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.

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.

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.

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.

Introgression of Gene for Non-Pollen Type Thermo-Sensitive Genic Male Sterility to Thai Rice Cultivars

For the two-line hybrid rice system, pol en sterility is regulated by recessive gene that responds to temperature. The recessive gene controlling thermo-sensitive genetic male sterility （TGMS） is expressed when the plants are grown in conditions with higher or lower critical temperatures. To transfer tgms gene（s） control ing TGMS to Thai rice cultivars by backcross breeding method, a male sterile line was used as a donor parent while Thai rice cultivars ChaiNat 1, PathumThani 1, and SuphanBuri 1 were used as recurrent parents. The BC2F2 lines were developed from backcrossing and selfing. Moreover, the simple sequence repeat （SSR） markers were developed for identifying tgms gene and the linked marker was used for assisting selection in backcrossing. The identification lines were confirmed by pol en observation. The results showed the success of introgression of the tgms gene into Thai rice cultivars. These lines will be tested for combining ability and used as female parent in hybrid rice production in Thailand.

Inheritance of the Male Sterility in a New Photo/Thermo-Sensitive Genie Male Sterile Line B06S of Rice

The major male sterile genes in a new photo/thermo-sensitive genie male sterile (PTGMS) line B06S of rice were analyzed by the manipulation of mixture distribution theory. The results indicated that a pair of major male sterile nuclear genes with large effects were responsible for controlling the male sterility of B06S.

Characterization of Reverse Thermo-sensitive Genic Male Sterile Lines in Cold Regions of China

Yannong S, a reverse thermo-sensitive genic male sterile （TGMS） line, is sterile at high temperature and fertile at low temperature. In the present study, the fertility of Yannong S and the japonica reverse TGMS lines derived from it was in- vestigated in Harbin, Heilongjiang from 2013 to 2015. The results showed that Yan- nong S was completely male sterile in July and August （the hottest months of the year in Harbin）, and the seed setting rate of bagged panicles was 0. We also in- ferred that the critical temperature for inducing the sterility of Yannong S was higher than 28 ℃. Most of the japonica reverse TGMS lines derived from Yannong S were completely aborted in July and August, with a seed setting rate of 0. It can be con- cluded that the reverse TGMS lines derived from Yannong S are genetically stable lines that have a long period of sterility in cold regions, so they may be the ideal genetic materials for the development and in-situ seed production of japonica hybrid rice.

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.

Characterizations and identification of the candidate gene of rice thermo-sensitive genic male sterile gene tms5 by mapping

Previous study indicated that the thermo-sensitive genic malesterile(TGMS) gene in rice was regulated by temperature.TGMS rice plays an important role in hybrid rice production,because the application of the TGMS system in two-line breeding is laborsaving,timesaving,simple,inexpensive,efficient,and eliminating the limitations of the cytoplasmic male sterility(CMS) system.'AnnongS' is the first discovered and deeply studied TGMS rice lines in China.'AnnongS-1' and 'Y58S',two derivatives of TGMS line AnnongS,were both controlled by a single recessive gene named tms5,which was genetically mapped on chromosome 2.In this study,three populations('AnnongS-1' × 'Nanjing11','Y58S' × 'Q611',and 'Y58S' × 'Guanghui122') were developed and used for the molecular fine mapping of the tms5 gene.By analyzing recombination events in the sterile individuals using a total of 125 probes covering the tms5 region,the tms5 gene was physically mapped to a 19-kb DNA fragment between two markers 4039-1 and 4039-2,which were located on the BAC clone AP004039.After the construction of the physical map between two markers 4039-1 and 4039-2,a member(ONAC023) of the NAC(NAM-ATAF-CUC-related) gene family was identified as the candidate gene of the tms5 gene.

Plant Temperature and Its Simulation Model of Thermo-Sensitive Genic Male Sterile Rice

Plant temperature （Tp） and its relations to the microclimate of rice colony and irrigation water were studied using a thermo-sensitive genic male sterile （TGMS） rice line, Pei＇ai 64S. Significant differences in the daily change of temperature were detected between Tp and air temperature at the height of 150 cm （TA）. From 8：00 to 20：00, Tp was lower than TA, but they were similar during 21：00 to next 7：00. The maximum Tp occurred one hour earlier than the maximum TA, though they both reached the minimum at 6：00. Tp fluctuated less than TA. At the same height, during 6：00-13：00, Tp was higher than air temperature （Ta）, and Tp reached the maximum one hour earlier than Ta. During the rest time on sunny day, Tp was close to or even a little lower than Ta. On overcast day, Tp was higher than Ta in the whole day, and both maximized at the same time. In addition, Tp was regulated by solar radiation, cloudage and wind speed in daytime, and by irrigation water at night. The present study indicated that a TA of 29.6℃ was the critical point, at which Tp was increased or decreased by irrigation water. Tp and the difference between water and air temperatures showed a conic relation. Tp fluctuation was also regulated by the absorption or reflection of solar radiation by leaves during daytime and release of heat energy during nighttime. By analysis on correlation and regression simulation, two models of Tp were established.

Characterization and fine mapping of RTMS10, a semi-dominant reverse thermo-sensitive genic male sterile locus in rice

The discovery and application of environment-sensitive genic male sterile(EGMS) rice germplasm provide an easy method for hybrid rice breeding and have made great contributions to hybrid rice production. Typically, the photoperiod-and thermosensitive GMS(P/TGMS) lines utilized in two-line hybrid systems are male sterile under long day or/and high temperature but fertile under short day or/and low temperature conditions. However, Yannong S(Yn S), a reverse TGMS(rTGMS) line, is sterile under low temperature(<29℃) and fertile under high temperature(>29.5℃). Here, we report a genetic study on the rTGMS trait in Yn S. Interestingly, the F1 plants of the cross between Yn S and a cultivar, L422, were male sterile at 22℃ and completely fertile at 27℃. Moreover, the segregation ratio of fertile and sterile individuals in Yn S/L422 F2 populations changed from 1:3.05 to 2.95:1 when the ambient temperature increased, showing that the rTGMS trait exhibits semidominance in Yn S. We further found a locus on chromosome 10, termed RTMS10, which controls the rTGMS trait in Yn S. We then finely mapped RTMS10 to a ~68 kb interval between markers ID13116 and ID1318 by Yn S/L422 BC6 F2 populations. A near iso-genic line(NIL) NL1 from the BC6 F3 generation was developed and the pollen of NL1 became abnormal from the meiosis stage under low temperature. In summary, we identified an rTGMS locus, RTMS10, and provided co-segregated markers, which could help to accelerate molecular breeding of rTGMS lines and better understand the rTGMS trait in rice.

Marker-Assisted Breeding of Thermo-Sensitive Genic Male Sterile Line 1892S for Disease Resistance and Submergence Tolerance

Rice line 1892S is an elite thermo-sensitive genic male sterile(TGMS)line for two-line hybrid rice production.However,1892S is susceptible to rice blast,bacterial blight and submergence.Here we reported the introduction of blast resistance(R)gene Pi9,bacterial blight R gene Xa21 and submergence tolerance gene Sub1A into 1892S genetic background through backcrossing and marker-assisted selection.The improved TGMS line 31892S and its hybrids conferred disease resistance to rice blast and bacterial blight,and showed submergence tolerance for over 14 d without significant loss of viability.The sterility-fertility conversion of 31892S was similar to that of 1892S.31892S and its derived hybrid rice had similar agronomic traits and grain quality with 1892S and the control hybrid rice,respectively.The newly developed 31892S provided an improved TGMS line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight,and submergence tolerance with no yield penalty or change in grain quality.

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S （9.09%）. On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

Elongation of the Uppermost Internode for Changxuan 3S,a Thermo-Sensitive Genic Male Sterile Rice Line

Changxuan 3S, a thermo-sensitive genic male sterile （TGMS） rice line with eui gene, is derived from the TGMS rice line Pei＇ai 64S by irradiation with 350 Gy of ^60Co γ-ray. To elucidate the uppermost internode elongation of the TGMS line with eui gene, Changxuan 3S and its parent Pei＇ai 64S were used to study the effects of temperature on panicle exsertion. At 24℃, the uppermost internode of Changxuan 3S elongated the fastest from the 4^th day before flowering to 0 day （flowering）, being 2.1-fold as that of Pei＇ai 64S, whereas it elongated slowly during the 12^th day to the 4^th day before flowering and the 1^st to the 3^rd day after flowering. The uppermost internode of Changxuan 3S exserted from the flag leaf sheath at 22℃, 24℃ and 26℃, and the length of elongated uppermost internode increased with the decreasing temperatures. At 28℃, though the panicles of Changxuan 3S were still enclosed in the leaf sheath, the degree of panicle enclosure was significantly lower compared with Pei＇ai 64S. Cytological studies on Changxuan 3S showed that the uppermost internode elongation was attributed to the increase of cell number and cell elongation, and the latter was more significant. Moreover, the numbers of outermost and innermost parenchyma cells and the cell length of the uppermost internode reduced with the increasing temperatures.

Differentially Expressed Genes Related to Fertility Conversion in Thermo-sensitive Genic Male Sterile(TGMS) Lines of Brassica juncea

[Objective] This study was performed to screen functional genes related to the fertility conversion of thermo-sensitive genic male sterile （TGMS） lines of Brassica juncea L. [Method] A B. juncea TGMS line K121S was selected as the experimental material. The total RNAs were isolated from fertile and sterile pollens at different development stages, including mother cell stage, tetrad stage, tricellular pollen stage and maturity stage. DDRT-PCR was carried out to identify differentially expressed genes. [Result] A total of 44 differentially expressed cDNA fragments were identified with Dot blot. And seven candidate genes related to fertility conversion of K121S were screened out by BLASTN, including callose synthase gene, aldehyde dehydrogenase gene and RNA polymerase I transcription factor RRN3 gene which were differentially expressed at the transcriptional level, H＇-ATPase gene, fructose diphosphate aldolase -class I gene, teucine-rich repeat receptor-Jike serine/threonine- protein kinase gene and alkaline/neutral invertase gene, which were differentially expressed at the post-transcriptional level. [Conclusion] The results of this study will help to explain the molecular mechanism of thermo-sensitive genic male sterility of B. juncea.

Thermo-sensitive genic male sterile (TGMS) rice line developed by population improvement

In this study population improvement or random polycross plus mass selection was adopted with using N8S, Huaizao4, Xiang2B and Zaoyoul as parents. To help comprehensively improve the important characters such as combining ability, grain quality, resistance, stigma exsertion rate, etc. The stigma exsertion rate of ZhunS, an elite TGMS line, is 78.6%, 72.4% higher than that of V20A. Hybrids produced by crossing ZhunS with R402, Minhui77 and Minhui63 increased the yield by 3.8%-5.8% and decreased grain chalkiness by 82.1%-84.4% compared with those from crossing V20A with the corresponding R lines.

Study on the Pollen Thermo-Sensitive and Stigma Exsertion Male Sterile Line Tomato (Lycopersicon esculentum Mill.) cv. Da107

To accelerate the breeding process, male sterile line is used to leave out the troublesome procedures of the artificial emasculation in tomato breeding. However, the fertility of the pollen thermo-sensitivity male sterile line (PTMSL) and the stigma exsertion male sterile line (SEMSL) are affected easily by the environments when used alone. The trial materials were Da107 and the control was First. This study was conducted to create a new male sterile line of tomato characterized by pollen thermo-sensitivity and long style by genic recombination through the hybridizing of the PTMSL and SEMSL. Research on the statistics of the pollen germination rate, the contamination rate in F1 and the flower organics indicated that Da107 was an ideal TS and SE male sterile line with the sterility of 95%, as well as it also could be used as fertile line at low temperature. Meanwhile, the results showed that hybrid-seed contamination risk with selfed seeds from residual fertility in Da107 was low.

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.