Interspecific amphiploid‐derived alloplasmic male sterility with defective anthers,narrow disc florets and small ray flowers in sunflower

The cytoplasmic male‐sterility (CMS)/fertility‐restoration system is important for hybrid sunflower (Helianthus annuus L.) seed production. The objective of this study was to characterize two novel alloplasmic CMSs, designated CMS GRO1 and CMS MAX3, with defective anthers, narrow disc florets with no swollen corolla, and short, narrow ray flowers derived from two tetraploid amphiploids (AMPs). Among 26 tested lines, only AMP Helianthus cusickii/P 21 and HA 410 failed to restore male‐fertility. Segregation of CMS, male‐fertile plants and plants with reduced male‐fertility was observed both in the testcross progeny of a six line half‐diallel cross of F₁s with CMS MAX3 and in an F₂ population of CMS GRO1 × RHA 274. Male‐fertility restoration was controlled by at least two dominant genes. Detailed analysis of the mitochondrial genes may provide insight into the differences between these CMSs and other CMS lines. The new CMSs will facilitate the studies of the incompatibility between cytoplasmic and nuclear genes, especially for the alloplasmic CMS involving perennial species, and also provide unique ornamental flower types and CMS sources for hybrid sunflower breeding.     

Molecular characterization of fertile and sterile cytoplasms in Beta spp.

Mitochondrial DNA (mtDNA) from sugar beet carrying fertile (F) and male sterile (CMS) cytoplasms, and from male sterile accession of Beta maritima collected in Brittany (France) were characterized and compared by restriction fragment length polymorphism (RFLP) and Southern hybridization with coxII. The F and CMS cytoplasms could be clearly distinguished from each other by RFLP when XhoI, EcoRI and BamHI endonucleases were used. Southern hybridization with the coxII gene provided further evidence that mitochondrial genome organization differs between fertile and sterile plants. All cytoplasmic male sterile lines from different breeding stations showed the same restriction and hybridization patterns, which confirms the uniformity of mitochondrial genomes within the materials used for hybrid seed production in several European countries. No visible differences were found between the maintainer lines studied. However, comparisons of XhoI restriction profiles of mtDNA from maintainer lines and from fertile monogerm populations revealed slight differences, which were reflected by the appearance of a unique 0.9 kb fragment in the latter. Analysis of mtDNA from male sterile plants of the wild beet B. maritima showed different restriction and hybridization patterns in comparison with normal and sterile sugar beet cytoplasms. This shows the unique nature of cytoplasmic male sterility in this species.     

Cytoplasmic male sterility in Brassicaceae crops

Brassicaceae crops display strong hybrid vigor, and have long been subject to F₁ hybrid breeding. Because the most reliable system of F₁ seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.     

Isolation and Characterization of the Cytoplasmic Male Sterility Associated Gene of Cotton (Gossypium harknessii)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the failure to produce functional pollen.It was identified in many plants,and it is widely used to exploit heterosis.Now,there are some genes associated with the CMS phenomena that have been identified in the mitochondrial genome.For example,specific genes implicated in CMS have been reported in maize,petunia,bean,Brassica,radish,sunflower,rice,carrot,sorghum,pepper,and many others.     

RFLP analysis of cytoplasmic male-sterile lines of pigeonpea [Cajanus cajan (L.) Millsp.] developed by interspecific crosses

Total DNA from three putative cytoplasmic male sterile (CMS) progenies derived from crosses between the wild species Cajanus sericeus and the cultivated species Cajanus cajan, five C. cajan, one accession of C. sericeus and two genetic male sterile lines of pigeonpea were compared for their RFLP patterns using maize mitochondrial DNA (mtDNA) specific probes. Three putative cytoplasmic male sterile (CMS) progenies from the multiple cross genome transfer of pigeonpea lines (CMS 7–1, CMS 12–3, and CMS 33–1) showed hybridization patterns identical to that of C. sericeus when DNA was digested with EcoRI and HindIII and probed with maize mtDNA clones. The results suggested that these putative CMS progenies have the mitochondria of the female wild species parent. The hybridization patterns of the three male parental lines used in the development of the CMS progenies were similar in all the restriction enzyme-probe combinations except HindIII-atp6. The genetic male sterile lines, MS Prabhat and QMS 1 differed from each other in their hybridization pattern. The genomic DNA hybridization pattern of HindIII digested DNA from ICPL 87 differed from the other pigeonpea lines when probed with the maize mtDNA clones. The cluster analysis of the hybridization data suggested the occurrence of variation in the mitochondrial genome even among the cultivated species. This revised version was published online in July 2006 with corrections to the Cover Date.     

向日葵细胞质雄性不育性的机制与利用

向日葵杂种生产采用三系配套系统完成。因此,向日葵细胞质雄性不育性的研究备受关注。其中研究较为清楚的是PET1细胞质,其不育机理是线粒体DNA重组形成嵌合基因,编码新的多肽,导致小孢子的败育。然而,这些基因如何影响小孢子发育的生理机制仍不清楚,有待进一步深入研究。丰富多样的向日葵CMS会大大降低因遗传单一性给大规模生产带来的风险;未发现恢复系的CMS可以用于无花粉观赏向日葵杂交种的生产。     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

A stable cytoplasmic male-sterile line of Brassica juncea carrying restructured organelle genomes from the somatic hybrid Trachystoma ballii+B. juncea

A cytoplasmic male-sterile (CMS) line of Brassica juncea has been developed by combining the cytoplasm originating from the somatic hybrid Trachystoma ballii+B. juncea, and the nucleus of B. juncea cv. Pusa Bold by repeated backcrossing. Male-sterile plants closely resembled the normal fertile B. juncea in general morphology, but had delayed flowering (5–7 days) when compared with fertile ‘Pusa Bold’ which flowered in 45 days. Stamens of the male-sterile line were transformed into petaloid structures. Pollen abortion occurred after tetrad formation. Female fertility of the male-sterile line was normal. Molecular analysis of organelle genomes indicated extensive mitochondrial DNA recombinations in the CMS line. Preliminary analysis of the chloroplast genome of the CMS line also indicated chloroplast DNA recombination.     

Introgression of a gene restoring fertility to CMS (Trachystoma) Brassica juncea and the genetics of restoration

A dominant gene restoring fertility to a cytoplasmic male sterile (CMS) line of Brassica juncea was derived from the somatic hybrid Trachystoma ballii+B. juncea. Its introgression resulted from forced pairing between chromosomes of the cultivar ‘Pusa Bold’ and chromosomes of the fusion hybrid. Segregation ratios of this fertility restorer gene followed a monogenic pattern. The introgression of the fertility restorer gene did not cause any abnormalities, such as reduced fertility; pollen and seed fertilities of the restored plants were over 90%. Restored fertile and CMS plants exhibited similar Southern hybridization patterns when probed with the mitochondrial probe atp6.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum

The orf138 gene, which is specific to Ogura male-sterile cytoplasm, was analysed in mitochondrial DNA (mtDNA) of the wild radish, Raphanus raphanistrum, by polymerase chain reaction (PCR), Southern hybridization and sequencing. The effect of R. raphanistrum cytoplasm on the expression of male sterility was also examined in progeny with R. sativus. A PCR-aided assay and Southern hybridization revealed that three out of six strains analysed included plants with orf138. The sequence of wild type orf138 was same as that of Ogura, except for one or two nucleotide substitutions. Southern hybridization showed a novel mtDNA configuration in R. raphanistrum, in addition to the normal and Ogura types identical to those in R. sativus. Among interspecific hybrids, all the F₁ had normal pollen fertility. In the F₂ progeny between female wild plants having orf138 and the maintainer of Ogura male sterility, male-sterile plants were segregated, fitting the ratio of 3 fertile: 1 sterile plant. R. raphanistrum has cytoplasm that induces male sterility in radishes, and contains a dominant fertility restorer gene.     

Induction of cytoplasmic male sterility in the seed progeny derived from artificially-synthesized interspecific chimera in Brassica

We produced artificial interspecific chimeras by in vitro grafting, and obtained cytoplasmic male sterile (CMS) variants in the seed progenies derived from backcrossing the chimera with one of the mother plants, B. campestris cv. Komatsuna. The induced CMS has been stably inherited by crossing it with `Komatsuna', not with `Ruby Ball' cabbage. The nuclear component of CMS is complete `Komatsuna' type in morphology, chromosome number (2n = 20) and Southern blot using ribosomal 17S RNA gene as a probe. PCR analysis by using mitochondrial atpA primer showed the complete `Ruby Ball' type, suggesting nuclear-cytoplasmic exchange. However, Southern blot patterns were different among those of the CMS and both parents by using atpA. Recombination or some unknown change is supposed in the mitochondrial genome via the processes of synthesis and propagation of the chimeras.     

Moricandia arvensis cytoplasm based system of cytoplasmic male sterility in Brassica juncea: Reappraisal of fertility restoration and agronomic potential

Cytoplasmic male sterility (CMS) system based on the cytoplasm from Moricandia arvensis (mori) was investigated for fertility restoration and agronomic potential. Fertility restorer gene for mori CMS was introgressed from cytoplasm donor species as all the evaluated Brassica juncea genotypes (155) acted as sterility maintainers. The allosyndetic pairing between M^a and the A/B genome chromosomes in the monosomic addition plants (2n= 18II + 1M^a) facilitated the gene introgression. Partial fertility restoration (43–52% pollen grain stainability) in F₁ hybrids and absence of segregation for male sterility in F₂ progenies suggested gametophytic control of fertility restoration. The pollen fertility in the F₁ hybrids was, however, sufficient to ensure complete seed set upon bag selfing. Introgression from M. arvensis also helped in correction of chlorosis associated with mori cytoplasm in CMS and fertile alloplasmic B. juncea plants. Yield evaluation of thirty F₁ hybrids having the same nuclear genotype but varied male sterilizing cytoplasms (mori, oxy, lyr, refined ogu), in comparison to respective euplasmic hand bred control hybrids, allowed an estimate of yield penalty associated with different CMS systems. It ranged from 1.8% to 61.6%. Hybrids based on cytoplasmically refined ogu were most productive followed by those based on cytoplasmically refined mori CMS. The male sterility systems emanating from somatic hybridization were found superior than those developed from sexual hybridization.     

Temperature related instability of a naturally induced cytoplasmic male sterility in industrial chicory (Cichorium intybus L.)

Natural alloplasmic cytoplasmic male sterile (CMS) clones of industrial chicory were obtained after crossing wild chicory with selected breeding lines. We investigated the CMS stability of 10 clones in various environmental growing conditions. CMS was stable under cool growing conditions in most of them. Fertility restoration, based on pollen production scores, was observed in all clones after a period of hot temperatures. The early flower bud stage was sensitive, resulting in fertile flowers 12–17 days after exposure to high temperatures. Experiments under controlled growing conditions at 15°C demonstrated that a heat shock of 2 days at 25 or 30°C was sufficient to restore fertility. Sterile flowers were formed when plants were again grown at lower temperatures. Significant differences between individual clones were observed, indicating the potential of genetic selection to obtain stable CMS parent lines.     

Genetic Control of Tetracycline Sensitivity of Cytoplasmic Male Sterility (cms) in Chives (Allium schoenoprasum L.)

The expression of cms in chives is controlled by a cytoplasmic factor (5), a restorer gene X/x (Tatlioglu 1982) and a temperature gene T/t (Tatlioglu 1987). In recent investigations (Tatlioglu 1986) it could be shown that tetracycline also influences the expression of 0731. To obtain information about the genetic control of tetracycline sensitivity of cms, tetracycline-sensitive and tetracycline-insensitive cms plants were crossed with each other as well as with maintainers and the reaction of their progeny to tetracycline base was investigated. The results indicated that the promotion of fertility due to high-temperature conditions (24°C/24 °C, day/night) and tetracycline · base are independent of each other. The results of crosses can be explained by a nuclear gene, A/a, causing the tetracycline sensitivity when recessive. By using different genotypes of the three loci for restoration, temperature and tetracycline sensitivity highly advantageous material can be constructed for the investigation of the interaction between the nuclear and the mitochondrial genome in higher plants, for the molecular and functional basis of cms and for its use in plant breeding.     

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

Cytoplasmic male sterility (CMS), a maternally transmitted failure in pollen formation, is an effective pollination control system in hybrid rapeseed (Brassica napus) breeding. However, CMS is not widely used in the related oilseed species Brassica rapa. In the past years, several male sterile plants have been isolated from the B. rapa landrace ‘0A193’, collected in Shaanxi, China, in 2011. It is noteworthy that the fertility expression of 0A193‐CMS was affected by temperature. In contrast to pol CMS, fertility tests with 18 B. rapa and 9 B. napus accessions suggest that a different system of maintaining and restoring is responsible for the observed phenotype. Further on, genetic investigation evidenced that fertility of 0A193‐CMS is controlled by both cytoplasmic and one pair of nuclear recessive genes. Interestingly, plants of the 0A193‐CMS type possess a highly specific fragment of the mitochondrial gene orf222, a crucial regulator of male sterility in nap CMS. Our study broadens the CMS resources in B. rapa and provides a highly applicable alternative to pol CMS and ogu CMS for hybrid breeding production.     

Expression of male sterility in alloplasmic Brassica juncea with Erucastrum canariense cytoplasm and the development of a fertility restoration system

An alloplasmic mustard, Brassica juncea, has been synthesized by placing its nucleus into the cytoplasm of the related wild species Erucastrum canariense to express cytoplasmic male sterility. To achieve this, the sexual hybrid E. canariense (2n=18, E^cE^c) ×Brassica campestris (2n= 20, AA) was repeatedly backcrossed to B. juncea (2n= 36, AABB). Cytoplasmic male‐sterile (CMS) plants were recovered in the BC₄ generation. These plants are a normal green and the flowers have slender, non‐dehiscing anthers that contain sterile pollen. Nectaries are well developed and female fertility is > 90%. The fertility restoration gene was introgressed to CMS B. juncea from the cytoplasmic donor E. canariense through pairing between chromosomes belonging to B. juncea with those of the E. canariense genome. The restorer plants have normal flowers, with well‐developed anthers containing fertile pollen. Meiosis proceeds normally. Pollen and seed fertility averaged 90% and 82%, respectively. F₁ hybrids between CMS and the restorer are fully pollen fertile and show normal seed set. Preliminary results indicate that restoration is achieved by a single dominant gene. The constitution of the organelle genomes of the CMS, restorer and fertility restored plants is identical, as revealed by Southern analysis using mitochondrial and chloroplast probes atp A and psb D, respectively.     

Molecular characterization of cytoplasmic male sterility conditioned by <Emphasis Type="Italic">Gossypium harknessii</Emphasis> cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F₁ seed lots.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enarthrocarpus lyratus-based cytoplasmic male sterility and fertility restorer system in Brassica rapa

Substituting the nuclear genome of Brassica rapa into the cytoplasmic background of Enarthrocarpus lyratus through backcross substitution helped in developing cytoplasmic male sterility (CMS). Alloplasmic male sterile plants had pale green leaves, small flowers with narrow petals and rudimentary anthers. Female fertility, low initially, improved considerably with advanced backcross generations. Male sterility expression was stable throughout the growing season. Except for EC 339014, all B. rapa accessions (38) evaluated were partial maintainers of the male sterility. Introgression of gene(s) for fertility restoration from the cytoplasm donor species was facilitated by homoeologous pairing between B. rapa and E. lyratus genomes, as was apparent from the very frequent occurrence of a trivalent in the monosomic addition plants (2n = 10 II + 1 I). Backcrossing of fertile monosomic addition plants with B. rapa led to the recovery of male fertile plants possessing the stable euploid chromosome number (2n = 20). These plants restored male fertility in crosses with different (lyr) CMS B. rapa genotypes, confirming the introgression of fertility restorer gene(s) from E. lyratus, the cytoplasm donor species.