Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. Latastei and V. seoanei by Illumina MiSeq sequencing.

BACKGROUND: European vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations. METHODS AND RESULTS: Genomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei). CONCLUSIONS: Our study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

PARSID: a Python script for automatic analysis of local BLAST results for a rapid molecular taxonomic identification.

OBJECTIVE: A reliable taxonomic identification of species from molecular samples is the first step for many studies. For researchers unfamiliar with programming, running a BLAST analysis, filtering, and organizing results for hundreds of sequences through the BLAST web interface can be difficult. Additionally, sequences deposited in GenBank can have outdated taxonomic identification. The use of reliable Reference Sequences Library (RSL) containing accurate taxonomically-identified sequences facilitates this task. Pending the availability of a RSL with the user, we developed a tool that automates the molecular taxonomic identification of sequences. RESULTS: We developed PARSID, a Python script running through the command-line that automates the routine workflow of blasting an input sequence file against the user's RSL, and retrieves the matches with the highest percentage of identity in five steps. PARSID accepts cut-off parameters and supplementary information in a.csv file for filtering the results. The final output is visualized in a spreadsheet. We tested its functioning using 10 input sequences simulating different situations of the molecular taxonomic identification of sequences against an example RSL containing 25 sequences. Step-by-step instructions and test files are publicly available at https://github.com/kokinide/PARSID.git .

An Ancestral Major Histocompatibility Complex Organization in Cartilaginous Fish: Reconstructing MHC Origin and Evolution.

Cartilaginous fish (sharks, rays, and chimeras) comprise the oldest living jawed vertebrates with a mammalian-like adaptive immune system based on immunoglobulins (Ig), T-cell receptors (TCRs), and the major histocompatibility complex (MHC). Here, we show that the cartilaginous fish "adaptive MHC" is highly regimented and compact, containing (i) a classical MHC class Ia (MHC-Ia) region containing antigen processing (antigen peptide transporters and immunoproteasome) and presenting (MHC-Ia) genes, (ii) an MHC class II (MHC-II) region (with alpha and beta genes) with linkage to beta-2-microglobulin (ß2m) and bromodomain-containing 2, (iii) nonclassical MHC class Ib (MHC-Ib) regions with 450 million-year-old lineages, and (iv) a complement C4 associated with the MHC-Ia region. No MHC-Ib genes were found outside of the elasmobranch MHC. Our data suggest that both MHC-I and MHC-II genes arose after the second round of whole-genome duplication (2R) on a human chromosome (huchr) 6 precursor. Further analysis of MHC paralogous regions across early branching taxa from all jawed vertebrate lineages revealed that Ig/TCR genes likely arose on a precursor of the huchr9/12/14 MHC paralog. The ß2m gene is linked to the Ig/TCR genes in some vertebrates suggesting that it was present at 1R, perhaps as the donor of C1 domain to the primordial MHC gene. In sum, extant cartilaginous fish exhibit a conserved and prototypical MHC genomic organization with features found in various vertebrates, reflecting the ancestral arrangement for the jawed vertebrates.

Identification of sex-linked SNP markers in wild populations of monomorphic birds.

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for population genetics, pedigree reconstruction and phenotypic trait mapping. However, the untapped potential of SNP markers to discriminate the sex of individuals in species with reduced sexual dimorphism or of individuals during immature stages remains a largely unexplored avenue. Here, we developed a novel protocol for molecular sexing of birds based on the detection of unique Z- and W-linked SNP markers. Our method is based on the identification of two unique loci, one in each sexual chromosome. Individuals are considered males when they show no calls for the W-linked SNP and are heterozygous or homozygous for the Z-linked SNP, while females exhibit both Z- and W-linked SNP calls. We validated the method in the Jackdaw (Corvus monedula). The reduced sexual dimorphism in this species makes it difficult to identify the sex of individuals in the wild. We assessed the reliability of the method using 36 individuals of known sex and found that their sex was correctly assigned in 100% of cases. The sex-linked markers also proved to be widely applicable for discriminating males and females from a sample of 927 genotyped individuals at different maturity stages, with an accuracy of 99.5%. Since SNP markers are increasingly used in quantitative genetic analyses of wild populations, the approach we propose has great potential to be integrated into broader genetic research programmes without the need for additional sexing techniques.

Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa.

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

Corrigendum: Uncovering a 500 million year old history and evidence of pseudogenization for TLR15.

[This corrects the article DOI: 10.3389/fimmu.2022.1020601.].

Extensive MHC class IIß diversity across multiple loci in the small-spotted catshark (Scyliorhinus canicula).

The major histocompatibility complex (MHC) is a multigene family responsible for pathogen detection, and initiation of adaptive immune responses. Duplication, natural selection, recombination, and their resulting high functional genetic diversity spread across several duplicated loci are the main hallmarks of the MHC. Although these features were described in several jawed vertebrate lineages, a detailed MHC IIß characterization at the population level is still lacking for chondrichthyans (chimaeras, rays and sharks), i.e. the most basal lineage to possess an MHC-based adaptive immune system. We used the small-spotted catshark (Scyliorhinus canicula, Carcharhiniformes) as a case-study species to characterize MHC IIß diversity using complementary molecular tools, including publicly available genome and transcriptome datasets, and a newly developed high-throughput Illumina sequencing protocol. We identified three MHC IIß loci within the same genomic region, all of which are expressed in different tissues. Genetic screening of the exon 2 in 41 individuals of S. canicula from a single population revealed high levels of sequence diversity, evidence for positive selection, and footprints of recombination. Moreover, the results also suggest the presence of copy number variation in MHC IIß genes. Thus, the small-spotted catshark exhibits characteristics of functional MHC IIß genes typically observed in other jawed vertebrates.

De Novo Assembly of the Dirofilaria immitis Genome by Long-Read Nanopore-Based Sequencing Technology on an Adult Worm from a Canine Cardiopulmonary Dirofilariosis Case.

Dirofilaria immitis is a zoonotic parasitic nematode that infects domestic and wild canids, among its vertebrate hosts. The genetic analysis of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such as the study of resistance to macrocyclic lactone. We expanded the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform, followed by the study of macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110 contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci previously characterized as being associated to macrocyclic lactone resistance selection pressure showed that four have a genotype associated with either some loss of efficacy or the resistance phenotype. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health.

Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers.

Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species' genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.

Uncovering a 500 million year old history and evidence of pseudogenization for TLR15.

Introduction: Toll like receptors (TLRs) are at the front line of pathogen recognition and host immune response. Many TLR genes have been described to date with some being found across metazoans while others are restricted to specific lineages. A cryptic member of the TLR gene family, TLR15, has a unique phylogenetic distribution. Initially described in extant species of birds and reptiles, an ortholog has been reported for cartilaginous fish. Methods: Here, we significantly expanded the evolutionary analysis of TLR15 gene evolution, taking advantage of large genomic and transcriptomic resources available from different lineages of vertebrates. Additionally, we objectively search for TLR15 in lobe-finned and ray-finned fish, as well as in cartilaginous fish and jawless vertebrates. Results and discussion: We confirm the presence of TLR15 in early branching jawed vertebrates - the cartilaginous fish, as well as in basal Sarcopterygii - in lungfish. However, within cartilaginous fish, the gene is present in Holocephalans (all three families) but not in Elasmobranchs (its sister-lineage). Holocephalans have long TLR15 protein sequences that disrupt the typical TLR structure, and some species display a pseudogene sequence due to the presence of frameshift mutations and early stop codons. Additionally, TLR15 has low expression levels in holocephalans when compared with other TLR genes. In turn, lungfish also have long TLR15 protein sequences but the protein structure is not compromised. Finally, TLR15 presents several sites under negative selection. Overall, these results suggest that TLR15 is an ancient TLR gene and is experiencing ongoing pseudogenization in early-branching vertebrates.

Pervasive hybridization with local wild relatives in Western European grapevine varieties.

Grapevine (Vitis vinifera L.) diversity richness results from a complex domestication history over multiple historical periods. Here, we used whole-genome resequencing to elucidate different aspects of its recent evolutionary history. Our results support a model in which a central domestication event in grapevine was followed by postdomestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties showing large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by gene flow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

Draft Genome Sequence of a Leptospira kirschneri Serovar Mozdok Type 2 Strain Isolated from a Horse in Portugal.

Leptospira kirschneri is an agent causing leptospirosis in animals and humans. We report the draft genome sequence of Leptospira kirschneri serovar Mozdok type 2 strain Horse 112, comprising 485 contigs and having a genome size of 4,301,784 bp. This genome will facilitate studying important mechanisms for clinical outcomes.

A Highly Complex, MHC-Linked, 350 Million-Year-Old Shark Nonclassical Class I Lineage.

Cartilaginous fish, or Chondrichthyes, are the oldest extant vertebrates to possess the MHC and the Ig superfamily-based Ag receptors, the defining genes of the gnathostome adaptive immune system. In this work, we have identified a novel MHC lineage, UEA, a complex multigene nonclassical class I family found in sharks (division Selachii) but not detected in chimaeras (subclass Holocephali) or rays (division Batoidea). This new lineage is distantly related to the previously reported nonclassical class I lineage UCA, which appears to be present only in dogfish sharks (order Squaliformes). UEA lacks conservation of the nine invariant residues in the peptide (ligand)-binding regions (PBR) that bind to the N and C termini of bound peptide in most vertebrate classical class I proteins, which are replaced by relatively hydrophobic residues compared with the classical UAA. In fact, UEA and UCA proteins have the most hydrophobic-predicted PBR of all identified chondrichthyan class I molecules. UEA genes detected in the whale shark and bamboo shark genome projects are MHC linked. Consistent with UEA comprising a very large gene family, we detected weak expression in different tissues of the nurse shark via Northern blotting and RNA sequencing. UEA genes fall into three sublineages with unique characteristics in the PBR. UEA shares structural and genetic features with certain nonclassical class I genes in other vertebrates, such as the highly complex XNC nonclassical class I genes in Xenopus, and we anticipate that each shark gene, or at least each sublineage, will have a unique function, perhaps in bacterial defense.

Cartilaginous fish class II genes reveal unprecedented old allelic lineages and confirm the late evolutionary emergence of DM.

Cartilaginous fish (chimaeras, rays and sharks) are the most basal extant jawed vertebrates with an adaptive immune system based on the Major Histocompatibility Complex (MHC). Despite being a key taxon in the evolution of vertebrate adaptive immunity, no comprehensive characterization of MHC class II genes has been undertaken for the group. We performed extensive bioinformatic searches on a taxonomically diverse dataset of transcriptomes and genomes of cartilaginous fish targeting MHC class II sequences. Class IIα and IIß sequences were retrieved from all taxa analyzed and showed typical features of classical class II genes. Phylogenetic trees of the immunoglobulin superfamily domain showed two divergent and remarkably ancient lineages of class II genes in Selachians (sharks), originating >350 million years ago. Close linkage of lineage-specific pairs of IIα and IIß genes was found, confirming previous results, with genes from distinct lineages segregating as alleles. Nonclassical class II DM sequences were not retrieved from these data and classical class II sequences lacked the conserved residues shown to interact with DM molecules, supporting claims that the DM system arose only in the lobe-finned fish lineage leading to tetrapods. Based on our search methods, other divergent class II genes are unlikely in cartilaginous fish.

Liver transcriptome resources of four commercially exploited teleost species.

The generation of omic resources is central to develop adequate management strategies for species with economic value. Here, we provide high-coverage RNA-seq datasets of liver tissue (containing between 80,2 and 88,4 million of paired-end reads) from four wildtype teleost species with high commercial value: Trachurus trachurus (TTR; Atlantic horse mackerel), Scomber scombrus (SSC; Atlantic mackerel), Trisopterus luscus (TLU; pout), and Micromesistius poutassou (MPO; blue whiting). A comprehensive assembly pipeline, using de novo single and multi-kmer assembly approaches, produced 64 single high-quality liver transcriptomes - 16 per species. The final assemblies, with N50 values ranging from 2,543-3,700 bp and BUSCO (Benchmarking Universal Single-Copy Orthologs) completeness values between 81.8-86.5% of the Actinopterygii gene set, were subjected to open reading frame (ORF) prediction and functional annotation. Our study provides the first transcriptomic resources for these species and offers valuable tools to evaluate both neutral and selected genetic variation among populations, and to identify candidate genes for environmental adaptation assisting in the investigation of the effects of global changes in fisheries.

High-Quality Draft Genome Sequences of Marine Fish Gut Bacillus sp. Strains ABP1 and ABP2 with Nonstarch Polysaccharide Hydrolytic Potential.

Here, we present the genome sequences of two environmental Bacillus strains with broad hydrolytic capacity toward different nonstarch polysaccharides (NSPs) that were isolated from the gut of marine fish fed NSP-rich diets. Several genes that may contribute to the NSP-degrading behavior were identified through in silico analysis.

Phylogenetic analysis of F-bZIP transcription factors indicates conservation of the zinc deficiency response across land plants.

Basic leucine zipper (bZIP) transcription factors control important developmental and physiological processes in plants. In Arabidopsis thaliana, the three gene F-bZIP subfamily has been associated with zinc deficiency and salt stress response. Benefiting from the present abundance of plant genomic data, we performed an evolutionary and structural characterization of plant F-bZIPs. We observed divergence during seed plant evolution, into two groups and inferred different selective pressures for each. Group 1 contains AtbZIP19 and AtbZIP23 and appears more conserved, whereas Group 2, containing AtbZIP24, is more prone to gene loss and expansion events. Transcriptomic and experimental data reinforced AtbZIP19/23 as pivotal regulators of the zinc deficiency response, mostly via the activation of genes from the ZIP metal transporter family, and revealed that they are the main regulatory switch of AtZIP4. A survey of AtZIP4 orthologs promoters across different plant taxa revealed an enrichment of the Zinc Deficiency Response Element (ZDRE) to which both AtbZIP19/23 bind. Overall, our results indicate that while the AtbZIP24 function in the regulation of the salt stress response may be the result of neo-functionalization, the AtbZIP19/23 function in the regulation of the zinc deficiency response may be conserved in land plants (Embryophytes).

Sma3s: A universal tool for easy functional annotation of proteomes and transcriptomes.

The current cheapening of next-generation sequencing has led to an enormous growth in the number of sequenced genomes and transcriptomes, allowing wet labs to get the sequences from their organisms of study. To make the most of these data, one of the first things that should be done is the functional annotation of the protein-coding genes. But it used to be a slow and tedious step that can involve the characterization of thousands of sequences. Sma3s is an accurate computational tool for annotating proteins in an unattended way. Now, we have developed a completely new version, which includes functionalities that will be of utility for fundamental and applied science. Currently, the results provide functional categories such as biological processes, which become useful for both characterizing particular sequence datasets and comparing results from different projects. But one of the most important implemented innovations is that it has now low computational requirements, and the complete annotation of a simple proteome or transcriptome usually takes around 24 hours in a personal computer. Sma3s has been tested with a large amount of complete proteomes and transcriptomes, and it has demonstrated its potential in health science and other specific projects.

De novo transcriptome assembly and polymorphism detection in two highly divergent evolutionary units of Bosca's newt (Lissotriton boscai) endemic to the Iberian Peninsula.

This article reports the de novo transcriptome assemblies of two highly divergent evolutionary units of the Iberian endemic Bosca's newt, Lissotriton boscai. These two units are distributed mostly allopatrically but overlap in the central-southwestern coastal region of Portugal. The resources we provide include the raw sequence reads, the assembled transcripts, the annotation and SNPs called for both lineages.