Identification of Cytoplasmic Male Sterility in Chinese Radish Following PCR Analysis of Mitochondrial DNA

In order to understand the molecular characteristics of the Chinese radish, the mitochondrial DNA structure and sequence were analyzed in Chinese wild radish and cultivated varieties. A total of four male-sterile lines, four maintainer lines, 17 cultivars, and a single Chinese wild radish were used, along with 25 male-sterile individuals and 159 fertile plants. We found that the cytoplasm of Chinese radishes could be classified into two types: the normal type and the Ogura type. The Ogura-type cytoplasm was detected in 25 male-sterile plants. The 159 fertile plants had normal cytoplasm. Both the Ogura cytoplasm and the normal cytoplasm were detected in the male-sterile ??RA??. The orf138 gene in mitochondrial DNA was detected in cultivated Chinese radish cultivars but not in the wild radish. The Chinese radish orf138 nucleotide sequence was determined in four male-sterile lines and displayed complete homology to the known orf138 type A nucleotide sequence. Three types of mitochondrial orfB (type 1, type 2 and type 3) were found in Chinese radishes. Type 1 was only present in the male-sterile lines. Chinese cultivated radishes were divided into type 2 and type 3, while the Chinese wild radish only had type 3 cytoplasm.     

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     

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.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. III. Distribution of Ogura-type cytoplasm among Japanese wild radishes and Asian radish cultivars

The distribution of Ogura male-sterile cytoplasm among Japanese wild radish populations and Asian cultivated radishes was studied by means of polymerase chain reaction (PCR)-aided assays using mitochondrial atp6 and orf138 loci as molecular markers. Three separate PCR experiments were performed to amplify the target sequences in normal-type atp6, Ogura-type atp6, and Ogura-specific orf138, and the cytoplasm of each plant was classified as either normal or Ogura. Among 217 wild radish plants, 93 had both Ogura-type atp6 and orf138 (or its modified form), whereas 124 had normal-type atp6. Of the 93 plants with Ogura-type cytoplasm, only a single plant showed male sterility. A complete linkage between Ogura-type atp6 and orf138 loci was found in Japanese wild radishes, confirming our findings that Ogura-type cytoplasm is distributed widely among Japanese wild radish populations. A modified form of orf138 (orf138-S) was identified in a few wild radish populations in a limited area of Japan, and the nucleotide sequence of the orf138-S revealed a 39-bp deletion shared in common with Kosena male-sterile cytoplasm. Among the 44 Asian cultivars analyzed, 40 were determined to have normal cytoplasm since all 4 plants tested in each cultivar showed the same PCR amplification profiles as that of Uchiki-Gensuke, a reference cultivar with normal cytoplasm. The plants with Ogura-type cytoplasm (or its modified form) were found in 1, 1, and 2 cultivars from Tibet, Japan, and Taiwan, respectively. Except for 1 cultivar from Taiwan, those with Ogura-type cytoplasm included a few plants having male sterility. The multiple and independent introduction of Ogura-type cytoplasm from the wild radish in Asia into these cultivars is suggested.     

Can feral weeds evolve from cultivated radish (Raphanus sativus, Brassicaceae)?

Cultivated plants that cannot survive on their own often have maladaptive domestication traits. Unharvested crop seeds may generate feral populations, at times causing serious weed problems, but little is known about the evolution of ferality. We explored the potential for cultivated radish, Raphanus sativus, to become feral, given that closely related taxa (e.g., R. raphanistrum and crop-wild hybrids) are well-documented weeds. First, we measured the population growth of five experimental, cultivated, self-seeding radish populations in Michigan, USA, for three generations. Three late-flowering populations went extinct, and two others apparently hybridized with local R. raphanistrum. A common garden experiment showed that the two surviving populations had earlier flowering, smaller root diameters, and greater individual fecundity than did nonhybridized populations. We also used artificial selection to measure the evolutionary potential for earlier flowering. After two generations of strong selection, two of three lineages flowered earlier and produced more seeds than control lineages, but insufficient genetic variation prevented dramatic evolution of crop phenotypes. In summary, it seems unlikely that radishes could spontaneously become feral in our study area without gene flow from R. raphanistrum. Applying these approaches to other cultivated species may provide a better understanding of mechanisms promoting the evolution of feral weeds.     

Genetic differentiation for nuclear,mitochondrial and chloroplast genomes in common wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.) and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The genetic differentiation of nuclear, mitochondrial (mt) and chloroplast (cp) genomes was investigated by Southern and PCR analysis using 75 varieties of cultivated rice ( Oryza sativa L.) and 118 strains of common wild rice (CWR, Oryza rufipogon Griff.) from ten countries of Asia. The distinguishing differences between the Indica and Japonica cultivars were detected both in the nuclear genome and the cytoplasmic genome, confirming that the Indica-Japonica differentiation is of major importance for the three different classes of genome in cultivated rice. This differentiation was also detected in common wild rice with some differences among the genome compartments and the various regions. For nuclear DNA variation, both Indica-like and Japonica-like types were observed in the Chinese CWR, with the latter more-frequent than the former. No Japonica-like type was found in South Asia, and only two strains of the Japonica-like type were detected in Southeast Asia, thus the Indica-like type is the major type among South and Southeast Asian CWR. For mtDNA, only a few strains of the Japonica-like type were detected in CWR. For cpDNA, the Japonica type was predominant among the CWR strains from China, Bangladesh and Burma, while the Indica type was predominant among the CWR strains from Thailand, Malaysia, Cambodia and Sri Lanka, and both types were found in similar frequencies among the Indian CWR. Altogether, however, the degree of Indica-Japonica differentiation in common wild rice was much-less important than that in cultivated rice. Cluster analyses for nuclear and mitochondrial DNA variation revealed that some CWR strains showed large genetic distances from cultivated rice and formed clusters distinct from cultivated rice. Coincidence in the genetic differentiation between the three different classes of genome was much higher in cultivated rice than in CWR. Among the 75 cultivars, about 3/4 entries were "homoeotype" showing congruent results for nuclear, mt and cpDNA regarding the Indica-Japonica differentiation. In CWR, the proportions of homoeotypes were 5.7%, 15% and 48.8% in China, South Asia and Southeast Asia, respectively. Based on the average genetic distance among all the strains of CWR and cultivated rice for nuclear and mitochondrial genomes, the variability of the nuclear genome was found to be higher than that of the mitochondrial genome. The global pattern based on all genomes shows much-more diversification in CWR than that in cultivated rice.     

ITS1 Sequences of Nuclear Ribosomal DNA in Wild Rices and Cultivated Rices of China and Their Phylogenetic Implications

he first internal transcribed spacer (ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3%to 72.7%. In pairwise comparison among the five taxa, sequence site divergence ranged from 1.5 % to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well-resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) of the genus Oryza.     

Mitochondrial restriction fragment length polymorphisms in wild Phaseolus vulgaris L.: insights on the domestication of the common bean

Summary Previous examination of intraspecific mitochondrial DNA (mtDNA) diversity in common bean, Phaseolus vulgaris, showed that five restriction fragment length polymorphisms (RFLPs) distinguish the mitochondrial genomes of the two major gene pools of cultivated beans, the Mesoamerican and the Andean. In the study presented here, mtDNA was used to compare the amount of diversity in cultivated beans to that in collections of wild beans to gain an understanding of how and when the mitochondrial genomes of the gene pools became distinct. The mtDNA of six wild bean accessions from Central and South America were digested with nine restriction endonucleases and analyzed by Southern hybridization. A total of twenty RFLPs were detected demonstrating a significantly higher amount of mtDNA variability in wild beans than in cultivated ones. All of the wild beans had the same mtDNA pattern for four out of the five inter-gene pool RFLPs, indicating that the polymorphism arose soon after domestication: two in the gene pool of the cultivated Mesoamerican beans and two in the gene pool of the cultivated Andean beans. The fifth RFLP must have occurred before domestication since the locus was also polymorphic in the wild beans. Wild beans from the south Andes were distinct and less variable than wild accessions of the north Andes and Mesoamerica. The distribution of mtDNA RFLPs among the wild beans supports the concept of two distinct domestication events for P. vulgaris.     

Patterns of weed invasion: evidence from the spatial genetic structure of Raphanus raphanistrum

Knowledge of the pathways of colonization is critical for risk assessment and management of weeds. In this study we adopted a landscape genetics approach to assess the impact of human disturbances and large-scale environmental features on the colonization of a global agricultural weed, Raphanus raphanistrum. We used nuclear microsatellite and chloroplast DNA sequence data to quantify the pattern of genetic diversity in 336 plants collected from 13 sites throughout the Cape Floristic Region, South Africa, one of the world’s recognized global biodiversity hotspots. The lack of strong spatial genetic structure suggests that R. raphanistrum colonized throughout the Cape Floristic Region via both local diffusive spread and long-distance jump dispersal. Furthermore, 47 % of analyzed plants contained Raphanus sativus (cultivated radish) chloroplast genomes, indicating historical and/or contemporary gene flow between wild and cultivated radish populations. The prevalence of high genetic diversity and long-distance gene flow are discussed in the context of ecological risk assessment.     

中国野生稻及栽培稻核糖体DNA第一转录间隔区序列分析及其系统学意义

用 PCR技术从产于我国的 3种野生稻和亚洲栽培稻的 2个亚种中特异地扩增和测序了 r DNA的第一转录间隔区。普通野生稻 (Oryza rufipogon)、药用野生稻 (O.officinalis)、疣粒野生稻 (O.granu-lata)和栽培稻的两个亚种 (O.sativa ssp.indica,O.sativa ssp.japonica)的 ITS1序列为 1 93bp、1 94bp、2 1 8bp、1 94bp和 1 94bp,它们的 G/ C含量为 69.3%～ 72 .7% ,序列中位点趋异率为 1 .5%～ 1 0 .6%。序列的相似性比较和简约性分支分析的结果表明 ,普通野生稻与栽培稻的两个亚种之间的亲缘关系最为密切 ;药用野生稻与普通野生稻和与栽培稻的两个亚种的相似性都为 82 % ,说明它与 AA基因组有一定的亲缘关系 ;疣粒野生稻与普通野生稻、药用野生稻和栽培稻两个亚种的亲缘关系相对较远 ,它在稻属中可能是一个系统地位较独特的类群。以 ITS1序列构建的 3种野生稻和 2个栽培稻亚种的系统发育关系与前人用同工酶、叶绿体 DNA、线粒体 DNA和核 DNA资料重建的稻属的系统发育关系基本一致     

香菇野生菌株线粒体DNA多态性

利用PCR技术扩增了我国 7个省的 2 0个野生香菇菌株的线粒体DNA的 2个小区段 ,利用限制性酶切技术研究这 2个片段长度多型性 (RFLPs) ,结果显示 ,2 0个野生香菇菌株间的线粒体DNA在研究的区段上分为 2大类型 ,相似性为 70 % ,与栽培种相比存在较大的遗传差异。     

The Influence of Foliage Habit on Competition Between Carrot Plants

Carrot plants of a cultivated variety (cv. Chantenay Red Cored)and those of a wild type were grown in both pure and mixed populationsin which root competition was prevented. During early growth,cultivated plants competed effectively against the wild plants.Fifty days after sowing chitted seed, the mean weight of thestorage organs of the cultivated variety grown in mixed populationswas 56 per cent greater than the mean weight of cultivated storageorgans in a pure population. Subsequently, however, this competitiveadvantage diminished, this being associated with the developmentof tall flowering stems by the wild plants. The foliage habitof the vegetative cultivated plants was more erect than thatof the vegetative wild plants. This difference in foliage habitmight explain why the cultivated plants out-compete the wildplants. Daucus carota L., carrot, foliage habit, competition     

Identification of a novel mitochondrial genome type and development of molecular markers for cytoplasm classification in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

Plant mitochondrial genomes have complex configurations resulting from the multipartite structures and highly rearranged substoichiometric molecules created by repetitive sequences. To expedite the reliable classification of the diverse radish (Raphanus sativus L.) cytoplasmic types, we have developed consistent molecular markers within their complex mitochondrial genomes. orf138, a gene responsible for Ogura male-sterility, was detected in normal cultivars in the form of low-copy-number substoichiometric molecules. In addition to the dominant orf138-atp8 Ogura mitochondrial DNA (mtDNA) organization, three novel substoichiometric organizations linked to the atp8 gene were identified in this study. PCR amplification profiles of seven atp8- and atp6-linked sequences were divided into three groups. Interestingly, the normal cytoplasm type, which had previously been considered a single group, showed two patterns by PCR amplification. The most prominent difference between the two normal mtDNAs was size variation within four short-repeat sequences linked to the atp6 gene. This variation appeared to be the result of a double crossover, mediated by these homologous, short-repeat sequences. Specific PCR amplification profiles reflecting the stoichiometry of different mtDNA fragments were conserved within cultivars and across generations. Therefore, the specific sequences detected in these profiles were used as molecular markers for the classification of diverse radish germplasm. Using this classification system, a total of 90 radish cultivars, or accessions, were successfully assigned to three different mitotypes.     

普通野生稻和亚洲栽培稻线粒体DNA的RFLP分析

通过７个探针、１７种内切酶探针组合对１１８份普通野生稻和７６份亚洲栽培稻的线粒体ＤＮＡ（ｍｔＤＮＡ）ＲＦＬＰ分析表明,籼粳分化是亚洲栽培稻线粒体基因组分化的主流,７６个栽培稻中,３６个品种ｍｔＤＮＡ为籼型,４０个品种ｍｔＤＮＡ为粳型。普通野生稻ｍｔＤＮＡ以籼型为主（８６份）,粳型较少（７份）,１份类型难以确定,还有２４份没有籼粳分化。     

肉质色不同萝卜遗传多样性的SSR分子标记分析

利用微卫星(SSR)标记技术,从600对SSR引物中筛选86对扩增条带清晰的引物,检测了来自我国不同地区37个肉质不同颜色萝卜品种的遗传多样性。86对引物共扩增到976个条带,每对引物扩增出2～17个条带,平均为10.7个,其中多态性条带892个,多态性条带比例为91.39%;共检测出753个基因型,每对引物检测2～20个基因型,平均8.7个,其中有效基因型443.99,有效基因型比例为58.96%;Shannon多态性指数变幅为0.44～2.77,平均1.76。当相似系数为0.81时,可将供试萝卜分成3类,第I类包括6份白色肉质萝卜,第II类包括3份红色肉质萝卜和6份白色肉质萝卜,第III类包括22份红色肉质萝卜。各红色肉质萝卜品种间的遗传相似系数有97%大于0.80,而各白色肉质萝卜品种间的遗传相似系数有91%大于0.80。红色肉质萝卜遗传多样性略低于白色肉质萝卜,红色肉质萝卜与白色肉质萝卜间平均相似系数为0.83,说明不同肉色的萝卜间亲缘关系较密切,在分类上红色肉质萝卜可能是白色萝卜的一个变种。     

The evolution of California's wild radish has resulted in the extinction of its progenitors

If two previously isolated taxa mutually assimilate through hybridization and subsequent biparental introgression, and if their introgressed descendants have the same or higher fitness than their parents, then gene flow should result in the local extinction of parental taxa via replacement by hybrid derivatives. These dramatic events may occur rapidly, even in a few generations. Given the speed at which such extinction by hybridization may occur, it may be difficult to identify that the process has occurred. Thus, documented instances of extinction by hybridization are rare, and especially so for cases in which both parents are replaced by the hybrid lineage. Here we report morphological and allozyme evidence for the local extinction of two Raphanus species in California via replacement by their hybrid-derived descendants. The results from a greenhouse experiment demonstrate that California wild radishes have a specific combination of traits from their progenitors, and comparison of our results to that of an earlier report indicate that pure parental types are no longer present in the wild. Our results also show the hybrid-derived lineage has transgressive fruit weight compared to its parents. Allozyme analysis demonstrates that California wild radishes are derived from hybridization between the putative parental species. However, that analysis also demonstrates that California wild radish has now become an evolutionary entity separate from both of its parents. We suggest that the aggressive colonizing behavior of the hybrid-derived lineage probably results from a novel combination of parental traits, rather than genetic variability of the population per se.     

TRANSMISSION GENETICS OF ISOZYME LOCI IN RAPHANUS SATIVUS (BRASSICACEAE): STRESS-DEPENDENT NON-MENDELIAN SEGREGATION

Deviations from Mendelian ratios are frequently treated as an intrinsic property of individuals, independent of the environment. We tested whether the environment of the parents could alter patterns of inheritance in the wild radish, Raphanus sativus. We demonstrated the genetic basis of 12 isozyme loci by controlled pollinations of unstressed plants. The frequency of deviant segregation detected was not different than that expected by chance. Controlled pollinations among stressed plants showed over 3 times as much deviant segregation as the unstressed controls. No genetic correlates of segregation bias were detected. Linkage was assessed for 64 of the 66 pairs of loci. Two linkage groups were detected, one involving four loci (PGM2–ACO–ACP–LAP), the other involving a single pair (EST-PRX). The second linkage group is apparently associated with a locus or tightly linked loci which may segregate for “balanced” lethals on the same chromosome. Deviant segregation did not appear to act primarily by selection on a particular gamete. Postzygotic selection was the probable source of at least some of the aberrant segregation. Because no particular allele was favored in such situations, selection is apparently operating on alleles at linked loci rather than on the allozyme loci per se. Data from other studies on wild radish support the suggestion that postzygotic selection might be an important influence on progeny segregation ratios. Because wild radishes often encounter a variety of stresses in the field, in this species, aberrant segregation may be common under natural conditions.     

Effects of substrate acidity and UV-B radiation on photosynthesis of radishes

The aim of this study was to assess the combined effect of substrate acidity (pH 4.8; pH 3.8) and 1 kJm⁻²d⁻¹ UV-B radiation on photosynthesis and growth of radishes (Raphanus sativus L.). Radishes were sown in a neutral pH 6.5 peat substrate. When the second true leaf unfolded, the growth substrate was acidified using different concentration of H₂SO₄ and exposed to UV-B radiation for a period of ten days. Gas exchange parameters were measured with the LI-6400 portable photosynthesis system. Content of chlorophyll was evaluated spectrophotometrically. The results showed that the greatest inhibition of net photosynthesis was observed when radishes were grown in an acidified pH 3.8 substrate. The decrease of the photosynthesis of radish plants treated with both investigated factors (substrate acidity and UV-B) were lower compared to the effect of substrate acidity alone. UV-B radiation stimulated both enzymatic reactions of photosynthesis and water use efficiency of radish plants grown in acidified peat substrates. Also, investigated factors had higher impact on biomass of tuber than biomass of foliage.     

Competition alters life history and increases the relative fecundity of crop-wild radish hybrids (Raphanus spp.)

The evolutionary impact of crop-to-wild gene flow depends on the fitness of hybrids under natural, competitive conditions. Here, we measured the performance of third-generation (F3) radish hybrids (Raphanus raphanistrum x Raphanus sativus) and weedy R. raphanistrum to understand how competitive interactions affect life history and relative fecundity. Three wild and three F1 crop-wild hybrid radish populations were established in semi-natural, agricultural conditions in Michigan, USA. The effects of competition on life-history traits and fecundity of F3 progeny were measured 2 yr later in a common garden experiment. Third-generation hybrid plants generally produced fewer seeds per fruit and set fewer fruits per flower than wild plants, resulting in lower lifetime fecundity. With increasing competition, age at reproduction was delayed, the relative number of seeds per fruit was reduced in wild plants and differences between hybrid and wild fecundity diminished. Competition may enhance the fecundity of advanced-generation hybrids relative to wild plants by reducing differences in life history, potentially promoting the introgression of crop alleles into weed populations.