Molecular characteristics and expression of cytoplasmic genes in cytoplasmic male sterility of pepper (Capsicum annuum L.)

This study compared the sequence variations and expressions of 12 chloroplastic and 8 mitochondrial genes in three pepper (Capsicum annuum L.) cytoplasmic male sterile (CMS) lines, their maintainers and two control cultivars. The results showed that the three CMS lines were highly similar in chloroplastic and mitochondrial fragment sequences, with average similarities of 96.81 and 98.73?%, respectively, and their chloroplastic trnH?CpsbA intergenic spacer, photosystem II 47?kDa protein (psbB) genes, mitochondrial apocytochrome b (cob) and RNAD fragments have 1, 9, 8 and 7 distinct sites from the maintainer lines, respectively, and could be used as informative sites to distinguish CMS lines from the maintainer lines. Meanwhile, the expressions of mitochondrial cob, RNAD and pvs in the reproductive organs (flowers) of CMS lines are different from those of the maintainer lines, but their expressions in the vegetative organs (roots and leaves) are similar. The results indicate that cytoplasmic DNA polymorphisms are rare in CMS lines, and mitochondrial cob, RNAD and pvs genes are closely related to pollen abortion.     

Characterisation of a cytoplasmic male-sterile hybrid line between Lycopersicon peruvianum Mill. ×Lycopersicon pennellii Corr. and its crosses with cultivated tomato

 The cytoplasmic male-sterile (CMS) line CMS-pennellii (BC₁₀P₂ L. peruvianum×L. pennellii) and its complex hybrids with L. esculentum were studied. The established sterility was classified as the sporogenous type. As a result of the interaction of the genome of L. pennellii and the cytoplasm of L. peruvianum clear changes were established in the profiles of malic enzyme and esterase. Restriction fragment length polymorphism (RFLP) was detected between the mitochondrial (mt) genomes of CMS-pennellii and the cytoplasm donor, L. peruvianum, for two mtDNA probes: atpA and nad3. The established differences in the isozyme pattern and mt genomes are considered as useful markers to distinguish fertile and sterile plants. A breakthrough in the unilateral incompatibility of CMS-pennellii and the incorporation of the genome of L. esculentum on a CMS background is reported. The analysis of the complex hybrids assumes the interaction of two dominant genes – a maintainer gene from L. pennellii and a restorer gene from cultivated tomato. The hybrids produced with L. esculentum provide the basis for the development of a CMS system in cultivated tomato. Received: 25 May 1998 / Accepted: 26 August 1998     

Mitochondrial DNA-RFLP Analysis Distinguishes New CMS Sources in Pearl Millet (Pennisetum glaucum (L.) R. Br.)

Restriction fragment length polymorphism (RFLP) of mitochondrial (mt) DNA provides a rapid and effective method to assess heterogeneity among male sterile cytoplasms. Six isonuclear A-lines (81 A₁, with Tift 23A₁, cytoplasm, ICMA 88001 (= 81A_v) with Violaceum cytoplasm, 81A (=81A₄) with monodli = violaceum cytoplasm, Pb 310A₂ and Pb 311A₂ with A₂ cytoplasm from L 66A, and Pb 406A₃ with A₃ cytoplasm from L 67A), nine cytoplasmic male-sterility sources from Large-Seeded Genepool (LSGP 6, LSGP 14, LSGP 17, LSGP 22, LSGP 28, LSGP 36, LSGP 43, LSGP 55 and LSGP 66) and two each from Early Genepool (EGP 33 and EGP 15) and Population Varieties (PV 1 and PV 2) were characterized for variation in their mitochondrial genomes following Southern blot hybridizations using homologous (pearl millet 13.6 kb, 10.9 kb, 9.7 kb and 4.7 kb clones) and heterologous (maize atp6 and coxl clones) mitochondrial DNA (mtDNA) probes. Following cluster analysis based on similarity indices for the RFLP banding patterns observed, we identified seven cytoplasmic groups within LSGP. Two (LSGP 43 and LSGP 66) of these were quite distinct from each other as well as from other cytoplasms. This clearly indicates that besides serving as a source of diversity for agronomic and adaptation traits, broad-based gene pools can also provide diverse sources of cytoplasmic male sterility. These new CMS sources were also compared with standard CMS systems and cytoplasm-specific restriction fragments were identified.     

Are the mitochondrial cox1 and cob genes suitable markers for species of Dinophysis Ehrenberg?

The identification of Dinophysis species with similar morphology but different toxic (Diarrhetic Shellfish Poisoning, DSP) potential is a crucial task in harmful algae monitoring programmes. The taxonomic assignment of Dinophysis species using molecular markers is a difficult task due to extremely low interspecific variability within their nuclear ribosomal genes and intergenic regions. Mitochondrial cox1 gene has been proposed as a better specific marker for Dinophysis species based on its higher resolution for two morphologically related species (Dinophysis acuminata and Dinophysis ovum) of the “Dinophysis acuminata complex”. In this study, the potential of two mitochondrial genes (mt cox1 and cob) to discriminate among six Dinophysis species (field isolates and cultures) associated with DSP events was explored. Neither mt cox1 nor cob genes provided enough resolution for all species of Dinophysis. The cob gene showed very poor resolution and grouped all Dinophysis spp. in a common clade. In contrast, the cox1 phylogeny distinguished 5 clades in the Dinophysiales – the “acuminata complex”, the “caudata group”, “acuta + norvegica” and Phalacromaspp. However, within the “D. acuminata complex” mtcox1 is so far the unique marker that differentiates D. acuminata from other species: isolates of D. ovum and Dinophysis sacculus had almost identical sequences (only four mismatches), but they were well separated from D. acuminata. D. acuminata and Dinophysis skagii (considered a life cycle stage of the former) showed identical cox1 sequences. Probes towards this gene can be useful in Mediterranean and Western Iberia sites where the co-occurrence of close morphotypes of D. acuminata and D. sacculus pose a problem for monitoring analyses. This is the first report on cultures of D. sacculus and its phylogenetic relation with other species of the D. acuminata complex.     

The Australian fresh water isopod (Phreatoicidea: Isopoda) allows insights into the early mitogenomic evolution of isopods

The complete mitochondrial (mt) genome sequence of the Australian fresh water isopod Eophreatoicus sp.-14 has been determined. The new species is a member of the taxon Phreatoicidea, a clade of particular interest, as it is often regarded as the sister group to all other Isopoda. Although the overall genome organization of Eophreatoicus sp.-14 conforms to the typical state of Metazoa—it is a circular ring of DNA hosting the usual 37 genes and one major non-coding region—it bears a number of derived characters that fall within the scope of “genome morphology”. Earlier studies have indicated that the isopod mitochondrial gene order is not as conserved as that of other crustaceans. Indeed, the mt genome of Eophreatoicus sp.-14 shows an inversion of seven genes (including cox1), which is as far as we know unique. Even more interesting is the derived arrangement of nad1, trnL(CUN), rrnS, control region, cob, trnT, nad5 and trnF that is shared by nearly all available isopod mt genomes. A striking feature is the close proximity of the rearranged genes to the mt control region. Inferable gene translocation events are, however, more suitable to trace the evolution of mt genomes. Genes like nad1/trnL(CUN) and nad5/trnF, which retained their adjacent position after being rearranged, were most likely translocated together. A very good example for the need to understand the mechanisms of translocations is the remolding of trnL(UUR) to trnL(CUN). Both tRNA genes are adjacent and have a high sequence similarity, probably the result of a gene duplication and subsequent anticodon mutation. Modified secondary structures were found in three tRNAs of Eophreatoicus sp.-14, which are all characterized by the loss of the DHU-arm. This is common to crustaceans for tRNA Serine(AGY), while the arm-loss in tRNA Cysteine within Malacostraca is only shared by other isopods. Modification of the third tRNA, Isoleucine, is not known from any other related species. Nucleotide frequencies of genes have been found to be indirectly correlated to the orientation of the mitochondrial replication process. In Eophreatoicus sp.-14 and in other Isopoda the associated nucleotide bias is inversed to the state of other Malacostraca. This is a strong indication for an inversion of the control region that most likely evolved in the isopod ancestor.     

The First Mitochondrial Genomes of Antlion (Neuroptera: Myrmeleontidae) and Split-footed Lacewing (Neuroptera: Nymphidae), with Phylogenetic Implications of Myrmeleontiformia

In the holometabolous insect order Neuroptera (lacewings), the cosmopolitan Myrmeleontidae (antlions) are the most species-rich family, while the closely related Nymphidae (split-footed lacewings) are a small endemic family from the Australian-Malesian region. Both families belong to the suborder Myrmeleontiformia, within which controversial hypotheses on the interfamilial phylogenetic relationships exist. Herein, we describe the complete mitochondrial (mt) genomes of an antlion (Myrmeleon immanis Walker, 1853) and a split-footed lacewing (Nymphes myrmeleonoides Leach, 1814), representing the first mt genomes for both families. These mt genomes are relatively small (respectively composed of 15,799 and 15,713 bp) compared to other lacewing mt genomes, and comprise 37 genes (13 protein coding genes, 22 tRNA genes and two rRNA genes). The arrangement of these two mt genomes is the same as in most derived Neuroptera mt genomes previously sequenced, specifically with a translocation of trnC. The start codons of all PCGs are started by ATN, with an exception of cox1, which is ACG in the M. immanis mt genome and TCG in N. myrmeleonoides. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1(AGN). The secondary structures of rrnL and rrnS are similar with those proposed insects and the domain I contains nine helices rather than eight helices, which is common within Neuroptera. A phylogenetic analysis based on the mt genomic data for all Neuropterida sequenced thus far, supports the monophyly of Myrmeleontiformia and the sister relationship between Ascalaphidae and Myrmeleontidae.     

Investigation of Mitochondrial Transmission in Selected Matings between Homokaryons from Commercial and Wild-Collected Isolates of Agaricus bisporus (= Agaricus brunnescens)

Ten heterokaryons of Agaricus bisporus (= Agaricus brunnescens) were shown to carry four different mitochondrial (mt) genotypes by analysis of mt restriction fragment length polymorphisms (RFLPs). Fifteen homokaryons derived from these strains were used to investigate mt inheritance in A. bisporus. One hundred eighty-nine pairings were performed in 25 different combinations. Pairings in 15 different combinations produced heterokaryons on the basis of nuclear RFLP analyses and/or fruiting trials. The mt genotype of each new intraspecies hybrid was examined by using mt RFLPs as genetic markers. Our results suggest the following. (i) Recombination between the mt genomes was not a common event. (ii) From most individual pairings, all heterokaryons carried the same mt genotype. (iii) Heterokaryons carrying either of the two possible mt genotypes were observed in certain crosses after modification of the pairing procedure. A biparental transmission pattern was demonstrated for some crosses, but there appears to be a preference for one of the mt genotypes to predominate in any specific pairing.     

Molecular diversity of male sterility inducing and male-fertile cytoplasms in the genus Helianthus

The organisation of mtDNA was investigated for 28 sources of cytoplasmic male sterility (CMS) and a fertile line (normal cytoplasm) of Helianthus annuus by Southern hybridisation. In addition to nine known mitochondrial genes (atp6, atp9, cob, coxI, coxII, coxIII, 18S, 5S and nd5) three probes for the open reading frames in the rearranged area of PET1, orfH522, orfH708 and orfH873, were used. Genetic similarities of the investigat-ed cytoplasms varied between 0.3 and 1. Cluster analyses using the UPGMA method allowed the distinction of ten mitochondrial (mt) types between the 29 investigated cytoplasms. Most mitochondrial types comprise two or more CMS sources, which could not be further separated, like the PET1-like CMS sources (with the exception of ANO1 and PRR1), or ANN1/ANN2/ANN3, ANN4/ ANN5, ARG3/RIG1, BOL1/EXI1/PEF1/PEP1 and GIG1/ PET2. ANL1, ANL2 and the fertile cytoplasms are also regarded as one mitochondrial type. Unique banding patterns were only observed for ANT1 (atp6), MAX1 (atp6, orfH522 and orfH708) and PRR1 (coxII). However, four of the mitochondrial types showed unique hybridisation signals: ANN4/ANN5 had characteristic bands for atp6 and orfH708, PEF1/PEP1/EXI1/BOL1 for atp6 and coxII, and PET2/GIG1 for atp9. The PET1-like cytoplasms all shared the same patterns for orfH522, orfH708 and cob (except ANO1). It could be demonstrated that CMS sources, like, e.g., PET2 and PEF1, are different from PET1 in mtDNA organisation and the CMS mechanism. Therefore, these CMS sources represent interesting candidates for the development of new hybrid breeding systems based on new CMS mechanisms. Received: 20 April 2001 / Accepted: 3 August 2001     

The Complete Mitochondrial Genome of Gossypium hirsutum and Evolutionary Analysis of Higher Plant Mitochondrial Genomes

Background

Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes.

Methodology/Principal Findings

We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes.

Conclusion

The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.     

Analyses of mitochondrial DNA structure and expression in three cytoplasmic male-sterile chicories originating from somatic hybridisation between fertile chicory and CMS sunflower protoplasts

Cytoplasmic male-sterile (CMS) chicories have been previously obtained by somatic hybridisation between fertile industrial chicory protoplasts and CMS sunflower protoplasts. In this study, we compared three different CMS chicory cybrids that originated from three different fusion events. The cybrids were backcrossed with different witloof chicories in order to transfer the three male-sterile cytoplasms from an industrial chicory nuclear environment to a witloof chicory nuclear context. Southern hybridisation, using different mitochondrial genes as probes, revealed that the three cybrid mitochondrial genomes were different and that they were stable throughout backcrossing generations regardless of the pollinator. However, pollinators were found to influence floral morphologies – with one being able to restore fertility – showing that nuclear context can affect the sterility of the cybrids. PCR and RFLP analyses revealed that the orf522 sequence, responsiblefor CMS in PET1 sunflower, was present in two out of the three cytoplasms studied, namely 411 and 523, but was absent from the other cytoplasm, 524. We thus concluded that orf522 is not responsible for CMS in the 524 cybrid. Although the orf522 gene is present in the 411 and 523 cytoplasms, it is probably not responsible for the sterile phenotype of these cybrids. Received: 3 June 1998 / Accepted: 30 April 1999     

细胞质雄性不育水稻线粒体基因组的RFLP分析

利用RFLP技术,比较研究了在农业生产上广泛应用的9种细胞质雄性不育体系的线粒体基因组,结果表明：1）9种水稻雄性不育细胞质的遗传相似性变化范围为0．615～1．000。所有配子体雄性不育细胞质的遗传相似性变化范围为0．6431．000,其中QXA,DY1A和YM15A这3种细胞质的遗传相似性为1．000。所有孢子体雄性不育细胞质的遗传相似性为1．000,2）在配子体细胞质雄性不育水稻中,用3个探针（atpa,atp9＋nad6,cox2）与2种内切酶（HindⅢ,BamHⅠ）的组合未发现不育系与保持系之间的多态性,但在探针atp6,cob,和had2的杂交带型中找到了不育系和保持系之间的一些差异。YTA和YTB在5个探针．内切酶组合（atp61HindⅢ,coblHindⅢ,atp61BamHⅠ,coblBamHⅠ,nad21BamHⅠ）中存在差异。3种细胞质（QX,SJ,DY1）的不育系和保持系之间的差异是相同的,都出现在atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ等3个组合中。YM15A和YM15B在4个组合（atp6／HindⅢ,atp6／BamHⅠ,cob／BamHⅠ,nad2／BamHⅠ）中存在差异。LYA和LYB的差异出现在cob／HindⅢ,cob／BamHⅠ,nad2／BamHⅠ这3个组合中;3）在孢子体细胞质雄性不育水稻中,所有不育系的带型是一样的,所有保持系的带型也一样。不育系和保持系的差异出现在atp6／HindⅢ,cob／HindⅢ,atp6／EcoR,Ⅰcob／EcoRⅠ,cox1／EcoRⅠ,atp6／BamHⅠ,cob／BamHⅠ,cox1／BamHⅠ,cox2／BamHⅠ等9个组合中。这些结果在分子水平上揭示了9种雄性不育细胞质的线粒体基因组存在结构多样性,并为其细胞质雄性不育分子机理的研究打下了基础。     

RFLP Analysis for Mitochondrial Genome of CMS-Rice

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.     

Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing

Background

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp.

Results

We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes.

Conclusions

Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-561) contains supplementary material, which is available to authorized users.     

Molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.