Development and characterization of microsatellite markers for Fusarium virguliforme and their utility within clade 2 of the Fusarium solani species complex

Clade 2 of the Fusarium solani species complex contains plant pathogens including Fusarium virguliforme and closely related species Fusarium brasiliense, Fusarium crassistipitatum, Fusarium tucumaniae, which are the primary causal agents of soybean sudden death syndrome (SDS), a significant threat to soybean production. In this study, we developed microsatellite markers from a F. virguliforme genome sequence and applied them to a F. virguliforme population collection of 38 isolates from Michigan and four reference strains from other locations. Of the 225 detected microsatellite loci, 108 loci were suitable for primer design, and 12 of the microsatellite markers were determined to be highly polymorphic, amplifying on average 5.7 alleles per locus. Using these markers, F. virguliforme isolates were partitioned into three distinct clusters, but isolates were not grouped based on relatedness of sampling sites. In addition, 11 out of 12 markers were demonstrated to be highly transferrable to other closely related species.     

Structure and evolution of the Fusarium mating type locus: new insights from the Gibberellafujikuroi complex

Mating type genes are central to sexual reproduction and compatibility in Ascomycete fungi. However the "MAT" loci experience unique evolutionary pressures that can result in rapid divergence and enhanced inter-specific gene-flow (lateral gene transfer). In this study, molecular evolution of MAT loci was considered using the genus Fusarium (Teleomorph: Gibberella) as a model. Both MAT1-1 and MAT1-2 "idiomorphs" from eleven species of the Gibberellafujikuroi species complex were sequenced. Molecular evolution of the MAT loci from these heterothallic (self-sterile) species was compared with that of the MAT loci from nine homothallic (self-fertile) species in the Fusariumgraminearum species complex. Although Fusarium has previously been thought to have the same complement of four MAT genes that are found in Neurospora, we found evidence of a novel gene, MAT1-2-3, that may be specific to the Hypocreales. All MAT genes share a similar set of cis-regulatory motifs, although homothallic species might have recruited novel regulatory elements, which could potentially facilitate alternate expression of MAT1-1-1 and MAT1-2-1. FusariumMAT loci displayed evidence consistent with historical lateral gene-flow. Most notably, the MAT1-1 idiomorph of Fusariumsacchari appears to be unrelated to those of other species in the G.fujikuroi complex. In general, FusariumMAT genes are highly divergent. Both positive selection and relaxed selective constraint could account for this phenomenon. However, the extent of both recombination and inter-specific gene-flow in the MAT locus also appears to affect the rate of divergence.     

Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex--F. virguliforme in North America and F. tucumaniae in South America

Soybean sudden-death syndrome has become a serious constraint to commercial production of this crop in North and South America during the past decade. To assess whether the primary etiological agent is panmictic in both hemispheres, morphological and molecular phylogenetic analyses were conducted on strains selected to represent the known pathogenic and genetic diversity of this pathogen. Maximum-parsimony analysis of DNA sequences from the nuclear ribosomal intergenic spacer region and the single copy nuclear gene translation elongation factor 1-α, together with detailed morphological comparisons of conidial features, indicate that SDS of soybean in North and South America is caused by two phylogenetically and morphologically distinct species. Fusarium virguliforme sp. nov., formally known as F. solani f. sp. glycines, is described and illustrated for the SDS pathogen in North America, and F. tucumaniae sp. nov. is proposed for the South American pathogen. The molecular phylogenetic results challenge the forma specialis naming system because pathogenicity to soybean might have evolved convergently in F. tucumaniae and F. virguliforme. Phylogenetic evidence indicates the two SDS pathogens do not share a most recent common ancestor, since F. tucumaniae was resolved as a sister to a pathogen of Phaseolus vulgaris, F. phaseoli comb. nov. All three pathogens appear to have evolutionary origins in the southern hemisphere since they are deeply nested within a South American clade of the F. solani species complex.     

Molecular and other biological evidence supports the recognition of at least three species of brown kiwi

The presence of morphologically crypticlineages within the threatened brown kiwi ofNew Zealand has confounded their taxonomy. Recent genetic studies (Herbert and Daugherty1994; Baker et al. 1995) revealed that atleast two phylogenetic species exist within thebrown kiwi, and suggested that further researchshould resolve the taxonomic problems. In thispaper we extend genetic analyses to includesequences from 58 brown kiwi representing fivephylogenetic lineages for four mitochondrialloci (control region, cytochrome b,ATPase 6 and ATPase 8). Major lineages ofbrown kiwi are shown to be reciprocallymonophyletic, and align with other biologicaldifferences in the ecology, behavior,morphology and parasites of kiwi. BecausemtDNA sequences of major lineages of kiwi arenot evolving in a clocklike manner, we used anew penalized likelihood method withrate-smoothing to date the divergence of NorthIsland brown kiwi and the geographicallyisolated Okarito population (rowi) at about 6.2Mya. These lineages diverged about 8.2 Myafrom the brown kiwi in Fiordland and Haast inthe southern part of the South Island, and arethus older than the species of spotted kiwi(5.8 Mya). Given their distinctness, long-termgeographical isolation, lack of hybridizationin introduced populations, and accumulation ofnew biological characters within theselineages, we hypothesize that reproductiveincompatibilities have probably arisen as well. We therefore recommend that these divergentlineages be formally recognized as fullspecies; Apteryx mantelli should bere-instated for the North Island brown kiwi,A. australis should be restricted to thetokoeka, and a new species A. rowiishould be erected to describe the rowi atOkarito. Tokoeka should be split into at leastthree conservation management units (Haast,Fiordland and Stewart Island [Rakiura]), butfurther research is required to determine theexact relationships and status of theselineages. Further investigation is alsorequired into the genetic structuring of theNorth Island brown kiwi to confirm conservationmanagement units on the North Island.     

Congruence between nuclear and mitochondrial genes in Demospongiae: a new hypothesis for relationships within the G4 clade (Porifera: Demospongiae)

The current morphological classification of the Demospongiae G4 clade was tested using large subunit ribosomal RNA (LSU rRNA) sequences from 119 taxa. Fifty-three mitochondrial cytochrome oxidase 1 (CO1) barcoding sequences were also analysed to test whether the 28S phylogeny could be recovered using an independent gene. This is the largest and most comprehensive study of the Demospongiae G4 clade. The 28S and CO1 genetrees result in congruent clades but conflict with the current morphological classification. The results confirm the polyphyly of Halichondrida, Hadromerida, Dictyonellidae, Axinellidae and Poecilosclerida and show that several of the characters used in morphological classifications are homoplasious. Robust clades are clearly shown and a new hypothesis for relationships of taxa allocated to G4 is proposed.     

The evolution of sperm competition genes: The effect of mating system on levels of genetic variation within and between species

It is widely established that proteins involved in reproduction diverge between species more quickly than other proteins. For male sperm proteins, rapid divergence is believed to be caused by postcopulatory sexual selection and/or sexual conflict. Here, we derive the expected levels of gene diversity within populations and divergence between them for male sperm protein genes evolving by postcopulatory, prezygotic fertility competition, i.e. the function imputed for some sperm and seminal fluid genes. We find that, at the mutation‐selection equilibrium, both gene diversity within species and divergence between them are elevated relative to genes with similar selection coefficients expressed by both sexes. We show that their expected level of diversity is a function of the harmonic mean number of mates per female, which affects the strength of fertility selection stemming from male–male sperm competition. Our predictions provide a null hypothesis for distinguishing between other selective hypotheses accounting for the rapid evolution of male reproductive genes.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

Novel primers for the amplification of nuclear DNA introns in the entomopathogenic nematode Heterorhabditis bacteriophora and their cross-amplification in seven other Heterorhabditis species

We describe 24 novel primers that amplify intron regions in housekeeping and structural genes of Heterorhabditis bacteriophora. The cross-amplification potential of these primers in seven other Heterorhabditis species was determined. The results obtained showed interspecific nucleotide, length and splice site variability in the sequenced introns and for one gene, an intron gain was observed. These primers will be useful tools for studying population genetics, genetic diversity and intron DNA evolution within the genus Heterorhabditis and other genera of rhabditid nematodes.     

Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes

The segregation of killer cell immunoglobulin-like receptor ( KIR) genes was determined for a panel of 21 Caucasoid families: 23 different KIR gene patterns were found and could be assigned to combinations of 16 different haplotypes. Four loci were held in common by all haplotypes: KIR2DL4, KIR3DL2, the putative pseudogene KIR3DL3 and KIR2DL2/KIR2DL3, the latter likely being alleles of one gene. Group A haplotypes, which have a unique combination of seven KIR genes, were found at 80% frequency in the family panel, the polygenic group B haplotypes at 65% frequency. KIR gene segregation was fully determined for the nine group B haplotypes, which occurred at highest frequencies in both the family panel and a panel of unrelated individuals. The group B haplotypes carried between seven and 11 KIR genes and encoded inhibitory KIR for one, two, or all three major HLA class I epitopes. Analysis of human leucocyte antigen (HLA) class I genotypes revealed that most, but not all, individuals possess an inhibitory KIR for a self HLA class I epitope. The number of stimulatory KIR genes in group B haplotypes varied considerably between one and five. The data show that group B haplotypes possess a broad spectrum of KIR gene patterns, which is largely complementary to the KIR gene set of group A haplotypes. The results suggest that rapid diversification of group B haplotypes is the result of pathogen-mediated selection for KIR genotypes that have more than the set of KIR genes provided by the group A haplotype.     

The nuclear export signal within the E4orf6 protein of adenovirus type 5 supports virus replication and cytoplasmic accumulation of viral mRNA

During the late phase of adenovirus infection, viral mRNA is efficiently transported from the nucleus to the cytoplasm while most cellular mRNA species are retained in the nucleus. Two viral proteins, E1B-55 kDa and E4orf6, are both necessary for these effects. The E4orf6 protein of adenovirus type 5 binds and relocalizes E1B-55 kDa, and the complex of the two proteins was previously shown to shuttle continuously between the nucleus and cytoplasm. Nucleocytoplasmic transport of the complex is achieved by a nuclear export signal (NES) within E4orf6. Mutation of this signal sequence severely reduces the ability of the E1B-55 kDa-E4orf6 complex to leave the nucleus. Here, we examined the role of functional domains within E4orf6 during virus infection. E4orf6 or mutants derived from it were transiently expressed, followed by infection with recombinant adenovirus lacking the E4 region and determination of virus yield. An arginine-rich putative alpha helix near the carboxy terminus of E4orf6 contributes to E1B-55 kDa binding and relocalization as well as to the synthesis of viral DNA, mRNA, and proteins. Further mutational analysis revealed that mutation of the NES within E4orf6 considerably reduces its ability to support virus production. The same effect was observed when nuclear export was blocked with a competitor. Further, a functional NES within E4orf6 contributed to the efficiency of late virus protein synthesis and viral DNA replication, as well as total and cytoplasmic accumulation of viral late mRNA. Our data support the view that NES-mediated nucleocytoplasmic shuttling strongly enhances most, if not all, intracellular activities of E4orf6 during the late phase of adenovirus infection.     

Markers linked to vegetative incompatibility (vic) genes and a region of high heterogeneity and reduced recombination near the mating type locus (MAT) in Cryphonectria parasitica

To find markers linked to vegetative incompatibility (vic) genes in the chestnut blight fungus, Cryphonectria parasitica, we constructed a preliminary linkage map. In general, this map is characterized by low levels of polymorphism, as evident from the more than 24 linkage groups observed, compared to seven expected from electrophoretic karyotyping. Nonetheless, we found markers closely linked to two vic genes (vic1 and vic2) making them candidates for positional cloning. Two markers were found to be linked to vic2: one cosegregated with vic2, i.e., it is 0.0 cM from vic2, the other was at a distance of 4.5 cM; a single marker was found 4.0 cM from vic1. The closest markers linked to three other vic genes (vic4, vic6, and vic7) were >15 cM away; additional markers are needed before efficient positional cloning of these three vic genes can be realized. In contrast to the low levels of polymorphism observed across most of the C. parasitica genome, the linkage group containing the MAT locus appears to harbor an extremely high level of RAPD heterogeneity and reduced recombination. Markers within this highly heterogeneous region are in linkage disequilibrium in some natural populations; however, recombination is clearly evident between this region and the MAT locus.     

Mining the Cicer arietinum genome for the mildew locus O (Mlo) gene family and comparative evolutionary analysis of the Mlo genes from Medicago truncatula and some other plant species

Journal of Plant Research - The mildew locus O (Mlo) gene family is ubiquitous in land plants. Some members of this gene family are involved in negative regulation of powdery mildew resistance,...     

Phylogenetic relationships between members of the crucifer pathogenic Leptosphaeria maculans species complex as shown by mating type (MAT1-2), actin, and beta-tubulin sequences

The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species differing in specificity and pathogenicity on Brassica napus. Twenty-eight isolates were investigated and compared to 20 other species of the Pleosporales order. Sequences of the mating type MAT1-2 (23), fragments of actin (48) and beta-tubulin (45) genes were determined and used for phylogenetic analyses inferred by maximum parsimony, distance, maximum likelihood, and Bayesian approaches. These different approaches using single genes essentially confirmed findings using concatanated sequences. L. maculans formed a monophyletic group separate from Leptosphaeria biglobosa. The L. biglobosa clade encompasses five sub-clades; this finding is consistent with classification made previously on the basis of internal-transcribed sequences of the ribosomal DNA repeat. The propensity for purifying and neutral evolution of the three genes was determined using sliding window analysis, a technique not previously applied to genes of filamentous fungi. For members of the L. maculans species complex, this approach showed that in comparison to actin and beta-tubulin, exonic sequences of MAT1-2 were more diverse and appeared to evolve at a faster rate. However, different regions of MAT1-2 displayed different degrees of sequence conservation. The more conserved upstream region (including the High Mobility Group domain) may be better suited for interspecies differentiation, while the more diverse downstream region is more appropriate for intraspecies comparisons.     

Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.     

Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells,fibroblasts, alveolar type II cells,airway epithelial cells and lymphocytes

Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC‐specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8⁺ T cells from bronchial lymph nodes (T‐BL) and CD8⁺ T cells from lungs (T‐LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up‐regulated and 70% down‐regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over‐expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types.     

Allopolyploid speciation and ongoing backcrossing between diploid progenitor and tetraploid progeny lineages in the <Emphasis Type="Italic">Achillea millefolium</Emphasis>species complex: analyses of single-copy nuclear genes and genomic AFLP

Background  

In the flowering plants, many polyploid species complexes display evolutionary radiation. This could be facilitated by gene flow between otherwise separate evolutionary lineages in contact zones. Achillea collina is a widespread tetraploid species within the Achillea millefolium polyploid complex (Asteraceae-Anthemideae). It is morphologically intermediate between the relic diploids, A. setacea-2x in xeric and A. asplenifolia-2x in humid habitats, and often grows in close contact with either of them. By analyzing DNA sequences of two single-copy nuclear genes and the genomic AFLP data, we assess the allopolyploid origin of A. collina-4x from ancestors corresponding to A. setacea-2x and A. asplenifolia-2x, and the ongoing backcross introgression between these diploid progenitor and tetraploid progeny lineages.