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.

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.

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.

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. Microsatel-lite 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 Xgwm273 were 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 IBS 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 IBS, 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.

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.

The cotton mitochondrial chimeric gene orf610a causes male sterility by disturbing the dynamic balance of ATP synthesis and ROS burst

Plant cytoplasmic male sterility(CMS)is maternally inherited and often manifested as aborted pollen development,but the molecular basis of abortion remains to be identified.To facilitate an investigation of CMS in cotton,the complete sequence of cotton mitochondrial(mt)genome for CMS-D2 line ZBA was determined.The mt genome was assembled as a single circular molecule with 634,036 bp in length.A total of 194 ORFs,36 protein-coding genes,six r RNAs,and 24 t RNAs were identified.Several chimeric genes encoding hypothetical proteins with transmembrane domains were identified.Among them,a previously unknown chimeric gene,orf610a,which is composed of atp1 and a 485-bp downstream sequence of unknown nature,was identified.RT-PCR and q RT-PCR validation indicated that orf610a was expressed specifically in a sterile line.Ectopic expression of orf610a in yeast resulted in excessive accumulation of reactive oxygen species and reduction in ATP content,in addition to inhibition of cellular growth.Transgenic A.thaliana overexpressing orf610a fused with a mitochondrial targeting peptide displayed partial male sterility.Interaction between ORF610a and the nuclear-encoded protein RD22 indicated an association between ORF610a and pollen abortion.Positive feedback during transcriptional regulation between nuclear regulatory factors and the mt CMS gene may account for the male sterility of ZBA.

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 BC, progeny, we found that the restoring line Fengkel had two duplicating restorer genes. The restorer gene R/5 in Fengkel 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 Rf4 was mapped on chromosome 8S linked with bnlg2307.

Genetic analysis of fertility restoring genes for AL-type male sterility in wheat

In order to screen molecular markers linked to fertility restoring genes and further improve the breeding efficiency of restorer lines, in this study, wheat varieties 18A, 18B and 99AR144-1 were used as experimental materials to establish F2 fertility-segregating population. Plant quantitative trait ＂major gene ＋ polygene mixed mo- del＂ separation analysis method and simple sequence repeat （SSR） molecular markers were adopted for genetic analysis of four generations, including the parents （P~ and P2）, and hybrid （G and G） populations. The results show that AL-type fertility restoring gene is controlled by two pairs of additive-dominant-epistatic genes and addi- tive-dominant polygene; two primers linked to fertility restoring genes were selected by SSR molecular markers, including Xgwm95 on chromosome 2A and Barc61 on chromosome 1B, with the linkage distance of 15.0 cM and 18.0 cM, respectively. Based on verification, these two markers are reliable for distinguishing AL-type wheat ste- rile lines and restorer lines.

Mapping of Rice Fertility-Restoring Genes for ID-Type Cytoplasmic Male Sterility in a Restorer Line R68

An F2 population derived from the cross Zhong 9A/R68 was used to map the fertility-restoring （Rf） gene for ID-type cytoplasmic male sterility （CMS）. Two bulks （a fertile bulk and a sterile bulk） were constructed by pooling equal amount of ten highly fertile lines and ten highly sterile lines, respectively. Four hundred and thirteen pairs of simple sequence repeat （SSR） primers, which evenly distributed on 12 chromosomes of rice, were selected for analyzing polymorphisms between the parents and between the two bulks. The primer RM283 on chromosome 1 and the primers RM5756, RM258, RM6100 and RM171 on chromosome 10 were found to be polymorphic between the parents and between the two bulks. These five SSR markers were linked to fertility-restoring genes. A total of 82 excessive sterile lines were selected from Zhong 9A/R68 F2 population to estimate the genetic distance between five SSR markers and fertility-restoring genes respectively. The results indicated that one Rf gene was linked to RM283 located on chromosome 1 at a distance of 6.7 cM, and the other Rfgene was mapped to the long arm of chromosome 10 flanked by RM258 and RM6100 at the distances of 8.0 cM and 2.4 cM, respectively.

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.

Isolation and Characterization of the Cytoplasmic Male Sterility Associated Gene of Cotton(Gossypium harknessii)

Cytoplasmic male sterility(CMS) is a maternally inherited trait that results in the failure to produce functional pollen.It was identified in many plants,and it is widely used to exploit heterosis.

Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice

Pingxiang-dominant genic male sterile rice （PDGMSR） was the first dominant genic male sterile mutant identified in rice （Oryza sativa L.）, and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to con- struct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection （MAS） of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future.

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.

Mapping of a gene for photoperiod-sensitive genic male sterility in Nongken 58s on chromosome 5 of rice

A photoperiod-sensitive genic male sterile (PGMS) rice was found in 1973 as a spontaneous mutant of Nongken 58, a japonica variety. Pollen fertility of Nongken 58s (N58s) is completely sterile when grown under long-day conditions, whereas fertile under short-day conditions. This PGMS was found to be controlled by one or two recessive gene(s), of which one gene(pms)was linked to a marker gene(d-1) on chromosome 5. In order to identify a more precise location of the pms, we analyzed the populations of BCFand BCFof N58s//N58s/KL211(v-10, virescent) and N58s//N58s/KL520 (gh-1, gold hull). The marker genes v-10 and gh-1 are located on the flanking region of d-1. The F, plants of two crosses were fertile. The number of fertile and sterile individuals in BCFfit

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.

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.

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.

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.

K-Domain Splicing Factor OsMADS1 Regulates Open Hull Male Sterility in Rice

We identified the rice floral organ development mutant, termed as open hull and male sterile 1 （ohms1）, from the progeny of the indica restorer line Zhonghui 8015 treated with 60Co γ-ray irradiation. The ohms1 mutant exhibited an open hull and lemma- and palea-like structure conversion between the anthers and stigma, which resulted in the ohms1 mutant spikelet showing‘tridentate lemma＇. The ohms1 mutant was entirely sterile but had 60%-70% fertile pollen. Genetic analysis and gene mapping showed that ohms1 was controlled by a single recessive gene, and the mutant gene was fine-mapped to a 42-kb interval on the short arm of chromosome 3 between markers KY2 and KY29. Sequence analysis of the four open reading frames in this region revealed that the mutant carried a single nucleotide transformation （A to G） at the last base of the fifth intron, which was likely corresponded to ohms1 phynotype, in an MIKC type MADS-box gene OsMADS1 （LOC_Os03g11614）. Enzyme digestion and cDNA sequencing further indicated that the variable splicing was responsible for the deletion of the sixth exon in ohms1, but no structural changes in the MADS domain or amino acid frame shifts appeared. Additionally, real-time fluorescent quantitative PCR analysis showed that the OsMADS1 expression level decreased significantly in the ohms1 mutant. The expression levels of rice flowering factors and floral glume development-related genes also changed significantly. These results demonstrate that OsMADS1 may play an important role in rice floral organ development, particularly in floral glume development and floret primordium differentiation.

Study on Agrobacterium-Mediated Transformation of Pepper with Barnase and Cre Gene

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper （Capsicum annuum L.）. Chili pepper inbred lines （A, D, E, and I） were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR[MB（MS mineral＋vitamine B5）＋BA（6-Benzyladenine） 5.0 mg·L^-1 ＋IAA（indoleacetic acid） 1.0 mg·L^-1＋GA3（gibberellic acid） 1.0mg·L^-1＋sucrose 3%＋agar 6.5g·L^-1] for 2d. The explants were infected by Agrobacterium tumefaciens when their OD600（optical density at 600 nm）reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB＋BA5.0 mg·L^-1＋IAA 1.0 mg·L^-1 ＋GA3 1.0 mg·L^-1＋sucrose 3% ＋agar 6.5 g·L^-1＋AS （acetosyringone） 200μmol·L^-1, these cotyledons with petiole were cultured on selective differentiation medium in the media MT[MB medium supplemented with BA [5.0 mg·L^-1＋ IAA 1.0 mg·L^-1＋ GA3 1.0 mg·L^-1＋ AgNO3 5.0 mg·L^-1＋ CW （coconut water） 5% ＋ Km （kanamycin） 65 mg·L^-1＋ Cb （carbenicillin） 500 mg·L^-1＋ 3% sucrose ＋ agar 6.5 g·L^-1].The Kmr （kanamycin resistant） bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen germination experiments.