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.

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.

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.

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 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.

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.

Fertility Expression of TGMS-Genes in the Backgrounds of indica CMS-lines,B-lines and R-lines of Hybrid Rice

The generation fertility of 51 F1, 19 F2 and 6 BC1 between 3 thermo-sensitive genic male sterile lines （TGMS-lines） Pei＇ai 64S, 6311S and 360S, and the three lines of hybrid rice including 7 indica cytoplasmic male sterile lines （CMS-lines） and their corresponding maintainer lines （B-lines） and 3 indica restorer lines （R-lines） were investigated to study the expression of TGMS-genes in the backgrounds of the three lines of hybrid rice. Pei＇ai 64S has stronger fertility restoring （Rf） genes for CMS-lines and its TGMS trait is governed by 2 pairs of independent recessive genes; The TGMS trait of 6311S is governed by a single recessive gene with weaker Rf-gene in 6311S and the TGMS trait of 360S is governed by a single recessive gene with no Rf-gene in 360S. The investigation on the fertility of F1 plants between 5 CMS-lines and 4 TGMS generations selected from F2 plants of 4 CMS-lines x 6311S confirmed that the expression of TGMS-gene was controlled by Rf-gene in the genetic background of cytoplasm of CMS-lines, but not affected by Rf-gene in the genetic background of normal fertile cytoplasm. The potential breeding strategies of TGMS-lines with cytoplasm of CMS-lines and CMS-lines with the nucleus of TGMS-genes were discussed.

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.

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%.

Analysis of restoring gene in Heizhenmi

The purple pigment is controlled by three domi-nant genes.According to the theory ofdominance-super dominance in Fgeneration, breeding purple black hybrid rice can be an effec-tive way to overcome lower yield and weak resist-ance to disease of purple black rice.

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.

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.

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.