Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

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.     

Molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.     

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.     

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.     

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     

<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.     

Genetic characterization of a pentatricopeptide repeat protein gene,orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.     

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.     

Characterization and Use of Male Sterility in Hybrid Rice Breeding

The hybrid rice （Oryza sativa L.） breeding that was Initiated In China in the 1970s led to a great improvement in rice productivity. In general, It increases the grain yield by over 20% to the inbred rice varieties, and now hybrid rice has been widely introduced into Africa, Southern Asia and America. These hybrid varieties are generated through either three-line hybrid and two-line hybrid systems; the former is derived from cytoplasmic male sterility （CMS） and the latter derived from genlc male sterility （GMS）. There are three major types of CMS （HL, BT and WA） and two types of GMS （photoperlod-sensitlve （PGMS） and temperature-sensitive （TGMS））. The BT- and HL-type CMS genes are characterized as orf79 and orfH79, which are chimeric toxic genes derived from mltochondrial rearrangement. Rf3 for CMS-WA Is located on chromosome 1, while Rf1, Rf4, Rf5 and Rf6 correspond to CMS-BT, CMSoWA and CMS- HL, located on chromosome 10. The Rfl gene for BT-CMS has been cloned recently, and encodes a mltochondriatargeted PPR protein. PGMS Is thought to be controlled by two recessive loci on chromosomes 7 and 12, whereas nine recessive alleles have been identified for TGMS and mapped on different chromosomes. Attention Is still urgently needed to resolve the molecular complexity of male sterility to assist rice breeding.     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.