Molecular markers linked to the blast resistance gene <Emphasis Type="Italic">Pi-z</Emphasis> in rice for use in marker-assisted selection

Rice blast, caused by the fungal pathogen Pyricularia grisea, is a serious disease affecting rice-growing regions around the world. Current methods for identification of blast-resistant germplasm and progeny typically utilize phenotypic screening. However, phenotypic screens are influenced by environmental conditions and the presence of one resistance gene can sometimes phenotypically mask other genes conferring resistance to the same blast race. Pi-z is a dominant gene located on the short arm of chromosome 6 that confers complete resistance to five races of blast. Using sequence data found in public databases and degenerate primer pairs based on the P-loop, nucleotide binding sites and kinase domain motifs of previously cloned resistance genes, we have developed PCR-based DNA markers that cosegregate with the gene. These markers are polymorphic in a wide range of germplasm, including the narrow crosses characteristic of applied rice-breeding programs. They can now be used as a low cost, high-throughput alternative to conventional phenotypic screening for direct detection of blast resistance genes, allowing rapid introgression of genes into susceptible varieties as well as the incorporation of multiple genes into individual lines for more-durable blast resistance.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by D. Mackill     

Novel sporophyte-like plants are regenerated from protoplasts fused between sporophytic and gametophytic protoplasts of<Emphasis Type="Italic"> Bryopsis plumosa</Emphasis>

Protoplasts of the marine coenocytic macrophyte Bryopsis plumosa (Hudson) C. Agardh. [Caulerpales] can easily be obtained by cutting gametophytes or sporophytes with sharp scissors. When a protoplast isolated from a gametophyte was fused with a protoplast isolated from a sporophyte of this alga, it germinated and developed into either one of two completely different forms. One plant form, named Type G, appeared quite similar to a gametophyte, and the other, named Type S, looked similar to a sporophyte. While the Type G plant contained many small nuclei of gametophyte origin together with a single giant nucleus of sporophyte origin, the Type S plant contained many large nuclei of uniform size. These large nuclei in the Type S plant had metamorphosed from the gametophytic nuclei, and were not formed through division of the giant nucleus of sporophyte origin. Fragments of the Type S plant, each having such a large nucleus, developed into creeping filaments that look very similar to sporophytes. While cell walls of gametophytes and Type G plants were stained by Congo-red, those of the thalli of regenerated Type S plants and sporophytes were not stained by the dye. This indicated that the large nuclei of the Type S plant did not express genes for xylan synthesis, which are characteristic of gametophytes. Two-dimensional gel electrophoretic analysis revealed that most of the proteins synthesized in the Type S plant were identical to those of sporophytes. These results strongly suggest that in the Type S plant, the gametophytic nuclei are transformed into sporophyte-like nuclei by an unknown factor(s) produced by the giant nucleus of sporophyte origin and that the transformed nuclei express the set of genes characteristic of sporophytes. Despite morphological similarity, however, the regenerated Type S plant could not produce zoospores, because its large nuclei did not divide normally. The transformed large nuclei of gametophyte origin still seemed to be in the haploid state.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - DIC Differential interference contrast - IEF Isoelectric focusing - PES Provasolis enriched seawater     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

A New Retroposed Gene in <Emphasis Type="Italic">Drosophila</Emphasis> Heterochromatin Detected by Microarray-Based Comparative Genomic Hybridization

A genomic pattern of new gene origination is often dependent on a genomic method that can efficiently identify a statistically adequate number of recently originated genes. The heterochromatic regions have often been viewed as genomic deserts with low coding potential and thus a low flux of new genes. However, increasing reports revealed unexpected roles of heterochromatic regions in the evolution of genes and genomes. We identified recently retroposed genes that originated in heterochromatic regions in Drosophila, by developing microarray-based comparative genomic hybridization (CGH) with multiple species. This new gene family, named Ifc-2h, originated in the common ancestor of the clade of D. simulans, D. mauritiana, and D. sechellia. The sequence features and phylogenetic distribution indicated that Ifc-2h resulted from the retroposition from its parental gene, Infertile crescent (Ifc), and integrated into heterochromatic region of common ancester of the three sibling species 2 million years ago. Expression analysis revealed that Ifc-2h had developed a new expression pattern by recruiting a putative regulatory element from its target sequence. The distribution of indel variation in Ifc-2h of D. simulans and D. mauritiana revealed a significant sequence constraint, suggesting that the Ifc-2h gene may be functional. These analyses cast fresh insight into the evolution of heterochromatin and the origin of its coding regions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

High-throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCRTM

Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole-genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high-throughput measurements of meiotic recombination rates in barley pollen nuclei without whole-genome amplification were performed through a Crystal Digital PCR^TM-based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCR^TM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.     

Mitosis and mitotic wave propagation in the coenocytic alga,<Emphasis Type="Italic"> Vaucheria terrestris</Emphasis> sensu Goetz

Mitosis and cytoplasmic microtubule (MT) dynamics were observed for the first time in Vaucheria terrestris sensu Goetz. Mitosis could occasionally be seen in part of the cylindrical coenocytic cell. The frequency of encountering cells with dividing nuclei was highest (ca 12%) 4 h after the onset of light in 12 h light/12 h dark regimes; it decreased thereafter and approached zero during the dark period. From the anterior end of every interphase nucleus a unique, long MT bundle extended. Differential-interference optics reveals that there is a filamentous structure in front of the moving nucleus. In prophase, the interphase bundle disappeared and shorter MT bundles emanated from both ends of the nucleus. In metaphase, the cytoplasmic MTs completely disappeared, probably being recycled to spindles. Continuous MTs elongated in anaphase and developed into an interzonal spindle in telophase; this elongated up to as much as 10 m. The daughter nuclei were pushed away from each other by the interzonal spindle. Mitosis started synchronously in a relatively narrow region, and the mitotic stage propagated as a mitotic wave to adjacent regions, most frequently from tip to base. The role of the mitotic wave in tip growth and morphogenesis of a coenocytic cell is discussed.This paper is dedicated to the memory of Dr. Eiji Kamitsubo who passed away on 25 April 2003.     

The gene <Emphasis Type="Italic">bap</Emphasis>, involved in biofilm production,is present in <Emphasis Type="Italic">Staphylococcus</Emphasis> spp. strains from nosocomial infections

This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Fusaric acid induces apoptosis in saffron root-tip cells: roles of caspase-like activity, cytochrome c, and H2O2

Programmed cell death (PCD), now known as apoptosis, is accompanied by specific morphological features. In this study, fusaric acid, a fusarium mycotoxin, was used to examine cell death in saffron (Crocus sativus Linnaeus) roots, using several apoptosis assays. Our results show that moderate FA doses (50–100 μM) induce apoptotic features while high FA doses (> 200 μM) stimulate necrosis. The apoptotic-like features induced by moderate doses of FA include chromatin condensation, formation of condensed chromatin spheres which bud from the nucleus, fragmentation of nucleosomal DNA into ∼ 180 bp fragments, exposure of phosphatidyl serine to the external membrane leaflet, delivery of cytochrome c to cytosol, and generation of H₂O₂. These apoptotic alterations in root cells are not observed in the presence of serine protease, caspase-1 or caspase-3 inhibitors. It is proposed that production of H₂O₂ and release of cytochrome c into the cytosol may activate caspase-like proteases and thus establish the apoptotic pathway. As nuclei budding spheres formed in plant root cells after exposure to 50–100 μM FA doses seem to be digested inside the cytosol, we suggest labeling them as internal apoptotic bodies (IAB) that may be more informative than previously used term, apoptotic-like bodies.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at     

The structure of the USP/RXR of <Emphasis Type="Italic">Xenos pecki</Emphasis> indicates that Strepsiptera are not closely related to Diptera

The receptor for the insect molting hormone, ecdysone, is a heterodimer consisting of the Ecdysone Receptor and Ultraspiracle (USP) proteins. The ligand binding domain sequences of arthropod USPs divide into two distinct groups. One group consists of sequences from members of the holometabolous Lepidoptera and Diptera, while the other arthropod sequences group with vertebrate retinoid-X-receptors (RXRs). We therefore wondered whether USP/RXR structure could be used to clarify the contentious phylogenetic position of the order Strepsiptera, which has proposed affinities with either Diptera or Coleoptera. We have cloned and sequenced the USP/RXR from the strepsipteran Xenos pecki. Phylogenetic analyses are not consistent with a close affinity between Strepsiptera and Diptera.Electronic Supplementary Material Supplementary material is available for this article at Edited by D. Tautz     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.     

Extreme nuclear disproportion and constancy of enzyme activity in a heterokaryon ofNeurospora crassa

Heterokaryons ofNeurospora crassa were generated by transformation of multinucleate conidia of ahistidine-3 auxotroph withhis-3 ⁺ plasmid. In one of the transformants, propagated on a medium with histidine supplementation, a gradual but drastic reduction occurred in the proportion of prototrophic nuclei that contained an ectopically integratedhis-3 ⁺ allele. This response was specific to histidine. The reduction in prototrophic nuclei was confirmed by several criteria: inoculum size test, hyphal tip analysis, genomic Southern analysis, and by visual change in colour of the transformant incorporating genetic colour markers. Construction and analyses of three-component heterokaryons revealed that the change in nuclear ratio resulted from interaction of auxotrophic nucleus with prototrophic nucleus that contained an ectopically integratedhis-3 ⁺ gene, but not with prototrophic nucleus that containedhis-3 ⁺ gene at the normal chromosomal location. The growth rate of heterokaryons and the activity of histidinol dehydrogenase—the protein encoded by thehis-3 ⁺ gene-remained unchanged despite prototrophic nuclei becoming very scarce. The results suggest that not all nuclei in the coenocytic fungal mycelium may be active simultaneously, the rare active nuclei being sufficient to confer the wild-type phenotype.     

Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination

Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This “discovery-oriented” pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column—a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker’s yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Franklin A. Hays and Zygy Roe-Zurz have contributed equally to this work.     

Behavior of nuclei during zoosporogenesis in Bryopsis plumosa (Bryopsidales, Chlorophyta)

The behavior of nuclei during zoosporogenesis in Bryopsis plumosa (Bryopsidales, Chlorophyta) was examined by fluorescence and electron microscopy. Each mature filamentous sporophyte had a single lenticular nucleus, which was about 25 m in diameter and embedded in a thick cytoplasmic layer. At the commencement of multinucleation, giant nuclei with large vacuolated nucleoli, giant nuclei containing chromosomes, and dumbbell-shaped nuclei were observed. Sometimes, two small nuclei also appeared in the thick cytoplasm where the giant nucleus had presumably been present. Electron microscopy revealed the existence of ribbon-like structures resembling synaptonemal complexes within the nucleus having a large vacuolated nucleolus. Nuclei extended their distribution by repetitive divisions. A pair of centrioles was adjacent to the interphase nucleus. When the nuclei were distributed throughout the cell, they became localized nearly equidistantly from one another, each being surrounded by several chloroplasts. At this stage, many centrioles lay along the nuclear surface. The bulk of cytoplasm was then divided into many masses of protoplasm, each of which developed into a uninucleate, stephanokontic zoospore with a whorl of flagella.     

Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.     

Unusual Origin of a Nuclear Pseudogene in the Italian Wall Lizard: Intergenomic and Interspecific Transfer of a Large Section of the Mitochondrial Genome in the Genus <Emphasis Type="Italic">Podarcis</Emphasis> (Lacertidae)

Two distinct cytochrome b-like sequences were discovered in the genome of Podarcis sicula. One of them represents a nuclear copy of a mitochondrial sequence (numt-sic) differing by 14.3% from the authentic mitochondrial (mt) sequence obtained from the same individual. This numt, however, differs by only 2.7% from the mt sequence found in one population of Podarcis muralis, a related species in which no corresponding numt was detected. The numt-sic sequence extends over at least 7637 bp and is homologous to a section of the mt genome spanning from the tRNA-Lys to the tRNA-Pro gene. Premature mt stop codons were detected in two of the nine protein coding genes of numt-sic. The distribution of substitutions among the three codon positions and the transition/transversion ratio of the numt-sic sequence resemble, with few exceptions, those of functional mt genes, indicating a rather recent transfer to the nucleus. Phylogenetic analyses performed on the data set including P. sicula numt-cytb sequences as well as mt-cytb sequences from the same individuals and mt sequences of various P. muralis populations suggest that numt-sic originated in P. muralis. In a geographic survey, P. sicula populations belonging to different mt lineages, covering most of the distribution area, were screened for the presence of numt-sic and for a 15-bp duplication polymorphism in the numt-nd5 sequence. Our results suggest that numt-sic has spread rapidly through the species range via sexual transmission, thereby being transferred to populations belonging to well-separated mt lineages that diverged 1–3 Mya. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Three dimensional confocal and electron microscopy imaging define the dynamics and mechanisms of diploidisation at early stages of barley microspore-derived embryogenesis

In order to determine the timing and mechanisms of the spontaneous diploidisation throughout microspore-derived embryogenesis in barley, we have estimated the ploidy level of individual nuclei within young pro-embryos, from the first androgenetic division up to multinuclear structures still surounded by the exine. Our methodological approach was based on the measure of the intensity of fluorescence after 4,6-Diamidino-2-phenylindole dihydrochloride staining, nuclear size and number of nucleoli in the confocal microscope. This method avoids the overlapping of the fluorescence signal in multinuclear pro-embryos, which cannot be studied using cytophotometer methods based on other types of fluorescence microscopes. The identification of haploid and diploid nuclei enabled us to determine the timing of diploidisation at early stages throughout androgenetic development. We found that diploidisation is an ongoing process that can start after the first embyogenic division and continues in multinuclear pro-embryos. Reconstruction of 3D-images of entire pro-embryos and the observation of cross and longitudinal sections across stacks of optical sections, together with correlative light and electron microscopy, provided evidences of nuclear fusion as the main mechanism of diploidisation.Electronic Supplementary Material Supplementary material is available for this article at This paper is dedicated to María Ángeles Ollacarizqueta (CCD and Confocal Service of the CIB) on her retirement