Cytoplasmic Male Sterile Rice Research Articles

The vesicle trafficking gene, OsRab7, is critical for pollen development and male fertility in cytoplasmic male-sterility rice

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.

Genetic Variability of Floral and Agronomic Characteristics that Influence Outcrossing Rate Percentage of Cytoplasmic Male Sterile Rice

Developing high-yielding hybrid rice varieties is much needed to ensure food security to meet the requirement of the increasing population across the world. Cytoplasmic male genetic sterility system is a valuable technology for the exploitation of heterosis and production of high-yielding hybrid rice with better grain quality. Large-scale commercial exploitation of hybrid vigor in rice largely depends on the availability of good CMS (Cytoplasmic Male Sterile) lines. A total of eighteen traits of five CMS lines of rice were evaluated to study the variability and correlation between floral and other yield-contributing traits with outcrossing rate using Randomized Complete Block Design with three replications. High significant variation was observed among the genotypes for all the studied traits. High GCV (Genotypic Coefficient of Variance) and PCV (phenotypic Coefficient of Variance) coupled with high heritability and high genetic advance in percent of mean were observed for number of panicles per plant, filled grain, flag leaf breadth, stigma length and breadth, filament length and outcrossing rate which indicated the broad genetic base, less environmental influence, and these traits are under the control of additive gene action. character association and path analysis indicated that number of grains per panicle, filled grains per panicle, stigma length, and breadth possessed both positive significant correlation and positive direct effects on outcrossing rate therefore, selection for the traits mentioned above will certainly bring in the improvement of the outcrossing percentage of the lines, which in turn enhances the seed quantity of the hybrid.

Precise genetic mapping of Rf18(t), a new fertility restorer gene from 'Nipponbare' for wild abortive cytoplasmic male sterility in rice (Oryza sativa L.).

We mapped Rf18(t), a Restorer-of-fertility gene for wild abortive cytoplasmic male sterility from the japonica maintainer 'Nipponbare', to chromosome 1. The best candidate gene, LOC_Os01g71320, is predicted to encode hexokinase. Three-line hybrid rice obtained through cytoplasmic male sterility (CMS) has helped increase the yield of rice globally, and the wild abortive (WA)-type cytoplasm from wild rice (Oryza rufipogon Griff.) is used widely in three-line indica hybrids. The identification and mapping of the Restorer-of-fertility (Rf) genes in maintainer lines aided in uncovering the genetic basis of fertility restoration of WA-type CMS and the development of WA-type hybrids. In this study, we identified a new Rf gene, Rf18(t), for WA-type CMS from the japonica maintainer line 'Nipponbare' using a chromosome segment substitution line population derived from a cross between the indica line 9311 and 'Nipponbare.' Using a substitution mapping strategy, Rf18(t) was delimited to a 48-kb chromosomal region flanked by molecular marker loci ID01M28791 and ID01M28845 on chromosome 1. By comparative sequence analyses, we propose that LOC_Os01g71320 is the most likely candidate gene for Rf18(t), and it is predicted to encode hexokinase. Furthermore, Rf18(t) was found to function in fertility restoration probably by a posttranscriptional mechanism and its function is dependent on the genetic background of 9311. These results broaden our knowledge on the mechanism of fertility restoration of WA-type CMS lines and will facilitate the development of WA-type rice hybrids.

Growth Regulators Improve Outcrossing Rate of Diverse Rice Cytoplasmic Male Sterile Lines through Affecting Floral Traits.

Cytoplasmic male sterility (CMS) provides an irreplaceable strategy for commercial exploitation of heterosis and producing high-yielding hybrid rice. The exogenous application of plant growth regulators could improve outcrossing rates of the CMS lines by affecting floral traits and accordingly increase hybrid rice seed production. The present study aimed at exploring the impact of growth regulators such as gibberellic acid (GA3), indole-3-acetic acid (IAA), and naphthalene acetic acid (NAA) on promoting floral traits and outcrossing rates in diverse rice CMS lines and improving hybrid rice seed production. The impact of foliar applications of growth regulators comprising GA3 at 300 g/ha or GA3 at 150 g/ha + IAA at 50 g/ha + NAA at 200 g/ha versus untreated control was investigated on floral, growth, and yield traits of five diverse CMS lines. The exogenously sprayed growth regulators, in particular, the combination of GA3, IAA, and NAA (T3) boosted all studied floral, growth, and yield traits in all tested CMS lines. Moreover, the evaluated CMS lines exhibited significant differences in all measured floral traits. L2, L3, and L1 displayed the uppermost spikelet opening angle, duration of spikelet opening, total stigma length, style length, stigma brush, and stigma width. In addition, these CMS lines exhibited the highest plant growth and yield traits, particularly under T3. Consequently, exogenous application of GA3, IAA, and NAA could be exploited to improve the floral, growth, and yield traits of promising CMS lines such as L2, L3, and L1, hence increasing outcrossing rates and hybrid rice seed production.

Improvement ability of male parent by gibberellic acid application to enhancing the outcrossing of cytoplasmic male sterility rice lines

<p class="042abstractstekst">The study quantified the effect of gibberellic acid (GA<sub>3</sub>) as a pre-flowering treatment for male parent Giza 178 R and the influence of male to female ratio (2R:10A, 2R:12A, 2R:14A and 2R:16A) between male (R) to female (A) for two Cytoplasmic Male Sterility (CMS) lines (‘IR69625’ and ‘G46’) on hybrid rice seed production. The main plots were occupied by CMS lines while; GA3 application for male parent Giza 178R were arranged in the sub plots and male to female ratio was arranged in the sub-sub plots. The results indicated that, the, duration of floret opening, angle of floret opening, filaments exsertion, filaments length<strong>, </strong>anther length, plant height and number of tiller hill<sup>-1 </sup>of male parent Giza 178R were significantly at 300 g GA<sub>3</sub> ha<sup>-1</sup> concentration. Plant height, panicle exsertion, panicle length, flag leaf angle and 1000-grain mass of CMS were not significantly affected by the GA<sub>3</sub> application for male parent and male to female ratio, while, number of fertile panicles hill<sup>-1</sup>, panicle mass, seed set, seed yield and harvest index of CMS lines were highly significantly affected. The highest seed yield (2.880 and 2.950 t ha<sup>-1</sup>) was obtained by CMS line IR69625A using 300 g GA<sub>3</sub> ha<sup>-1</sup> of male parent Giza 178R with male to female ratio of 2R:14A during both seasons.</p>

Improvement ability of male parent by gibberellic acid application to enhancing the outcrossing of cytoplasmic male sterility rice lines

The study quantified the effect of gibberellic acid (GA3) as a pre-flowering treatment for male parent Giza 178 R and the influence of male to female ratio (2R:10A, 2R:12A, 2R:14A and 2R:16A) between male (R) to female (A) for two Cytoplasmic Male Sterility (CMS) lines (‘IR69625’ and ‘G46’) on hybrid rice seed production. The main plots were occupied by CMS lines while; GA3 application for male parent Giza 178R were arranged in the sub plots and male to female ratio was arranged in the sub-sub plots. The results indicated that, the, duration of floret opening, angle of floret opening, filaments exsertion, filaments length, anther length, plant height and number of tiller hill-1 of male parent Giza 178R were significantly at 300 g GA3 ha-1 concentration. Plant height, panicle exsertion, panicle length, flag leaf angle and 1000-grain mass of CMS were not significantly affected by the GA3 application for male parent and male to female ratio, while, number of fertile panicles hill-1, panicle mass, seed set, seed yield and harvest index of CMS lines were highly significantly affected. The highest seed yield (2.880 and 2.950 t ha-1) was obtained by CMS line IR69625A using 300 g GA3 ha-1 of male parent Giza 178R with male to female ratio of 2R:14A during both seasons.

Disruption of mitochondrial open reading frame 352 partially restores pollen development in cytoplasmic male sterile rice.

Plant mitochondrial genomes sometimes carry cytoplasmic male sterility (CMS)-associated genes. These genes have been harnessed in various crops to produce high-yielding F1 hybrid seeds. The gene open reading frame 352 (orf352) was reported to be an RT102-type CMS gene in rice (Oryza sativa), although the mechanism underlying its role in CMS is unknown. Here, we employed mitochondrion-targeted transcription activator-like effector nucleases (mitoTALENs) to knockout orf352 from the mitochondrial genome in the CMS rice RT102A. We isolated 18 independent transformation events in RT102A that resulted in genome editing of orf352, including its complete removal from the mitochondrial genome in several plants. Sequence analysis around the mitoTALEN target sites revealed their induced double-strand breaks were repaired via homologous recombination. Near the 5ʹ-target site, repair involved sequences identical to orf284, while repair of the 3ʹ-target site yielded various new sequences that generated chimeric genes consisting of orf352 fragments. Plants with a chimeric mitochondrial gene encoding amino acids 179–352 of ORF352 exhibited the same shrunken pollen grain phenotype as RT102A, whereas plants either lacking orf352 or harboring a chimeric gene encoding amino acids 211–352 of ORF352 exhibited partial rescue of pollen viability and germination, although these plants failed to set seed. These results demonstrated that disruption of orf352 partially restored pollen development, indicating that amino acids 179–210 from ORF352 may contribute to pollen abortion.

Degradation of mitochondrial structure and deficiency of complex I were associated with the transgenic CMS of rice

BackgroundMitochondria play a significant role in plant cytoplasmic male sterility (CMS). In our previous study, mitochondrial complex I genes, nad4, nad5, and nad7 showed polymorphisms between the transgenic CMS line M2BS and its wild type M2B. The sterility mechanism of the M2BS at cytological, physiological, biochemical, and molecular level is not clear.ResultsCytological observation showed that the anthers were light yellow, fissured, invalid in KI-I2, and full of irregularly typical abortion pollen grains in M2BS. Transmission electron microscopic (TEM) observation revealed no nucleus and degraded mitochondria with obscure cristae in anther cells of M2BS. The results of staining for H2O2 presented a large number of electron dense precipitates (edp) in intercellular space of anther cells of M2BS at anthesis. Moreover, the anther respiration rate and complex I activity of M2BS were significantly lower than those of wild type M2B during pollen development. Furthermore, RNA editing results showed only nad7 presented partially edited at 534th nucleotides. The expression of nad5 and nad7 revealed significant differences between M2B and M2BS.ConclusionsOur data demonstrated that mitochondrial structural degradation and complex I deficiency might be associated with transgenic CMS of rice.

Development of High Yielding Glutinous Cytoplasmic Male Sterile Rice (Oryza sativa L.) Lines through CRISPR/Cas9 Based Mutagenesis of Wx and TGW6 and Proteomic Analysis of Anther

Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx, major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T0 mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased (P < 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T1 plants were re-edited but T2 generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC2F1. The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD+-dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development.

The chimeric mitochondrial gene orf182 causes non-pollen-type abortion in Dongxiang cytoplasmic male-sterile rice.

D1-cytoplasmic male sterility (CMS) rice is a sporophytic cytoplasmic male-sterile rice developed from Dongxiang wild rice that exhibits a no-pollen-grain phenotype. A mitochondrial chimeric gene (orf182) was detected by mitochondrial genome sequencing and a comparative analysis. Orf182 is composed of three recombinant fragments, the largest of which is homologous to Sorghum bicolor mitochondrial sequences. In addition, orf182 was found only in wild rice species collected from China. Northern blot analysis showed that orf182 transcripts were affected by Rf genes in the isocytoplasmic restorer line DR7. Western blot analysis showed that the ORF182 product was localized in the mitochondria of the CMS line. An expression cassette containing orf182 fused to a mitochondrial transit peptide induced the maintainer line of male sterility, which lacked pollen grains in the anthers. Furthermore, the invivo expression of orf182 also inhibited the growth of Escherichia coli, with lower respiration rate, excess accumulation of reactive oxygen species and decreased ATP levels. We conclude that the mitochondrial chimeric gene orf182 possesses a unique structure and origin differing from other identified mitochondrial CMS genes, and this gene is connected to non-pollen type of sporophytic male sterility in D1-CMS rice.

Crystal structure of WA352 provides insight into cytoplasmic male sterility in rice

Plant cytoplasmic male sterility (CMS) is an important phenomenon and is widely utilized in hybrid crop breeding. The Wild Abortive CMS (CMS-WA), a well-known CMS type, has been successfully applied in the commercial production of hybrid rice seeds for more than 40 years. The CMS-WA causal gene WA352 encodes a novel transmenbrane protein and the interacts with the mitochondrial copper chaperone COX11, triggering reactive oxygen species production and resulting in male sterility in CMS-WA lines. However, the structure of WA352 is currently unknown, and the structural mechanism whereby WA352 perturbs COX11 function to cause CMS remains largely unknown. Here, we report the crystal structure of the C-terminal functional domain of WA352 at 1.3 Å resolution. This functional domain, consisting of five α helices, is spindle-shaped with a length of 42 Å, and a diameter of 28 Å. Notably, the absence of any structural similarity to a known protein structure suggests that the WA352 functional domain is a novel fold. In addition, surface conservation analysis and structural modeling of the WA352-COX11 complex revealed details about the WA352-COX11 interaction. Further structural analysis suggested that the WA352-COX11 interaction blocks the copper ion transportation activity of COX11, which is essential for the assembly of cytochrome c oxidase, resulting in male sterility in CMS-WA lines. Our study paves the way toward structural determination of the WA352-COX11 complex and provides new insight into the mechanism of plant CMS.

The Genetic Variability of Floral and Agronomic Characteristics of Newly-Bred Cytoplasmic Male Sterile Rice

Male sterility enabled commercialization of heterosis in rice but low seed set remains a constraint on hybrid dissemination. We evaluated 216 F6 maintainer lines for agronomic and floral characteristics in augmented design and selected 15 maintainer lines, which were testcrossed with IR58025A. Five backcrosses were conducted to transfer cytoplasmic male sterility (CMS) to select maintainer lines. Newly-bred BC5:6 CMS lines were evaluated for outcrossing rates and agronomic characteristics. There were highly significant differences among 216 F6 maintainer lines for characteristics whose genotypic variance was higher than environmental variance. The phenotypic coefficient of variation was almost the same as the genotypic coefficient of variation, indicating that most phenotypic variation was due to genetics. There were highly significant differences among CMS lines for number of days to 50% flowering and maturity; stigma exertion; panicle exertion, length and weight; spikelet fertility; tillers per plant; plant height; grains per panicle; grain yield per plant; and 1000-grain weight, but not for pollen and panicle sterility during dry and wet seasons. Three CMS lines (CMS3, CMS12, and CMS14), exhibited high outcrossing rates (56.17%, 51.42% and 48.44%, respectively), which had a highly significant, positive correlation with stigma exertion (0.97), spikelet opening angle (0.82), and panicle exertion (0.95).

Conversion of a rice CMS maintainer into a photo- or thermo-sensitive genetic male sterile line

A maintainer line of 3-line hybrid rice commonly presents a certain genetic distance to a 2-line restorer line, but in many cases, 2-line restorer lines present defects upon recovery of the object cytoplasmic male sterile (CMS) line of the maintainer line, which impedes the utilization of their heterosis. Here, we report a strategy and an example of converting a maintainer into a photoperiod/temperature-sensitive genic male sterile (P/TGMS) line with an almost identical genetic background, thus maximizing the heterosis. Firstly, through treatment of maintainer line T98B with 60CO-γ irradiation, we identified the TGMS line T98S, which is sterile at higher temperatures and fertile at lower temperatures. Secondly, the T98S line was proven to be identical to T98B with regard to genetic background via an examination of 48 parental polymorphous SSR markers and exhibited excellent blossom traits similar to those of T98B, with an extensive forenoon flowering rate of 75.92% and a high exertion rate of 64.59%. Thirdly, in a combination test, three out of six hybrids from T98S crossed with 2-line restorer lines showed a yield increase of 6.70–15.69% for 2 consecutive years. These results demonstrated that the strategy can generate a new P/TGMS line with strong general combining ability (converted from a maintainer line), thus helping to increase the genetic diversity of male sterile heterotic groups.

Choice of microsatellite markers for isolation of fertility restorers of wild abortive (WA) type cytoplasmic male sterility in rice

Simple sequence length polymorphism was examined in a set of eight effective restorers, four partial restorers, two weak maintainers and four complete maintainers of WA type CMS system in rice using a set of thirty-two microsatellite primer pairs in order to assess the nature and extent of genetic variation among them at molecular level. Altogether 248 allelic variants were detected with an average of 7.75 alleles per primer pairs. Polymorphic information content ranged from 0.508 for RM 6737 to 0.882 for RM 1 with an average value of 0.740 per primer pair. Amongst the primer pairs used, RM 1, RM 206, RM 591 RM 3873 and RM 8146 had remarkably higher number of allelic variants with greater gene diversity and genotype discrimination ability. Using similarity coefficient in numerical taxonomic approach of classification, the entries were differentiated and classified into different groups. Analysis based on molecular profiles allowed discrimination of effective fertility restorers from rest of the entries including partial fertility restorers, weak sterility maintainers and complete sterility maintainers. Principal coordinate analysis based two dimensional plotting of genetic profiles completely supported the results obtained from hierarchical classification of entries and effective fertility restorers were distinctly discriminated from the remaining entries. Four microsatellite primer pairs namely, RM 171, RM 315, RM 3873 and RM 6100, which allowed unambiguous discrimination of the eight effective fertility restorers from rest of the entries. These microsatellite primer pairs, may be further utilized in identification of effective fertility restorers of WA based CMS system in rice.

Jasmonic acid deficiency leads to scattered floret opening time in cytoplasmic male sterile rice Zhenshan 97A.

Cytoplasmic male sterile (CMS) rice has been widely used for hybrid rice seed production in China. However, CMS rice suffers from undesirable flowering habits including scattered floret opening time (FOT), which causes different FOTs among parental rice plants and greatly reduces hybrid rice seed production. Little is known about the mechanism of scattered FOT in CMS rice. Our results demonstrate that scattered FOT in CMS rice Zhenshan 97A (ZS97A) resulted from the lack of a driving force to open florets, which was directly caused by retarded lodicule expansion. Our results indicate that retarded lodicule expansion in ZS97A was caused by reduced water accumulation due to retarded accumulation of osmotic regulation substances (ORSs). Further, the retardation in accumulation of ORSs and water were caused by jasmonic acid (JA) deficiency, resulting from down-regulation of OsAOC expression. Applying JA restored scattered FOT in ZS97A by promoting ORS and water accumulation, and inducing the expansion of the lodicules. Taken together, JA deficiency inhibited lodicule expansion by retarding the accumulation of ORSs and water, leading to scattered FOT in CMS rice ZS97A.

Origins of rice cytoplasmic male sterility genes.

Cytoplasmic male sterility (CMS) factors have long been known in some wild plants, and also in some domesticated species, where they are used to produce plants to be used as maternal parents, for example to breed hybrids that display hybrid vigor. Their origins have been mystifying, and now a study recently published in Cell Research helps understand how one widely-used rice CMS factor evolved.

Discovery, utilization and perspective of Honglian cytoplasmic male sterile rice

Hybrid rice has been researched and utilized in China for 50 years. Three mainly cytoplasmic male sterile rice are wild abortive (WA), boro-II (BT), and Honglian (HL). Honglian (HL) type hybrid rice has made important contributions to the development of hybrid rice by combining scientific research, application research and industrial utilization. We identified Honglian cytoplasmic male sterile (HL-CMS) gene, orfH79 , which located downstream of atp6 in mitochondrial genome. CMS RNA, atp6-orfH79 , were translated into a cytotoxic protein, ORFH79. Data showed ORFH79 interacts with P61, which is a subunit of complex III of mitochondrial electron transfer chain. This interaction may impair energy production and leads to the accumulation of reactive oxygen species (ROS) in mitochondria, consequently resulting in abnormal pollen development. Furthermore, we cloned two restorer of fertility ( Rf ) genes, Rf5 and Rf6 , both of which are PPR genes. Neither RF5 nor RF6 binds to CMS RNA, atp6-orfH79 , directly. RF5 interacts with glycine rich protein 162 (GRP162) and RFC3 (restoration of fertility complex subunit 3) thus forming a restoration of fertility complex (RFC) to process CMS RNA. GRP162 can form homodimer and bind to CMS RNA with two binding sites directly. RCF3 is transported into the mitochondria as a membrane protein and functions as a scaffold protein for the assembly of the RFC complex. The RFC complex was detected with a size ranging from 400 to 500 kD. Moreover, Rf6 encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3 to 5. Results show RF6 is also targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS RNA, atp6-orfH79 , to restore the fertility of CMS line. To create more available germplasm in hybrid rice, we developed a molecular marker of orfH79 to screen wild rice and bred some novel CMS lines. We also developed molecular markers of Rf5 and Rf6 to breed novel stronger restore lines which contain two Rf genes. Consequently, we bred several combinations such as Honglian you 6, Yueyou 938, Luoyou 8 and Luoyou 9348 with high yield, good quality, multiple resistance and wide adaptation. HL hybrid rice has been widely planted in Yangzte river areas and South China with the yield of 10 to 12 tons per hectare. HL hybrid rice combinations also have been planted in Southeast Asian countries, such as Vietnam, Indonesia, Malaysia, Bangladesh and Phillippine. Most combinations exhibited 140% or more of yield compared with the local rice cultivars in Southeast Asian countries. HL hybrid rice even more is becoming the leading hybrid rice and priority choice in most of Asian countries and other Africa countries.

A Gene Encoding Pentatricopeptide Repeat Protein Partially Restores Fertility in RT98-Type Cytoplasmic Male-Sterile Rice.

Cytoplasmic male sterility (CMS) lines in rice, which have the cytoplasm of a wild species and the nuclear genome of cultivated rice, are of value for the study of genetic interactions between the mitochondrial and nuclear genomes. The RT98-type CMS line RT98A and the fertility restorer line RT98C carry the cytoplasm of the wild species Oryza rufipogon and the nuclear genome of the Taichung 65 cultivar (Oryza sativa L.). Based on a classical crossing experiment, fertility is reported to be restored gametophytically by the presence of a tentative single gene, designated Rf98, which is derived from the cytoplasm donor. Fine mapping of Rf98 revealed that at least two genes, which are closely positioned, are required for complete fertility restoration in RT98A. Here, we identified seven pentatricopeptide repeat (PPR) genes that are located within a 170 kb region as candidates for Rf98 Complementation tests revealed that the introduction of one of these PPR genes, PPR762, resulted in the partial recovery of fertility with a seed setting rate up to 9.3%. We conclude that PPR762 is an essential fertility restorer gene for RT98-type CMS. The low rate of seed setting suggested that some other genes near the Rf98 locus are also necessary for the full recovery of seed setting.

Whole Mitochondrial Genome Sequencing and Re-Examination of a Cytoplasmic Male Sterility-Associated Gene in Boro-Taichung-Type Cytoplasmic Male Sterile Rice.

Nuclear genome substitutions between subspecies can lead to cytoplasmic male sterility (CMS) through incompatibility between nuclear and mitochondrial genomes. Boro-Taichung (BT)-type CMS rice was obtained by substituting the nuclear genome of Oryza sativa subsp. indica cultivar Chinsurah Boro II with that of Oryza sativa subsp. japonica cultivar Taichung 65. In BT-type CMS rice, the mitochondrial gene orf79 is associated with male sterility. A complete sequence of the Boro-type mitochondrial genome responsible for BT-type CMS has not been determined to date. Here, we used pyrosequencing to construct the Boro-type mitochondrial genome. The contiguous sequences were assembled into five circular DNA molecules, four of which could be connected into a single circle. The two resulting subgenomic circles were unable to form a reliable master circle, as recombination between them was scarcely detected. We also found an unequal abundance of DNA molecules for the two loci of atp6. These results indicate the presence of multi-partite DNA molecules in the Boro-type mitochondrial genome. Expression patterns were investigated for Boro-type mitochondria-specific orfs, which were not found in the mitochondria from the standard japonica cultivar Nipponbare. Restorer of fertility 1 (RF1)-dependent RNA processing has been observed in orf79-containing RNA but was not detected in other Boro-type mitochondria-specific orfs, supporting the conclusion that orf79 is a unique CMS-associated gene in Boro-type mitochondria.

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.