lodging in all combinations, which can be used as an effective index in lodging resistance breeding in rice.

水稻新质源(CMS-FA)雄性不育恢复基因的遗传

发掘水稻新型雄性不育细胞质源CMS-FA,育成系列优质米不育系和系列新质源恢复系,在组配成强优势杂交稻组合的基础上,研究新质源雄性不育恢复系的恢复基因遗传。采用新质源(CMS-FA)不育系金农1A与恢复系金恢3号杂交获得杂交F1和F2代种子。用F1分别与不育系或保持系回交,获得(不育系//不育系/恢复系和不育系/恢复系//保持系)2个测交群体。同时种植P1、P2、P3、F1、F2、B1F1和B2F1等群体,考察花粉染色率、套袋结实率和自然结实率,卡平方测验遗传分离适合度。结果表明,不育系与恢复系杂交F1代正常可育,育性恢复(可育)基因为显性遗传。F2代分离出可育︰不育适合3︰1,育性恢复(可育)基因为1对显性基因控制。B1F1和B2F1代2个测交群体的可育︰不育都适合1︰1分离规律,验证了F2代育性恢复(可育)单基因的遗传模式。暂时确定新质源(CMS-FA)核质互作三系的基因型为不育系S(SS)、保持系F(SS)和恢复系S(FF)。

烟草线粒体基因coxⅡ的SNP检测及其与CMS的相关性分析

对烟草胞质雄性不育性(CMS)的分子机理进行了研究。以7个CMS系及其相应的保持系为材料,利用特异引物PCR法扩增其线粒体细胞色素氧化酶亚基Ⅱ(coxⅡ),通过直接测序和比对,检测到coxⅡ中有3个核苷酸位点存在碱基变异,分别是:C-770G、G-772A和G-773C,其中第770位碱基的变化导致了相应位点编码氨基酸的改变,第772和773位碱基的变化共同导致了1个编码氨基酸的改变。对coxⅡ基因中第770位C→G的突变进行了240个烟草植株个体的PCR-RFLP检测及分析,结果表明,所有保持系单株的线粒体coxⅡ基因片段都可以被HapⅡ酶切,酶切后出现2种条带;而全部雄性不育系单株的线粒体coxⅡ基因片段由于第770位C→G的突变都不能被HapⅡ酶切,电泳图中仅有1条未被切开的条带。说明coxⅡ基因第770位的SNP位点与烟草CMS特性存在极显著的相关性。

2个油菜CMS系统的酯酶和过氧化物酶同工酶分析

以甘蓝型油菜细胞质雄性不育系(CMS)Polima A及其保持系Polima B、陕2A及其保持系陕2B 4个材料初花期的叶片、叶柄以及花蕾组织为材料,采用聚丙烯酰胺垂直板凝胶电泳比较它们在酯酶(EST)和过氧化物酶(POD)同工酶酶谱的差异。结果表明,甘蓝型油菜不育系与其对应保持系的叶片、叶柄的EST同工酶谱均无明显差异,但两系花蕾的EST同工酶谱有一定的差异;Polima A与陕2A对应器官的EST同工酶谱均表现出明显差异。不育系与其对应保持系的叶片、叶柄的POD同工酶谱差异不明显,而两系花蕾的POD同工酶谱有明显的差异;两个不育系之间的POD同工酶谱明显不同。花蕾的EST和POD同工酶的条带数目明显多于相应材料的叶片和叶柄,且EST同工酶的条带数目明显多于相应的POD同工酶。因此,甘蓝型油菜CMS系统Polima A和陕2A有着不同的遗传背景。