Advances in DNA methylation and its role in cytoplasmic male sterility in higher plants

The impact of epigenetic modifications like DNA methylation on plant phenotypes has expanded the possibilities for crop development.DNA methylation plays a part in the regulation of both the chromatin structure and gene expression,and the enzyme involved,DNA methyltransferase,executes the methylation process within the plant genome.By regulating crucial biological pathways,epigenetic changes actively contribute to the creation of the phenotype.Therefore,epigenome editing may assist in overcoming some of the drawbacks of genome editing,which can have minor off-target consequences and merely facilitate the loss of a gene’s function.These drawbacks include gene knockout,which can have such off-target effects.This review provides examples of several molecular characteristics of DNA methylation,as well as some plant physiological processes that are impacted by these epigenetic changes in the plants.We also discuss how DNA alterations might be used to improve crops and meet the demands of sustainable and environmentally-friendly farming.

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.

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.

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.

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 of Japonica Male Sterile Lines Integrating Cytoplasmic Male Sterility and Photosensitive Genic Male Sterility

Acknowledgement It has been previously established that the BT type of cytoplasmic male sterility （CMS） is induced by high temperatures, while photosensitive genic male sterility （PGMS） seed sets by low temperatures induce. In the current study, we have bred photosensitive cytoplasmic male sterility （PCMS） lines （2308SA and 2310SA） by crossing the CMS line with the PGMS japonica line with maintainer genes. The sterility of PCMS japonica was consequently controlled by two groups of male sterile genes resulting from the integration of PGMS and CMS genes. The results on plant fertility, at different sowing times, were as follows： （a） Under conditions of natural long-day photoperiod and at temperatures above 35~C, the PGMS gene regulated PCMS japonica sterility - the higher the temperature, the lower the pollen fertility. However, bagged seed sets of PCMS japonica, not exposed to high temperatures, induced the CMS seed set. （b） Exposure to long-day photoperiod and temperature conditions between 35℃ and the critical sterility inducing temperature of PGMS resulted in both PGMS and CMS gene controlled sterility of PCMS japonica, which exhibited stable characteristics. （c） When exposed to critical sterility inducing temperatures or short-day photoperiod and daily high temperatures below 32℃, the BT type of the CMS gene regulated PCMS sterility. Under these conditions, the PGMS gene rendered male sterility insusceptible to occasional cool summer days when this PCMS line, adopted for hybrid seed production, develops into panicle differentiation stage. The present study also investigated the fertility restoration, seed production and combining ability of PCMS japonica so as to optimize its use.

Comparative Transcriptome Analysis between Cytoplasmic Male-sterile Line and Its Maintainer During the Floral Bud Development of Pepper

The male-sterile line has been largely used in the hybrid seed production of pepper, which can effectively improve the efficiency of hybrid seed production. However, the formation mechanism of male sterility in pepper remains unclear. In the present study, we compared the gene expression patterns between pepper cytoplasmic male sterile line 9704 A and its maintainer 9704 B during floral bud development using RNA sequencing technology. A total of 547 976 and 2 416 Differentially Expressed Genes(DEGs) were identified in the stage S1, S2 and S3, respectively,and more than 70% of the DEGs were down-regulated in the sterile line. Gene Ontology(GO), and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed to further understand the functions of these identified DEGs. The results showed that the DEGs were mainly enriched in pathways of starch and sucrose metabolism, pentose and glucuronate interconversions. A number of genes, such as MS1, PME5, ATPB, and lots of transcription factors were found down-regulated in the sterile line, and we also identified a series of genes with large differences in expression patterns between sterile line and maintainer line. Collectively, our findings laid a foundation for further molecular breeding in pepper and provided new insights into its mechanism underlying the male sterility.

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.

Molecular identification of the cytoplasmic male sterile source from Dongxiang wild rice(Oryza rufipogon Griff.)

The use of cytoplasm male sterility （CMS） is crucial for three-line hybrid seed production. Two types of CMS have been discovered from Dongxiang wild rice, namely the wild abortion type CMS （CMS-WA） and the Dongxiang wild type CMS （CMS-DW）. In this study, we show the molecular identification of the two types of CMS in Dongxiang wild rice. WA352, which conferred CMS-WA, was not detected in Dongxiang wild iice, implying Dongxiang wild rice does not carry the CMS- WA source. Further analysis of WA352 in DYIA, a CMS-DW line, by PCR amplification and sequencing, revealed two insertion-deletion polymorphisms occurred in CMS-DW compared to WA352 of CMS-WA. It was reported that WA352 was comprised of an unknown origin sequence and partial sequences of three open reading frames （ORFs）, orf284, orf224 and orf288. The 42-bp insertion was located between the two segments of orf224 and orf288, which created a new chimeric ORF, orf216. This new ORF was also detected in CMS-HL. Based on the 9-bp deletion in orf284, a specific mitochon- drial marker of DW-M1 was developed, which could be used to specifically distinguish the DW-type source. Moreover, semi-quantitative RT-PCR analysis preliminarily suggested that both orf216 and orf284 could be considered as candidates for CMS-DW. These findings present a preliminary understanding of CMS-DW at the molecular level.

Metabolism of Reactive Oxygen Species in the Cytoplasmic Male-Sterile Cotton Anther

Reactive oxygen species （ROS） in plant cell, including superoxide （O2^-）, hydrogen peroxide （H2O2）, and malondialdehyde （MDA）, are thought to be important inducible factors of cell apoptosis if excessively accumulated in cells. To elucidate the metabolic mechanism of ROS production and scavenging in anthers of the cytoplasmic male-sterile （CMS） cotton, CMS line, maintainer, and hybrid F1 anthers, were employed for studying the relationship between CMS and metabolism of ROS, by comparing ROS changes in the sterile and fertile anthers at different developmental stages. The results showed that during the abortion preliminary stage （sporogenous cell division stage）, anthers of CMS line had higher contents of O2^-, H2O2, and MDA than those of maintainer or hybrid F1. Simultaneously, the higher activities of superoxide dismutase （SOD）, catalase （CAT）, and peroxidase （POD） in scavenging ROS were measured in the anthers of the CMS line, indicating that an increase of ROS in anthers of abortion preliminary stage had an inducible effect on the antioxidant enzymes. But during the abortion peak of CMS anther （pollen mother cell meiosis stage）, on the one hand, contents of O2^-, H2O2, and MDA were extraordinarily high in CMS anthers, on the other hand, the activities of SOD, CAT, and POD were excessively low, which disrupted the balance between the production and elimination of ROS and led to pollen mother cells apoptosis at this stage. In the following two stages （uninucleate microspore stage and mature pollen stage）, the contents of O2^- and H2O2 in the aborted anthers were approximated to contents in the fertile anthers of the maintainer and hybrid F1. However, MDA contents were continuously raised and enzymic activities of SOD, CAT, and POD were consistently decreased in sterile anthers, which indicated that ROS still had harmful effects on the anthers after the apoptosis of the male cells. Excessive accumulation of O2^-, H2O2, and MDA and significant reduction of ROS scavengingenzyme activities were coinstantaneous with male cells apoptosis in the anthers of the cotton CMS line. But when the restorer gene was transferred into the CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F1.

Changes in Physiological and Biochemical Characteristics of Floral Organ Development in a Soybean Cytoplasmic-nuclear Male Sterile Line

[Objectives]This study was conducted to explore the mechanism of soybean cytoplasmic-nuclear male sterility.[Methods]With soybean cytoplasmic-nuclear male sterile line JLCMS9 A and its homotype maintainer line JLCMS9 B as experimental materials,the activity of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT),malondialdehyde(MDA)content,starch content,soluble protein content,soluble sugar content and free proline content in flower buds,alabastrums and mature flowers were determined,and the contents and changes of auxin(IAA),gibberellin(GA3),isopentenyl adenosine(iPA)and abscisic acid(ABA)at the three stages were analyzed.[Results]The activity of SOD and CAT and the contents of MDA and free proline in the sterile line at the flower bud stage were lower than those of the maintainer line,but the opposite was true at the alabastrum stage and the flowering stage,and their values were higher than those of the maintainer line;the POD activity of the sterile line was significantly lower than that of the maintainer line at the flower bud stage,and the opposite was true at the alabastrum stage and the flowering stage,and its values were higher than those of the maintainer line;and the starch content and soluble sugar content of sterile line 9 A showed an overall upward trend,and were significantly lower than those of the maintainer line 9 B at the alabastrum stage and the flowering stage.During the whole development process of floral organs,the content of IAA in sterile line 9 A showed a trend of first increasing and then decreasing,and the content of iPA increased gradually.The contents of hormones in the sterile line were lower than those in the maintainer line.The ratios of IAA/ABA,IAA/GA3,IAA/iPA and ABA/GA3 in the sterile line were significantly different from those in the maintainer line.It is inferred that the abnormal physiological characteristics of floral organ development are related to the cytoplasmic-nuclear male sterility of soybean.The alabastrum stage may be a critical period for the occurrence of abnormal physiological and biochemical indexes in the floral organs of soybean cytoplasmic-nuclear male sterile lines.[Conclusions]This study provides a theoretical basis for the breeding of fine sterile lines of soybean and the research on the mechanism of sterility.

棉花CMS-D2和CMS-D8不育系线粒体全基因组比较分析

【目的】探究哈克尼西棉不育胞质(CMS-D2)和三裂棉不育胞质(CMS-D8)的不育系线粒体基因组之间的序列结构差异,为筛选鉴定不育相关基因奠定基础。【方法】根据D2A和D8A这2个不育系的线粒体基因组测序组装结果,使用Synteny and Rearrangement Identifier(Sy RI)软件鉴定结构变异,用Plotsr可视化分析包含共线性及非共线性区域的重组变异位点。以D8A线粒体基因组注释结果为参考,用D2A线粒体基因组注释的开放阅读框(open reading frame,ORF)编码的氨基酸序列进行tblastn比对,筛选出D2A线粒体基因组中特有的ORF,并进行聚合酶链式反应(polymerase chain reaction,PCR)验证、相对表达量分析和生物信息学分析。【结果】D2A和D8A这2个不育系线粒体基因组之间存在2个部分重叠且相邻的倒易位区域。在D2A中发现17个特异的ORF,PCR验证了D2A中存在的6个特异ORF(orf114e、orf121b-1、orf121b-2、orf138b-2、orf186a-2和orf317a-2)。3~4 mm花蕾中orf121b-1和orf121b-2的相对表达量较高。orf114e、orf186a-2和orf317a-2具有典型的跨膜结构域和嵌合基因结构,符合不育基因的部分特征。【结论】D2A和D8A线粒体基因组间存在2个相邻的倒易位区域。D2A中存在6个特异的ORF,其中orf114e、orf186a-2和orf317a-2可能与棉花CMS-D2孢子体败育有关。

Differential RNA Editing of Mitochondrial Genes in WA-Cytoplasmic Based Male Sterile Line Pusa 6A, and Its Maintainer and Restorer Lines

RNA editing changes the nucleotides at the transcript level of mitochondrial genes which results in synthesis of functional proteins.This study was designed to find the editing sites which could be implicated in male fertility restoration and to develop editing based markers for differentiation of cytoplasmic male sterility and maintainer lines from each other.DNA and RNA from young panicles were isolated from three-line system of hybrid rice PRH10,wild abortive(WA)cytoplasm based male sterile(A line Pusa 6A),maintainer(B line Pusa 6B)and restorer(R line PRR78)lines.Pusa 6A and PRR78 having the same WA cytoplasm are allo-nuclear and iso-cytpolasmic lines.The genomic and cDNA amplicons for eight mitochondrial genes(18SrRNA,atp6,atp9,cobII,coxI,coxIII,nadI and rps3)were sequenced and compared.Differences in genomic and cDNA sequences were considered as editing.Two hundred and thirty editing sites having base substitution or insertion/deletion were identified with the highest in 18SrRNA(5.74%)and the lowest in coxI(0.60%).The highest editing sites were observed in fertile maintainer Pusa 6B followed by PRR78 and Pusa 6A,of which random five editing sites in five different rice mitochondrial transcripts namely atp9,cobII,coxIII,rps3 and 18SrRNA were chosen and validated through cleaved amplified polymorphism sequence(CAPS)analysis and found to be partially edited in four genes.The identical editing sites of different mitochondrial genes from maintainer and restorer lines might reflect their possible contribution to fertility restoration of sterile WA cytoplasm.

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.

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.

Identifying Cytoplasmic Male Sterile Types of Chinese Cabbage(Brassica campestris L.)by Molecular Markers

Different sterile cytoplasm types of nine cabbage cytoplasmic male sterile materials were identified by molecular marker in the study, in order to better use molecular marker to conduct the assisted breeding in the future. Genomic DNA was isolated from Chinese cabbage by CTAB method. The design of two pairs of specific primers was performed on conserved flanking region of orf138 gene in the GenBank. PCR was performed with genomic DNA of the nine Chinese cabbage materials. The bands were sequenced. The homologous comparison was conducted in NCBI, and finally, the type of sterile cytoplasm was determined. The results showed that the bands were amplified only in four Chinese cabbage male sterile materials with two pairs of specific primers PUPIl and PIII/PIV, while the other five materials did not obtain the relative bands. The result was consistent with the field sterility identification. And then four molecular markers of Chinese cabbage Ogura cytoplasmic male sterility （CMS） were obtained. After conducting a homologous comparative analysis with BLAST in GenBank, it was found that the homologous degree was 100% in specific segments of tbe tbree sterility materials （L1-CI, L3-CI and L3- F1 ） and Ogu orf138 gene （GenBank accession No. ： HQ149728） of the reported broccoli Ogu CMS. The homologous degree of L1-F1 was 99% with a variation point. The type of cytoplasmic male sterility of the other five materials needed further research. Four materials of the nine were identified as the radish cytoplasmic male sterility materials and four molecular markers were obtained.

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.