Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence.

We sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short- and long-read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated almond genome size of 238 Mb, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27 969 protein-coding genes and 6747 non-coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (Prunus persica) diverged around 5.88 million years ago. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions per kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). Transposable elements have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. Transposable elements may also be at the origin of important phenotypic differences between both species, and in particular for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach.

Phylogenomics Identifies an Ancestral Burst of Gene Duplications Predating the Diversification of Aphidomorpha.

Aphids (Aphidoidea) are a diverse group of hemipteran insects that feed on plant phloem sap. A common finding in studies of aphid genomes is the presence of a large number of duplicated genes. However, when these duplications occurred remains unclear, partly due to the high relatedness of sequenced species. To better understand the origin of aphid duplications we sequenced and assembled the genome of Cinara cedri, an early branching lineage (Lachninae) of the Aphididae family. We performed a phylogenomic comparison of this genome with 20 other sequenced genomes, including the available genomes of five other aphids, along with the transcriptomes of two species belonging to Adelgidae (a closely related clade to the aphids) and Coccoidea. We found that gene duplication has been pervasive throughout the evolution of aphids, including many parallel waves of recent, species-specific duplications. Most notably, we identified a consistent set of very ancestral duplications, originating from a large-scale gene duplication predating the diversification of Aphidomorpha (comprising aphids, phylloxerids, and adelgids). Genes duplicated in this ancestral wave are enriched in functions related to traits shared by Aphidomorpha, such as association with endosymbionts, and adaptation to plant defenses and phloem-sap-based diet. The ancestral nature of this duplication wave (106-227 Ma) and the lack of sufficiently conserved synteny make it difficult to conclude whether it originated from a whole-genome duplication event or, alternatively, from a burst of large-scale segmental duplications. Genome sequencing of other aphid species belonging to different Aphidomorpha and related lineages may clarify these findings.

Occurrence of the p019 Gene in the blaKPC-Harboring Plasmids: Adverse Clinical Impact for Direct Tracking of KPC-Producing Klebsiella pneumoniae by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study, we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated Klebsiella pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or noncarbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by whole-genome sequencing (WGS) and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid but that it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K), and IncX3. In addition, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), and IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used by clinical laboratories for identification of antibiotic-resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae isolates (112 CPK and 50 non-CPK) were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages; the first is denominated the "reproducibility stage" and the second "CPK identification." The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage to assess the final accuracy of MALDI-TOF MS for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS Data Analysis Software (Clover Biosoft, Spain) that is designed to reduce variability, guarantee interlaboratory reproducibility, and maximize the information selected from the bacterial proteome. Using the random forest (RF) algorithm, 100% of CPK isolates were correctly identified when all the peaks in the spectra were selected as input features and total ion current (TIC) normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

Genomic evidence for recurrent genetic admixture during the domestication of Mediterranean olive trees (Olea europaea L.).

BACKGROUND: Olive tree (Olea europaea L. subsp. europaea, Oleaceae) has been the most emblematic perennial crop for Mediterranean countries since its domestication around 6000 years ago in the Levant. Two taxonomic varieties are currently recognized: cultivated (var. europaea) and wild (var. sylvestris) trees. However, it remains unclear whether olive cultivars derive from a single initial domestication event followed by secondary diversification, or whether cultivated lineages are the result of more than a single, independent primary domestication event. To shed light into the recent evolution and domestication of the olive tree, here we analyze a group of newly sequenced and available genomes using a phylogenomics and population genomics framework. RESULTS: We improved the assembly and annotation of the reference genome, newly sequenced the genomes of twelve individuals: ten var. europaea, one var. sylvestris, and one outgroup taxon (subsp. cuspidata)-and assembled a dataset comprising whole genome data from 46 var. europaea and 10 var. sylvestris. Phylogenomic and population structure analyses support a continuous process of olive tree domestication, involving a major domestication event, followed by recurrent independent genetic admixture events with wild populations across the Mediterranean Basin. Cultivated olives exhibit only slightly lower levels of genetic diversity than wild forms, which can be partially explained by the occurrence of a mild population bottleneck 3000-14,000 years ago during the primary domestication period, followed by recurrent introgression from wild populations. Genes associated with stress response and developmental processes were positively selected in cultivars, but we did not find evidence that genes involved in fruit size or oil content were under positive selection. This suggests that complex selective processes other than directional selection of a few genes are in place. CONCLUSIONS: Altogether, our results suggest that a primary domestication area in the eastern Mediterranean basin was followed by numerous secondary events across most countries of southern Europe and northern Africa, often involving genetic admixture with genetically rich wild populations, particularly from the western Mediterranean Basin.

Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture.

The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell-line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9-day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12- and 24-hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription- and promoter-specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non-coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA-DNA-DNA triplexes. Such triplex-forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex-forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA-triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes.

European sea bass brain DLB-1 cell line is susceptible to nodavirus: A transcriptomic study.

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the fish central nervous system. It is considered one of the most serious viral diseases in marine aquaculture, the European sea bass (Dicentrarchus labrax) being amongst the most susceptible. We have evaluated the European sea bass brain derived cell line (DLB-1) susceptibility to NNV genotypes and evaluated its transcriptomic profile. DLB-1 cells supported NNV gene transcription and replication since strains belonging to the four NNV genotypes produce cytopathic effects. Afterwards, DLB-1 cells were infected with an RGNNV strain, the one which showed the highest replication, for 12 and 72 h and an RNA-seq analysis was performed to identify potential genes involved in the host-NNV interactions. Differential expression analysis showed the up-regulation of many genes related to immunity, heat-shock proteins or apoptosis but not to proteasome or autophagy processes. These data suggest that the immune response, mainly the interferon (IFN) pathway, is not powerful enough to abrogate the infection, and cells finally suffer stress and die by apoptosis liberating infective particles. GO enrichment also revealed, for the first time, the down-regulation of terms related to brain/neuron biology indicating molecular mechanisms causing the pathogenic effect of NNV. This study opens the way to understand key elements in sea bass brain and NNV interactions.

Landscape of transcription in human cells.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

The genome of melon (Cucumis melo L.).

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3% of the estimated melon genome. We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site-leucine-rich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies.

Genomics of the expanding pine pathogen Lecanosticta acicola reveals patterns of ongoing genetic admixture.

Lecanosticta acicola is the causal agent for brown spot needle blight that affects pine trees across the northern hemisphere. Based on marker genes and microsatellite data, two distinct lineages have been identified that were introduced into Europe on two separate occasions. Despite their overall distinct geographic distribution, they have been found to coexist in regions of northern Spain and France. Here, we present the first genome-wide study of Lecanosticta acicola, including assembly of the reference genome and a population genomics analysis of 70 natural isolates from northern Spain. We show that most of the isolates belong to the southern lineage but show signs of introgression with northern lineage isolates, indicating mating between the two lineages. We also identify phenotypic differences between the two lineages based on the activity profiles of 20 enzymes, with introgressed strains being more phenotypically similar to members of the southern lineage. In conclusion, we show undergoing genetic admixture between the two main lineages of L. acicola in a region of recent expansion. IMPORTANCE: Lecanosticta acicola is a fungal pathogen causing severe defoliation, growth reduction, and even death in more than 70 conifer species. Despite the increasing incidence of this species, little is known about its population dynamics. Two divergent lineages have been described that have now been found together in regions of France and Spain, but it is unknown how these mixed populations evolve. Here we present the first reference genome for this important plant pathogenic fungi and use it to study the population genomics of 70 isolates from an affected forest in the north of Spain. We find signs of introgression between the two main lineages, indicating that active mating is occurring in this region which could propitiate the appearance of novel traits in this species. We also study the phenotypic differences across this population based on enzymatic activities on 20 compounds.

Chromosome-level assembly and annotation of the Xyrichtys novacula (Linnaeus, 1758) genome.

The pearly razorfish (Xyrichtys novacula), commonly known as raor in the Balearic Islands, is a wrasse within the family Labridae. This fish species has particular biological and socio-cultural characteristics making it an ideal model organism in the fields of behavioural ecology, molecular ecology and conservation biology. In this study, we present the first annotated chromosome-level assembly for this species. Sequencing involved a combination of long reads with Oxford Nanopore Technologies, Illumina paired-end short reads (2 × 151 bp), Hi-C and RNA-seq from different tissues. The nuclear genome assembly has a scaffold N50 of 34.33 Mb, a total assembly span of 775.53 Mb and 99.63% of the sequence assembled into 24 superscaffolds, consistent with its known karyotype. Quality metrics revealed a consensus accuracy (QV) of 42.92 and gene completeness > 98%. The genome annotation resulted in 26,690 protein-coding genes and 12,737 non-coding transcripts. The coding regions encoded 39,613 unique protein products, 93% of them with assigned function. Overall, the publication of the X. novacula's reference genome will broaden the scope and impact of genomic research conducted on this iconic and colourful species.

De novo transcriptome sequencing and gene co-expression reveal a genomic basis for drought sensitivity and evidence of a rapid local adaptation on Atlas cedar (Cedrus atlantica).

Introduction: Understanding the adaptive capacity to current climate change of drought-sensitive tree species is mandatory, given their limited prospect of migration and adaptation as long-lived, sessile organisms. Knowledge about the molecular and eco-physiological mechanisms that control drought resilience is thus key, since water shortage appears as one of the main abiotic factors threatening forests ecosystems. However, our current background is scarce, especially in conifers, due to their huge and complex genomes. Methods: Here we investigated the eco-physiological and transcriptomic basis of drought response of the climate change-threatened conifer Cedrus atlantica. We studied C. atlantica seedlings from two locations with contrasting drought conditions to investigate a local adaptation. Seedlings were subjected to experimental drought conditions, and were monitored at immediate (24 hours) and extended (20 days) times. In addition, post-drought recovery was investigated, depicting two contrasting responses in both locations (drought resilient and non-resilient). Single nucleotide polymorphisms (SNPs) were also studied to characterize the genomic basis of drought resilience and investigate a rapid local adaptation of C. atlantica. Results: De novo transcriptome assembly was performed for the first time in this species, providing differences in gene expression between the immediate and extended treatments, as well as among the post-drought recovery phenotypes. Weighted gene co-expression network analysis showed a regulation of stomatal closing and photosynthetic activity during the immediate drought, consistent with an isohydric dynamic. During the extended drought, growth and flavonoid biosynthesis inhibition mechanisms prevailed, probably to increase root-to-shoot ratio and to limit the energy-intensive biosynthesis of secondary metabolites. Drought sensitive individuals failed in metabolism and photosynthesis regulation under drought stress, and in limiting secondary metabolite production. Moreover, genomic differences (SNPs) were found between drought resilient and sensitive seedlings, and between the two studied locations, which were mostly related to transposable elements. Discussion: This work provides novel insights into the transcriptomic basis of drought response of C. atlantica, a set of candidate genes mechanistically involved in its drought sensitivity and evidence of a rapid local adaptation. Our results may help guide conservation programs for this threatened conifer, contribute to advance drought-resilience research and shed light on trees' adaptive potential to current climate change.

Chromosome-level genome assembly of Lilford's wall lizard, Podarcis lilfordi (Günther, 1874) from the Balearic Islands (Spain).

The Mediterranean lizard Podarcis lilfordi is an emblematic species of the Balearic Islands. The extensive phenotypic diversity among extant isolated populations makes the species a great insular model system for eco-evolutionary studies, as well as a challenging target for conservation management plans. Here we report the first high-quality chromosome-level assembly and annotation of the P. lilfordi genome, along with its mitogenome, based on a mixed sequencing strategy (10X Genomics linked reads, Oxford Nanopore Technologies long reads and Hi-C scaffolding) coupled with extensive transcriptomic data (Illumina and PacBio). The genome assembly (1.5 Gb) is highly contiguous (N50 = 90 Mb) and complete, with 99% of the sequence assigned to candidate chromosomal sequences and >97% gene completeness. We annotated a total of 25,663 protein-coding genes translating into 38,615 proteins. Comparison to the genome of the related species Podarcis muralis revealed substantial similarity in genome size, annotation metrics, repeat content, and a strong collinearity, despite their evolutionary distance (~18-20 MYA). This genome expands the repertoire of available reptilian genomes and will facilitate the exploration of the molecular and evolutionary processes underlying the extraordinary phenotypic diversity of this insular species, while providing a critical resource for conservation genomics.

A chromosome-level reference genome for the common octopus, Octopus vulgaris (Cuvier, 1797).

Cephalopods are emerging animal models and include iconic species for studying the link between genomic innovations and physiological and behavioral complexities. Coleoid cephalopods possess the largest nervous system among invertebrates, both for cell counts and brain-to-body ratio. Octopus vulgaris has been at the center of a long-standing tradition of research into diverse aspects of cephalopod biology, including behavioral and neural plasticity, learning and memory recall, regeneration, and sophisticated cognition. However, no chromosome-scale genome assembly was available for O. vulgaris to aid in functional studies. To fill this gap, we sequenced and assembled a chromosome-scale genome of the common octopus, O. vulgaris. The final assembly spans 2.8 billion basepairs, 99.34% of which are in 30 chromosome-scale scaffolds. Hi-C heatmaps support a karyotype of 1n = 30 chromosomes. Comparisons with other octopus species' genomes show a conserved octopus karyotype and a pattern of local genome rearrangements between species. This new chromosome-scale genome of O. vulgaris will further facilitate research in all aspects of cephalopod biology, including various forms of plasticity and the neural machinery underlying sophisticated cognition, as well as an understanding of cephalopod evolution.

Contrasting transcriptomic patterns reveal a genomic basis for drought resilience in the relict fir Abies pinsapo Boiss.

Climate change challenges the adaptive capacity of several forest tree species in the face of increasing drought and rising temperatures. Therefore, understanding the mechanistic connections between genetic diversity and drought resilience is highly valuable for conserving drought-sensitive forests. Nonetheless, the post-drought recovery in trees from a transcriptomic perspective has not yet been studied by comparing contrasting phenotypes. Here, experimental drought treatments, gas-exchange dynamics and transcriptomic analysis (RNA-seq) were performed in the relict and drought-sensitive fir Abies pinsapo Boiss. to identify gene expression differences over immediate (24 h) and extended drought (20 days). Post-drought responses were investigated to define resilient and sensitive phenotypes. Single nucleotide polymorphisms (SNPs) were also studied to characterize the genomic basis of A. pinsapo drought resilience. Weighted gene co-expression network analysis showed an activation of stomatal closing and an inhibition of plant growth-related genes during the immediate drought, consistent with an isohydric dynamic. During the extended drought, transcription factors, as well as cellular damage and homeostasis protection-related genes prevailed. Resilient individuals activate photosynthesis-related genes and inhibit aerial growth-related genes, suggesting a shifting shoot/root biomass allocation to improve water uptake and whole-plant carbon balance. About, 152 fixed SNPs were found between resilient and sensitive seedlings, which were mostly located in RNA-activity-related genes, including epigenetic regulation. Contrasting gene expression and SNPs were found between different post-drought resilience phenotypes for the first time in a forest tree, suggesting a transcriptomic and genomic basis for drought resilience. The obtained drought-related transcriptomic profile and drought-resilience candidate genes may guide conservation programs for this threatened tree species.

Development of a novel streamlined workflow (AACRE) and database (inCREDBle) for genomic analysis of carbapenem-resistant Enterobacterales.

In response to the threat of increasing antimicrobial resistance, we must increase the amount of available high-quality genomic data gathered on antibiotic-resistant bacteria. To this end, we developed an integrated pipeline for high-throughput long-read sequencing, assembly, annotation and analysis of bacterial isolates and used it to generate a large genomic data set of carbapenemase-producing Enterobacterales (CPE) isolates collected in Spain. The set of 461 isolates were sequenced with a combination of both Illumina and Oxford Nanopore Technologies (ONT) DNA sequencing technologies in order to provide genomic context for chromosomal loci and, most importantly, structural resolution of plasmids, important determinants for transmission of antimicrobial resistance. We developed an informatics pipeline called Assembly and Annotation of Carbapenem-Resistant Enterobacteriaceae (AACRE) for the full assembly and annotation of the bacterial genomes and their complement of plasmids. To explore the resulting genomic data set, we developed a new database called inCREDBle that not only stores the genomic data, but provides unique ways to filter and compare data, enabling comparative genomic analyses at the level of chromosomes, plasmids and individual genes. We identified a new sequence type, ST5000, and discovered a genomic locus unique to ST15 that may be linked to its increased spread in the population. In addition to our major objective of generating a large regional data set, we took the opportunity to compare the effects of sample quality and sequencing methods, including R9 versus R10 nanopore chemistry, on genome assembly and annotation quality. We conclude that converting short-read and hybrid microbial sequencing and assembly workflows to the latest nanopore chemistry will further reduce processing time and cost, truly enabling the routine monitoring of resistance transmission patterns at the resolution of complete chromosomes and plasmids.

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis.

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.

The origins, evolution, and functional potential of alternative splicing in vertebrates.

Alternative splicing (AS) has the potential to greatly expand the functional repertoire of mammalian transcriptomes. However, few variant transcripts have been characterized functionally, making it difficult to assess the contribution of AS to the generation of phenotypic complexity and to study the evolution of splicing patterns. We have compared the AS of 309 protein-coding genes in the human ENCODE pilot regions against their mouse orthologs in unprecedented detail, utilizing traditional transcriptomic and RNAseq data. The conservation status of every transcript has been investigated, and each functionally categorized as coding (separated into coding sequence [CDS] or nonsense-mediated decay [NMD] linked) or noncoding. In total, 36.7% of human and 19.3% of mouse coding transcripts are species specific, and we observe a 3.6 times excess of human NMD transcripts compared with mouse; in contrast to previous studies, the majority of species-specific AS is unlinked to transposable elements. We observe one conserved CDS variant and one conserved NMD variant per 2.3 and 11.4 genes, respectively. Subsequently, we identify and characterize equivalent AS patterns for 22.9% of these CDS or NMD-linked events in nonmammalian vertebrate genomes, and our data indicate that functional NMD-linked AS is more widespread and ancient than previously thought. Furthermore, although we observe an association between conserved AS and elevated sequence conservation, as previously reported, we emphasize that 30% of conserved AS exons display sequence conservation below the average score for constitutive exons. In conclusion, we demonstrate the value of detailed comparative annotation in generating a comprehensive set of AS transcripts, increasing our understanding of AS evolution in vertebrates. Our data supports a model whereby the acquisition of functional AS has occurred throughout vertebrate evolution and is considered alongside amino acid change as a key mechanism in gene evolution.

Chromosome-level assembly, annotation and phylome of Pelobates cultripes, the western spadefoot toad.

Genomic resources for amphibians are still hugely under-represented in vertebrate genomic research, despite being a group of major interest for ecology, evolution and conservation. Amphibians constitute a highly threatened group of vertebrates, present a vast diversity in reproductive modes, are extremely diverse in morphology, occupy most ecoregions of the world, and present the widest range in genome sizes of any major group of vertebrates. We combined Illumina, Nanopore and Hi-C sequencing technologies to assemble a chromosome-level genome sequence for an anuran with a moderate genome size (assembly span 3.09 Gb); Pelobates cultripes, the western spadefoot toad. The genome has an N50 length of 330 Mb with 98.6% of the total sequence length assembled into 14 super scaffolds, and 87.7% complete BUSCO genes. We use published transcriptomic data to provide annotations, identifying 32,684 protein-coding genes. We also reconstruct the P. cultripes phylome and identify 2,527 gene expansions. We contribute the first draft of the genome of the western spadefoot toad, P. cultripes. This species represents a relatively basal lineage in the anuran tree with an interesting ecology and a high degree of developmental plasticity, and thus is an important resource for amphibian genomic research.

A New Variant of the aadE-sat4-aphA-3 Gene Cluster Found in a Conjugative Plasmid from a MDR Campylobacter jejuni Isolate.

Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera.