The Application of ISSR Markers to Identify the Fertility Restorer Gene Rf6 in T. timopheevii Cytoplasmic MaleSterile Wheat(Triticum aestivum)

Inter-simple sequence repeat (ISSR) analysis was carried out on a F2 population of 147 plants derived from a cross between a wheat male fertility restorer line 2114 and a male sterile line ND44A. Out of 43 primers examined, 18 primers produced distinguishable, polymorphic bands between the two parents. Linkage analysis in the mapping population showed that two markers UBC-808 and UBC-848 were closely linked with the restorer gene Rf6 of the Triticum timopheevii CMS system. The distance between the two markers and the restorer gene was 7.9 cM and 4.9 cM, respectively. Also two parents were screened with 181 pairs of SSR primers, of which, 34.3% showed polymorphisms. But no locus was found linked with the restorer gene. Compared with the SSR technique, the ISSR approach used in the experiment provided more information and proved to be a valuable method to identify alien fragments.

Microsatellite-Aided Screening for Fertility Restoration Genes (Rf) Facilitates Hybrid Improvement

DNA markers enabled to determine the chromosomal locations of the two Rf genes(Rf3 and Rf4) in the wild-abortive cytoplasmic male sterility(WA-CMS) system. Four simple sequence repeats(SSRs) RM171, RM258, RM315 and RM443 were used to detect the allelic status with respect to the fertility restoration genes(Rf3 and Rf4) in 300 rice cultivars or breeding lines. The results revealed that out of 300 lines, 90 lines screened had Rf3, 65 lines had Rf4, and 45 lines had Rf3 and Rf4 alleles. Furthermore, 45 lines selected using SSR markers were mated with a CMS line(IR58025A) to analyze their restoring ability. Offspring of all the test lines except HHZ8-SAL9DT1-Y1, HHZ5-SAL9-Y3-1 and IDSA77 exhibited higher pollen and spikelet fertility(> 80%), thus confirming they bear the Rf alleles. The hybrid offspring of ARH12-6-1-1-B-3-1, IR32307-10-3-2-1 and Sahel 329 had the highest pollen fertility(97.39%, 98.30% and 97.10%, respectively) and spikelet fertility(95.10%, 97.07% and 96.10%, respectively).

Breeding and Characterization of a New Rice Restorer Line Containing Bt Gene

Bt5198, a new rice restorer line containing Bt gene, was developed from the cross and backcross of the elite restorer line Chenghui 177 with Bt Minghui 63, a transgenic Bt restorer line. The inbred lines were evaluated using PCR amplification, test paper evaluation, insect resistance evaluation in both the laboratory and paddy fields, nursery evaluation of rice blast resistance and pedigree selection of agronomic traits. Larval mortalities on Bt5198 and Bt Minghui 63 were 100% when rice culms were inoculated with the eggs of the striped stem borer （SSB） in the laboratory. Bt5198 was highly resistant against SSB and the yellow stem borer （YSB） under field conditions. The F1 hybrids derived from Bt5198 and four cytoplasmic male sterile （CMS） lines were also highly resistant to SSB and YSB and had a significant heterosis. Two-year evaluation of rice blast resistance confirmed that the resistance levels of Bt5198 to leaf blast and neck blast were similar to those of Chenghui 177 and significantly better than those of Bt Minghui 63. Seed germination ability and pollen yield of Bt5198 were similar with Chenghui 177, suggesting that the introduction of the Bt gene into the new restorer line had no significant effects on seed vitality or the yield of seed production. To identify the presence of the Bt gene, it was effective to combine test paper examination with the evaluation of insect-resistance, both in the laboratory and under field conditions.

Genetic Analysis of Stripe Disease Resistance in Rice Restorer Line C224 Using Major Gene plus Polygene Mixed Effect Model

The inheritance of stripe disease resistance in a rice restorer line C224 was analyzed using the mixed effect model of major gene plus polygene for quantitative traits.In addition,the resistance was investigated in seven crosses of C224 with maintainer lines.The results showed that the stripe resistance of C224 was controlled by two major genes with additive-dominance-epistasis effects plus polygenes with additive-dominance effects （E-1 model）.These two genes had additive effects of-12.47% and-24.75%,respectively,showing negative dominance effects.There were significant epistasis and interaction effects between the two major genes.The heritability of the two major genes was 92.12%,while that of polygenes was 2.74%,indicating that the stripe resistance had dominant major gene effect.Of the seven crosses,five displayed high or medium resistance to the stripe disease.

Significant association of the novel Rf4-targeted SNP marker with the restorer for WA-CMS in different rice backgrounds and its utilization in molecular screening

In the rice cytoplasmic-genetic male sterility （CMS） system, the combination of a CMS line, maintainer line and restorer line carrying the restorer gene to restore fertility, is indispensable for the development of hybrids. However, the process of screening for the trait of fertility restoration is laborious and time-consuming. In the present study, we analyzed the nucleotide sequence of the Rf4 gene, which is the major locus controlling fertility restoration, to identify allele-specific variation. A single nucleotide polymorphism （SNP） A/C at ＋474 in the coding sequence （CDS） was found to be capable of strictly distinguishing groups of alleles Rf4 （A） and rf4 （C）. Using KASP genotyping, this valuable SNP was converted to an allele-specific PCR marker. We evaluated and validated the marker among three-line parents with different backgrounds, and the results revealed a complete correlation between SNP alleles and the fertility restoration phenotype. Molecular screening was subsequently carried out for the presence of alleles of Rf4 and Rf3 among 328 diverse rice cultivars with worldwide distribution. The results demonstrate that this SNP marker could be the optimal choice for the molecular identification of potential restorers.

Identification and fine mapping of a fertility restorer gene for wild abortive cytoplasmic male sterility in the elite indica rice non-restorer line 9311

The wild abortive(WA)-type cytoplasmic male sterility(CMS)derived from the wild rice species Oryza rufipogon Griff.is used widely in three-line indica hybrids.The identification and mapping of restorer of fertility(Rf)genes aided in the development of WA-type hybrids.Here we report that testcross F1 plants from the WA-type CMS line and 9311 exhibited stainable pollen grains with no seed set,indicating that 9311 carries minor-effect Rfs for WA-type CMS.We developed an advanced backcross population consisting of plants harboring small regions of donor chromosomal segments from 9311 in the WATianfeng A genetic background with moderate seed setting rates.Genetic analysis showed that the pollen fertility levels of the backcross individuals are governed by a single gene from 9311 that we named Rf19(t).By use of the RICE 40 K gene chip,three introduced segments were identified in the fertile lines,and a candidate region spanning 4.37–8.29 Mb on chromosome 1 was identified for Rf19(t).Finally,Rf19(t)was fine-mapped to a region of 90 kb between the DNA marker loci STS1-163 and STS1-183,in which eight ORFs were predicted.Also,using relative expression analyses,comparative sequence analyses and functional domain analyses,we identified LOC_Os01g10530 as the most likely candidate gene for Rf19(t).Furthermore,Rf19(t)was found to function in fertility restoration,most probably by regulating the degradation of m RNA transcribed from the mitochondrial gene WA352.These results increase our knowledge of fertility restoration in WA-type CMS lines and will facilitate the development of high-quality pairs of WAtype CMS and maintainer 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.

Influence of Genetic Drift of Restoring Gene (Rf) on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

The seed samples of Yuetai A, a Honglian （HL） type cytoplasmic male sterile （CMS） line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing, Jiangsu Province and Lingshui, Hainan Province during 2004-2006. The seed impurity of Yuetai A mainly resulted from the genetic drift of restoring fertility gene （Rt） after biological contamination but not from its sterility unstability. All of the ten maintainer lines, five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants, directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating ＇iso-cytoplasm restoring-generations＇. Furthermore, high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination. After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A, the seed purity of Yuetai A and Yuetai B had been raised to 100%.

Breeding of Rice Restorer Line Haihui 818 of Multi-resistant Gene Pyramiding

Haihui 818 is a new restorer line by cross breeding with Hua 23 S and Huahui 8131.Good plant type,high tillering ability,fine grain quality,strong restoring ability and high yield were shown in hybrid production.After artificial inoculation and field stress and molecular market-assisted selection,the results showed that rice-blast resistance genes Pi1,BB resistance genes Xa7,and brown planthopper resistance genes Bph3,Bph14 and Bph15 were pyramided together.Among its hybrids,Bo II You 818(Bo II A/Haihui 818)was approved for commercial production by Hainan Provincial Crop Variety Approval Committee in 2015.

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.

The Restoring Ability of Normal Indica Red Rice Ruby and Its Restoring Gene Mapping

[Objective] This study was to investigate the restoring ability of normal indica red rice Ruby and to carry out its restoring gene mapping. [Method] Normal indica red rice Ruby was hybridized with the sterile lines Zhenxian 97A, D62A, G46A and D702A to prepare their F1, BC1 and F2 progenies, and the pollen fertilities of these progenies were investigated. Meanwhile the restoring genes were mapped using SSLP. [ Result] For the sterile lines tested, Ruby has a gene to restore their fertilities. This gene is located on the chromosome 7 and shows a genetic distance of 7.4 cM with RM182. Unlike the clustering distribution of the restoring genes on chromosome 10, it is a specific restoring gene. [ Conclusion] it is feasible to breed restoring genes controlling red color characters via transgene and backcross.

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.

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.

Genome-wide analysis of Rf-PPR-like(RFL)genes and a new InDel marker development for Rf1 gene in cytoplasmic male sterile CMS-D2 Upland cotton

Background: Cytoplasmic male sterility in flowering plants is a convenient way to use heterosis via hybrid breeding and may be restored by nuclear restorer-of-fertility(Rf) genes. In most cases, Rf genes encoded pentatricopeptide repeat(PPR) proteins and several Rf genes are present in clusters of similar Rf-PPR-like(RFL) genes. However, the Rf genes in cotton were not fully characterized until now.Results: In total, 35 RFL genes were identified in G. hirsutum, 16 in G. arboreum, and 24 in G. raimondii. Additionally,four RFL-rich regions were identified; the RFL-rich region in Gh05 is the probable location of Rf-PPR genes in cotton and will be studied further in the future. Furthermore, an insertion sequence was identified in the promoter sequence of Gh05 G3392 gene in the restorer line, as compared with the CMS-D2 line and maintainer lines. An InDel-R marker was then developed and could be used to distinguish the restorer line carrying Rfl from other genotypes without the Rf1 allele.Conclusion: In this study, genome-wide identification and analysis of RFL genes have identified the candidate Rf-PPR genes for CMS in Gossypium. The identification and analysis of RFL genes and sequence variation analysis will be useful for cloning Rf genes in the future and also for three-line hybrid breeding in cotton.

Breeding Rice Restorer Lines with High Resistance to Bacterial Blight by Using Molecular Marker-Assisted Selection

Two bacterial blight （BB） resistance genes, Xa21 and Xa4, from IRBB24 were introduced into hybrid rice restorer line Mianhui 725, which is highly susceptible to BB, by using hybridization and molecular marker-assisted selection technology. Four homologous restorer lines were obtained through testing the R target genes with molecular markers and analyzing parental genetic background. Inoculation of the four lines and their hybrids with the specific strains of Xanthomonas oryzae pv. oryzae, P1, P6 and seven representative strains of Chinese pathotype, C Ⅰ -CⅦ, showed that all of the four lines and their hybrids were highly resistant and presented broad resistance-spectrum to BB. The hybrids of G46A / R207-2 displayed good agronomic characters and high yield potential, and R207-2 was named Shuhui 207.

Inheritance of Fertility Restoration for Cytoplasmic Male Sterility in a New Gossypium barbadense Restorer

In order to clarify inheritance mechanism of fertility restoration for cytoplasmic male sterility （CMS） in a new Gossypium barbadense restorer line Hai R which was found in the fertility test crossing of G. hirsutum CMS lines with G. barbadense germplasms. 23 fertility segregation populations of F2 and backcross were used to analyze the inheritance of fertility restoring gene（s） of Hai R. The result showed that Hai R had one major dominant gene （RfB） to control the CMS fertility restoration and this fertility restoration gene functioned at the sporophytic level. The sterile cytoplasm background might not only influence the transmission rate of male gamete but also that of female gamete when the restorer gene was recessive. It could be deduced that this fertility restoration gene might come from G. harknessii cotton, Hai R is of value in the application of cotton interspecific hybrid breeding.

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.

Molecular cloning and expression analysis of the Rf-m candidate genes encoding pentatricopeptide repeat proteins in soybean

Cytoplasmic male sterility(CMS)-restorer system is a useful tool to exploit heterosis in soybean.The major restorer gene for the M-type CMS is known as Rf-m,located in the 162.4-kb region on chromosome 16.Sequence analysis has revealed that the Rf-m locus in Glycine max consists of seven penta tricopeptide repeat(GmPPR)genes.The deduced amino acid sequences contain 8 to 14 PPR motifs,and a phylogenetic analysis grouped these GmPPR proteins into two PPR subfamilies:Glyma.16G161800 belongs to the PLS subfamily,and the P subfamily consists.of Glyma.16G161900,Glyma 16G162000,Glyma.16G162100,Glyma.16G162700,Glyma.16G162800,and Gly-ma 16G163100.The phylogenetic analysis of seven GmPPR proteins and 27 other plant PPR proteins also showed that proteins in the same subfamilies cluster together.Comparative sequence analysis was conducted using the seven Rf-m candidate GmPPR genes from the sterile line W931A,the maintainer line W931B,and the restorer line WR016,the result showed that Glyma 16G161900 had higher polymorphism than the other candidate genes.Based on real-time quantitative RT-PCR data,all seven GmPPR genes were differentially expressed but showed constitutive expression in roots,stems,leaves,and pollen grains.Additionally,the expression level of Gly-ma 16G161900 in the sterile line W931 A was significantly higher in all tissues than in the restorer line WR016.Taken together,these results suggest that Glyma 16G161900 is the most likely candidate for the restorer gene Rf-m.This study is the first report and analysis of candidate fertility restorer(Rf)genes encoding PPR proteins in soybean.

Development of New InDel Marker to Detect Genotypes of Rf-1a Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight （BB） diseases, the two most prevalent diseases jeopardizing rice production. Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food. An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions. Only one pair of parents, DH146 ×TM487, showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes, Xa21, xa13 and xa5, thus it was used in the marker-assisted selection （MAS）. F2 individuals of DH146× TM487 were genotyped using flanking markers of RM3330 and sequence tagged site （STS） marker SA7 for Pi25. The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21, xa13 and xa5 identified by the markers pTA248, RM264 and RM153, respectively in subsequent generations. Finally, after selection for agronomic traits and restoration ability among 12 pyramided lines, we acquired an elite restorer line, R8012 including all four target genes （Pi25＋Xa21＋xa13＋xa5）. Hybrid Zhong 9NR8012 derived from the selected line showed stronger resistance to blast and BB, and higher grain yield than the commercial checks uniformally in experimental plots, 2007 state-wide yield trial and 2008 nation-wide yield trial. This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes. The resultant restoring line and its hybrid would play an important role in securing rice production in China.