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July 7, 2019

Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.).

Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n?=?2x?=?50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes.The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides.Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation suggesting its recent origin and/or intensive homogenisation processes. The dense methylation of units indicates that powerful epigenetic mechanisms have evolved in this group of fish to silence amplified genes. We discuss how the higher-order repeat structures impact on homogenisation of 5S rDNA in the genome.

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July 7, 2019

Divergent and convergent modes of interaction between wheat and Puccinia graminis f. sp. tritici isolates revealed by the comparative gene co-expression network and genome analyses.

Two opposing evolutionary constraints exert pressure on plant pathogens: one to diversify virulence factors in order to evade plant defenses, and the other to retain virulence factors critical for maintaining a compatible interaction with the plant host. To better understand how the diversified arsenals of fungal genes promote interaction with the same compatible wheat line, we performed a comparative genomic analysis of two North American isolates of Puccinia graminis f. sp. tritici (Pgt).The patterns of inter-isolate divergence in the secreted candidate effector genes were compared with the levels of conservation and divergence of plant-pathogen gene co-expression networks (GCN) developed for each isolate. Comprative genomic analyses revealed substantial level of interisolate divergence in effector gene complement and sequence divergence. Gene Ontology (GO) analyses of the conserved and unique parts of the isolate-specific GCNs identified a number of conserved host pathways targeted by both isolates. Interestingly, the degree of inter-isolate sub-network conservation varied widely for the different host pathways and was positively associated with the proportion of conserved effector candidates associated with each sub-network. While different Pgt isolates tended to exploit similar wheat pathways for infection, the mode of plant-pathogen interaction varied for different pathways with some pathways being associated with the conserved set of effectors and others being linked with the diverged or isolate-specific effectors.Our data suggest that at the intra-species level pathogen populations likely maintain divergent sets of effectors capable of targeting the same plant host pathways. This functional redundancy may play an important role in the dynamic of the “arms-race” between host and pathogen serving as the basis for diverse virulence strategies and creating conditions where mutations in certain effector groups will not have a major effect on the pathogen’s ability to infect the host.

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July 7, 2019

Population genomics of picophytoplankton unveils novel chromosome hypervariability.

Tiny photosynthetic microorganisms that form the picoplankton (between 0.3 and 3 µm in diameter) are at the base of the food web in many marine ecosystems, and their adaptability to environmental change hinges on standing genetic variation. Although the genomic and phenotypic diversity of the bacterial component of the oceans has been intensively studied, little is known about the genomic and phenotypic diversity within each of the diverse eukaryotic species present. We report the level of genomic diversity in a natural population of Ostreococcus tauri (Chlorophyta, Mamiellophyceae), the smallest photosynthetic eukaryote. Contrary to the expectations of clonal evolution or cryptic species, the spectrum of genomic polymorphism observed suggests a large panmictic population (an effective population size of 1.2 × 10(7)) with pervasive evidence of sexual reproduction. De novo assemblies of low-coverage chromosomes reveal two large candidate mating-type loci with suppressed recombination, whose origin may pre-date the speciation events in the class Mamiellophyceae. This high genetic diversity is associated with large phenotypic differences between strains. Strikingly, resistance of isolates to large double-stranded DNA viruses, which abound in their natural environment, is positively correlated with the size of a single hypervariable chromosome, which contains 44 to 156 kb of strain-specific sequences. Our findings highlight the role of viruses in shaping genome diversity in marine picoeukaryotes.

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July 7, 2019

Maize defective kernel mutant generated by insertion of a Ds element in a gene encoding a highly conserved TTI2 cochaperone.

We have used the newly engineered transposable element Dsg to tag a gene that gives rise to a defective kernel (dek) phenotype. Dsg requires the autonomous element Ac for transposition. Upon excision, it leaves a short DNA footprint that can create in-frame and frameshift insertions in coding sequences. Therefore, we could create alleles of the tagged gene that confirmed causation of the dek phenotype by the Dsg insertion. The mutation, designated dek38-Dsg, is embryonic lethal, has a defective basal endosperm transfer (BETL) layer, and results in a smaller seed with highly underdeveloped endosperm. The maize dek38 gene encodes a TTI2 (Tel2-interacting protein 2) molecular cochaperone. In yeast and mammals, TTI2 associates with two other cochaperones, TEL2 (Telomere maintenance 2) and TTI1 (Tel2-interacting protein 1), to form the triple T complex that regulates DNA damage response. Therefore, we cloned the maize Tel2 and Tti1 homologs and showed that TEL2 can interact with both TTI1 and TTI2 in yeast two-hybrid assays. The three proteins regulate the cellular levels of phosphatidylinositol 3-kinase-related kinases (PIKKs) and localize to the cytoplasm and the nucleus, consistent with known subcellular locations of PIKKs. dek38-Dsg displays reduced pollen transmission, indicating TTI2’s importance in male reproductive cell development.

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July 7, 2019

MYB transcription factor gene involved in sex determination in Asparagus officinalis.

Dioecy is a plant mating system in which individuals of a species are either male or female. Although many flowering plants evolved independently from hermaphroditism to dioecy, the molecular mechanism underlying this transition remains largely unknown. Sex determination in the dioecious plant Asparagus officinalis is controlled by X and Y chromosomes; the male and female karyotypes are XY and XX, respectively. Transcriptome analysis of A. officinalis buds showed that a MYB-like gene, Male Specific Expression 1 (MSE1), is specifically expressed in males. MSE1 exhibits tight linkage with the Y chromosome, specific expression in early anther development and loss of function on the X chromosome. Knockout of the MSE1 orthologue in Arabidopsis induces male sterility. Thus, MSE1 acts in sex determination in A. officinalis.© 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

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July 7, 2019

Genome sequencing reveals the origin of the allotetraploid Arabidopsis suecica.

Polyploidy is an example of instantaneous speciation when it involves the formation of a new cytotype that is incompatible with the parental species. Because new polyploid individuals are likely to be rare, establishment of a new species is unlikely unless polyploids are able to reproduce through self-fertilization (selfing), or asexually. Conversely, selfing (or asexuality) makes it possible for polyploid species to originate from a single individual-a bona fide speciation event. The extent to which this happens is not known. Here, we consider the origin of Arabidopsis suecica, a selfing allopolyploid between Arabidopsis thaliana and Arabidopsis arenosa, which has hitherto been considered to be an example of a unique origin. Based on whole-genome re-sequencing of 15 natural A. suecica accessions, we identify ubiquitous shared polymorphism with the parental species, and hence conclusively reject a unique origin in favor of multiple founding individuals. We further estimate that the species originated after the last glacial maximum in Eastern Europe or central Eurasia (rather than Sweden, as the name might suggest). Finally, annotation of the self-incompatibility loci in A. suecica revealed that both loci carry non-functional alleles. The locus inherited from the selfing A. thaliana is fixed for an ancestral non-functional allele, whereas the locus inherited from the outcrossing A. arenosa is fixed for a novel loss-of-function allele. Furthermore, the allele inherited from A. thaliana is predicted to transcriptionally silence the allele inherited from A. arenosa, suggesting that loss of self-incompatibility may have been instantaneous.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Recombination-dependent replication and gene conversion homogenize repeat sequences and diversify plastid genome structure

There is a misinterpretation in the literature regarding the variable orientation of the small single copy region of plastid genomes (plastomes). The common phenomenon of small and large single copy inversion, hypothesized to occur through intramolecular recombination between inverted repeats (IR) in a circular, single unit-genome, in fact, more likely occurs through recombination-dependent replication (RDR) of linear plastome templates. If RDR can be primed through both intra- and intermolecular recombination, then this mechanism could not only create inversion isomers of so-called single copy regions, but also an array of alternative sequence arrangements.We used Illumina paired-end and PacBio single-molecule real-time (SMRT) sequences to characterize repeat structure in the plastome of Monsonia emarginata (Geraniaceae). We used OrgConv and inspected nucleotide alignments to infer ancestral nucleotides and identify gene conversion among repeats and mapped long (>1 kb) SMRT reads against the unit-genome assembly to identify alternative sequence arrangements.Although M. emarginata lacks the canonical IR, we found that large repeats (>1 kilobase; kb) represent ~22% of the plastome nucleotide content. Among the largest repeats (>2 kb), we identified GC-biased gene conversion and mapping filtered, long SMRT reads to the M. emarginata unit-genome assembly revealed alternative, substoichiometric sequence arrangements.We offer a model based on RDR and gene conversion between long repeated sequences in the M. emarginata plastome and provide support that both intra-and intermolecular recombination between large repeats, particularly in repeat-rich plastomes, varies unit-genome structure while homogenizing the nucleotide sequence of repeats.© 2017 Botanical Society of America.

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July 7, 2019

A novel inversion in the chloroplast genome of marama (Tylosema esculentum).

Tylosema esculentum (marama bean) is being developed as a possible crop for resource-poor farmers in arid regions of Southern Africa. As part of the molecular characterization of this species, the chloroplast genome has been assembled from next-generation sequencing using both Illumina and Pac-Bio data. The genome is of typical organization with a large single-copy region and a small single-copy region separated by a pair of inverted repeats and covers 161537 bp. It contains a unique inversion not present in any other legumes, even in the closest relatives for which the complete chloroplast genome is available, and two complete copies of the ycf1 gene. These data extend the range of variability of legume chloroplast genomes. The sequencing of multiple individuals has identified two different chloroplast genomes which were geographically separated. The current sampling is limited so that the extent of the intraspecific variation is still to be determined, leaving open the question of legume chloroplast genomes adapted to particular arid environments.© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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July 7, 2019

Molecules to ecosystems: Actinomycete natural products in situ.

Actinomycetes, filamentous actinobacteria found in numerous ecosystems around the globe, produce a wide range of clinically useful natural products (NP). In natural environments, actinomycetes live in dynamic communities where environmental cues and ecological interactions likely influence NP biosynthesis. Our current understating of these cues, and the ecological roles of NP, is in its infancy. We postulate that understanding the ecological context in which actinomycete metabolites are made is fundamental to advancing the discovery of novel NP. In this review we explore the ecological relevance of actinomycetes and their secondary metabolites from varying ecosystems, and suggest that investigating the ecology of actinomycete interactions warrants particular attention with respect to metabolite discovery. Furthermore, we focus on the chemical ecology and in situ analysis of actinomycete NP and consider the implications for NP biosynthesis at ecosystem scales.

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July 7, 2019

Whole-genome restriction mapping by “subhaploid”-based RAD sequencing: An efficient and flexible approach for physical mapping and genome scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based “in vitro” linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of “subhaploid” fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (~816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies. Copyright © 2017 Dou et al.

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July 7, 2019

Genome-wide analysis of WOX genes in upland cotton and their expression pattern under different stresses.

WUSCHEL-related homeobox (WOX) family members play significant roles in plant growth and development, such as in embryo patterning, stem-cell maintenance, and lateral organ formation. The recently published cotton genome sequences allow us to perform comprehensive genome-wide analysis and characterization of WOX genes in cotton.In this study, we identified 21, 20, and 38 WOX genes in Gossypium arboreum (2n = 26, A2), G. raimondii (2n = 26, D5), and G. hirsutum (2n = 4x = 52, (AD)t), respectively. Sequence logos showed that homeobox domains were significantly conserved among the WOX genes in cotton, Arabidopsis, and rice. A total of 168 genes from three typical monocots and six dicots were naturally divided into three clades, which were further classified into nine sub-clades. A good collinearity was observed in the synteny analysis of the orthologs from At and Dt (t represents tetraploid) sub-genomes. Whole genome duplication (WGD) and segmental duplication within At and Dt sub-genomes played significant roles in the expansion of WOX genes, and segmental duplication mainly generated the WUS clade. Copia and Gypsy were the two major types of transposable elements distributed upstream or downstream of WOX genes. Furthermore, through comparison, we found that the exon/intron pattern was highly conserved between Arabidopsis and cotton, and the homeobox domain loci were also conserved between them. In addition, the expression pattern in different tissues indicated that the duplicated genes in cotton might have acquired new functions as a result of sub-functionalization or neo-functionalization. The expression pattern of WOX genes under different stress treatments showed that the different genes were induced by different stresses.In present work, WOX genes, classified into three clades, were identified in the upland cotton genome. Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of gene numbers during the evolution. Moreover, the expression patterns suggested that the duplicated genes might have experienced a functional divergence. Together, these results shed light on the evolution of the WOX gene family, and would be helpful in future research.

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July 7, 2019

Hybrid assembly with long and short reads improves discovery of gene family expansions.

Long-read and short-read sequencing technologies offer competing advantages for eukaryotic genome sequencing projects. Combinations of both may be appropriate for surveys of within-species genomic variation.We developed a hybrid assembly pipeline called “Alpaca” that can operate on 20X long-read coverage plus about 50X short-insert and 50X long-insert short-read coverage. To preclude collapse of tandem repeats, Alpaca relies on base-call-corrected long reads for contig formation.Compared to two other assembly protocols, Alpaca demonstrated the most reference agreement and repeat capture on the rice genome. On three accessions of the model legume Medicago truncatula, Alpaca generated the most agreement to a conspecific reference and predicted tandemly repeated genes absent from the other assemblies.Our results suggest Alpaca is a useful tool for investigating structural and copy number variation within de novo assemblies of sampled populations.

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July 7, 2019

Frameshift mutation confers function as virulence factor to leucine-rich repeat protein from Acidovorax avenae.

Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (K?Lrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (N?Lrp) did not interfere with infection of finger millet. In addition, N?Lrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain a, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain a was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation.

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July 7, 2019

Genomics and comparative genomic analyses provide insight into the taxonomy and pathogenic potential of novel Emmonsia pathogens.

Over the last 50 years, newly described species of Emmonsia-like fungi have been implicated globally as sources of systemic human mycosis (emmonsiosis). Their ability to convert into yeast-like cells capable of replication and extra-pulmonary dissemination during the course of infection differentiates them from classical Emmonsia species. Immunocompromised patients are at highest risk of emmonsiosis and exhibit high mortality rates. In order to investigate the molecular basis for pathogenicity of the newly described Emmonsia species, genomic sequencing and comparative genomic analyses of Emmonsia sp. 5z489, which was isolated from a non-deliberately immunosuppressed diabetic patient in China and represents a novel seventh isolate of Emmonsia-like fungi, was performed. The genome size of 5z489 was 35.5 Mbp in length, which is ~5 Mbp larger than other Emmonsia strains. Further, 9,188 protein genes were predicted in the 5z489 genome and 16% of the assembly was identified as repetitive elements, which is the largest abundance in Emmonsia species. Phylogenetic analyses based on whole genome data classified 5z489 and CAC-2015a, another novel isolate, as members of the genus Emmonsia. Our analyses showed that divergences among Emmonsia occurred much earlier than other genera within the family Ajellomycetaceae, suggesting relatively distant evolutionary relationships among the genus. Through comparisons of Emmonsia species, we discovered significant pathogenicity characteristics within the genus as well as putative virulence factors that may play a role in the infection and pathogenicity of the novel Emmonsia strains. Moreover, our analyses revealed a novel distribution mode of DNA methylation patterns across the genome of 5z489, with >50% of methylated bases located in intergenic regions. These methylation patterns differ considerably from other reported fungi, where most methylation occurs in repetitive loci. It is unclear if this difference is related to physiological adaptations of new Emmonsia, but this question warrants further investigation. Overall, our analyses provide a framework from which to further study the evolutionary dynamics of Emmonsia strains and identity the underlying molecular mechanisms that determine the infectious and pathogenic potency of these fungal pathogens, and also provide insight into potential targets for therapeutic intervention of emmonsiosis and further research.

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