Genetic analysis of fertility-restorer genes in rice

Wild-abortive (WA), Honglian (HL) and Baro-II (BT) are three important cytoplasmic male sterility (CMS) types in rice. It is essential to investigate genetic mode and allelism of fertility restorer (Rf) genes and the relationship between Rf and CMS. Fertility of the all test-cross F₁ plants shows that the restorer-maintainer relationship is similar for HL-CMS and BT-CMS, while that is variance for WA-CMS and HL-CMS (or BT-CMS), respectively. Genetic analysis of Rf genes indicates that HL-or BT-CMS are controlled by single dominant Rf gene and WA-CMS is controlled by one or two pairs of dominant Rf genes, which reflects the characters of the gametophytic and sporophytic restoration CMS type. It is concluded that there are at least three Rf loci in different accessions with Rf genes for each CMS type.     

Distribution of fertility-restorer genes for wild-abortive and Honglian CMS lines of rice in the AA genome species of genus Oryza

BACKGROUND AND AIMS: Rice (Oryza sativa) is one of the most important cereal plants in the world. Wild-abortive (WA) and Honglian (HL) cytoplasmic male sterility (CMS) have been used extensively in the production of hybrid seeds. Although a variable number of fertility-restorer genes (Rf) for WA and HL-CMS have been identified in various cultivars, information on Rf in Oryza species with the AA-genome is sparse. Therefore the distribution and heredity of Rf for WA and HL-CMS in wild rice species of Oryza with the AA-genome were investigated. METHODS: Fertility-restorer genes for WA and HL-CMS in wild rice species with the AA-genome were investigated by following the fertility of microspores identified by I2-KI staining and by following the seed-setting rate of spikelets. A genetic model of Rf in some selected restorer accessions was analysed based on the fertility segregation of BC1F1 populations. KEY RESULTS: Fertility analysis showed that 21 out of 35 HL-type F1s, and 13 out of 31 WA-type F1s were scored as fertile. The frequency of Rf in wild rice was 60% for HL-CMS and 41.9% for WA-CMS, respectively. The fertility-restorer accessions, especially those with complete restoring ability, aggregated mainly in two species of O. rufipogon and O. nivara. The wild rice accessions with Rf for HL-CMS were distributed in Asia, Oceania, Latin American and Africa, but were centered mainly in Asia, whilst the wild restorer accessions for WA-CMS were limited only to Asia and Africa. Apart from one restorer accession that possessed two pairs of Rf for WA-CMS, all of the other nine tested wild restorer accessions each contained only a single Rf for WA-CMS or HL-CMS. Allele analysis indicated that there existed at least three Rf loci for the WA and HL-CMS systems. CONCLUSIONS: These data support the hypothesis that fertility-restorer genes exist widely in Oryza species with the AA-genome, and that Rf in Oryza sativa originated from the Oryza rufipogon/Oryza nivara complex, the ancestor of cultivated rice in Asia. The origin and evolution of Rf is tightly linked to that of CMS in wild rice, and fertility of a given CMS type is controlled by several Rf alleles in various wild restorer accessions.     

<Emphasis Type="Italic">Rf5</Emphasis> is able to partially restore fertility to Honglian-type cytoplasmic male sterile <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) lines

Three-line japonica hybrids have been developed mainly on Chinsurah Boro II (BT)-type cytoplasmic male sterile (CMS) lines of Oryza sativa L., but the unstable sterility of some BT-type CMS lines, and the threat of genetic vulnerability when using a single cytoplasm source, have inhibited their use in rice cultivation. Previously, the sterility of Honglian (HL)-type japonica CMS lines derived from common red-awned wild rice (Oryza rufipogon) has been proven to be more stable than that of BT-type japonica CMS lines. Here, we genetically characterized HL-type japonica CMS lines and the restorer-of-fertility (Rf) gene for breeding HL-type japonica hybrids. HL-type japonica CMS lines displayed stained abortive pollen grains, unlike HL-type indica CMS lines. The BT-type japonica restorer lines, which contain Rf, had different capabilities to restore HL-LiuqianxinA (HL-LqxA), an HL-type japonica CMS line, and the restorers for the HL-type japonica CMS lines could be selected from the preexisting BT-type japonica restorers in rice production. A genetic analysis showed that the restoration of normal fertility to HL-LqxA was controlled by a major gene and was affected by minor effector genes and/or modifiers. The major Rf in SiR2982, a BT-type japonica restorer, was mapped to a ~100-kb physical region on chromosome 10, and was demonstrated to be Rf5 (Rf1a) by sequencing. Furthermore, Rf5 partially restored fertility and had a dosage effect on HL-type japonica CMS lines. These results will be helpful for the development of HL-type japonica hybrids.     

Genetical and molecular analysis reveals a cooperating relationship between cytoplasmic male sterility- and fertility restoration-related genes in <Emphasis Type="Italic">Oryza</Emphasis> species

Although the characterization of genes associated with cytoplasmic male sterility (CMS) and fertility restoration (Rf) has been well documented, the evolutionary relationship between nuclear Rf and CMS factors in mitochondria in Oryza species is still less understood. Here, 41 accessions from 7 Oryza species with AA genome were employed for analyzing the evolutionary relationships between the CMS factors and Rf candidates on chromosome 10. The phylogenetic tree based on restriction fragment length polymorphism patterns of CMS-associated mitochondrial genes showed that these 41 Oryza accessions fell into 3 distinct groups. Another phylogenetic tree based on PCR profiles of the nuclear Rf candidates on chromosome 10 was also established, and three groups were distinctively grouped. The accessions in each subgroup/group of the two phylogenetic trees are well parallel to each other. Furthermore, the 41 investigated accessions were test-crossed with Honglian (gametophytic type) and Wild-abortive (sporophytic type) CMS, and 5 groups were classified according to their restoring ability. The accessions in the same subgroup of the two phylogenetic trees shared similar fertility restoring pattern. Therefore, we conclude that the CMS-associated mitotypes are compatible to the Rf candidate-related nucleotypes, CMS and Rf have a parallel evolutionary relation in the Oryza species.     

Inheritance and molecular mapping of two fertility-restoring loci for Honglian gametophytic cytoplasmic male sterility in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

The Honglian cytoplasmic male sterility (cms-HL) system, a novel type of gametophytic CMS in indica rice, is being used for the large-scale commercial production of hybrid rice in China. However, the genetic basis of fertility restoration (Rf) in cms-HL remains unknown. Previous studies have shown that fertility restoration is controlled by a single locus located on chromosome 10, close to the loci Rf1 and Rf4, which respond to cms-BT and cms-WA, respectively. To determine if the Rf locus for cms-HL is different from these Rf loci and to establish fine-scale genetic and physical maps for map-based cloning of the Rf gene, high-resolution mapping of the Rf gene was carried out using RAPD and microsatellite markers in three BCF₁ populations. The results of the genetic linkage analysis indicated that two Rf loci respond to cms-HL, and that these are located in different regions of chromosome 10. One of these loci, Rf5 , co-segregates with the SSR marker RM3150, and is flanked by RM1108 and RM5373, which are 0.9 cM and 1.3 cM away, respectively. Another Rf locus, designated as Rf6(t), co-segregates with RM5373, and is flanked by RM6737 and SBD07 at genetic distances of 0.4 cM. The results also demonstrated these loci are distinct from Rf1 and Rf4. A 105-kb BAC clone covering the Rf6(t) locus was obtained from a rice BAC library. The sequence of a 66-kb segment spanning the Rf6(t) locus was determined by a BLASTX search in the genomic sequence database established for the cultivar 93-11.Communicated by R. Hagemann     

Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear‐encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)‐type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD–CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS‐associated gene in LD–CMS rice, similar to its role in BT–CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT–CMS rice. We also show that RF2 promotes degradation of atp6–orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT–CMS rice. The amount of ORF79 protein in LD–CMS rice was one‐twentieth of the amount in BT–CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD–CMS and BT–CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD–CMS and BT–CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD–CMS and BT–CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

Comparative analysis of fertility restoration genes for WA,Y, and DA cytoplasmic male sterility in rice

Rice chromosome single segment substitution line (SSSL) W23-19-06-06-11 with the genotype Rf3Rf3/Rf4Rf4, a strong restorer line for wild-abortive (WA) cytoplasmic male sterility (CMS), was recently identified from the SSSL library. To investigate the genetic mode of Rf genes and the genetic relationship among WA, yegong (Y), and dwarf-wildabortive (DA) CMS systems, the plants derived from three BC3F2 populations involving W23-19-06-06-11 and the three CMS lines, that carried the Rf3Rf3/Rf4Rf4, Rf3Rf3/rf4rf4, and rf3rf3/Rf4Rf4 genotypes and WA-, Y-, and DA-CMS cytoplasm, were selected and their pollen and spikelet fertility were evaluated. The results show that the genetic effect displayed a trend of Y-CMS > WA-CMS > DA-CMS in the genetic background of W23-19-06-06-11, the effect of Rf4 appeared to be slightly larger than that of Rf3, and their effects were additive for the three CMS systems. Two pairs of dominant genes governed the fertility restoration in pollen and spikelet in the W23-19-06-06-11 which indicates that the genetic mode of the Rf genes was a qualitative character for the three CMS systems.     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4. Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F₁s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F₁s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363 and PRR 396 possessing both Rf4 and Rf3 genes and good fertility restoration have been identified which could be used further in hybrid rice breeding.     

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

Genetic analysis and molecular mapping of a new fertility restorer gene Rf8 for Triticum timopheevi cytoplasm in wheat (Triticum aestivum L.) using SSR markers

A study on mode of inheritance and mapping of fertility restorer (Rf) gene(s) using simple sequence repeat (SSR) markers was conducted in a cross of male sterile line 2041A having Triticum timopheevi cytoplasm and a restorer line PWR4099 of common wheat (Triticum aestivum L.). The F₁ hybrid was completely fertile indicating that fertility restoration is a dominant trait. Based on the pollen fertility and seed set of bagged spikes in F₂ generation, the individual plants were classified into fertile and sterile groups. Out of 120 F₂ plants, 97 were fertile and 23 sterile (based on pollen fertility) while 98 plants set ≥5 seeds/spike and 22 produced ≤4 or no seed. The observed frequency fits well into Mendelian ratio of 3 fertile: 1 sterile with χ² value of 2.84 for pollen fertility and 2.17 for seed setting indicating that the fertility restoration is governed by a single dominant gene in PWR4099. The three linked SSR markers, Xwmc503, Xgwm296 and Xwmc112 located on the chromosome 2DS were placed at a distance of 3.3, 5.8 and 6.7 cM, respectively, from the Rf gene. Since, no known Rf gene is located on the chromosome arm 2DS, the Rf gene in PWR4099 is a new gene and proposed as Rf8. The closest SSR marker, Xwmc503, linked to the Rf8 was validated in a set of Rf, maintainer and cytoplasmic male sterile lines. The closely linked SSR marker Xwmc503 may be used in marker-assisted backcross breeding facilitating the transfer of fertility restoration gene Rf8 into elite backgrounds with ease.     

Phylogenetic Genomewide Comparisons of the Pentatricopeptide Repeat Gene Family in indica and japonica Rice

More than 400 pentatricopeptide repeat (PPR) genes have been found in higher plants, but most of them have not been functionally analyzed and their origins are still obscure. In this study, we performed phylogenetic genomewide comparisons of the PPR gene family in indica and japonica rice to explore the expansion mechanisms of these genes in higher plants. The functions of PPR genes in plant CMS/Rf systems are also discussed. The results indicate that (1) unequal crossing over participated in the expansion of the newly evolved PPR genes in indica and japonica rice genomes, (2) CMS/Rf systems are different in monocots and dicots, (3) the BT-type CMS/Rf system exists in both indica and japonica rice, and (4) both the PPR gene family and the BT-type CMS/Rf system may have existed before the divergence of indica and japonica rice.     

Development of a candidate gene marker for Rf 1 based on a PPR gene in cytoplasmic male sterile CMS-D2 Upland cotton

The cytoplasmic male sterility (CMS) system is convenient and efficient for hybrid seed production in Upland cotton (Gossypium hirsutum L.). However, it has not been widely used because of limited restorer lines carrying the restorer gene Rf ₁ in the CMS-D2 system. In this study, the fertility segregation in a backcross (BC8F1) population of 409 individuals and an F2 population of 695 plants confirmed that the fertility restoration was determined by one dominant restorer gene (Rf ₁ ). A sequence alignment showed that 13 Rf ₁ -linked simple sequence repeat marker sequences were distributed on nine scaffolds of chromosome 9 in the sequenced D5 genome of G. raimondii Ulbrich. Ten pentotricopeptide repeat (PPR)-like genes were identified on two scaffolds, including Scaffold 333 where nine PPR-like genes were clustered in a region of about 160 kb. Among them, PPR-like gene Cotton_D_gene_10013437 was identified as the candidate for the Rf ₁ gene through a comparative sequence analysis of the homologous gene among sterile (A), maintainer (B) and restorer (R) lines, and co-segregation analysis. Compared with the non-restoring lines, the restorer had a 9-nucleotide (nt) insertion and a single nucleotide polymorphism (SNP) 8 nt upstream of the insertion at the 3′ untranslated regions (3′ UTRs) in this gene. A cleaved amplified polymorphic sequence (CAPS) marker named CAPS-R was developed from the SNP site using the restriction enzyme DraI, and was further used to track the restorer gene and its homozygous or heterozygous status in molecular breeding for restorer lines. A marker-assisted selection system using the Rf ₁ -specific CAPS-R marker and a CMS-D2 cytoplasm-specific SCAR marker was established to distinguish the three-line hybrids from other genotypes.     

Mapping and Genetic Characterization of Loci Controlling the Restoration of Male Fertility in Ogura CMS Radish

Three Raphanus populations (BC1, F2 and R8) each segregating for the restoration of Ogura CMS were used tomap restorer loci. The three restorer loci, Rf1, Rf2 and Rf3, each exhibited dominant restoring alleles and wereeach mutually epistatic. Rf1 was mapped to the upper region of Rs1 using data from each population. Rf2 wasmapped to the middle of Rs2 using both the F2 and R8 populations. Rf3 was mapped to the upper region of Rs7using the R8 population. The marker analysis and linkage mapping of the BC1 and F2 populations were describedpreviously (Bett and Lydiate, 2003). Scoring at 114 marker loci in R8 population allowed a new map ofthe Raphanus genome to be integrated with the consensus map. The complex genetic control of the restoration ofOgura CMS in Raphanus is compared with the more simple genetic control of this trait previously described inB. napus. Markers linked to each of the three restorer loci will allow the routine generation and verification ofdefined restorer and maintainer lines for various combinations of defined restorer loci. Although the restorationof Ogura CMS in Raphanus probably involves additional loci, the identification of three loci and diagnosticmarkers for each provides a solid foundation for the development of a holistic model for the genetic control ofthis trait through mapping in additional populations.     

Diversifying Sunflower Germplasm by Integration and Mapping of a Novel Male Fertility Restoration Gene

The combination of a single cytoplasmic male-sterile (CMS) PET-1 and the corresponding fertility restoration (Rf) gene Rf₁ is used for commercial hybrid sunflower (Helianthus annuus L., 2n = 34) seed production worldwide. A new CMS line 514A was recently developed with H. tuberosus cytoplasm. However, 33 maintainers and restorers for CMS PET-1 and 20 additional tester lines failed to restore the fertility of CMS 514A. Here, we report the discovery, characterization, and molecular mapping of a novel Rf gene for CMS 514A derived from an amphiploid (Amp H. angustifolius/P 21, 2n = 68). Progeny analysis of the male-fertile (MF) plants (2n = 35) suggested that this gene, designated Rf₆, was located on a single alien chromosome. Genomic in situ hybridization (GISH) indicated that Rf₆ was on a chromosome with a small segment translocation on the long arm in the MF progenies (2n = 34). Rf₆ was mapped to linkage group (LG) 3 of the sunflower SSR map. Eight markers were identified to be linked to this gene, covering a distance of 10.8 cM. Two markers, ORS13 and ORS1114, were only 1.6 cM away from the gene. Severe segregation distortions were observed for both the fertility trait and the linked marker loci, suggesting the possibility of a low frequency of recombination or gamete selection in this region. This study discovered a new CMS/Rf gene system derived from wild species and provided significant insight into the genetic basis of this system. This will diversify the germplasm for sunflower breeding and facilitate understanding of the interaction between the cytoplasm and nuclear genes.     

Chromosomal polymorphism of ribosomal genes in the genus Oryza

The genes encoding for 18S–5.8S–28S ribosomal RNA (rDNA) are both conserved and diversified. We used rDNA as probe in the fluorescent in situ hybridization (rDNA-FISH) to localized rDNAs on chromosomes of 15 accessions representing ten Oryza species. These included cultivated and wild species of rice, and four of them are tetraploids. Our results reveal polymorphism in the number of rDNA loci, in the number of rDNA repeats, and in their chromosomal positions among Oryza species. The numbers of rDNA loci varies from one to eight among Oryza species. The rDNA locus located at the end of the short arm of chromosome 9 is conserved among the genus Oryza. The rDNA locus at the end of the short arm of chromosome 10 was lost in some of the accessions. In this study, we report two genome specific rDNA loci in the genus Oryza. One is specific to the BB genome, which was localized at the end of the short arm of chromosome 4. Another may be specific to the CC genome, which was localized in the proximal region of the short arm of chromosome 5. A particular rDNA locus was detected as stretched chromatin with bright signals at the proximal region of the short arm of chromosome 4 in O. grandiglumis by rDNA-FISH. We suggest that chromosomal inversion and the amplification and transposition of rDNA might occur during Oryza species evolution. The possible mechanisms of cyto-evolution in tetraploid Oryza species are discussed.     

Gametophytically alloplasmic CMS line of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) with variant <Emphasis Type="Italic">orfH79</Emphasis> haplotype corresponds to specific fertility restorer

For years discovery and identification of the cytoplasmic male sterility (CMS) resource in wild rice is the most intriguing events in breeding field. orfH79, a chimeric gene in mitochondria, has been suggested being the determinant for Honglian CMS in rice. In this report orfH79 gene as molecular marker to screen the wild rice, we found eight accessions with orfH79 gene in the total 42 investigated objects. Sequence analysis revealed that there were a total of nine nucleotide substitutions resulting in the change of nine amino acids in the newly identified orfH79 in wild rice, which further fell into seven haplotypes. In order to investigate the underlying relationship between orfH79 haplotypes and the corresponding fertility restorers, four accessions were selected with different orfH79 haplotype as female parents to hybridize the Honglian maintainer line, Yuetai B. After eight consecutive recurrent backcrosses, four alloplasmic CMS lines with different orfH79 haplotype were developed. Microscopic observation exhibited that their pollen grains were spherical and clear in 1% I₂–KI solution same as that of Honglian CMS line. Moreover, these four CMS lines displayed various fertility restoring model through test cross, suggesting that each orfH79 haplotye represents a new CMS type and corresponds to their specific Rf allele.