Cytoplasmic Male Sterile Wheat Research Articles

Mapping of wheat mitochondrial mRNA termini and comparison with breakpoints in DNA homology among plants

Mitochondrial DNA rearrangements occur very frequently in flowering plants and when close to genes there must be concomitant acquisition of new regulatory cis-elements. To explore whether there might be limits to such DNA shuffling, we have mapped the termini of mitochondrial mRNAs in wheat, a monocot, and compared them to the known positions for counterpart genes in the eudicot Arabidopsis. Nine genes share homologous 3' UTRs over their full-length and for six of them, the termini map very close to the site of wheat/Arabidopsis DNA rearrangements. Only one such case was seen for comparisons of 5' UTRs, and the 5' ends of mRNAs are typically more heterogeneous than 3' termini. Approximately half of the thirty-one wheat mitochondrial transcriptional units are preceded by CRTA promoter-like motifs, and of the potential stem-loop or tRNA-like structures identified as candidate RNA processing/stability signals near the 5' or 3' ends, several are shared with Arabidopsis. Comparison of the mitochondrial gene flanking sequences from normal fertile wheat (Triticum aestivum) with those of Aegilops kotschyi which is the source of mitochondria present in K-type cytoplasmic male sterile wheat, revealed six cases where mRNAs are precluded from sharing full-length homologous UTRs because of genomic reorganization events, and the presence of short repeats located at the sites of discontinuity points to a reciprocal recombination-mediated mode of rearrangement.

A ras GTPase-activating protein-binding protein, TaG3BP, associated with the modulation of male fertility in a thermo-sensitive cytoplasmic male sterile wheat line

Hybrid wheat development may contribute to higher, more stable yield and could result in greater food security for much of the world's growing population. YS type thermo-sensitive cytoplasmic male sterile (TCMS) wheat lines were developed for use in hybrid wheat breeding in China. To investigate the molecular mechanism of modulation of male fertility in the YS-type TCMS wheat lines, a ras GTPase-activating protein-binding protein (TaG3BP) was examined. The deduced amino acid sequence encoded by TaG3BP was conserved in the sequenced genomes of Embryophyte. TaG3BP expression in the anthers of YS-type TCMS lines taken at the critical fertility reversion stage of pollen development from male fertile anthers was higher than that from male-sterile anthers, either by quantitative real-time PCR or by western blot analysis. Sequence analysis on the cDNA and genomic DNA of TaG3BP in three kinds of K-type CMS wheat lines and their maintainers indicated that there were no significant difference between the genes or in their 5' flanking sequences. The TaG3BP expression revealed by quantitative real-time RT-PCR was lower in the young spikes of these CMS lines than that of their maintainers. This indicates that TaG3BP expression is associated with the modulation, from male-sterile to fertile, of the TCMS wheat line, and TaG3BP might be a key factor in the pathway responsible for the fertility reversion.

Cloning and Expression Analysis of cMDH Gene Related to Cytoplasmic Male Sterile Wheat with <I>Aegilops kotschyi</I> Cytoplasm

Cloning and Expression Analysis of cMDH Gene Related to Cytoplasmic Male Sterile Wheat with <I>Aegilops kotschyi</I> Cytoplasm

Unique High Molecular Weight Proteins expressed in Cytoplasmic Male Sterile Wheat (Triticum aestivum) as determined by Two‐Dimensional Electrophoresis (2DE)

When careful computer analysis of the patterns produced during 3 separate growth years by 2DE was performed the separation of expressed proteins at the young spike stage of growth of male sterile wheat plants indicated there were 18 proteins missing when compared to male fertile plants at the same growth stage. Surprisingly, there were found 12 high molecular weight (over 90 kDa) proteins present in the male sterile plant patterns that were not present in the male fertile plant patterns. Molecular weight and isoelectric points of the affected proteins will be presented. Leaf 2DE patterns at the corresponding developmental growth stages indicated there were no differences to be found. This suggested that the protein pattern differences seen are specifically expressed at the spike stage of growth. Searching of the literature suggests this is the first study of expressed proteins by 2DE of the rather common cytoplasmic male sterility phenomenon in nature. Because of this it is tentatively proposed that male cytoplasmic sterility in wheat at least may have a quite unique genetic program controlling expressed proteins in specific structures of wheat in at least part of its life cycle. Supported in part by institutional research grants from Harbin Normal University.

Development of photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines showing high male sterility under long-day conditions and high seed fertility under short-day conditions

A “two-line system” using photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under long-day photoperiods (≧15 h) has been proposed as a means of producing hybrid varieties in common wheat (Triticum aestivum). The PCMS line is maintained by self-pollination under short-day conditions, and hybrid seeds can be produced through outcrossing of the PCMS line with a pollinator line under long-day conditions. Our previous studies revealed that PCMS lines showing complete male sterility under long-day conditions are necessary for practical hybrid wheat breeding, especially to obtain high hybrid purity in F1 seeds. Furthermore, practical PCMS lines should have high seed fertility under short-day conditions, which is associated with female fertility. Wheat cv. Norin 26 with Ae. crassa cytoplasm exhibits high seed fertility under short-day conditions, and cv. Fujimikomugi with Ae. crassa cytoplasm shows high male sterility under long-day conditions. Here we developed practical PCMS lines derived from the F1 generation of Norin 26 and Fujimikomugi (with Ae. crassa cytoplasm) that were then backcrossed to elite wheat lines.

Comparison of two fertility restoration systems against photoperiod‐sensitive cytoplasmic male sterility in wheat

AbstractA ‘two‐line system’ using photoperiod‐sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under a long‐day photoperiod ( 15 h) has been proposed as a new means of producing hybrid varieties in common wheat. The PCMS line is maintained by self‐pollination under short‐day conditions, and hybrid seeds can be produced through outcrossing of the PCMS line with a pollinator under long‐day conditions. Two kinds of fertility restoration systems against the PCMS are known. One is involved with a set of multiple fertility‐restoring (Rf) genes in the wheat cultivar ‘Norin 61’ located on (at least) chromosomes 4A, 1D, 3D and 5D. The other is controlled by a single dominant major Rf gene, Rfd1, located on the long arm of chromosome 7B in the wheat cultivar ‘Chinese Spring’. To examine the degree of fertility restoration by these two systems, nine PCMS lines were crossed with ‘Norin 61’ and ‘Chinese Spring’ as the restorer lines, and the F1 hybrids were investigated. The degree of fertility restoration was estimated by comparing the seed set rates in the F1 hybrids having the Ae. crassa cytoplasm and those with normal cytoplasm. The results revealed that the fertility restoration ability of a set of multiple Rf genes in ‘Norin 61’ was higher than that of the Rfd1 gene in ‘Chinese Spring’.

QTL analysis of fertility-restoration against cytoplasmic male sterility in wheat.

The cytoplasm of Triticum timopheevi causes cytoplasmic male sterility (CMS) in common wheat (T. aestivum) cv. 'Chinese Spring' (CS), and that of Aegilops kotschyi causes CMS in spelt wheat (T. spelta) var. duhamelianum (Sp). CS has fertility-restoring (Rf) genes against the latter cytoplasm and Sp has the ones against the former. To know the genetic system concerning to CMS, we crossed 66 F8 recombinant inbred lines (RILs) derived from a cross between CS and Sp as males to the alloplasmic lines of CS and Sp having the cytoplasms of T. timopheevi and Ae. kotschyi, respectively. The fertilities of respective F1 plants derived from the crosses were examined for QTL analysis. The major QTLs detected in both systems were located on the short arm of chromosome 1B. One minor QTL on chromosome 2B was also commonly detected in both of the systems, while other minor QTLs against T timopheevi cytoplasm were distributed on the chromosomes 2A, 4B, and 6A.

Factors responsible for levels of male sterility in photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines

A `two-line system' using photoperiod-sensitivecytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm has been proposed as a newmeans of producing hybrid wheat. The PCMS line ismaintained by self-pollination under short-dayconditions (≦14.5 h light period), and F1 seedscan be produced by outcrossing of the PCMS line witha pollinator under long-day conditions (≧15 h lightperiod). As the levels of male sterility in PCMSlines under the long-day conditions is a crucialfactor in determining hybrid purity of the F1seeds, a study was conducted into the effect ofseeding rate on male sterility in PCMS lines. Threedifferent density levels were tested using analloplasmic line of Japanese wheat cultivar `Norin 26'which exhibits PCMS. Levels of male sterility of thePCMS line increased at sparse planting, because tiller(ear) number per plant increased at low seedingdensity and late-appearing ears tended to exhibithigher levels of male sterility than early-appearingears. On the other hand, male sterility levels of thePCMS lines depended on genotype, e.g., the PCMS`Fujimikomugi' was completely male sterile, whereasthe PCMS `Norin 26' showed partial male sterility. APCMS line showing complete male sterility, such as thePCMS `Fujimikomugi', should produce F1 seeds withhigh purity. However, the PCMS `Fujimikomugi' showeda lower female fertility. For practical use, it isnecessary to produce PCMS lines having high malesterility with high female fertility under long-dayconditions.

Photoperiod-sensitive cytoplasmic male sterility in wheat: nuclear-mitochondrial incompatibility results in differential processing of the mitochondrial orf25 gene.

An alloplasmic wheat line with the cytoplasm of Aegilops crassa expresses photoperiod-sensitive cytoplasmic male sterility (PCMS). Southern- and Northern-hybridization analyses showed that this line contains alterations in both the gene structure and transcription patterns of the mitochondrial gene orf25. In this study, the nucleotide sequence around the orf25 gene of Ae. crassa (CR-orf25) and common wheat (AE-orf25) was determined, and we found that the upstream region of CR-orf25 had been replaced by that of rps7 of common wheat (AE-rps7) through recombination. A novel open reading frame (orf48) is present upstream of CR-orf25. In these three genes, transcription was initiated from the consensus promoter motif of plant mitochondrial genes located in the upstream regions. Processing enzymes in Ae. crassa and common wheat cleave the respective precursor mRNAs, namely CR-orf25 and AE-rps7, at sites similar to that of the premature mitochondrial 26S rRNA. In contrast, the precursor mRNA is not effectively processed at the target sequence of CR-orf25 in the alloplasmic wheat line. Because major transcripts of the euplasmic CR-orf25 and AE-rps7 genes would result in a truncated orf48 product, one possibility is that the orf48 protein might disturb mitochondrial function at a specific stage and hence affect the expression of the PCMS trait.

Studies on the effects of nuclear background and tissue specificity on RNA editing of the mitochondrial ATP synthase subunits α, 6 and 9 in fertile and cytoplasmic male-sterile (CMS) wheat

RNA editing occurs in transcripts encoded by the mitochondrial genome in all major groups of land plants and is characterised by C-to-U editing. When editing occurs, the amino acid is usually altered because the nucleotide modification is generally in the first or second position and the amino acid change improves the conservation of the predicted protein as compared with other organisms. In order to assess the effect of the nuclear background and tissue specificity of the RNA editing, we have analysed the RNA editing of the transcripts encoding for the mitochondrial ATPsynthase subunits atpα, atp6, and atp9 in euplasmic and alloplasmic cytoplasmic male-sterile (CMS) wheat lines. The editing of atpα was detected in 6 codons. Although partially edited transcripts were found in all the cytoplasms, a larger number of partially edited clones were present in the CMS cytoplasm. The RNA editing of atp6 occurred in 12 codons, and whereas in the euplasmic Triticum timopheevi all the clones were fully edited, in the CMS line 17% of the clones were only partially edited, suggesting an effect of the nuclear background on RNA editing efficiency. In atp9 transcripts, 8 codons were modified by RNA editing. When the RNA editing of the atp9 transcripts extracted from various tissues was compared, only fully edited clones were detected in embryos, roots, shoots, and anthers in the euplasmic wheat lines, whereas in the transcripts obtained from the CMS line, 19% of the clones were partially edited. This study reveals that RNA editing efficiency can be affected by the tissue and nuclear background. The mechanism by which incomplete and complete edited transcripts accumulate may indicate a link between the transcription kinetics and RNA editing which can be affected by nuclear background and tissue specificity.

Genetic Analysis of Fertility Restoration in Wheat Using Restriction Fragment Length Polymorphisms

Fertility restoration for Triticum timopheevi Zhuk. cytoplasmic male sterility in wheat (Triticum aestivum L.) is controlled by both major fertility restorer (Rf) genes and modifier genes together with environment effects. Therefore, the breeding of efficient and stable malefertility restorer lines (R‐lines) should be facilitated by marker‐assisted selection. This study was conducted to identify restriction fragment length polymorphism (RFLP) markers for the Rf genes, Rf1 and Rf4 in restorer line R113, and Rf3 and a Rf gene on 5D in cultivar Primepi.Polymorphic markers were mapped in testcross F1 populations and their association with fertility restoration was analyzed by analysis of variance. The results indicated that Rf4 was linked to Xksug48 located on 6BS in Rl13, Rf3 was linked to Xbcd249 and Xcdo442 on 1BS, and Xcdo786 may be associated with the 5D Rf gene in Primepi. In addition, in Rl13 two other chromosome regions with fertility restoration‐associated loci were detected by CD0786 and H1 on 5AL and CDOl189 on 7BS. A locus on 5DS, detected by BCD1871, showed significant epistatic effect on the expression of Rf genes. In Primepi, a locus on 5AL, detected by Xbcd183 and Xbcd876, had effects similar to Xcdo786. Two loci in ‘Qu Xian Early A’, identified by Xbcd871 and Xwg1026, may be associated with genes controlling ease of restoration in this male‐sterile line (A‐fine). Except for Xbcd1871 and Xwg1026, the effects of other significant RFLP loci were additive in both populations.

A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat.

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.

Photoperiod-sensitive cytoplasmic male sterility in wheat with Aegilops crassa cytoplasm

Triticum aestivum cv. Norin 26 with Aegilops crassa, Ae. juvenalis or Ae. vavilovii cytoplasm (all D2 type) has been studied relative to its photoperiodic response of male sterility and fertility restoration patterns. Alloplasmic lines of ‘Norin 26’ with a D2 type cytoplasm showed almost complete male sterility under long-day conditions (≥15 h), but high male fertility under short-day conditions (≤14.5 h). No significant influence of temperature on reduction in male fertility was observed. Thus, this type of male sterility is called ‘photoperiod-sensitive cytoplasmic male sterility’ (PCMS). The PCMS is expressed in the form of pistillody of stamens. Histological studies revealed that there were incomplete ovule-like structures instead of tapetal cells and pollen grains in the pistillate stamens. The floret differentiation stage of the plant is the stage that is sensitive to photoperiod. The PCMS can be used as a new means for hybrid wheat production, named ‘two-line system’. The PCMS line is maintained and multiplied by self-fertilization under short-day conditions, and hybrid seed can be produced by crossing the PCMS line with a pollinator line under long-day conditions. In contrast to the system of hybrid wheat production using the T. timopheevi cytoplasm, the present system requires only PCMS and pollinator lines.

Maize S1 and S2 sequences in wheat mitochondrial DNA

The mitochondrial DNA (mtDNA) of fertile ( Triticum aestivum) and cytoplasmic male sterile (cms) wheat ( T. aestivum nucleus; T. timopheevi mitochondria) was isolated and subjected to restriction enzyme analysis. Restriction fragments of the mtDNAs were probed with DNA specific to the S-episomes of maize mitochondria. Small, independent episomal DNA was not found in the mtDNA of wheat. Sequences related to maize S1 DNA are present in fertile and cms wheat mtDNA but do not appear to be contiguous. Restriction enzyme analyses do not show detectable differences between fertile and cms mtDNA around the maize-like S1 sequences. Sequences related to maize S2 mtDNA may be located at two different positions in both fertile and cms mitochondrial genomes. The fertile and cms mtDNA show restriction fragment length polymorphism at one of two sites.

A chimeric open reading frame in the 5' flanking region of coxI mitochondrial DNA from cytoplasmic male-sterile wheat.

A chimeric open reading frame in the 5' flanking region of coxI mitochondrial DNA from cytoplasmic male-sterile wheat.

Studies on isoenzymes of anther tissue of fertile and cytoplasmic male sterile wheat plants

We studied the isoenzyme pattern of peroxidase, esterase, malate dehydrogenase and leucine aminopeptidase from anther tissue of fertile and male sterile wheat plants by means of polyacrylamide gel electrophoresis.Three of the tested enzymes (peroxidase, esterase and leucine aminopeptidase) show differences in their isoenzyme pattern.The pattern of proteins extracted from sterile anthers lacks isoenzymes present in the extracts of fertile anthers.The differences in enzyme activity of fertile and sterile plants seem to be a consequence rather than a primary cause of cytoplasmic male sterility.

Evaluation of three sources of pollen fertility restoration genes and natural outcrossing of cytoplasmic male-sterile wheat

Fertility restoration genes in Triticum aestivum L. in Texas Restorer Composite (TRC), D6301, and four CIMMYT restorer lines were studied, and selection was made for higher fertility in TRC. Mean-while, outcrossing percentages of seed set for 27 spring habit cytoplasmic male sterile (cms) varieties were evaluated for 3 to 5 years at Davis. The winter-habit TRC material did not restore reasonably good fertility, and the response to selection for higher fertility seemed to be slow. This poor fertility could be partly due to its late winter growth habit causing flowering at a period of high temperature and low humidity at Davis. The highest F1 fertility was 46.6% in the cross cms Ramona x TRC-6, and its F2 segregated into the ratio of 15 fertile to 1 sterile, with fertility ranging from 3.2 to 100%. Suggested for its improvement was intensive selection in the original TRC material and in the segre-gating F2 population, followed by intercrossing. D6301 has 2 fertility restoration genes with different strengths which restore fertility up to 45.2% when both genes are heterozygous. D6301 is quite likely heterogeneous for these genes. Four CIMMYT restorer lines, D7464, D7465, D7466, and D7467, had satisfactory F1 fertility restoration after crossing with cms Ramona 50. In 1975, the fertilities of the F1's ranged from 71 to 85% and were over 90% in 1976. The F2 population of the cross cms Ramona 50 × D7464 segregated into a ratio of 3 fertile to 1 sterile, indicating that D7464 has a single dominant gene for fertility restoration. The F2's of crosses cms Ramona 50 × D7465, cms Ramona 50 × D7466, and cms Ramona 50 × D7467 gave a ratio of 15 fertile to 1 sterile, indicating that two gene pairs in these three lines were responsible for the fertility restoration. The best of this group was D7467 which restored fertility fully after being crossed with cms Ramona 50 (T. timopheevi cytoplasm).

Heterogeneous population of mitochondrial DNA molecules in higher plants

THE physicochemical properties of mitochondrial (mt)-DNA of higher plants have been reported1–4. mt-DNAs occur as closed circles with molecular weights of (60–70) × 106 and it is likely that each mitochondria contains several of these circles. mt-DNAs isolated from different higher plants exhibit similar physicochemical parameters: all mt-DNAs so far analysed1–5 in higher plants band at a unique buoyant density of 1.706 g cm−3 in neutral CsCl gradients and the few published melting points and reassociation kinetics are not significantly different2–4. Not enough values have been reported to state on possible differences in the contour length measured by electron microscopy. Thus, mt-DNAs isolated from different higher plants cannot be specifically identified by conventional physicochemical techniques. We have recently reported that the analysis of restriction nuclease digests by gel electrophoresis provides a very sensitive test to identify specifically chloroplastic (cp)-DNAs isolated from different plant genera and species6. We show here that this technique also allows the characterisation and the specific identification of mt-DNAs isolated from various higher plants. We used EcoRI specific cleavage to evidence the general occurrence of heterogeneous population of mt-DNA molecules within an higher plant and to detect clear differences between mt-DNAs extracted from normal and cytoplasmic male-sterile wheat.

Single gene restoration of cytoplasmic male sterility in wheat and its implications in the breeding of restorer lines

Cultivars of T. aestivum crossed onto two lines with male sterility induced by the cytoplasm of T. timopheevi gave a high level of restoration in the F1 generation. The ratio of fertile to sterile plants segregating in the F2 generation was consistent with that expected for a single dominant restorer gene. The possible association between this gene and mildew resistance or some other desirable character derived from 'CI 12633', a common ancestor of the cultivars used, is discussed.

Electron-microscopy on anther tissue and pollen of male sterile and fertile wheat (Triticum aestivum L.)

1. Anther tissue and pollen grains of cytoplasmic male sterile and fertile wheat were studied with the aid of an electron-microscope for differences in cytoplasmic structures.