Cytoplasmic Male Sterility Types Research Articles

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

AbstractThe so‐called “wild abortive” (WA) type of cytoplasmic male sterility (CMS) derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS‐associated gene(s) remain unknown. In this study, we exploited a mitochondrial genome‐wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondrial genomes of a CMS‐WA line and two normal fertile rice lines were amplified by Long‐polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondrial expression profile among the CMS‐WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS‐WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.

A Honglian CMS line of rice displays aberrant F0 of F0F1-ATPase

Honglian (HL) is one of the three major types of cytoplasmic male sterility (CMS) of rice (Oryza sativa L) that has been commercially used in hybrid production. In previous studies, the CMS in HL is shown to be associated with a chimeric gene orfH79 that is cotranscribed with an extra atp6 in mitochondria. This study demonstrated that the intact F0F1-ATPase in HL CMS line was specifically reduced in both of its protein quantity and enzyme activity, whereas its F1 sector was not affected. It implies that the F0 sector presents a labile linkage with F1. In the presence of fertility restorer gene, F0F1-ATPase can be recovered. Furthermore, orfH79 transcripts were preferentially polyadenylated, and consequently degraded rapidly in florets of the restored hybrid plants, indicating that the atp6-orfH79 is involved in the sterile phenotype. With inhibition of cytochrome pathway of electron transfer chain, the biomass of the sterile plants grown in dark was significantly lower than that of the fertile lines. However, the respiration measurements showed an increase in the electron transferring capacity in the sterile plants, suggesting that the reduction of biomass in sterile line was caused by the disruption of F0F1-ATPase.

Cytological and molecular characterization of a novel monogenic dominant GMS in Brassica napus L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5'-CTATGCCGAC-3') gave a repeatable 1500-bp DNA polymorphic segment S243(1500) between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243(1500) is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.

Penampilan Fenotipik dan Tingkat Kemandulan Tepungsari Calon Galur Mandul Jantan Tipe Wild Abortive

Wild Abortive (WA) type of cytoplasmic male sterility (CMS) was developed by Indonesian Center of Rice Research, Sukamandi through backcross method. Pollen sterility and phenotypic acceptability evaluation of cytoplasmic male sterile (CMS) candidates were considered important in CMS development process. Both of evaluation were done during dry season 2002 and wet season 2002/2003. The materials were 18 CMS candidates and their resembled maintainers. Each line planting on two rows (2.5 m each) with 20 x 20 cm spacing. Observations were done for 50% flowering time, pollen sterility and phenotypic acceptability. The results of observations showed that : (a) the variation of pollen sterility among 18 CMS candidates were 82.5 - 100%; (b) Nine CMS candidates were consistent in sterility (100%) and good in phenotypic acceptability during the two seasons. Those lines were derived from IR62829A/BP1082, IR68897A/S3613F, IR66707A/Barumun, IR58025A/S24731, IR62829A/BP68C, IR68886A/IR71605, IR66707A/A2790, IR69622A/IRBB5 and IR68886A/T12357. The male parent as donor were elite lines with some good characters such as good eating quality, resistant to Rice Tungro Virus (RTV) and Bacterial Leaf Blight (BLB). Key words : Rice, cytoplasmic male sterile, pollen sterility, phenotypic acceptability

Effects of Rf-1, Rf-3 and Rf-6(t) Genes on Fertility Restoration in Rice (Oryza sativa L.) with WA- and BT-type Cytoplasmic Male Sterility

Genes involved in the fertility restoration (Rf) of wild-abortive (WA)-type cytoplasmic male sterility (CMS) in rice have not been clearly identified, because of the inconsistency of their number and map position. Although the Rf-1 genes, which restore Chinsurah Boro (BT)-type CMS, have been recently isolated, their effects on WA-type CMS are still unclear. In the present study, near-isogenic lines (NILs) of japonica rice, differing at Rf loci, were developed using marker-assisted selection (MAS) to determine the effect of each Rf gene on WAor BT-cytoplasm in a japonica background. The NILs were crossed with a japonica CMS line, then the pollen and seed fertility of the hybrid plants was analyzed. The Rf-1 locus consisting of two Rf genes from IR36 was sufficient to restore fertility in the hybrids with BT-type CMS but conferred very little fertility to those with WA-type CMS. Rf-3 on chromosome 1, a weak Rf gene, and Rf-6(t) on chromosome 10 were unable to confer seed fertility to plants with BT-type or WA-type CMS. Both Rf genes, however, could accelate the development of abortive pollen in the case of WA-type CMS and their effect was additive. Crossing of CMS rice with chromosome segment substitution lines carrying Rf loci from IR24 resulted in similar pollen development and seed fertility.

Mechanism of cytoplasmic male-sterility modulated by mitochondrial retrograde regulation in higher plants

The type of cytoplasmic male-sterility, cytoplasmic male-sterility related mitochondrial factors, and nuclear restored gene-regulated mitochondrial factors in higher plants are reviewed in this paper. Based on gene regulation network for cytoplasmic male-sterility occurrence, we discussed and hypothesized that the interactions between nucleus and mitochondria determine cytoplasmic male-sterility occurrence modulated by mitochondrial retrograde regulation through possible mitochondrial retrograde signaling.

Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice

One of the cytoplasmic male sterility (CMS) types used for hybrid rice (Oryza sativa L.) production in China is the Honglian (HL)-CMS. Previous studies suggested that pollen abortion of the sterile plants was resulted from a special programmed cell death (PCD) program started at meiosis in the microspores. To elucidate the molecular basis of the pollen abortion, we compared the biochemical and physiological properties such as content of reactive oxygen species (ROS), ATP, NADH, total glutathione and ascorbate acid, the activities of dehydroascrbate reductase, glutathione reductase, ascorbate peroxides and superoxide dismutase, and the integrity of mitochondrial genome DNA isolated from an HL-CMS line, Yuetai A and its maintainer line, Yuetai B. Our results indicated that the mitochondria of the HL-CMS line suffered from a serious oxidative stress during microspores development. Oxidative stress induced by abnormal increased ROS at meiosis stage resulted in the depletion of ATP and NADH, and the degradation of mitochondrial genomic DNA. This suggests that the presence of redox signal originated in mitochondria affects the rest of the cell. Therefore, it is possible that the abortion of premature microspores in HL-CMS line is induced by the chronic oxidative stress in mitochondria in the early stage of pollen development.

Spatial Distributions of Cytoplasmic Types and Sex Expression in Alaskan Populations ofSilene acaulis

Sex expression in gynodioecious plants is often governed by an interaction between maternally inherited cytoplasmic male sterility (CMS) genes and nuclear male fertility restorer genes. Previous studies have documented an association between females and maternally inherited molecular markers in the cytoplasmic genomes in natural populations, suggesting that these markers are associated with different CMS types. We analyzed the spatial distributions of mitochondrial and chloroplast alleles and the sexual phenotype of individuals within five Alaskan populations of Silene acaulis. Within two populations, mitochondrial alleles, but not sex, were clustered at a scale ≤2 m. We suggest that this pattern is generated by short‐distance dispersal of mitochondrial alleles (and CMS) through seed and longer‐distance dispersal of nuclear restorers through both pollen and seed. We also investigated whether the association between mitochondrial alleles and sex ratio changed through time (across size classes), as might be...

Genome-wide analysis of maize cytoplasmic male sterility-S based on QTL mapping

Three types of sterile cytoplasm in cytoplasmic-male-sterility (CMS) maize, T, C and S, can be identified according to their fertility-restoration and mitochondrial DNA RFLP patterns. CMS-S, which is the least stable among the three types of CMS, is controlled by sterile cytoplasm interactions with certain nuclear-encoded factors. We constructed a high-resolution map of loci associated with male-restoration of CMS-S in BC1 populations of maize. The map covers 1730.29 cM, including 32 RFLP, 51 SSR 62 RAPD and 21 AFLP markers. Genome-wide QTL analysis detected 6 QTLs with significant effects on male fertility as assessed by their starch-filled pollen ratios. Four QTLs out of six were located between the SSR markers MSbnlg1633-Mrasg20, MSbnlg1662-Msume1126, MSume1230-MSumc1525, and RAPD marker MraopQ07-2-MraopK06-2 on chromosome 2. Two other minor loci were mapped between MraopK16-1-Mraopi4-1, on chromosome 9, and between Msuncbnlg1139-MraopR10-2, on chromosome 6. The Rf3 nuclear restoring gene was precisely located on the chromosome 2, 2.29 cM to the left of umc1525 and 8.9 cM to the right of umc1230. The results provide important markers for marker-assisted selection of stable CMS-S maize.

Estimation of stoichiometry of marker sequences of the sugar beet (Beta vulgaris) mitochondrial genome by real-time PCR

47 Plant mitochondrial genomes exhibit some features distinguishing them from the mitochondrial genomes of animals and fungi, the main of which are a large size (with the coding capacity being similar) and a complex organization. It was shown that the mitochondrial DNA fraction (mtDNA) contains concatemers, circular molecules with linear fragments, and subgenomic circular and linear variants whose stoichiometry may vary [1‐5]. The traditional representation of full-length sequences of plant mitochondrial genomes in the form of the “master” chromosome apparently does not reflect their state in vivo , because it does not take into account the lowcopy-number and recombinant sequences located in untypical environment. The trait of cytoplasmic male sterility (CMS) is related to the presence in mitochondria of a special genome type. In the case of Owen’s type of CMS in sugar beet ( Beta vulgaris ), this is the Svulg genome. This trait is expressed only in a special nuclear environment and consists in the inability of the plant to produce fertile pollen. CMS is usually caused by the formation of chimerical sequences, with this process being facilitated by a high recombination activity of plant mitochondrial genome. To date, two variants of the main chromosome of B. vulgaris mitochondrial genome have been sequenced: the N type, which ensures the formation of fertile pollen, and the Svulg -type [6, 7]. A large number of regions unique for each genome type does not make it possible to assign CMS to a loss or acquisition of a certain sequence in sugar beet. Additionally, the analysis of translation patterns of N- type and Svulg type mitochondria did not allow the CMS determinant to be determined at the protein level [8]. The relationship between CMS and selective amplification of certain subgenomic variants has been demonstrated for the haricot ( Phasoleus vulgaris ) [9]. Probably, the expression of CMS in B. vulgaris is also ensured not only by the presence of the genome (the “master” chromosome) but also by the composition and stoichiometry of subgenomic variants. In this case, the number of copies of subgenomic variants of mtDNA in the cytoplasm of sterile and fertile plants should be determined to find the relationship between CMS and the mitochondrial genome structure. In this study, we performed a comparative analysis of the number of copies of marker sequences in N -type and Svulg -type mitochondrial genomes. For this purpose, we for the first time used the real-time PCR—one of the most sensitive and precise methods of determination of the amount of certain sequences in DNA samples [10].

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3' region of the atp6 gene (5'-cgcttggactatgctatgtatga-3') and the 5' region of the nad3 gene (5'-tcatagagaaatccaatcgtcaa-3') produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.

Distribution of Fertility-restorer Genes for Wild-abortive and Honglian CMS Lines of Rice in the AA Genome Species of Genus Oryza

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. 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. 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. 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.

Evaluasi Daya Pemulih Kesuburan Padi Lokal dari Kelompok Tropical Japonica

Twenty seven land races of tropical japonica rice were test-crossed with a 'WA' type cytoplasmic male sterility (CMS) line IR58025A to evaluate their fertility restoration ability. Based on pollen fertility and spikelet fertility of their F1 hybrids, genotypes are classified into maintainer or restorer. The result showed frequency of maintainers among genotypes was higher than restorers. Four genotypes, i.e., Ase Lapan, Ase Mandi, Hawara Bunar and Lampung Kuning were designated as maintainers. These maintainers possess a number of desirable traits such as pest resistance and abiotic stresses tolerance; thus they will be useful for improving parental lines of hybrid rice. Key words: Land race, test cross, fertility restoration

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs.

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.

New type of cytoplasmic male sterility in sunflower

An alteration in the cytoplasm reaction was provoked during the transfer of sunflower line M-C-5 into the cytoplasmic male sterility ARG-3. The new source of CMS was different from CMS ARG-3. The differences were proved by investigating the fertility restoration in hybrid plants produced with the participation of line M-C-5A in CMS ARG-3 as well as the restorer lines for CMS PET-1 and CMS ARG-3 - lines 147R, 7015R, 7042R and 7045R. Combinations M-C-5A in CMS ARG-3 and lines 7015R and 7045R gave 100% sterile hybrid plants, while combinations M-C-5A in CMS ARG-3 and lines147R and 7042R gave 60.3% and 23.8% sterile plants, respectively. As a result of the combinations between other nine lines in CMS ARG-3 and these four R lines, only fertile hybrid plants were produced. The hybrid plants obtained from combinations between line M-C-5A in CMS DEB-1 and CMS ANN-16 with line 7015R were also fertile. These facts led us to conclude that the mutation has been provoked in the process of transfer of line M-C-5A into CMS ARG-3. Thus a new different CMS source has arisen out of an existing CMS source and it was named CMS ARG-3-M-1. The newly obtained CMS source seems to be a separate type. The way of origination of CMS ARG-3-M-1 was entirely different from the already existing methods for obtaining CMS in sunflower.

Obtaining an ogura-type CMS line from asymmetrical protoplast fusion between cabbage (fertile) and radish (fertile)

Cytoplasmic male sterile (CMS) plants were obtained by asymmetrical protoplast fusion between red cabbage (fertile) and normal radish (fertile). The CMS plants showed restriction fragment length polymorphism (RFLP) patterns of mitochondrial (mt) DNA that were different from those of both parental lines. PCR analysis of mtDNA of the CMS plants and though Southern blot analysis showed that the mtDNA of the CMS line had the characteristics of the ogura CMS type. The results suggested that the orf138 gene and the ogura type atp6 gene are present in normal fertile cabbage and radish at a low copy level.

A PCR Assay for Rapid Discrimination of Sterile Cytoplasm Types in Maize

A single‐seed, multiplex polymerase chain reaction (PCR) protocol is presented to distinguish the three major types of cytoplasmic male sterility (CMS‐T, ‐C, and ‐S) in maize (Zea mays L.). Three pairs of PCR primers were designed, corresponding to the chimeric regions of mitochondrial DNA sequences specific for the three types of CMS cytoplasm. The specificity of these primers was tested by means of the inbred line B37 with male‐sterile (T, C, and S) and normal (N) cytoplasm as reference material. Subsequently, a single‐seed PCR assay was developed for the determination of cytoplasm types in a single reaction and applied to a wide range of inbred lines with normal and sterile cytoplasm (T, C, and S) and with predominantly identified subtypes of the major sterile cytoplasm types. Our protocol proved to be a rapid, inexpensive, and reliable procedure for the molecular classification of male sterile maize cytoplasm to the three major categories T, C, and S.

A PCR Assay for Rapid Discrimination of Sterile Cytoplasm Types in Maize

A single-seed, multiplex polymerase chain reaction (PCR) protocol is presented to distinguish the three major types of cytoplasmic male sterility (CMS-T, -C, and -S) in maize (Zea mays L.). Three pairs of PCR primers were designed, corresponding to the chimeric regions of mitochondrial DNA sequences specific for the three types of CMS cytoplasm. The specificity of these primers was tested by means of the inbred line B37 with male-sterile (T, C, and S) and normal (N) cytoplasm as reference material. Subsequently, a single-seed PCR assay was developed for the determination of cytoplasm types in a single reaction and applied to a wide range of inbred lines with normal and sterile cytoplasm (T, C, and S) and with predominantly identified subtypes of the major sterile cytoplasm types. Our protocol proved to be a rapid, inexpensive, and reliable procedure for the molecular classification of male sterile maize cytoplasm to the three major categories T, C, and S.

PCR-based markers to differentiate the mitochondrial genomes of petaloid and male fertile carrot (Daucus carota L.)

Cytoplasmic male sterility (CMS) is essential for the development of highly adapted and uniform hybrid varieties of carrot and other crops. The most widely used type of CMS in carrot is petaloidy, in which the stamens are replaced by petals or bract-like structures. We have developed a series of mitochondria-specific PCR markers to distinguish cytoplasms inducing petaloidy (Sp) and male-fertility (N). The markers target the atp1,atp6, atp9, orfB (atp8), nad6 and cob loci from the mitochondrial genomes of a diverse collection of male fertile and petaloid carrots. We report 14 primer pairs that amplify marker fragments from either Sp or N cytoplasms and three primer pairs that amplify fragments with length polymorphism. The amplification products span sites of insertions, deletions or recombinations adjacent to or within the coding regions of the targeted genes. The markers reported here are useful tools to identify the type of cytoplasm in cultivated carrot and to evaluate variation in the mitochondrial genomes within the genus Daucus.

The spatial structure of sexual and cytonuclear polymorphism in the gynodioecious Beta vulgaris ssp. maritima: I/ at a local scale.

We have analyzed the spatial distribution of the sex phenotypes and of mitochondrial, chloroplast, and nuclear markers within two gynodioecious populations of Beta vulgaris ssp. maritima. Within both populations, sexual phenotype variation is controlled mainly by the cytoplasmic genotype, although in one study population a joint polymorphism of cytonuclear factors is clearly involved. In spite of contrasts in the ecology (mainly due to different habitats), a clear common feature in both populations is the highly patchy distribution of cytoplasmic haplotypes, contrasting with the wide distribution of nuclear diversity. This high contrast between cytoplasmic vs. nuclear spatial structure may have important consequences for the maintenance of gynodioecy. It provides opportunities for differential selection since nuclear restorer alleles are expected to be selected for in the presence of their specific cytoplasmic male sterile (CMS) type, but to be neutral (or selected against if there is a cost of restoration) in the absence of their CMS type. Selective processes in such a cytonuclear landscape may explain the polymorphism we observed at restorer loci for two CMS types.