Identification of male sterility maintainer lines for Ogura radish (Raphanus sativus L.)

‘Ogura radish’, a cytoplasmic genetic male sterile line, was crossed with four local and three Japanese cultivars to identify maintainer lines. Out of seven F₁ families, one cross involving a local cultivar, Aushi, produced 100% male sterile (MS) progeny. The crosses involving the other two local cultivars, Tangail Local and Kuni, produced about 90% MS progeny, indicating the presence of maintainer gene(s) for male sterility. The fourth local cultivar, Tasaki, produced 100% male fertile (MF) progeny. All three exotic cultivars appeared to possess the chromosomal gene(s) for controlling the male sterility. In BC₁, BC₂ and BC₃ generations, segregation of MS plants were more frequent when ‘Aushi’ was used as recurrent parent. The expression of male sterility was not affected by seasonal influences. Thus the local cultivar ‘Aushi’ may be used as maintainer line for ‘Ogura radish’. To produce hybrid seed, ‘Tasaki’ can be used as pollinator line as it exhibit high heterosis with ‘Aushi’. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular and biological studies on male sterile cytoplasm in Cruciferae. II. The origin of Ogura male sterile cytoplasm inferred from the segregation pattern of male sterility in the F1 progeny of wild and cultivated radishes (Raphanus sativus L.)

Summary To determine the origin of Ogura male sterile cytoplasm in radish (Raphanus sativus L.), wild and cultivated radishes were crossed. Three types of progeny resulted from the F₁ hybrids between the wild radish from Kushikino with Ogura-type mtDNA and the cultivars (Uchiki-Gensuke or Comet). The segregation patterns of the male sterility were compared with those of Ogura cytoplasm. The male sterility induced in the F₁ hybrid was maintained by crossing with Uchiki-Gensuke, that maintains Ogura male sterility. In the two types of progeny, in which Comet (a restorer of Ogura cytoplasm) was used as one of the parents, both fertile and sterile plants segregated at the predicted ratio on the assumption that a single dominant fertility restoring gene exists in the restorer. From these results, we concluded that the Ogura cytoplasm is identical to that of the wild radish, and the former originated in a population of Japanese wild radish.     

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.     

Identification of a sequence-tagged site (STS) marker linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L.) by non-radioactive AFLP analysis

To identify DNA markers linked to a fertility restorer (Rf) genefor Ogura cytoplasmic male sterility in radish (Raphanus sativus L.),a non-radioactive, amplified fragment length polymorphism (AFLP) analysiswas performed on bulked DNA samples from male-sterile and male-fertileradishes. Ten male-fertile and 10 male-sterile plants selected arbitrarilyfrom an F₂ population made by selfing of F₁ plant from a crossbetween a male-sterile (`MS-Gensuke') plant and a restorer (`Comet') plantwere used as material. Using 32 AFLP primer pairs, one AFLP fragment(AFLP190) which is specific to the bulked DNA samples from male-fertileF₂ plants was identified. AFLP190 was characterized by molecularcloning and nucleotide sequencing, and was converted to a sequence-taggedsite (STS) marker, STS190. A linkage analysis performed in 126individuals of two independent F₂ populations showed tight linkageof STS190 to the Rf gene. The rate of recombination between themarker and Rf was estimated to be less than 1%, making STS1901.2 cM from the gene.     

Identification of self-incompatibility alleles (S) by PCR-RFLP in radish (Raphanus sativus L.)

From 16 inbred lines of cultivated radishes (Raphanus sativus L.), 6 S-alleles tentatively named S²⁰¹ to S²⁰⁶ were identified, and their dominance relationships were examined. Among the S-alleles, S²⁰¹, S²⁰², S²⁰³ and S²⁰⁴ were found to be co-dominant. These 4 S-alleles showed dominance with S²⁰⁵ in pollen and with S²⁰⁶ in both pollen and stigma, while S²⁰⁵ and S²⁰⁶ were co-dominant. Polymerase chain reaction (PCR) was performed using the radish inbred lines randomly selected from the 6 S-allele groups. The primers were based on the highly conserved sequences of the S-locus specific glycoprotein (SLG) genes in Brassica oleracea. As a result of the PCR, a single DNA fragment of about 1.16kb was amplified as expected from the original sequence of B.oleracea. The S-allele specific pattern in the restriction fragments of the PCR products (PCR-RFLP) was confirmed for the first group of S-alleles (S²⁰¹, S²⁰², S²⁰³ and S²⁰⁴). However, for the second group of the S-alleles (S²⁰⁵ and S²⁰⁶), no PCR products were obtained. The usefulness of the PCR-RFLP in a radish breeding program is described. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytoplasmic-genetic male sterility in radish (Raphanus sativus L.). Identification of maintainers,inheritance of male sterility and effect of environmental factors

Summary Research has been carried out on identification of maintainers for cytoplasmic-genetic male sterile lines in Japanese and European radish, on the mode of inheritance of male sterility and on the effect of environmental factors on the expression of this character.In a Japanese radish population and in most early European radish populations maintainers were found in high frequency. Segregations for male sterility in full-sib families, obtained by crossing male sterile and male fertile plants, and in backcross generations, indicated that male sterility is probably determined by one dominant and two recessive independently acting genes, but also minor genes may be involved.The expression of male sterility was not affected by seasonal influences. In some populations a reversible temperature effect was found, most ms plants occurred at 10, 14 and 26°C and most mf plants at 17 and 20°C.     

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.     

Chromosome pairing in haploid plants of radish derived from alien monosomic addition lines

Six haploid plants of radish were obtained via alien monosomic addition lines (2n = 19). One plant was derived from anther culture of an Raphanus sativus‐Brassica oleracea addition line, one plant from the selected smaller seed of an R. sativus‐Sinapis arvensis addition line and four plants from an R. sativus‐B. rapa addition line. During metaphase I of pollen mother cells, two plants exhibited the chromosome pairing of (0‐3)II+ (3‐9)I, three showed (0‐1)III + (0‐3)II + (3‐9)I and the remaining plant (0‐2)II + (5‐9)I. Trivalents seemed to be formed by the pairing between two larger chromosomes and the smaller one with somewhat loose pairing. All haploid plants were inferior to the radish cv. ‘Shogoin’ (2n = 18) with respect to vegetative growth. Their flowers were smaller, with sterile pollen grains, but a few normal flowers with fertile pollen sporadically developed and then produced a few seeds. It is suggested that the radish genome (R, n = 9) might comprise three pairs of homoeologous chromosomes, with the remaining three chromosomes carrying the homologous region(s) that results in a trivalent formation.     

Early-bolting trait and RAPD markers in the specific monosomic addition line of radish carrying the e-chromosome of Brassica oleracea

The specific monosomic addition line of radish, Raphanus sativus, carrying the e chromosome of Brassica oleracea (2n = 19, e‐type MAL) with the genetic background of the late‐bolting cv.‘Tokinashi’ was produced by successive backcrossing of the original e‐type MAL of radish that showed early bolting in the genetic background of the cv. ‘Shogoin’. The early‐bolting trait specific to the e‐type MAL was constantly expressed in the backcrossed progenies (BC₂, BC₃ and BC₄), whereas the reverted radish‐like plants (2n =18) were gradually converted to bolting as late as ‘Tokinashi’. The added e‐chromosome expressed an epistatic effect against the genome of Japanese radish. Its early‐bolting trait was dominant to the late‐bolting trait of ‘Tokinashi’ which may be under the control of a few genes. Moreover, e‐type specific RAPD markers detected in eight primers were invariably transmitted in the backcrossed progenies by ‘Tokinashi’. From the analysis of the characteristics to the e‐type MAL and e‐type specific RAPD markers, it is suggested that the e‐added chromosome of kale (B. oleracea) was transmitted from generation to generation without any recombination with the radish chromosome. The gene(s) for the early‐bolting trait detected in this study may be useful for breeding work in radish, especially in the tropical areas.     

New insights into genotypic thermodependency of cytoplasmic male sterility for hybrid barley breeding

The cytoplasmic male sterility (CMS) system msm1 in barley is known to be thermosensitive, sometimes resulting in spontaneous fertility restoration in the absence of the corresponding restorer gene Rfm1. Here, we investigated genotypic differences concerning temperature sensitivity and the plant developmental stage at which elevated temperature induces spontaneous fertility restoration in three CMS mother lines. While one line stayed completely male sterile, a significantly higher fertility was observed in two lines after treatment from growth stage DC 41 until maturation. Microscopic analysis revealed that sterile anthers contained neither intact pollen, nor remains of aborted pollen grains, whereas pollen was visible in anthers of potentially fertile plants. We conclude that the barley CMS system affects anther and pollen development prior to meiosis. Elevated temperature during heading and flowering can lead to a spontaneous fertility restoration by reactivating pollen growth. Nevertheless, genotypic variation exists enabling the selection for stable CMS mother lines and the development of F₁ hybrids with high hybridity. As spontaneous fertility restoration due to environmental effects is difficult to phenotype, further investigations will focus on the development of molecular markers for marker‐assisted selection.     

Effect of Ogura male-sterile cytoplasm on the performance of cabbage hybrid variety. II. Commercial characteristics

Summary The main constraint of using Ogura male-sterile cytoplasm in Brassica is the induction of leaf yellowing at low temperature and the low seed set. But whether or not the cytoplasm can disturb the general commerical performance of the plants is not well-known. Therefore, this work was carried out with the purpose of evaluating Ogura cytoplasmic male-sterile cabbage hybrids and compare them to genomic similar male-fertile ones and to the most popular cabbage hybrid cultivated in Brazil. Harvest data showed an effect of the cytoplasm on reducing plant and head weight, core length and head length and width, although not altering heading index, shape and core indexes, nor head compactness. On the other hand, it was observed also that a careful selection of the parental lineages can produce male-sterile hybrids as good as their male-fertile analogues. All the evaluated hybrids were similar or better than the check for the characteristics under study.Abbreviations dat - days after transplanting - CNPH - National Center for Vegetable Crops Research     

Characterization of transgenic male sterility in alfalfa

Dependable male sterility would help to make hybrid cultivar development a reality in alfalfa once higher levels of heterosis are attained. Alfalfa plants obtained by genetic transformation with a construct containing the Barnase gene under the control of a tobacco anther tapetum specific promoter were studied. Vacuolization and degeneration of the tapetal cell cytoplasm at a premeiotic stage of development were observed in all five transformed plants (T₀)examined, but the severity of the abnormalities varied greatly among pollen sacs of a genotype. During the meiotic stage, some pollen sacs showed reduction in size, and the tapetum generally appeared thinner when compared to those of the non transgenic plants; tapetal cells showed abnormal vacuolization and signs of cytoplasm degeneration. Despite this, some microspores were formed and some pollen grains were shed in all the T₀ plants, but these were highly variable in size and had very low in vitro germinability. Self-fertility was negligible. The T₀ plants were crossed with one or two unrelated non transgenic male-fertile plants. Mendelian segregation was observed with two exceptions. Instability of the trait in F₁ progenies was noticed, varying for different T₀ parents. F₁ plants exhibiting higher sterility than the primary transformants were observed, indicating that it should be possible to obtain good male sterile plants by backcrossing this trait into different genetic backgrounds. The possible use of this transgenic male sterility in alfalfa breeding is briefly discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic and molecular analysis of the restoration of fertility ( Rf ) genes for Ogura male-sterility from a Japanese wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino)

Ogura male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. In this study, in order to gain better understanding of the variation and evolution of the restoration of the fertility (Rf) gene for Ogura male-sterile cytoplasm, the nucleotide sequence of the orf687 homologue in the Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino) was analyzed using an F₂ population made with a cross between a Japanese wild radish plant containing the Rf gene and ‘Uchiki-Gensuke’ (a maintainer of Ogura-male sterility). Segregation of male-fertile/-sterile plants in the F₂ generation suggested that another unidentified Rf gene unlinked to orf687 exists in the Japanese wild radish. The genotype of orf687 was determined for each F₂ plant by Southern hybridization with an orf687 gene probe, mismatch-specific endonuclease digestion of PCR products, and direct sequencing of a PCR product. Genotyping revealed that some fertility-restored plants are homozygotic for the ‘Uchiki-Gensuke’ type orf687 allele, supporting the idea that another gene different from orf687 also functions as an Rf gene for Ogura male-sterility. Protein analysis using an antibody raised against the Ogura-specific ORF138 protein suggests a mechanism of fertility restoration by the unidentified Rf similar to that by orf687. Sequence analysis of orf687 from a Japanese wild radish plant and ‘Uchiki-Gensuke’ revealed that both orf687 regions encode a mitochondrially-targeted protein consisting of 687 amino acids with 16 PPR motifs. Comparison of the deduced amino acid sequences with those of the known orf687 sequences from ‘Yuan hong’ and ‘Kosena’ containing Rf and recessive one (rf), respectively, showed that three unique amino acid replacements are present in ORF687 of the Japanese wild radish. Two of the three replacements, that from lysine to isoleucine at position 232 and from asparagine to asparate at position 240, confer negative charges to the protein. Since the Rf of ‘Yuan hong’ was reported to have a unique replacement that confers a negative charge to ORF687 (from asparagine to aspartate at position 170), it is proposed that the amino acid replacements conferring a negative charge to ORF687 are important for determining the status of the Rf/rf gene.     

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

A ‘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’.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

Development of the components of a cytoplasmic male sterility hybrid system in rye through anther culture

Anther culture was applied as a method to develop the essential components of a cytoplasmic male sterility hybrid system in rye (Secale cereale L.). These components are the male sterile seed parent (A line), its isogenic maintainer counterpart (B line) and the restorer pollen parent (R line). Australian rye cultivars were crossed reciprocally to the cultivar ‘Luchs’ which carries the Pampa male sterile cytoplasm (cms-P). Anthers of the F1s in the cms-P cytoplasm (primary cross) and their reciprocals in the normal cytoplasm (reciprocal cross) were cultured in a modified C17 medium. Male sterile and male fertile doubled haploids were obtained from the anther culture of the F1s in the cms-P cytoplasm. Testcrosses indicated that the male sterile doubled haploids were A lines and the male fertile doubled haploids were R lines (restorers). The anther culture of genotypes in the normal cytoplasm (reciprocal cross) gave all male fertile doubled haploids. Testcrosses indicated that the male fertile doubled haploids were R lines (restorers) in the normal cytoplasm. The expected maintainer B lines were not identified because of the limited number of doubled haploids obtained from the anther culture of reciprocal crosses. Experimental single cross hybrids between male sterile and restorer male fertile doubled haploids showed high levels of heterosis. The results of this study have significant economic implications especially in the production of hybrids in several species. This revised version was published online in July 2006 with corrections to the Cover Date.     

萝卜D染色体在7号连锁群的定位研究

（1北京蔬菜研究中心,北京 100089;2德国栽培作物育种研究中心,Quedlinburg,D-06484）     

Molecular markers for cytoplasm in potato: male sterility and contribution of different plastid-mitochondrial configurations to starch production

Distinct parental cytoplasms were combined in symmetric tetraploid hybrids of potato by somatic cell fusion. This allowed, in the presence of nearly isogenic nuclear genomes, to estimate the contribution of mitochondrial (mt) and chloroplast (cp) genomes to starch content. Analysis of mt-cp configurations in the complete gene pool of german potato cultivars [2n=4x], in a reciprocal dihaploid population [2n=2x],in di-haploid fusion parents [2n=2x] and in their respective hybrids [2n=4x] made visible the effects of different cytoplasmic backgrounds and mitochondrial subgenomic rearrangements. Genotypes identified by markers as cytoplasm Wγ were associated with cytoplasmic male sterility. Evaluation of cytoplasmic types leads to the conclusion, that in starch content the ‘wild type’ cytoplasms Wα and Wγ have a significant advantage to other cytoplasmic types(Tβ, Wδ, Sε).This results from the experiments with a reciprocal population, 180 di-haploids, and from cultivar comparisons. In hybrids an interaction between starch content and different mt-cp combinations could be found. In general the highest field performance, measured in starch content and yield was associated with such cytoplasmic configurations which appeared to a high frequency within a population, when the segregation process was completed. This fact is explained by a selection advantage of clones with optimized organellar segregation already during in vitro phase. PCR markers for cytoplasm differentiation are actualized on a website, http://www.flg.tum.de/pbpz/mm/mt/hybrid.html This revised version was published online in July 2006 with corrections to the Cover Date.     

Conversion of the RAPD OPC021150 marker of the Rfo restorer gene into a SCAR marker for rapid selection of oilseed rape

The Rfo fertility restorer gene for the Ogura cytoplasmic male sterility (CMS) applied for oilseed rape hybrid seed production can be monitored with the use of the RAPD OPC02₁₁₅₀ marker while molecular breeding. The aim of this work was to convert the RAPD marker into a more suitable SCAR marker. Total DNA was isolated from a doubled haploid line derived from the line BO20 (INRA, France). A fragment of 1150‐bp linked to the Rfo gene was PCR amplified with the use of the RAPD OPC02 primer, cloned and sequenced. A pair of primers was designed and PCR amplification was performed to develop a SCAR marker for the Rfo gene. The new marker was applied for analysis of 220 oilseed rape lines comprising doubled haploid and inbred restorer lines, restored hybrids as well as F₁ and F₂ recombinant generations involving restorer lines. Simultaneously, the RAPD OPC02 marker was used and it revealed that the markers are equivalent to each other. However, the developed new SCAR marker has made the analysis more practical, rapid and efficient.     

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F₂ plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.