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.

Fine mapping and cloning of the sterility gene Bra2Ms in nonheading Chinese cabbage(Brassica rapa ssp.chinensis)

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage(Brassica rapa ssp.chinensis).However,the molecular mechanisms underlying male sterility in B.rapa are still largely unclear.We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus,Bra2Ms,on the A2 chromosome.Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation.Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B.rapa.The Bra039753 gene encodes a plant homeodomain(PHD)-finger protein and is considered a potential candidate gene for Bra2Ms.Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage.Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants,which led to an amino acid insertion in the Bra039753 protein.In addition,the 369 bp fragment insertion was found to cosegregate with the male sterility trait.This study identified a novel locus related to male sterility in non-heading Chinese cabbage,and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.

Jasmonic acid-mediated stress responses share the molecular mechanism underlying male sterility induced by deficiency of ZmMs33 in maize

Plant male reproduction is a fine-tuned developmental process that is susceptible to stressful environments and influences crop grain yields.Phytohormone signaling functions in control of plant normal growth and development as well as in response to external stresses,but the interaction or crosstalk among phytohormone signaling,stress response,and male reproduction in plants remains poorly understood.Cross-species comparison among 514 stress-response transcriptomic libraries revealed that ms33-6038,a genic male sterile mutant deficient in the Zm Ms33/Zm GPAT6 gene,displayed an excessive drought stress-like transcriptional reprogramming in anthers triggered mainly by disturbed jasmonic acid(JA)homeostasis.An increased level of JA appeared in Zm Ms33-deficient anthers at both meiotic and postmeiotic stages and activated genes involved in JA biosynthesis and signaling as well as genes functioning in JA-mediated drought response.Excessive accumulation of JA elevated expression level of a gene encoding a WRKY transcription factor that activated the Zm Ms33 promoter.These findings reveal a feedback loop of Zm Ms33-JA-WRKY-Zm Ms33 in controlling male sterility and JA-mediated stress response in maize,shedding light on the crosstalk of stress response and male sterility mediated by phytohormone homeostasis and signaling.

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.

Mapping of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat Using SSR Markers

LK783 was found to be a good fertility restorer for K-type male sterility of wheat. Microsatellite markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among (KJ5418A//911289/LK783)F1 population,respectively. Seventy-nine sets of SSR primers were screened for polymorphism between the two pools, 6 of which were found polymorphic. Linkage analysis showed that Xgwm11, Xgwm18 , Xgwm264a and Xgwm273were linked to the restoring gene in LK783, while Xgwm11, Xgwm18 and Xgwm273 were co-segregated. The distance between the Rf gene in LK783 and the three co-segregated markers was 6.54±4.37 cM, the distance between Rf gene and Xgwm264a was 5.71±4.10 cM. The four SSR markers were located on chromosome 1BS by amplifying the DNA of nulli-tetrasomics and ditelosomics of CS with the 4 sets of primers, indicating that the major restoring gene in LK783 was located on 1BS, but the relative location of the gene was different from Rfv1, allelism of the two genes should be further investigated. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility in wheat would be facilitated by using the four polymorphic markers.

Mapping Major Restore Genes for C-type Cytoplasmic Male Sterility in Maize with SSR Marker

Through observation about the restoration of male fertility of F2 and BC1 progeny, we found that the restoring line Fengke1 had two duplicating restorer genes. The restorer gene R f5 in Fengke1 background was located on chromosome 5L by SSR method; it linked with bnlg1711, bnlg1346 and phi058,the genetic distances with bnlg1711, bnlg1346, and phi058 were 7.51cM, 1.68cM, and 9.87cM respectively;the restorer gene R f4 was mapped on chromosome 8S linked with bnlg2307.

Mitochondrial localization of ORF346 causes pollen abortion in alloplasmic male sterility

The Nsa cytoplasmic male sterility(CMS) system confers stable male sterility and offers great potential for production of hybrid seeds in oilseed rape. However, genes responsible for male sterility in Nsa CMS have not been identified. By mitochondrial genome sequencing of Nsa CMS and its maintainer line,we identified in an Nsa CMS line several chimeric genes encoding hypothetical proteins harboring transmembrane domains. One novel chimeric gene orf346 showed high identity with cox1 at the 50 terminal region and was co-transcribed with nad3 and rps12 genes. Transgenic plants of orf346 fused with or without mitochondrial targeting peptide conferred complete male sterility in Arabidopsis. ORF346 was mitochondrion-localized. Expression of orf346 in Escherichia coli inhibited bacterial growth, with excessive accumulation of reactive oxygen species and decreased ATP content. These results reveal a link between the newly identified mitochondrial gene orf346 and the abortion of Nsa CMS. Inadequate energy supply and excessive accumulation of reactive oxygen species may account for pollen abortion in Nsa CMS plants.

Identification of a locus associated with genic male sterility in maize via EMS mutagenesis and bulked-segregant RNA-seq

Genic male sterility(GMS) is one of the most important resources for exploiting heterosis in crop breeding, so that identifying genomic loci regulating GMS is desirable. However, many regulatory genes controlling GMS have not yet been characterized in maize, owing partly to a lack of genetic materials. We generated a recessive male-sterile maize mutant in the Jing 724 genetic background via ethyl methanesulfonate treatment, and found the male sterility to be due to a single gene mutation. Bulk-segregant RNA sequencing of three replicates indicated that one genomic region located at the end of chromosome 4 was associated with the observed mutant phenotype. Among genes with nonsynonymous mutations,Zm00001 d053895(bHLH51) showed abolished expression in the sterile bulks and was annotated as a bHLH transcription factor orthologous to Arabidopsis AMS, suggesting an association with the male sterility of the mutant. Kompetitive Allele-Specific PCR assays further validated the exclusive correlation of male sterility with the single C-to-T mutation in the fifth exon. The new maize mutant and the potential SNP locus provide novel genetic material for investigating the molecular mechanism underlying tapetal development and may facilitate the improvement of hybrid production systems.

Development of Transgenic Restorer of Cytoplasmic Male Sterility in Upland Cotton

A glutathione S-transferase gene (gst) has been introduced into restorers of cytoplasmic male sterility in upland cotton ( Gossypium hirsutum L. ) using Agrobacterium-mediated transformation. A transviable pollens of hybrid (sterile line×restorer) F1. The hybrid gave 3.6 more bolls per plant, 10.1% less aborted seeds and 10.6% more lint yield when ‘Zheda strong restorer’ was used as male parent than when ‘DES-HAF277’ was. Southern and Northern bloting analysis showed that the foreign gst gene was detectable and highly expressed in ‘Zheda strong restorer’.

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.

Mechanism of Sterility and Breeding Strategies for Photoperiod/Thermo-Sensitive Genic Male Sterile Rice

To understand the male sterility mechanism of photoperiod/thermo-sensitive genic male sterile [P(T)GMS] lines in rice, the research progress on genetics of photoperiod and/or temperature sensitive genic male sterility in rice was reviewed. A new idea was proposed to explain the sterility mechanism of P(T)GMS rice. The fertility transition from sterile to fertile is the result of cooperative regulation of major-effect sterile genes with photoperiod and/or temperature sensitive genes, but not the so-called pgms gene in P(T)GMS rice. The minor-effect genes, which exhibit accumulative effect on sterility, are the important factors for the critical temperature of sterility transition. The more minor-effect genes the sterile line holds, the lower the critical temperature of sterility transition is. The critical temperature of sterility transition will be invariable if all the minor-effect genes are homozygous. The strategies for breeding P(T)GMS rice were also proposed. The selective indices of critical photoperiod and temperature for sterility transition should be set according to varietal type and ecological region. Imposing selection pressure is a key technology for breeding P(T)GMS rice with lower critical temperature for sterility, and improving the comprehensive performance of the whole traits and combining ability is vital for breeding P(T)GMS rice lines.

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

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 35oC, 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 35oC 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 32oC, 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.

A Fragment Substitution in Promoter of MS92/PTC1 Causes Male Sterility in Rice

Persistent tapetal cell1(PTC1) plays a curial role in pollen development, and is thought to function as a transcriptional activator in rice. However, the molecular mechanism of PTC1 in regulating pollen development and its cis-elements are not well understood. We identified a novel weak male sterility mutant(ms92) which exhibited expanded tapetum and shrink pollen grains. Map-based cloning and allelic analysis suggested that the male sterility of ms92 was caused by a DNA fragment substitution in the promoter of PTC1. The decreased expression of MS92/PTC1 in ms92 and cis-element analysis indicated that the substituted sequence contained several potential binding cis-element of negative feedback. MS92/PTC1 was specifically expressed in tapetum and microspores at the young microspore stage, and its protein was localized in nucleus. We further found that MS92/PTC1 functions as a transcription activator by recognizing H3K4me3. Transcriptomic analysis revealed that a number of genes involved in tapetum degeneration and pollen wall formation were down-regulated in ms92, which are the potential targets of MS92/PTC1. The substitution fragment in MS92/PTC1 promoter was essential for pollen development, and we provided a novel mutant for further identifying the cis-elements in promoter and the molecular network of MS92/PTC1.

Characterization and mapping of a new male sterility mutant of anther advanced dehiscence(t) in rice

Anther dehiscence is very important for pollen maturation and release. The mutants of anther dehiscence in rice (Oryza sativa L.) are few, and related research remains poor. A male sterility mutant of anther dehiscence in advance, add(t), has been found in Minghui 63 and its sterility is not sensitive to thermo-photo. To learn the character of sterilization and the function of the add(t) gene, the morphological and cytological studies on the anther and pollen, the ability of the pistil being fertilized, inheritance of the mutant, and mapping of add(t) gene have been conducted. The anther size is normal but the color is white in the mutant against the natural yellow in the wild-type. The pollen is malformed, unstained, and small in the KI-I2 solution. The anther dehiscence is in advance at the bicellular pollen stage. A crossing test indicated that the grain setting ratio of the add(t) is significantly lower than that of the CMS line 2085A. The ability of the pistil being fertilized is most probably decreased by the add(t) gene. The male sterility is controlled by a single recessive gene of add(t). This gene is mapped between the markers of R02004 (InDel) and RM300 (SSR) on chromosome 2, and the genetic distance from the add(t) gene to these markers is 0.78 cM and 4.66 cM, respectively.

Genetic Analysis and Primary Mapping of pms4, a Photoperiod-Sensitive Genic Male Sterility Gene in Rice (Oryza sativa)

To understand the genetic characteristics of a new photoperiod-sensitive genic male sterile line Mian 9S, some reciprocal crosses were made between Mian 9S and six indica rice materials, Yangdao 6, Luhui 602, Shuihui 527, Mianhui 725, Fuhui 838 and Yixiang 1B. Genetic analysis results suggested that the photoperiod-sensitive genic male sterility (PGMS) of Mian 9S was controlled by a single recessive nuclear gene. Thus, the F2 population derived from the cross of Yangdao 6/Mian 9S was used to map the PGMS gene in Mian 9S. By using SSR markers, the PGMS gene of Mian 9S was mapped on one side of the markers, RM6659 and RM1305, on rice chromosome 4, with the genetic distances of 3.0 cM and 3.5 cM, respectively. The gene was a novel PGMS gene and designated tentatively as pms4. In addition, the application of the pms4 gene was discussed.

Nectar secretion of RN-type cytoplasmic male sterility three lines in soybean [Glycine max(L.) Merr.]

Significant progress has been achieved in the use of heterosis in soybean and several soybean hybrids have been released in China. However, broad use of hybrid soybean seed is limited due to low seed setting of female parents. Breeding cytoplasmic male sterile(CMS) lines with high out-crossing rate is necessary to solve the problem. The objective of this study was to determine the relationship between out-crossing rate of CMS lines and their nectar secretion. The daily nectar secretion rhythm, meteorological effect on nectar secretion, and differences in nectar secretion among genotypes and years were investigated in 27 soybean CMS lines(A-lines) with their maintainers(B-lines) and restorers(R-lines). The correlation between out-crossing rate of CMS lines and nectar production was also evaluated. Nectar secretion had diurnal variation. Secretion initiated at about 06:00 for most materials and reached a peak at 07:00–08:30 after flower opened, then the nectar secretion decreased gradually. A sub-peak appeared at about 13:00, while the nectar could not be detected at 17:00. Nectar secretion was greatly influenced by the weather conditions. The amount of nectar secretion increased gradually over time during periods of high temperature and no rainfall for several days. Rainy weather and low temperatures inhibited nectar secretion. There were obvious variations of nectar amount among different genotypes tested. Significant nectar variation within a genotype among years was also observed, and the highest nectar secretion was 3-fold higher than the lowest. The amount of nectar secretion from R-lines was significantly higher than that of A-and B-lines. There was no significant difference in nectar secretion between A-and B-lines. A-and B-lines with higher out-crossing rates secreted more nectar. The amount of nectar secretion of A-and B-lines were significantly positively correlated with the out-crossing rate of A-lines.

Application of hybrids with cytoplasmic male-sterility in Zea mays L.in China

The abortive behaviour and the cytoplasm quality of cytoplasmic male-sterility(CMS)lines of homocaryon were investigated and the existence of C race of Bipolaris maydiswere discussed from the point of view of genetic breeding,physiology and pathology in this paper.Then the countermeasures to prevent the danger from disease of B. maydis in CMS hybrid pro-duction were proposed.

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-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was sterile with a shortened filament and defl ated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A 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-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

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.

Plant Male Sterility Induced by Anti-Gene CYP86MF in Brassica oleracea var. italica

An anti-gene CYP86MF was introduced into hypocotyls of broccoli (Brassica oleracea L.var. italica Plenck) with Agrobacterium tumefaciens, and the transgenic plants were obtained by kanamycin selection. The results of PCR, Southern blot and Northern blot indicated that the anti-CYP86MF has been integrated into chromosome of the transgenic plant. And also, plants with hypogenetic stamina or ungerminated pollen were observed. The transgenic male sterility plant could fructify via artificial pollination with normal pollen. Thus it was proved that the pistil of male sterility plant was normally developed, and the sterility originated from anti-CYP86MF.