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The mitochondrial gene orf108, which is co-transcribed with atp1 and causes cytoplasmic male sterility in Brassica crops, is widely distributed across wild species and genera of Brassicaceae. However, to date, intraspecific variations in the presence of orf108 have not yet been studied, and the mechanisms underlying the wide distribution of the gene remain unclear. We analyzed the presence and sequence variations of orf108 in two wild species, Brassica maurorum and Moricandia arvensis. After polymerase chain reaction amplification of the 5 (0) region of atp1 and the coding sequence of orf108, we determined the DNA sequences. Brassica maurorum and M. arvensis showed variations in the presence of orf108 or orf117 (orf108 (V117)) both between and within accessions and were not fixed to the mitochondrial type with the male sterile genes. Sequencing of the amplicons showed that B. maurorum had orf108 (V117) instead of orf108. Sequencing also indicated mitochondrial heteroplasmy in the two species; in particular, in B. maurorum, one plant possessed both orf108 and orf108 (V117) sequences. These results suggest that substoichiometric shifting of mitochondrial genomes leads to the acquisition or loss of orf108. Furthermore, fertility restorer genes of the two species were involved in the processing of the mRNA of male sterility genes at different sites. Key words: orf108, orf117, intraspecific variation, Brassica maurorum, Moricandia arvensis. Le gene mitochondrial orf108, lequel est co-transcrit avec atp1 et cause l'androsterilite cytoplasmique chez les Brassica cultives, est largement distribue au sein des especes sauvages et divers genres chez les cruciferes. Cependant, a ce jour, aucune etude de la variation intraspecifique quant a la presence de l'orf108 n'a ete realisee et les mecanismes assurant une large distribution de ce gene restent inconnus. Les auteurs ont examine la presence et les variations dans la sequence de l'orf108 chez deux especes sauvages, Brassica maurorum et Moricandia arvensis. Apres amplification PCR de la region 5 (0) du gene atp1 et des sequences codantes de l'orf108, les auteurs en ont realise le sequencage. Au sein de B. maurorum et de M. arvensis, de la variation a ete observee pour la presence des orf108 et orf117 (orf108 (V117)) tant entre les accessions qu'au sein de celles-ci et ces variations n'etaient pas fonction de la presence des genes de sterilite. Le sequencage des amplicons a permis d'etablir que le B. maurorum possede l'orf108 (V117) plutot que l'orf108. Le sequencage a aussi revele une heteroplasmie mitochondriale chez ces deux especes, en particulier chez le B. maurorum ou une plante possedait les deux sequences (orf108 et orf108 (V117)). Ces resultats suggerent que des changements substoichiometriques dans les genomes mitochondriaux menent a l'acquisition ou a la perte de l'orf108. De plus, les genes de restauration de la fertilite chez les deux especes etaient impliques dans la maturation de l'ARNm des genes d'androsterilite a differents sites. Mots-cles : orf108, orf117, variation intraspecifique, Brassica maurorum, Moricandia arvensis., Introduction Cytoplasmic male sterility (CMS) is a phenomenon in which plants fail to produce fertile pollen due to a mitochondrial gene. The phenotypic expression of CMS is suppressed by the [...]

Crop species of Brassica (Brassicaceae) consist of three monogenomic species and three amphidiploid species resulting from interspecific hybridizations among them. Until now, mitochondrial genome sequences were available for only five of these species. We sequenced the mitochondrial genome of the sixth species, Brassica nigra (nuclear genome constitution BB), and compared it with those of Brassica oleracea (CC) and Brassica carinata (BBCC). The genome was assembled into a 232145 bp circular sequence that is slightly larger than that of B. oleracea (219 952 bp). The genome of B. nigra contained 33 protein-coding genes, 3 rRNA genes, and 17 tRNA genes. The cox2-2 gene present in B. oleracea was absent in B. nigra. Although the nucleotide sequences of 52 genes were identical between B. nigra and B. carinata, the second exon of rps3 showed differences including an insertion/deletion (indel) and nucleotide substitutions. A PCR test to detect the indel revealed intraspecific variation in rps3, and in one line of B. nigra it amplified a DNA fragment of the size expected for B. carinata. In addition, the B. carinata lines tested here produced DNA fragments of the size expected for B. nigra. The results indicate that at least two mitotypes of B. nigra were present in the maternal parents of B. carinata. Key words: Brassica nigra, Brassica carinata, complete mitochondrial genome, intraspecific mitotype variation, maternal parent of alloploid. Les especes cultivees au sein du genre Brassica (Cruciferes) comprennent trois especes monogenomiques et trois especes amphidiploides, les secondes etant le resultat d'hybridations interspecifiques entre les premieres. Jusqu'a maintenant, les sequences des genomes mitochondriaux etaient disponibles pour seulement cinq de celles-ci. Les auteurs ont sequence le genome mitochondrial de la sixieme espece, Brassica nigra (genome nucleaire BB), et ont compare celui-ci a ceux de Brassica oleracea (CC) et de Brassica carinata (BBCC). Le genome a ete assemble en une sequence circulaire de 232 145 pb qui est legerement plus grande que celle du B. oleracea (219 952 pb). Le genome du B. nigra comprend 33 genes codant pour des proteines, 3 genes d'ARNr et 17 genes d'ARNt. Le gene cox2-2 present chez le B. oleracea est absent chez le B. nigra. Bien que les sequences nucleotidiques des 52 genes soient identiques entre le B. nigra et le B. carinata, le deuxieme exon du gene rps3 presentait certaines differences dont une insertion/deletion (indel) et des substitutions nucleotidiques. Un test PCR a ete mis au point pour detecter l'indel et celui-ci a permis de montrer une variation intraspecifique au sein de rps3. Au sein d'une lignee du B. nigra, le fragment amplifie etait de la taille attendue chez le B. carinata. De plus, certaines lignees testees chez le B. carinata ont produit des amplicons de la taille attendue chez le B. nigra. Ces resultats indiquent qu'il existait au moins deux mitotypes du B. nigra au sein des parents maternels du B. carinata. [Traduit par la Redaction] Mots-cles : Brassica nigra, Brassica carinata, genome mitochondrial complet, variation intraspecifique pour le mitotype, parent maternel d'un alloploide., Introduction One of unique features of higher plant evolution is the occurrence of spontaneous interspecific hybridization followed by polyploidization. These processes result in establishment of new plant species referred to [...]

In wheat (Triticum) and Aegilops, chloroplast and mitochondrial genomes have been studied for over three decades to clarify the phylogenetic relationships among species, and most of the maternal lineages of polyploid species have been clarified. Mitochondrial genomes of Emmer (tetraploid with nuclear genome AABB) and Dinkel (hexaploid with AABBDD) wheat are classified into two different types, VIIa and VIIb, by the presence-absence of the third largest HindIII fragment (named H3) in the mitochondrial DNA. Although the mitochondrial genome in the genera often provides useful information to clarify the phylogenetic relationship among closely related species, the phylogenetic significance of this dimorphism has yet not been clarified. In this study, to facilitate analysis using a large number of accessions, a sequence characterized amplified region (SCAR) marker that distinguishes the type VIIb mitochondrial genome from type Vila was first developed. Mitochondrial genome type was determined for each of 30 accessions of wild and cultivated Emmer wheat and 25 accessions of Dinkel wheat. The mitochondrial genome type for each accession was compared with the plastogroup that had been determined using chloroplast microsatellite markers. Unexpectedly, the distribution of mitochondrial genome type was not in accordance with that of the plastogroups, suggesting occasional paternal leakage of either the mitochondrial or chloroplast genome during speciation and differentiation of Emmer and Dinkel wheat. An alternative possibility that substoichiometric shifting is involved in the observed dimorphism of the mitochondrial genome is also discussed. Key words: wheat, mitochondrial DNA, SCAR, linkage disequilibrium, substoichiometric shifting. Chez le ble (Triticum)etl'Aegilops, les genomes chloroplastiques et mitochondriaux ont ete etudies depuis plus de trois decennies pour eclairer les relations phylogenetiques entre les especes et la plupart des lignages maternels des especes polyploides ont ete determines. Les genomes mitochondriaux du ble amidonnier (tetraploide avec un genome nucleaire AABB) et de l'epeautre (hexaploide, AABBDD) appartiennent aux types Vila et VlIb sur la base de la presence-absence du troisieme plus grand fragment HindIII (H3) au sein de l'ADN mitochondrial. Bien que le genome mitochondrial procure souvent des informations utiles pour eclairer les relations phylogenetiques au niveau du genre entre especes proches, la signification phylogenetique de ce dimorphisme n'a pas encore ete determinee. Dans ce travail, afin de faciliter l'analyse d'une grand nombre d'accessions, un marqueur PCR de type SCAR (<< sequence characterized amplified region >>) permettant de distinguer les genomes mitochondriaux des types VIIb et VIIa a ete developpe. Le type de genome mitochondrial a ete determine pour chacune de 30 accessions de ble amidonnier sauvage ou cultive ainsi que pour 25 accessions d'epeautre. Le type de genome mitochondrial rencontre chez chaque accession a ete compare au groupe plastidique qui avait ete determine a l'aide de marqueurs microsatellites chloroplastiques. Contrairement aux attentes, la distribution du type de genome mitochondrial ne concordait pas avec celui des groupes plastidiques, ce qui suggere une transmission paternelle occasionnelle de l'un ou l'autre genome au cours de la speciation et de la differenciation des bles amidonniers et des epeautres. Les auteurs discutent de la possibilite alternative que des fluctuations substoechiometriques soient responsables du dimorphisme observe au sein du genome mitochondrial. [Traduit par la Redaction] Mots-cles : ble, ADN mitochondrial, SCAR, desequilibre de liaison, fluctuations substoechiometriques., Introduction Chloroplast and mitochondria contain their own genomes, collectively called plasmon. Genetic information stored in the plasmon is a rich source of study for revealing phylogenetic relationships among taxa at [...]

To reveal the molecular and genetic mechanism of fertility restoration in Ogura male sterility in Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides), we investigated fertility restoration of a plant that lacks the dominant type of orf687, a previously identified fertility restorer gene. A total of 100 [F.sub.2] plants were made from the cross between a male-sterile strain with the Ogura cytoplasm, 'MS-Gensuke', and a Japanese wild radish plant. Segregation of pollen fertility in the [F.sub.2] plants led us to assume that 2 dominant complementary genes controlled the fertility restoration of the plants. However, the fertility of 27 of 59 male-fertile plants was not completely restored, resulting in a group of plants with partial male fertility. Northern blot analysis of the CMS-associated gene orf138 indicated that one restorer allele (termed Rft) was involved in the processing of orf138 RNA. Rapid amplification of cDNA ends (RACE) and subsequent Northern blot analysis confirmed that the orf138 transcript lost a 5' part of the coding region of the orf138 gene in the restored plants. The accumulation of ORF138 protein was significantly reduced by Rft, but trace amounts of the protein were recognized in both partially male-fertile and male-sterile plants with Rft. The relationship of pollen fertility and segregation of co-dominant sequence tagged site (STS) markers in the [F.sub.2] generation suggested that the penetrance of Rft was so low that Rft needs suitable conditions to function sufficiently for the complete restoration of fertility. Key words: Raphanus sativus, Japanese wild radish, Rf gene, cytoplasmic male sterility (CMS), RNA processing, STS marker. Afin de mettre en evidence les mecanismes moleculaires et genetiques impliques dans la restauration de la fertilite chez le systeme d'androsterilite Ogura du radis sauvage japonais (Raphanus sativus var. hortensis f. raphanistroides), les auteurs ont investigue la restauration de la fertilite chez une plante depourvue du type dominant de l'orf687, un gene de restauration decrit anterieurement. Au total, 100 plantes [F.sub.2] ont ete obtenues en croisant une lignee androsterile ayant le cytoplasme Ogura, 'MS-Gensuke', et un plant de radis sauvage japonais. La segregation pour la fertilite du pollen chez les plantes [F.sub.2] a permis d'avancer l'hypothese que deux genes complementaires dominants controleraient la restauration de la fertilite chez les plantes. Cependant, la fertilite chez 27 des 59 plantes fertiles n'etaient pas completement restauree de telle sorte qu'un groupe a fertilite partielle a ete observe. Des analyses northern du gene orf138, associe a l'androsterilite, a revele qu'un des alleles restaurateurs (appele Rft) etait implique dans la maturation du transcrit de l'orf138. Une amplification des extremites des ADNc (RACE) et une analyse northern subsequente ont confirme que le transcrit de l'orf138 avait perdu une portion en 5' de sa region codante chez les plants restaures. L'accumulation de la proteine ORF138 etait significativement reduite par Rft, bien que des traces de proteine aient ete observees chez les plantes possedant Rft et qui etaient partiellement fertiles ou steriles. La relation entre la fertilite pollinique et la segregation de marqueurs STS (<< sequence tagged sites >>) codominants au sein de la generation [F.sub.2] suggere que la penetrance de Rft est si faible que Rft necessite des conditions propices a un fonctionnement suffisant qui permet la restauration complete de la fertilite. Mots-cles: Raphanus sativus, radis sauvage japonais, gene Rf, androsterilite cytoplasmique (CMS), maturation de l'ARN, marqueur STS. [Traduit par la Redaction], Introduction Cytoplasmic male sterility (CMS) is a maternally inherited trait that prevents the production of functional pollen. CMS is successfully exploited in the commercial production of [F.sub.1] hybrid seeds in [...]

To study the origin and maintenance mechanisms of the PGI allozyme polymorphism of a wild plant, Dioscorea tokoro, DNA sequences of the entire coding region (1701 bp) and two intronic regions (total 2049 bp) of the Pgi gene as well as a part of the Adh gene (590 bp) were analyzed. Two replacement substitutions were revealed to be responsible for the differentiation of three allozymes alleles (Pgi-a, Pgi-b and Pgi-c) that occur in natural population in intermediate frequencies. Interspecific comparison of DNA sequences identified Pgi-b as the oldest allele, from which two other alleles were derived probably within the last 150,000 years. The level of DNA polymorphism at D. tokoro Pgi locus was low. No elevated level of DNA polymorphism was detected in the close vicinity of the two replacement sites differentiating the three allozymes. Departures from the neutral mutation hypothesis were detected by Fu and Li's and MK tests. The observed patterns of DNA polymorphism are explainable by both (1) the neutral mutation hypothesis with an assumption of small effective size of D. tokoro population, and (2) the positive selection hypothesis that the allele frequencies of Pgi-a and Pgi-c have increased in a short time by their selective advantages.

Abstract Background Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. Results Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. Conclusions Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.