Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome

Background

It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Results

We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.

Conclusions

Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.     

GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists

Background

Although expression microarrays have become a standard tool used by biologists, analysis of data produced by microarray experiments may still present challenges. Comparison of data from different platforms, organisms, and labs may involve complicated data processing, and inferring relationships between genes remains difficult.

Results

S TAR N ET 2 is a new web-based tool that allows post hoc visual analysis of correlations that are derived from expression microarray data. S TAR N ET 2 facilitates user discovery of putative gene regulatory networks in a variety of species (human, rat, mouse, chicken, zebrafish, Drosophila, C. elegans, S. cerevisiae, Arabidopsis and rice) by graphing networks of genes that are closely co-expressed across a large heterogeneous set of preselected microarray experiments. For each of the represented organisms, raw microarray data were retrieved from NCBI's Gene Expression Omnibus for a selected Affymetrix platform. All pairwise Pearson correlation coefficients were computed for expression profiles measured on each platform, respectively. These precompiled results were stored in a MySQL database, and supplemented by additional data retrieved from NCBI. A web-based tool allows user-specified queries of the database, centered at a gene of interest. The result of a query includes graphs of correlation networks, graphs of known interactions involving genes and gene products that are present in the correlation networks, and initial statistical analyses. Two analyses may be performed in parallel to compare networks, which is facilitated by the new H EAT S EEKER module.

Conclusion

S TAR N ET 2 is a useful tool for developing new hypotheses about regulatory relationships between genes and gene products, and has coverage for 10 species. Interpretation of the correlation networks is supported with a database of previously documented interactions, a test for enrichment of Gene Ontology terms, and heat maps of correlation distances that may be used to compare two networks. The list of genes in a S TAR N ET network may be useful in developing a list of candidate genes to use for the inference of causal networks. The tool is freely available at http://vanburenlab.medicine.tamhsc.edu/starnet2.html, and does not require user registration.     

Conserved features of cohesin binding along fission yeast chromosomes

Background

Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.

Results

Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.

Conclusions

P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.     

Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species

Background

Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group.

Results

Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes - genes present in more than one strain but not in all strains - was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence.

Conclusions

Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan-genome guarantees the species a quick and economical response to diverse environments.     

An AFLP-based genetic linkage map of Plasmodium chabaudi chabaudi

Background

Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.

Methods

The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers (used as chromosomal anchors) were organized into linkage groups using Map Manager software.

Results

614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, Plasmodium falciparum

Conclusion

The P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.     

Divergent evolution and molecular adaptation in the Drosophila odorant-binding protein family: inferences from sequence variation at the OS-E and OS-F genes

Background

Lineage-specific genes, the genes that are restricted to a limited subset of related organisms, may be important in adaptation. In parasitic organisms, lineage-specific gene products are possible targets for vaccine development or therapeutics when these genes are absent from the host genome.

Results

In this study, we utilized comparative approaches based on a phylogenetic framework to characterize lineage-specific genes in the parasitic protozoan phylum Apicomplexa. Genes from species in two major apicomplexan genera, Plasmodium and Theileria, were categorized into six levels of lineage specificity based on a nine-species phylogeny. In both genera, lineage-specific genes tend to have a higher level of sequence divergence among sister species. In addition, species-specific genes possess a strong codon usage bias compared to other genes in the genome. We found that a large number of genus- or species-specific genes are putative surface antigens that may be involved in host-parasite interactions. Interestingly, the two parasite lineages exhibit several notable differences. In Plasmodium, the (G + C) content at the third codon position increases with lineage specificity while Theileria shows the opposite trend. Surface antigens in Plasmodium are species-specific and mainly located in sub-telomeric regions. In contrast, surface antigens in Theileria are conserved at the genus level and distributed across the entire lengths of chromosomes.

Conclusion

Our results provide further support for the model that gene duplication followed by rapid divergence is a major mechanism for generating lineage-specific genes. The result that many lineage-specific genes are putative surface antigens supports the hypothesis that lineage-specific genes could be important in parasite adaptation. The contrasting properties between the lineage-specific genes in two major apicomplexan genera indicate that the mechanisms of generating lineage-specific genes and the subsequent evolutionary fates can differ between related parasite lineages. Future studies that focus on improving functional annotation of parasite genomes and collection of genetic variation data at within- and between-species levels will be important in facilitating our understanding of parasite adaptation and natural selection.     

Fusion to chicken C3d enhances the immunogenicity of the M2 protein of avian influenza virus

Background

Infectious hematopoietic necrosis virus (IHNV) is the type species of the genus Novirhabdovirus, within the family Rhabdoviridae, infecting several species of wild and hatchery reared salmonids. Similar to other rhabdoviruses, IHNV has a linear single-stranded, negative-sense RNA genome of approximately 11,000 nucleotides. The IHNV genome encodes six genes; the nucleocapsid, phosphoprotein, matrix protein, glycoprotein, non-virion protein and polymerase protein genes, respectively. This study describes molecular characterization of the virulent IHNV strain 220-90, belonging to the M genogroup, and its phylogenetic relationships with available sequences of IHNV isolates worldwide.

Results

The complete genomic sequence of IHNV strain 220-90 was determined from the DNA of six overlapping clones obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of 220-90 comprises 11,133 nucleotides (GenBank GQ413939) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. An additional uracil nucleotide was found at the end of the 5'-trailer region, which was not reported before in other IHNV strains. The first 15 of the 16 nucleotides at the 3'- and 5'-termini of the genome are complementary, and the first 4 nucleotides at 3'-ends of the IHNV are identical to other novirhadoviruses. Sequence homology and phylogenetic analysis of the glycoprotein genes show that 220-90 strain is 97% identical to most of the IHNV strains. Comparison of the virulent 220-90 genomic sequences with less virulent WRAC isolate shows more than 300 nucleotides changes in the genome, which doesn't allow one to speculate putative residues involved in the virulence of IHNV.

Conclusion

We have molecularly characterized one of the well studied IHNV isolates, 220-90 of genogroup M, which is virulent for rainbow trout, and compared phylogenetic relationship with North American and other strains. Determination of the complete nucleotide sequence is essential for future studies on pathogenesis of IHNV using a reverse genetics approach and developing efficient control strategies.     

Sequence and structure of Brassica rapa chromosome A3

Background

The species Brassica rapa includes important vegetable and oil crops. It also serves as an excellent model system to study polyploidy-related genome evolution because of its paleohexaploid ancestry and its close evolutionary relationships with Arabidopsis thaliana and other Brassica species with larger genomes. Therefore, its genome sequence will be used to accelerate both basic research on genome evolution and applied research across the cultivated Brassica species.

Results

We have determined and analyzed the sequence of B. rapa chromosome A3. We obtained 31.9 Mb of sequences, organized into nine contigs, which incorporated 348 overlapping BAC clones. Annotation revealed 7,058 protein-coding genes, with an average gene density of 4.6 kb per gene. Analysis of chromosome collinearity with the A. thaliana genome identified conserved synteny blocks encompassing the whole of the B. rapa chromosome A3 and sections of four A. thaliana chromosomes. The frequency of tandem duplication of genes differed between the conserved genome segments in B. rapa and A. thaliana, indicating differential rates of occurrence/retention of such duplicate copies of genes. Analysis of 'ancestral karyotype' genome building blocks enabled the development of a hypothetical model for the derivation of the B. rapa chromosome A3.

Conclusions

We report the near-complete chromosome sequence from a dicotyledonous crop species. This provides an example of the complexity of genome evolution following polyploidy. The high degree of contiguity afforded by the clone-by-clone approach provides a benchmark for the performance of whole genome shotgun approaches presently being applied in B. rapa and other species with complex genomes.     

Segregate or cooperate- a study of the interaction between two species of Dictyostelium

Background

Plastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. Although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive.

Results

Here we show an extended phylogenetic analysis including novel gnd gene sequences from Excavata and Glaucophyta. Our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. The Diplonema gene was related to cytosol-type genes in red algae and Opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. Statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. In addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in Euglenida were robustly monophyletic with Stramenopiles, and this monophyletic clade was moderately separated from those of red algae. These data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses.

Conclusion

We propose an evolutionary scenario in which plastid-lacking Excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene.     

In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals

Background

Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals.

Results

The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations.

Conclusion

The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.