Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. I. The origin and distribution of Ogura male-sterile cytoplasm in Japanese wild radishes (Raphanus sativus L.) revealed by PCR-aided assay of their mitochondrial DNAs

Ogura male-sterile cytoplasm was surveyed in common Japanese radish cultivars and in wild radishes growing in various localities in Japan. Mitochondrial (mt) DNA rearrangement involving the atp6 gene was used as a molecular marker. To detect the mtDNA rearrangement, polymerase chain reactions (PCR) were designed to amplify the upstream region of the atp6 gene. The oligonucleotides homologous to the following three regions were synthesized: (1) trnfM, (2) ORF105 and (3) atp6. PCRs were conducted with a pair of the first and the third primers to detect normal mtDNA, and with the second and the third primers for Ogura-type mtDNA. All 15 Japanese cultivars yielded an amplification product which was the same as that of normal mtDNA, whereas some wild radishes gave the product specific to Ogura mtDNA. Twenty-four populations of wild radish were classified into three groups according to the frequency of Ogura-type mtDNA: (1) in ten populations, all four plants analyzed per population had normal type mtDNA, (2) in five populations, only plants with Ogura-type mtDNA were found, and (3) nine populations included both normal and Oguratype mtDNAs. There were no geographical restrictions and no cline in the distribution of the plants with Ogura-type mtDNA. These results suggested that the Ogura-type male-sterile cytoplasm originated in wild radishes.     

Intra- and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cytoplasm from Raphanus sativus and Raphanus raphanistrum

In order to gain a better understanding of the evolution of Ogura male-sterile cytoplasm in radish, a large-scale sequence analysis of mitochondrial orf138 was conducted using 107 Japanese wild radishes, 29 cultivated radishes and seven Raphanus raphanisturum. A single approximately 0.8-kb fragment containing the orf138 locus was amplified from each plant by PCR, and the nucleotide sequence of an entire coding region of orf138 was determined by direct-sequencing procedures. An identical sequence to the published orf138 (Type A) was identified in Japanese wild radish, including a single plant in a population near Kagoshima prefecture where Ogura (1968) first found ’Ogura male-sterile radish’. Thus, it was confirmed that the ’Ogura male-sterile cytoplasm’ was derived from Japanese wild radish, with a Type A orf138 sequence, growing in this area. A total of six nucleotide changes and a single insertion/deletion (indel) were found in orf138 from both wild and cultivated radishes. By a combination of mutations, the orf138 sequences of the 143 radish plants were classified into nine types. Based on the pattern of mutations and the distribution of orf138 variants, it was concluded that the orf138 variants are derived from Type B or C, after Ogura-type cytoplasm was introduced from R. raphanistrum into Japanese wild radish. Received: 19 December 2000 / Accepted. 26 January 2001     

Micropropagation of Raphanus sativus L. var. longipinnatus (Japanese radish) cv. Gungjung

Shoot cultures were established from seedling shoot tips of Raphanus sativus var. longipinnatus Bailey cv. Gungjung, (Japanese radish) cultured on a Murashige-Skoog medium supplemented with ca. 4.5–135 M kinetin or N⁶-benzyladenine. The latter cytokinin supported overall better growth, and 22.2 M was adopted for maintenance of established cultures. The nitrate: ammonium levels in the medium proved optimal for growth and shoot proliferation and both these parameters were significantly increased by addition of adenine sulfate or sodium phosphate. Rooting of excised shoots was achieved on auxin containing medium. Indole-3-butyric acid (ca. 5 or 10 M) also enhanced shoot growth. Plants were easily established in soil, appeared morphologically normal, and flowered.     

The impact of domestication on distribution of allozyme variation within and among cultivars of radish,Raphanus sativus L.

Summary Allozyme surveys of cultivated plant species generally report little within-cultivar variation, but considerable among-cultivar variation. This trend contrasts with natural plant populations in which most allozyme variation resides within, rather than among, populations. The difference may be an artifact of the extreme inbreeding techniques used to develop and propagate these crops, rather than a consequence of domestication per se. To test this hypothesis, we compared the population genetic structure of 24 lines of radish cultivars — a domesticated species developed and maintained as open-pollinated, outcrossed populations — with four wild radish populations in California. Although the wild populations displayed more overall allozyme variation than the cultivars, most of the allozyme variation in the cultivars remains partitioned within, rather than among, lines. Apparently, how a crop is developed and maintained can have a profound influence on the organization of genetic variation of that species.     

Numbers of sporophytic self-incompatibility alleles in populations of wild radish

Summary To estimate the numbers of sporophytic S-alleles in two adjacent populations of wild radish, we performed 701 reciprocal crosses among 50 individuals. Each cross was replicated five times in each direction. Sixteen plants were fully intercompatible, indicating the presence of at least 32 S-alleles in the two populations. A minimum of 22 S-alleles occur in a single population. The frequency of incompatibility was significantly higher for within-population crosses (14.5%) than for between-population crosses (7.8%). This suggests that the two populations differ in the composition and frequency of alleles at the S-locus.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Production and characterization of somatic hybrids between the Japanese radish and cauliflower

Summary Somatic hybrids between the Japanese radish and cauliflower (Brassica oleracea) were produced by protoplast electrofusion in order to introduce clubroot disease resistance in the Japanese radish (Raphanus sativus) into Brassica crops. After electrofusion of iodoacetamide-treated cauliflower protoplasts with untreated radish ones, culture was performed under conditions, that allowed only cauliflower protoplasts to regenerate. Out of 40 regenerated plants, 37 were morphologically of a hybrid type and 3 of a cauliflower type. On the basis of isozyme and RFLP analysis, all of the hybrid-type plants tested proved to be true hybrids. Of the 10 true hybrids tested, 9 were found to contain chloroplasts similar to those found in the Japanese radish, while only 1 contained those of the cauliflower. Using two mitochondrial genes as probes, we were able to show that 3 hybrids contained mitochondria of the Japanese radish, with some modification, while 7 hybrids had either parental or new patterns. All of the hybrid-type plants showed resistance to clubroot disease as high as that found in the Japanese radish. Some hybrids were self-fertile. All of the self-fertile hybrids were found to contain 36 chromosomes, indicating that they were amphidiploids. In addition, a few seeds were obtained from a backcross of the self-fertile hybrids to both parents.     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasaharaet al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+20g) but not under acropetal hypergravity (H-13g), as compared to growth under the control conditions (C+1g and C-1g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Thiocyanate content in relation to the quality features of radish Raphanus sativus L

Relationships between radish thiocyanate content and its dry weight, the content of sugar, protein, fibre, ascorbic acid, some minerals, the incidence of plant shooting, the firmness and pithiness of storage-roots, and the ratio of leaves to storageroot (wt/wt) were investigated. The analysis of linear correlation was based on numerous data from the 4-year field experiment with six radish cultivars and different sowing and harvest dates. The content of thiocyanate in radish roots was found to be positively correlated with their dry weight, and the content of total protein, crude fibre, and soluble sugar. A strong relationship was found between the content of thiocyanate and dry weight of radish leaves. The negative correlation between the thiocyanate content in the leaves and the firmness of storageroots and the positive correlation with their pithiness might indicate the translocation of this compounds into green plant parts during the ageing of root tissue. The root thiocyanate content and the percentage of shooting correlated significantly only in the case of Tokinashi. The closeness of relations between the ratio of leaves/storage-root and thiocyanate content, though in general small, was affected also by a cultivar. A similar effect was observed for the correlations between the thiocyanate contents in leaves and storage-roots.     

Effect of polyamines and guanidines on the growth,nitrogen assimilation and reserve mobilization in germinating radish seeds

Polyamines and guanidines enhanced the growth of radish seedlings grown in dark or light, irrespective of the supply of nitrogen. All the compounds inhibited ntirate reducatase and glutamine synthetase in the cotyledons of light-grown but not in dark-grown seeds. Nitrite reductase and glutamate dehydrogenase were not affected. Protease activity was enhanced by all the compounds in dark-as well as in light-grown seeds. Alanine aminotransferase activity was increased only in the light-grown seeds. The inhibition of nitrate reductase was not due to decreased nitrate uptake but was due to a decreased metabolic pool of nitrate and a decline in enzyme synthesis. The inhibition of glutamine synthetase and activation of alanine aminotransferase by the compounds was found only in the chloroplast fraction. The activation of protease was due to the release or activation of preexisting enzyme while that of alanine aminotransferase was dependent on the de novo protein synthesis which was abolished by cycloheximide.     

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F₁ seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC₃ generations.     

Linkage between an isozyme marker and a restorer gene in radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Summary Co-segregation studies of isozyme markers and male fertility restoration showed that a restorer gene from radish was introduced into rapeseed along with an isozyme marker (Pgi-2). The radish chromosome segment carrying these genes was introgressed into rapeseed through homoeologous recombination, substituting for some of the rapeseed alleles. By crossing heterozygous restored plants to male-sterile lines and to maintainers, tight linkage was found between the restorer gene and the marker. The recombination fraction was estimated at 0.25 ± 0.02%. Although few restored plants lacked the radish isozyme marker, it was still possible to distinguish male-fertile from male-sterile plants by their PGI-2 patterns. Furthermore, homozygous and heterozygous restored plants could be separated by specific PGI-2 phenotypes. Thus, the Pgi-2 marker is now currently used in restorer breeding programs.     

Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus)

Background and Aims

Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development.

Methods

Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages.

Key Results

In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress.

Conclusions

The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be times in development when selection on the maternal, paternal or embryo contributions to development are more and less likely.     

Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm

Summary The structure of anther tissues has been studied during microsporogenesis in male-sterile and -fertile onions. Three types of abnormal tapetal behaviour have been observed within the single line II/3ms containing the cms-S cytoplasm: type 1, the premature breakdown of the tapetum at the tetrad stage, type 2, the hypertrophy of the tapetum after the diad stage followed by its premature autolysis and, type 3, in which the tapetum remains in good condition but for an abnormally long period of time. Tapetal autolysis proceeds in the same manner in both male-steriles and -fertiles with only the stage at which it occurs differing between the types of plants. Mitochondria were prominent in the tapetal tissue of all onion types throughout all stages of microsporogenesis and were still visible during the last stages of tapetal autolysis. In a detailed study of type 2 behaviour, no differences in mitochondrial volumes were found until the tapetum hypertrophied.     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

萝卜TALE转录因子家族的鉴定与分析

TALE (three-amino acid loop extension)转录因子在植物生长发育及细胞分化过程中起重要作用.在多种植物中均已鉴定出TALE转录因子的家族成员,但是萝卜TALE转录因子家族的研究鲜有报道.文中通过生物信息学手段在象牙白萝卜全基因组中鉴定出了分布于9条染色体上的33个TALE家族基因.研究...     

Mitochondrial genome diversity among cultivars of daucus carota (ssp. sativus) and their wild relatives

Summary Restriction fragment patterns of mitochondrial DNAs (mtDNAs) from 13 carrot cultivars (Daucus carota ssp. sativus), wild carrot (ssp. carota), ssp. gummifer, and D. capillifolius were compared with each other using four restriction endonucleases. The mtDNAs of the 13 carrot cultivars could be classified into three distinct types — I, II and III — and were also clearly distinguishable from the mtDNAs of wild carrot (type IV), gummifer (V) and D. capillifolius (VI). The proportions of common restriction fragments (F values) shared by two of the three mtDNA types (I, II and III) of carrot cultivars were approximately 0.5–0.6. The F values were 0.4–0.5 for mitochondrial genomes between wild carrot, ssp. gummifer and D. capillifolius. The mitochondrial genomes between wild carrot and the carrot cultivars showed closer homologies those between wild carrot, ssp. gummifer, and D. capillifolius. The diversity of the mitochondrial genomes among the carrot cultivars is too high to presume that it was generated from the cytoplasm of only one common ancestor during the relatively short history of carrot breeding. These results suggested that the three types of cytoplasms found in the carrot cultivars might have existed in a prototype of D. carota in pre-historical times.