Crassa Cytoplasm Research Articles

Comparative transcriptome analysis indicates conversion of stamens into pistil-like structures in male sterile wheat (Triticum aestivum L.) with Aegilops crassa cytoplasm

BackgroundAegilops crassa cytoplasm is an important source for investigating cytoplasmic male sterility (CMS). Moreover, the stamens of line C303A exhibit a high degree of pistillody, turning almost white. However, the molecular mechanism that underlies pistillody in C303A remains unclear. Therefore, to obtain a better understanding of pistillody in C303A, the phenotypic and cytological features of C303A were observed to identify the key stage for the homeotic transformation of stamens into pistil-like structures. Transcriptome profiles were determined for stamens using Illumina RNA sequencing.ResultsMorphological observations of the CMS wheat line with Aegilops crassa cytoplasm C303A showed that the pistils developed normally, but the stamens were ultimately aborted and they released no pollen when mature. According to paraffin section observations, the stamens began to transform into pistils or pistil-like structures in the binucleate stage (BNS). Therefore, the stamens were collected from line C303A and its maintainer 303B in the BNS for transcriptome sequencing. In total, 20,444 wheat genes were determined as differentially expressed in C303A and 303B stamens, with 10,283 upregulated and 10,161 downregulated genes. Gene Ontology enrichment analyses showed that most of the differentially expressed genes (DEGs) were annotated with GO terms comprising metabolic process, cell, cellular process, catalytic activity, and cell part. Analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed that the enriched DEGs were mainly associated with energy metabolism. We also found several essential genes that may contribute to pistillody in C303A. These findings suggest that disrupted energy metabolism and reactive oxygen metabolism induce pistillody and eventually lead to abortion in C303A.ConclusionWe determined the complex transcriptome profiles for C303A stamens and demonstrated that disrupted energy metabolism and class B MADS-box genes are related to pistillody. These findings may facilitate future studies of the mechanistic response of the wheat stamen and pollen development in CMS.

Chemically induced male sterility in common wheat mediated by Aegilops cytoplasm

Several approaches have been proposed, in the past, for the development of hybrid wheat varieties. Nevertheless, common wheat hybrids today account for less than 1% of the world's wheat production. The reason for the limited success of hybrid wheat varieties is the fact that to date, a simple and efficient system for the production of hybrid seed has not been developed. A two-line system using photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa Boiss. cytoplasm seems to be very promising way to produce hybrid wheat seed on large scale. Non-desirable side effects of PCMS system are the unreliable sterility of the male sterile lines under different latitudes. Therefore, we tried to connect male sterility system based on Ae. crassa cytoplasm with chemical induction of male sterility. The presented results reveal that the majority of male sterility genes are conserved in the Ae. crassa cytoplasm and that their effects can be promoted by chemical signals, such as synthetic auxins (e.g., 2,4-Dichlorophenoxyacetic acid). Our research work represents the development of a novel male sterility system which is controlled by chemical signals (e.g., synthetic auxins and their pro-herbicide analogues) and mediated by Ae. crassa cytoplasm.

Photoperiod-sensitive cytoplasmic male sterile elite lines for hybrid wheat breeding, showing high cross-pollination fertility under long-day conditions

AbstractPhotoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under long-day (LD) photoperiods (≧15 h) has been proposed as a two-line system for producing hybrid varieties in common wheat (Triticum aestivum). The PCMS line is maintained by self-pollination under short-day (SD) conditions, and hybrid seeds can be produced through outcrossing of the PCMS line with a pollinator line under LD conditions. Maintainer lines of the PCMS lines are not necessary in this system. In our previous study, we developed two PCMS lines with the genetic background of the Japanese wheat cultivar ‘Fukuotome’, which showed high male sterility under LD conditions and high seed fertility under SD conditions. These PCMS lines were from a BC2 generation and were not genetically pure. Therefore, we screened the progeny of the BC2 lines, and identified BC2F8 lines that showed high male sterility and high cross-pollination fertility under LD conditions and high seed fertility under SD conditions. Furthermore, we screened the pollinator lines suitable for the PCMS system. These PCMS elite lines and selected pollinator lines will be useful for developing hybrid wheat varieties.

Characterization of wheat Bell1-type homeobox genes in floral organs of alloplasmic lines with Aegilops crassa cytoplasm

BackgroundAlloplasmic wheat lines with Aegilops crassa cytoplasm often show homeotic conversion of stamens into pistils under long-day conditions. In the pistillody-exhibiting florets, an ectopic ovule is formed within the transformed stamens, and female sterility is also observed because of abnormal integument development.ResultsIn this study, four wheat Bell1-like homeobox (BLH) genes were isolated and named WBLH1 to WBLH4. WBLH1/WBLH3/WBLH4 expression was observed in the basal boundary region of the ovary in both normal pistils and transformed stamens. WBLH2 was also strongly expressed in integuments not only of normal ovules in pistils but also of the ectopic ovules in transformed stamens, and the WBLH2 expression pattern in the sterile pistils seemed to be identical to that in normal ovules of fertile pistils. In addition, WBLH1 and WBLH3 showed interactions with the three wheat KNOX proteins through the BEL domain. WBLH2, however, formed a complex with wheat KNOTTED1 and ROUGH SHEATH1 orthologs through SKY and BEL domains, but not with a wheat LIGULELESS4 ortholog.ConclusionsExpression of the four WBLH genes is evident in reproductive organs including pistils and transformed stamens and is independent from female sterility in alloplasmic wheat lines with Ae. crassa cytoplasm. KNOX-BLH interaction was conserved among various plant species, indicating the significance of KNOX-BLH complex formation in wheat developmental processes. The functional features of WBLH2 are likely to be distinct from other BLH gene functions in wheat development.

Altered expression of wheat AINTEGUMENTA homolog, WANT-1, in pistil and pistil-like transformed stamen of an alloplasmic line with Aegilops crassa cytoplasm

Homeotic transformation of stamens into pistil-like structures, called pistillody, has been reported in some alloplasmic common wheat lines with Aegilops crassa cytoplasm. An alloplasmic line of Chinese Spring ditelosomic 7BS (CSdt7BS) with Ae. crassa cytoplasm lacking the long arm of the chromosome 7B shows pistillody, and the pistils and transformed stamens are sterile due to abnormal ovule development. To elucidate the molecular mechanism of the ovule abnormality, we compared the expression profiles of floral organs between euplasmic and alloplasmic CSdt7BS lines. Two differential display methods of mRNA profiling demonstrated that Ae. crassa cytoplasm largely affects nuclear gene expression profiles of common wheat. Of the differentially expressed genes, a wheat AINTEGUMENTA (ANT) homolog, WANT-1, was preferentially expressed in pistils but not in stamens, and accumulation of the transcript was limited to ovule primordia at the floral organ development stage. In alloplasmic wheat, WANT-1 expression was patchy and weak at the ovule-development stages. On the other hand, no significant difference in gene expression patterns of wheat AGAMOUS (AG) homologs (WAG-1 and WAG-2) was observed between fertile and sterile pistils. These results indicated that alteration of gene expression after initiation of ovule primordia results in abnormal ovule development, and that the aberrant ovule formation is at least partly associated with the weak expression of WANT-1 around ovule primordia in alloplasmic wheat with Ae. crassa cytoplasm.

Orf260cra, a Novel Mitochondrial Gene, is Associated with the Homeotic Transformation of Stamens into Pistil-Like Structures (Pistillody) in Alloplasmic Wheat

Homeotic transformation of stamens into pistil-like structures (pistillody) can occur in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) that have the cytoplasm of the related species, Aegilops crassa. Previously we showed that pistillody results from altered patterns of expression of class B MADS-box genes mediated by mitochondrial gene(s) in the Ae. crassa cytoplasm. The wheat cultivar Chinese Spring does not show pistillody when Ae. crassa cytoplasm is introduced. The absence of an effect is due to a single dominant gene (designated Rfd1) located on the long arm of chromosome 7B. To identify the mitochondrial gene involved in pistillody induction, we performed a subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. We found that mitochondrial cDNA clone R04 was abundant in the young spikes of the pistillody line but was down-regulated in the normal line that carried nuclear Rfd1. Sequencing of the full-length cDNA corresponding to clone R04 showed that two genes were present, cox I (cytochrome c oxidase subunit I) and orf260(cra). orf260(cra) shows high sequence similarity to orf256, the T. timopheevii mitochondrial gene responsible for cytoplasmic male sterility (CMS). orf260(cra) was also present in the cytoplasms of Ae. juvenalis and Ae. vavilovii, which induce pistillody, but not in the cytoplasms of other species not associated with pistillody. Furthermore, Western blot analysis revealed that the ORF260cra protein was more abundant in the pistillody line than in the normal line. We suggest therefore that orf260(cra) is associated with pistillody induction.

Identification of a protein kinase gene associated with pistillody, homeotic transformation of stamens into pistil-like structures, in alloplasmic wheat

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.

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.

WAG, a wheat AGAMOUS homolog, is associated with development of pistil-like stamens in alloplasmic wheats

Pistillody, homeotic transformation of stamens into pistil-like structures, has been reported in cytoplasmic substitution (alloplasmic) lines of common wheat (Triticum aestivum) with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by the Rfd1 gene detected on the long arm of chromosome 7B in wheat cultivar 'Chinese Spring' (CS). Because of the absence of Rfd1, the alloplasmic line CS ditelosomic 7BS [(cr)-CSdt7BS] lacking the long arm of chromosome 7B exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with normal cytoplasm forms normal stamens. To study the molecular mechanism of pistillody caused by nuclear-cytoplasm interaction in alloplasmic wheat, we cloned and characterized a wheat AGAMOUS homolog, WAG. The WAG gene copy number in the wheat genome was estimated at three, located on the homoeologous group 1 chromosomes. Northern blot analysis with a normal wheat line revealed that the transcription level of WAG was lower in young spikes and increased during spike development. The highest expression was observed in spikes at the booting to heading stage. The 1.1 kb WAG transcript accumulated in both the reproductive (pistil and stamen) and non-reproductive (palea and lemma) portions of spikes at the heading stage, whereas an extra transcript of different molecular size (1.3 kb) was observed in pistil-like stamens of line (cr)-CSdt7BS as well as in pistils of CSdt7BS and (cr)-CSdt7BS. These results suggest that the product of the 1.3 kb WAG transcript is involved in pistil development and is associated with pistillody caused by a nuclear-cytoplasm interaction in alloplasmic wheat.

Value and utilization of alloplasmic common wheats with Aegilops crassa cytoplasm

AbstractDifferences between alloplasmic lines and euplasmic controls indicated consistent beneficial effects of Aegilops crassa cytoplasm on common wheats. In general, the agronomic performance of alloplasmic lines was superior to that of euplasmic controls; the significant differences observed were ascribed to nucleus‐cytoplasmic (NC) interactions. A number of useful genetic attributes, for example, high yield, good quality and salt tolerance, were identified. A new NC hybrid variety ‘Xiaoshan 2134’ was bred. Field trials showed that the yield NC heterosis of ‘Xiaoshan 2134’ was 13.9% and the yield of ‘Xiaoshan 2134’ was at least 20% higher than that of a control variety widely grown in North China. The results suggested that Ae. crassa cytoplasm could broaden the genetic base of common wheat and improve common wheat cultivars by utilizing NC heterosis.

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’.

Pistillody, homeotic transformation of stamens into pistil-like structures, caused by nuclear-cytoplasm interaction in wheat.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal flowers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all florets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule deficiency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.

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.

Breeding technology of alloplasmic wheat

Novel wheat bearing alloplasm was bred via nucleo substitution backcross. Through comparative study between the hornonucleo-heterocytoplasmic lines and homocytoplasm-heteronuclear lines, it was shown that a portion of the alloplasm types were able to produce useful genetic effects on wheat yield, grain quality, tolerance of salt and resistance to diseases. Among the species studied, the cytoplasm of Aegilops crassa, Ae. squarrosa and Ae. vcntricosa contributed the best effect. Specific combination of nuclear genes and cytoplasmic genes conferred specific characteristics and degree of nucleoplasmic heterosis. The alloplasmic wheat lines with cytoplasm substituted were successfully bred with good quality and resistance to disease and harsh conditions. In particular, the new variety of nucleoplasmic hybrid having Ae. crassa cytoplasm proved the best in production test, demonstration and regional test and was consequently pre-approved for large-scale plantation. The probing of the genetic variations and the utilization of alloplasmic wheat was proven to be valuable for both genetic study and breeding of new varieties.

F1 Seed Production Efficiency by Using Photoperiod-sensitive Cytoplasmic Male Sterility and Performance of F1 Hybrid Lines in Wheat.

Photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm to common wheat (Triticum aestivum) under long-day conditions (≥15 h light period) has been reported as a new means of producing hybrid wheat, that is a “two-line system”. The PCMS Iine is Inaintained by self-pollination under short-day conditions (≤14.5 h light period), and F1 seeds can beproduced by outcrossing of the PCMS Iine with a pollinator under long-day conditions. A cross pollination study for producing F1 seeds was carried out using three PCMS Iines and three restorer lines under a long-day condition at Tanno, Hokkaido, Japan. Seed setting rate was 14-33%, and 19-55 g/m2 F1 seeds with 47-88% hybrid purity were obtained. Variation in seed setting rate attributable to male parents was highly significant. High seed setting rate was achieved by using a pollinator taller than the PCMS Iine. Shriveled and preharvest sprouting kernels were found in the out-crossed F1 seeds, resulting in lower volume weight and germination rate than the selfed seeds of original cultivars of the PCMS Iines. A field test of evaluating four F1 hybrids with high hybrid purity (>65 %) was conducted under a short-day condition at Kasai, Hyogo, Japan. All F1 hybrids exhibited higher grain yield than their respective better-parent because of more spikelet number/ear and higher 1000 grain weight. The top F1 hybrid yielded 40% more than the mid-parent and 37% more than the better-parent, suggesting a possibility of the practical use of this system for hybrid wheat breeding. F1 hybrids tended to show higher volume weight than their parental cultivars. This indicates that grain quality of the F1 hybrids with the Ae. crassa cytoplasm is not inferior to those of parental cultivars with the wheat cytoplasm.

Genetic analysis of fertility restoration against photoperiod‐sensitive cytoplasmic male sterility in Triticum aestivum cv. Norin 61

Abstract Triticum aestivum cv. Norin 26 with the Aegilops crassa cytoplasm becomes almost completely male sterile when grown under a long‐day condition (15 h of light or longer), but is highly male fertile under a short‐day condition (14.5h or less). This type of male sterility is called photoperiod‐sensitive cytoplasmic male sterility (PCMS). Genetic analyses were made of the fertility‐restoring (Rf) genes effective against PCMS that are present in T. aestivum cv. Norin 61. Conventional and monosomic studies indicated that restoration of fertility is controlled by multiple Rf genes located on at least four chromosomes: 4 A, 1D, 3D and 5D. The genetic mechanism of fertility restoration by the genes of‘Norin 61’differs from the mechanisms reported for‘Chinese Spring’and a‘Norin 26’mutant line.

Alloplasmic wheats with Aegilops crassa cytoplasm which express photoperiod-sensitive homeotic transformations of anthers, show alterations in mitochondrial DNA structure and transcription.

Alloplasmic wheat. Triticum aestivum cv. Norin 26, with Aegilops crassa cytoplasm, shows photoperiod-sensitive cytoplasmic male sterility (PCMS). This alloplasmic line expresses pistillody of anthers only when grown in long-day conditions (> 15 h light). To assess the molecular basis of the PCMS, we carried out Southern and Northern hybridization analyses on mitochondrial DNAs and RNAs isolated from seedlings of alloplasmic lines showing various PCMS phenotypes using probes for twelve mitochondrial genes. All RFLP patterns of mitochondrial DNA from alloplasmic lines greatly differed from those of common wheat, and were slightly changed from those of the parental species, i.e., Ae. crassa. This indicates that nuclear substitutions between related plant species induce structural alterations in the mitochondrial genome. Furthermore, RFLP patterns of (cr)-N61 and FR-mutant probed with coxIII and orf25 were identical with each other, but different from those of the other alloplasmic lines, indicating that the nuclei of N61 and FR-mutant harbor some gene(s) that induces structural alterations of the mitochondrial genome in the coxIII and orf25 regions. The transcription patterns of atp6 and cob in Ae. crassa type were different from those of T. aestivum type. Furthermore, the orf25 transcript in alloplasmic wheats was about 300 nucleotides longer than that of euplasmic lines, including the Ae. crassa pure line, suggesting that transcription patterns of orf25 are associated with recovery from the PCMS phenomenon. These data clearly show the mutual cross-talk between the nuclear genome and chondriome. These observations raise the possibility that the dysfunction of mitochondria caused by the failure of a cooperative control of mitochondrial gene(s) expression influences the pathway of flower morphogenesis, especially in the process that determines organ identity.

Effects of Aegilops crassa Cytoplasm on the Agronomic Characters in Photoperiod-sensitive CMS Wheat Lines and F1 Hybrids.

Aegilops crassa cytoplasm causes photoperiod-sensitive Introductron cytoplasmic male sterility (PCMS) to some Japanese common wheat (Triticum aestivum ) cultivars: the PCMS line with the Ae. crassa cytoplasm is almost completely male sterile under long-day conditions (≥l5 h light period), but highly male fertile under short-day conditions (≤14.5 h). Using the PCMS, a “two-line systerm” is applicable for hybrid wheat production. To examine the effects of Ae. crassa cytoplasm on the agronomic characters in the PCMS Iines and F1 hybrids, five PCMS Iines and 23 F1 hybrids with the Ae. crassa cytoplasm were compared their agronomic characters to the corresponding lines with the wheat cytoplasm under a short-day condition (natural condition at Kasai, Hyogo, Japan). In the PCMS Iines, the Ae. crassa cytoplasm delayed heading date and reduced spikelet number/ear. Shriveled kernels were found in the PCMS seeds, resulting in decreased volume weight and germination rate. Although the Ae. crassa cytoplasm reduced selfed and open-pollinated seed fertilities in the PCMS Iines, the averaged 69 % open-pollinated seed fertility indicates that the PCMS Iines can be maintained by self-pollination under the natural condition at Kasai. In the F1 hybrids, selfed and open-pollinated seed fertilities were also reduced by the effect of Ae. crassa cytoplasm. The reduced seed fertility resulted in decreased grain numberlear and grain weight/plant, but increased 1, 000-grain weight. No significant effect of the cytoplasm was detected on volume weight of the seeds produced by the F1 hybrids, suggesting no seriously deleterious effects of Ae. crassa cytoplasm on the grain quality. In spite of reduced seed fertility, the F1 hybrids with the Ae. crassa cytoplasm showed on the average 14 % heterosis for grain weight/plant over the mid-parent value of the female and male parents with the wheat cytoplasm. Correlation analysis revealed that grain weight/plant was more strongly related with the ear number/plant and grain numberlear than the 1, 000-grain weight in both F1 hybrids with the Ae. crassa and wheat cytoplasm.

An EMS‐induced wheat mutant restoring fertility against photoperiod‐sensitive cytoplasmic male sterility

Triticum aestivum cv. ‘Norin 26’ with Aegilops crassa cytoplasm shows photoperiod‐sensitive cytoplasmic male sterility (PCMS). This alloplasmic line is almost completely male‐sterile under long‐day conditions (≥ 15h), but highly male‐fertile under short‐day conditions (≤ 14.5h). To obtain male—fertile mutants against PCMS, seeds of the alloplasmic line were treated with ethyl methane sulfonate (EMS). The M3 generation was evaluated for PCMS expression, and one fertility‐restoring (FR‐mutant) line showing high male fertility under the long‐day conditions was selected. Reciprocal F, hybrids between the FR‐mutant and the alloplasmic ‘Norin 26’ showed male sterility under the long‐day conditions, and continuous segregation with respect to the degree of fertility restoration occurred in their F2 generations. These results indicate that multiple recessive mutations with minor effects, induced in the nuclear genome, are involved in the fertility restoration. In fact, no restriction‐fragment‐length polymorphisms of mitochondrial DNA between the FR‐mutant and the alloplasmic ‘Norin 26’ are found.

Photoperiod-sensitive Cytoplasmic Male Sterility Induced in Japanese Wheat Cultivars by Transferring Aegilops crassa Cytoplasm.

Triticum aestivum cv. Norin 26 with Aegilops crassa cytoplasm shows photoperiodsensitive cytoplasmic male sterility (PCMS): The alloplasmic line is almost completely male sterile under longday conditions (≥l5 h light period), but highly male fertile under shortday conditions (≤14.5 h). To examine the effects of the Ae. crassa cytoplasm on other wheat cultivars, this cytoplasm was introduced into 17 Japanese wheat cultivars by repeated backcrosses. Among their alloplasmic lines which reached B5 (11 lines) or B4 (six lines) generation, those of nine cultivars showed PCMS caused by the Ae. crassa cytoplasm, while those of other eight cultivars did not. Thus, the former nine cultivars are converted to the PCMS Iines by transfer of the Ae. crassa cytoplasm, and the latter eight cultivars have demonstrated to carry fertilityrestoring (Rf) gene(s) against PCMS. Pedigrees of the Japanese wheat cultivars suggest monophyletic origin of the Rf gene(s).