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.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

Background Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources.However,only a few cytoplasmic male sterility(CMS)lines of cotton have been produced due to several challenges,like inadequate variation of agronomic traits,incomplete sterility,weak resilience of restorer lines,and difficulty in combining strong dominance.Therefore,the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.Results Two F_(2) segregating populations of cotton were constructed from cytoplasmic male sterile lines(HaA and 01A,maternal)and restorer lines(HaR and 26R,paternal).Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants.Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants.However,sterile plants exhibited smaller floral organs,shortened filament lengths,and anther atrophy on the flowering day in comparison with the fertile plants.When performed scanning electron microscopy(SEM),the two F_(2) populations revealed morphological variations in the anther epidermis.Cellular analysis showed no significant differences in pollen development before pollen maturation.Interestingly,between the pollen maturation and flowering stages,the tapetum layer of sterile plants degenerated prematurely,resulting in abnormal pollen grains and gradual pollen degradation.Conclusion The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene.Sterile plants exhibit distinctive floral morphology,which is characterized by stamen atrophy and abnormal anthers.Pollen abortion occurs between pollen maturity and flowering,indicating that premature tapetum degradation may be the primary cause of pollen abortion.Overall,our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations,mechanisms of sterility,and the role of sterile and restorer genes.

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.

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.

Genic male and female sterility in vegetable crops

Vegetable crops are greatly appreciated for their beneficial nutritional and health components.Hybrid seeds are widely used in vegetable crops for advantages such as high yield and improved resistance,which require the participation of male(stamen)and female(pistil)reproductive organs.Male-or female-sterile plants are commonly used for production of hybrid seeds or seedless fruits in vegetables.In this review we will focus on the types of genic male sterility and factors affecting female fertility,summarize typical gene function and research progress related to reproductive organ identity and sporophyte and gametophyte development in vegetable crops[mainly tomato(Solanum lycopersicum)and cucumber(Cucumis sativus)],and discuss the research trends and application perspectives of the sterile trait in vegetable breeding and hybrid production,in order to provide a reference for fertility-related germplasm innovation.

RAPD and ISSR Markers of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat

LK783 was found to be a good fertility restorer for K-type male sterility of wheat (Triticum aestivum L.). RAPD and ISSR (inter-simple sequence repeat polymorphism) 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 F 1 population, respectively. Four hundred and eighteen RAPD primers and 33 ISSR primers were used for screening polymorphisms between the two pools, and amplification bands using a RAPD primer of OPK18 and an ISSR primer of UBC-845 were found polymorphic between the two pools. Linkage analysis showed that OPK18 450 and UBC-845 800 were linked to the restoring gene in LK783. The distance between the restoring gene and OPK18 450 was (15.07±6.28) cM (centiMorgan), with the distance between the restoring gene and UBC-845 800 being (8.20±4.85) cM. The marker of UBC-845 800 was located on chromosome 1BS by amplifying nulli-tetrasomics and 1B ditelosomics of Chinese Spring with the primer of UBC-845, indicating that the restoring gene in LK783 was located on 1BS. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility of wheat would be facilitated by using the two markers.

Mapping of S-b Locus for F_1 Pollen Sterility in Cultivated Rice Using PCR Based Markers

In cultivated rice ( Oryza sativa L.), F-1 pollen sterility is controlled by at least 6 loci of the F, pollen sterility genes. To map S-b, one of loci, rice variety Taichung 65 (T65) carrying S-b(j)/S-b(j) and its near-isogenic line TIST2 carrying S-b(i)/S-b(i) were used to develop the mapping population. One hundred and fifty-eight microsatellite markers were selected to survey T65 and TISL2. RM13 on chromosome 5 was found to be polymorphic between them. Cosegregation indicated that RM13 was closely linked with locus S-b. Eleven RFLP markers were selected on the corresponding region from the genetic map of Rice Genome Research Program (RGP) of Japan to convert into sequence-tagged site (STS) markers. Amplicon length polymorphism (ALP) was carried out, but none of them was found to be polymorphic between T65 and TISL2. Then PCR-based RFLP (PBR) was done using six 4-nucleotide recognizing restriction endonucleases. Polymorphism was detected when PCR products of R830STS and R2213SSTS were digested with Taq I. Genetic analysis indicated that the distance between locus S-b and markers, R830STS, RM13 and R2213SSTS were 3.3 cM (centi-Morgan), 5.2 cM and 5.5 cM, respectively. These PCR-based markers could be directly used in marker-assisted selection. The technical system combining genetic mapping and PCR-based marker-assisted selection will facilitate the development of molecular breeding.

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Microtubule Structure and Male Sterility in a Gene-Cytoplasmic Male Sterile Line of Rice, Zhen Shan 97A

Histological changes that occur during microsporogenesis are documented in a gene-cytoplasmic male sterile rice ( Oryza saliva L.) line, Zhen Shan 97A, its maintainer line, Zhen Shan 97B, and the restorer line, Ce64 of a Mine hybrid rice production system. In the restorer line, Ce64, the developing microsporocytes have dense cytoplasm and a distinct set of circumferential microtubules around the nucleus. Successive cytokinesis results in the formation of tetrads. The microtubules within the cells of tetrads and microspores radiate from the surface of the nucleus towards the outer edge of the cytoplasm. Subsequent pollen development is normal. During the course of microspore formation tubulin speckles can be found in the cytoplasm. The general pattern of development and microtubule organization in the maintainer lined Zhen Shan 97B, is similar to Ce64, except that a few more tubulin speckles appear during microspore formation. In the case of the mate sterile line, Zhen Shan 97A, a number of abnormalities can be discerned during early microsporogenesis. These include vacuoles forming within the developing microsporocyte and faintly stained microtubules with no defined distribution pattern. Prominent tubulin speckles are common within the cytoplasm. For those microsporocytes that undergo meiosis, no defined organizational patterns of microtubules can be found within the tetrad. All microspores abort soon after. Abnormalities and defects in microtubule organization observed in Zhen Shan 97A showed that complex interactions between the cytoplasm and the nucleus began at very early stage of microsporocyte development.

Investigation on the Sterility Changeover of Male Sterility Line CMS7311 in Heading Chinese Cabbage

Sterility changeover induced by low temperature in male sterility line CMS7311 of heading Chinese cabbage ( Brassica campestris L. ssp. penkinsis) was investigated under constant temperature, day/night alternating temperature in light-growth-incubator and outdoor natural day/night alternating temperature respectively. It showed that the sterility of CMS7311 has significant change with temperature. The constant temperature of 6 - 12 degreesC was suitable to induce the changeover of sterility (CGS) of CMS7311 ; the shortest treatment time for CGS induction at this temperature was 3 d. The significant value of sterility changeover of CMS7311 occurred at the constant temperature of 6 - 9 degreesC after treatment for 6 - 9 d. The intensity of CGS was stronger under constant temperature than under alternate temperature, but the shortest treatment time inducing CGS has no difference between them. When treated for 9 d under the day/night alternating temperature of 11/7 degreesC, its sterility could also turn to normal fertility (near to sterile grade I or zero). In normal condition, the interval from the end of temperature treatment to the beginning of CGS induction was 10 - 16 d. In the condition of natural day/night alternating temperature, the lower limit of temperature for effective CGS induction was at the daily mean temperature ( DMT) of 3. 5 - 6. 8 degreesC, and the shortest treatment time which could induce the sterility changeover was also longer than that under constant temperature, being 11d at DMT of 1.5 - 3.1 degreesC. In addition, for the apical dominance, the flower bud on the first lateral branch showed the CGS 8 - 10 d later than the flower bud on the main stem under the same temperature with the same days of treatment. The intensity of CGS of CMS7311 on the first lateral branch was also weaker than the flower bud on the main stem.

Construction and Identification of HSP70 Antisense RNA Expression Vector for Genetic Engineering Male Sterility in Plant

[ Objective] In order to study the relation between the HSPTO gene and male sterility of plant further. [ Methods ] Anther specific expression promoter Osg6B of rice was coloned by PCR then connected with HSP70 antisense fragment to construct HSPTO antisense expression vector. The expression vector was identified by PCR experiment and enzyme digestion. [ Result] The sequence of coloned Osg6B promoter had 97% homology to the published sequence, and the cis-regulatory element in promoter area was integrated. HSP70 antisense expression vector driven by the promoter Osg6B was confired by colony PCR and enzyme digestion. [ Conclusion] The construction of expression vector would lay solid foundation for utilization of genetic engineering male sterility of plant.

Research of Fertility Standard on Male Sterility of Lycium chinensis

Botanical features of "YX-1" reproductive organ and bearing rate by number of pollen granules in pollen sacs of Lycium chinensis was observed and researched through field investigation, smear preparation and paraffin sections. YX-1 of Lycium chinensis was determined as incomplete male sterility and further classified into complete sterile type, sterile type I and sterile type II. The fertility standard of male sterility of Lycium chinensis was preliminarily proposed to establish a basis for crossbreeding of male sterility of Lycium chinensis.

Study on the Agroclimatic Division of Thermosensitive Genetic Male Sterility Rice Breeding

Using the meteorological data and geographical information during January-March（1961-2010） accumulated by 18 stations of Hainan,a suitability zoning map for thermo-sensitive genetic male sterility（TGMS） rice was made by GIS technology based on temperature indicators required by TGMS rice during fertility sensitive period and heading-flowering period,aiming to provide reasonable layout and scientific basis for sustainable development of TGMS rice in Hainan Island under the background of global warming.The results indicated that the suitable planting zones covered the south regions of Wuzhishan,Jianfengling and Diaoluoshan;subordinate suitable zones expanded northward to central parts of Wuzhishan,Dongfang,Changjiang,Qiongzhong and southern parts of Wanning;the other regions were unsuitable for breeding.

Genetic Study on Barley Male Sterility

[Objective] The aim was to research the genetic mechanism of barley male sterility, and provide the theoretical reference for breeding strong heterosis hybrid of barley. [ Method] Fertility segregation phenomenon, morphological characteristics and main agronomic characters of male sterility character of 4 kinds of barley male sterility materials 2001 - 17, 2001 - 37, 2001 - 84 and 2001 - 116 and their dedved lines were observed and researched.[ Result] Barley male sterility existed genetic phenomenon of single dominant nuclear gene caused by environmental stimulation, its sterility controlled by MS, the sterile genotype was MSms. There was stable nucleo-cytoplasmic inheritance on barley male sterility which was controlled by cytoplasmic mate sterile gene S and nuclear gene rr, and its genotype was S（rr）, this male sterility belonged to CMS type sterility. Temperature had no effect on fertility. [ Conclusion] There was stable nucleo-cytoplasmic inheritance on barley male sterility, this result played a positively promoting role in barley practical production.

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.

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.

Breeding and Cultivation Technique of Recessive Genic Male Sterility Hybrid Wheat New Variety Xinan 112

Xinan 112 is the hybrid combination（11S12） by wheat recessive genic male sterility line 2011Z1 （08L5070） and restoring line K152-2, particpating in the regional test of 2011-2012, 2012-2013 and production test of 2013-2014 in Chongqing. The results showed that 3 years average yield was 4 167.5 kg/hm2, which was increased 10.5% compared with CK Yumai 7, 1 000-grain weight was 45.8 g, which was 1.7 g heavier than the control; grain number per ear was 39.3, more than 1.6 grains compared with CK. The results of 2 years quality determination were： bulk density of 811 g/L, falling number of 353 s, crude protein of 15.15%, wet gluten of 31.2%, water adsorption of 62.9 ml/100 g, formation time of 5.5 min, stable time of 6.5 min, softening degree of 90 F.U., and powder quality coefficient of 96 mm. Therefore, Xinan 112 is a high yield and high protein gluten wheat variety and is suitable for cultivated in Chongqing and climate contion similar areas.

Technical Specification for Producing Hybrid Pepper Seed Using Three-line Male Sterility

This paper described the whole process of three line hybrid pepper seed production in detail, including requirement of the seed production base, parent culti- vation, field management, and specified the key operation techniques in seed pro- duction, such as parental impurity removal to preserve pure state, pollen collection, pollination and seed collecting essentials. This specification is of guiding significance for the production of hybrid pepper seed and ensuring the purity of hybrid pepper seed.

Research Advances in Molecular Mechanism of Thermo-sensitive Genic Male Sterility in Plants

Thermo-sensitive genic male sterile （TGMS） lines have specific superiority in heterosis utilization of crops. So far, thermo-sensitive genic male sterile lines have been found in many plants and are widely used in two-line hybrid breeding. With the rapid development of molecular biology, the molecular nature of thermo-sensitive genic male sterility has been revealed, which lays the foundation for further devel- opment and utilization of thermo-sensitive genic male sterile lines. In this study, the molecular mechanisms of fertility conversion of plant thermo-sensitive genic male sterile lines were reviewed from gene molecular mapping and gene differential ex- pression, and the mechanisms of gene differential expression in thermo-sensitive genic male sterile lines were further discussed.