Species-specific duplications driving the recent expansion of NBS-LRR genes in five Rosaceae species

Background

Disease resistance (R) genes from different Rosaceae species have been identified by map-based cloning for resistance breeding. However, there are few reports describing the pattern of R-gene evolution in Rosaceae species because several Rosaceae genome sequences have only recently become available.

Results

Since most disease resistance genes encode NBS-LRR proteins, we performed a systematic genome-wide survey of NBS-LRR genes between five Rosaceae species, namely Fragaria vesca (strawberry), Malus × domestica (apple), Pyrus bretschneideri (pear), Prunus persica (peach) and Prunus mume (mei) which contained 144, 748, 469, 354 and 352 NBS-LRR genes, respectively. A high proportion of multi-genes and similar Ks peaks (Ks = 0.1- 0.2) of gene families in the four woody genomes were detected. A total of 385 species-specific duplicate clades were observed in the phylogenetic tree constructed using all 2067 NBS-LRR genes. High percentages of NBS-LRR genes derived from species-specific duplication were found among the five genomes (61.81% in strawberry, 66.04% in apple, 48.61% in pear, 37.01% in peach and 40.05% in mei). Furthermore, the Ks and Ka/Ks values of TIR-NBS-LRR genes (TNLs) were significantly greater than those of non-TIR-NBS-LRR genes (non-TNLs), and most of the NBS-LRRs had Ka/Ks ratios less than 1, suggesting that they were evolving under a subfunctionalization model driven by purifying selection.

Conclusions

Our results indicate that recent duplications played an important role in the evolution of NBS-LRR genes in the four woody perennial Rosaceae species. Based on the phylogenetic tree produced, it could be inferred that species-specific duplication has mainly contributed to the expansion of NBS-LRR genes in the five Rosaceae species. In addition, the Ks and Ka/Ks ratios suggest that the rapidly evolved TNLs have different evolutionary patterns to adapt to different pathogens compared with non-TNL resistant genes.

Electronic supplementary material

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A genetic map of cassava (Manihot esculenta Crantz) with integrated physical mapping of immunity-related genes

Background

Cassava, Manihot esculenta Crantz, is one of the most important crops world-wide representing the staple security for more than one billion of people. The development of dense genetic and physical maps, as the basis for implementing genetic and molecular approaches to accelerate the rate of genetic gains in breeding program represents a significant challenge. A reference genome sequence for cassava has been made recently available and community efforts are underway for improving its quality. Cassava is threatened by several pathogens, but the mechanisms of defense are far from being understood. Besides, there has been a lack of information about the number of genes related to immunity as well as their distribution and genomic organization in the cassava genome.

Results

A high dense genetic map of cassava containing 2,141 SNPs has been constructed. Eighteen linkage groups were resolved with an overall size of 2,571 cM and an average distance of 1.26 cM between markers. More than half of mapped SNPs (57.4%) are located in coding sequences. Physical mapping of scaffolds of cassava whole genome sequence draft using the mapped markers as anchors resulted in the orientation of 687 scaffolds covering 45.6% of the genome. One hundred eighty nine new scaffolds are anchored to the genetic cassava map leading to an extension of the present cassava physical map with 30.7 Mb. Comparative analysis using anchor markers showed strong co-linearity to previously reported cassava genetic and physical maps. In silico based searching for conserved domains allowed the annotation of a repertory of 1,061 cassava genes coding for immunity-related proteins (IRPs). Based on physical map of the corresponding sequencing scaffolds, unambiguous genetic localization was possible for 569 IRPs.

Conclusions

This is the first study reported so far of an integrated high density genetic map using SNPs with integrated genetic and physical localization of newly annotated immunity related genes in cassava. These data build a solid basis for future studies to map and associate markers with single loci or quantitative trait loci for agronomical important traits. The enrichment of the physical map with novel scaffolds is in line with the efforts of the cassava genome sequencing consortium.

Electronic supplementary material

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The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Background

Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens.

Results

The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation.

Conclusions

Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.

Electronic supplementary material

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Evolutionary mode and functional divergence of vertebrate NMDA receptor subunit 2 genes

Background

Ionotropic glutamate receptors in the central nervous system play a major role in numerous brain functions including learning and memory in many vertebrate species. NR2 subunits have been regarded as rate-limiting molecules in controlling the optimal N-methyl-D-aspartate (NMDA) receptor''s coincidence-detection property and subsequent learning and memory function across multi-species. However, its evolutionary mode among vertebrate species remains unclear.

Results

With extensive analysis of phylogeny, exon structure, protein domain, paralogon and synteny, we demonstrated that two-round genome duplication generated quartet GRIN2 genes and the third-round fish-specific genome duplication generated extra copies of fish GRIN2 genes. In addition, in-depth investigation has enabled the identification of three novel genes, GRIN2C_Gg, GRIN2D-1_Ol and GRIN2D-2_Tr in the chicken, medaka and fugu genome, respectively. Furthermore, we showed functional divergence of NR2 genes mostly occurred at the first-round duplication, amino acid residues located at the N-terminal Lig_chan domain were responsible for type I functional divergence between these GRIN2 subfamilies and purifying selection has been the prominent natural pressure operating on these diversified GRIN2 genes.

Conclusion and Significance

These findings provide intriguing subjects for testing the 2R and 3R hypothesis and we expect it could provide new insights into the underlying evolution mechanisms of cognition in vertebrate.     

Using the taxon-specific genes for the taxonomic classification of bacterial genomes

Background

The correct taxonomic assignment of bacterial genomes is a primary and challenging task. With the availability of whole genome sequences, the gene content based approaches appear promising in inferring the bacterial taxonomy. The complete genome sequencing of a bacterial genome often reveals a substantial number of unique genes present only in that genome which can be used for its taxonomic classification.

Results

In this study, we have proposed a comprehensive method which uses the taxon-specific genes for the correct taxonomic assignment of existing and new bacterial genomes. The taxon-specific genes identified at each taxonomic rank have been successfully used for the taxonomic classification of 2,342 genomes present in the NCBI genomes, 36 newly sequenced genomes, and 17 genomes for which the complete taxonomy is not yet known. This approach has been implemented for the development of a tool ‘Microtaxi’ which can be used for the taxonomic assignment of complete bacterial genomes.

Conclusion

The taxon-specific gene based approach provides an alternate valuable methodology to carry out the taxonomic classification of newly sequenced or existing bacterial genomes.

Electronic supplementary material

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Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products

Background

In recent years, the genus Pestalotiopsis is receiving increasing attention, not only because of its economic impact as a plant pathogen but also as a commonly isolated endophyte which is an important source of bioactive natural products. Pestalotiopsis fici Steyaert W106-1/CGMCC3.15140 as an endophyte of tea produces numerous novel secondary metabolites, including chloropupukeananin, a derivative of chlorinated pupukeanane that is first discovered in fungi. Some of them might be important as the drug leads for future pharmaceutics.

Results

Here, we report the genome sequence of the endophytic fungus of tea Pestalotiopsis fici W106-1/CGMCC3.15140. The abundant carbohydrate-active enzymes especially significantly expanding pectinases allow the fungus to utilize the limited intercellular nutrients within the host plants, suggesting adaptation of the fungus to endophytic lifestyle. The P. fici genome encodes a rich set of secondary metabolite synthesis genes, including 27 polyketide synthases (PKSs), 12 non-ribosomal peptide synthases (NRPSs), five dimethylallyl tryptophan synthases, four putative PKS-like enzymes, 15 putative NRPS-like enzymes, 15 terpenoid synthases, seven terpenoid cyclases, seven fatty-acid synthases, and five hybrids of PKS-NRPS. The majority of these core enzymes distributed into 74 secondary metabolite clusters. The putative Diels-Alderase genes have undergone expansion.

Conclusion

The significant expansion of pectinase encoding genes provides essential insight in the life strategy of endophytes, and richness of gene clusters for secondary metabolites reveals high potential of natural products of endophytic fungi.

Electronic supplementary material

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Deletions of chromosomal regulatory boundaries are associated with congenital disease

Background

Recent data from genome-wide chromosome conformation capture analysis indicate that the human genome is divided into conserved megabase-sized self-interacting regions called topological domains. These topological domains form the regulatory backbone of the genome and are separated by regulatory boundary elements or barriers. Copy-number variations can potentially alter the topological domain architecture by deleting or duplicating the barriers and thereby allowing enhancers from neighboring domains to ectopically activate genes causing misexpression and disease, a mutational mechanism that has recently been termed enhancer adoption.

Results

We use the Human Phenotype Ontology database to relate the phenotypes of 922 deletion cases recorded in the DECIPHER database to monogenic diseases associated with genes in or adjacent to the deletions. We identify combinations of tissue-specific enhancers and genes adjacent to the deletion and associated with phenotypes in the corresponding tissue, whereby the phenotype matched that observed in the deletion. We compare this computationally with a gene-dosage pathomechanism that attempts to explain the deletion phenotype based on haploinsufficiency of genes located within the deletions. Up to 11.8% of the deletions could be best explained by enhancer adoption or a combination of enhancer adoption and gene-dosage effects.

Conclusions

Our results suggest that enhancer adoption caused by deletions of regulatory boundaries may contribute to a substantial minority of copy-number variation phenotypes and should thus be taken into account in their medical interpretation.

Electronic supplementary material

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Distance-based assessment of the localization of functional annotations in 3D genome reconstructions

Background

Recent studies used the contact data or three-dimensional (3D) genome reconstructions from Hi-C (chromosome conformation capture with next-generation sequencing) to assess the co-localization of functional genomic annotations in the nucleus. These analyses dichotomized data point pairs belonging to a functional annotation as “close” or “far” based on some threshold and then tested for enrichment of “close” pairs. We propose an alternative approach that avoids dichotomization of the data and instead directly estimates the significance of distances within the 3D reconstruction.

Results

We applied this approach to 3D genome reconstructions for Plasmodium falciparum, the causative agent of malaria, and Saccharomyces cerevisiae and compared the results to previous approaches. We found significant 3D co-localization of centromeres, telomeres, virulence genes, and several sets of genes with developmentally regulated expression in P. falciparum; and significant 3D co-localization of centromeres and long terminal repeats in S. cerevisiae. Additionally, we tested the experimental observation that telomeres form three to seven clusters in P. falciparum and S. cerevisiae. Applying affinity propagation clustering to telomere coordinates in the 3D reconstructions yielded six telomere clusters for both organisms.

Conclusions

Distance-based assessment replicated key findings, while avoiding dichotomization of the data (which previously yielded threshold-sensitive results).

Electronic supplementary material

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Fungal artificial chromosomes for mining of the fungal secondary metabolome

Background

With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30–80 kb in size, breakthrough techniques are needed to characterize this SM wealth.

Results

Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery.

Conclusion

The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

Electronic supplementary material

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A novel model of double replications and random loss accounts for rearrangements in the Mitogenome of Samariscus latus (Teleostei: Pleuronectiformes)

Background

Although more than one thousand complete mitochondrial DNA (mtDNA) sequences have been determined in teleostean fishes, only a few gene rearrangements have been observed, and genome-scale rearrangements are even rarer. However, flatfishes (Pleuronectiformes) have been identified as having diverse types of mitochondrial gene rearrangements. It has been reported that tongue soles and the blue flounder mitogenomes exhibit different types of large-scale gene rearrangements.

Results

In the present study, the complete mitochondrial genome of another flatfish, Samariscus latus, was sequenced, and genome-scale rearrangements were observed. The genomic features of this flounder are different from those of any other studied vertebrates, including flatfish species too. The mitogenome of S. latus is characterized by the duplication and translocation of the control region (CR). The genes located between the two CRs are divided into two clusters in which their relative orders are maintained.

Conclusions

We propose a “Double Replications and Random Loss” model to explain the rearrangement events in S. latus mitogenome. This model consists of the following steps. First, the CR was duplicated and translocated. Subsequently, double replications of the mitogenome were successively initiated from the two CRs, leading to the duplication of the genes between the two CRs. Finally, one of each pair of duplicated genes was lost in a random event.

Electronic supplementary material

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Genome sequence of Erinnyis ello granulovirus (ErelGV), a natural cassava hornworm pesticide and the first sequenced sphingid-infecting betabaculovirus

Background

Cassava (Manihot esculenta) is the basic source for dietary energy of 500 million people in the world. In Brazil, Erinnyis ello ello (Lepidoptera: Sphingidae) is a major pest of cassava crops and a bottleneck for its production. In the 1980s, a naturally occurring baculovirus was isolated from E. ello larva and successfully applied as a bio-pesticide in the field. Here, we described the structure, the complete genome sequence, and the phylogenetic relationships of the first sphingid-infecting betabaculovirus.

Results

The baculovirus isolated from the cassava hornworm cadavers is a betabaculovirus designated Erinnyis ello granulovirus (ErelGV). The 102,759 bp long genome has a G + C content of 38.7%. We found 130 putative ORFs coding for polypeptides of at least 50 amino acid residues. Only eight genes were found to be unique. ErelGV is closely related to ChocGV and PiraGV isolates. We did not find typical homologous regions and cathepsin and chitinase homologous genes are lacked. The presence of he65 and p43 homologous genes suggests horizontal gene transfer from Alphabaculovirus. Moreover, we found a nucleotide metabolism-related gene and two genes that could be acquired probably from Densovirus.

Conclusions

The ErelGV represents a new virus species from the genus Betabaculovirus and is the closest relative of ChocGV. It contains a dUTPase-like, a he65-like, p43-like genes, which are also found in several other alpha- and betabaculovirus genomes, and two Densovirus-related genes. Importantly, recombination events between insect viruses from unrelated families and genera might drive baculovirus genomic evolution.

Electronic supplementary material

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Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Background

In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results

Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions

The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Electronic supplementary material

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