miR-430 microRNA Family in Fishes: Molecular Characterization and Evolution.

The miR-430 microRNA family has been described in multiple fish species as one of the first microRNAs expressed by the zygote. It has been suggested that this family is implicated in maternal mRNA elimination, but may also play a role in steroidogenesis, sexual differentiation, and flatfish metamorphosis. The miR-430 sequences have been found in multiple-copy tandem clusters but evidence of their conservation outside of teleost fishes is scarce. In the present study, we have characterized the tandem repeats organization of these microRNAs in different fish species, both model and of interest in aquaculture. A phylogenetic analysis of this family has allowed us to identify that the miR-430 duplication, which took place before the Chondrostei and Neopterygii groups' divergence, has resulted in three variants ("a", "b", and "c"). According to our data, variant "b" is the most closely related to the ancestral sequence. Furthermore, we have detected isolated instances of the miR-430 repeat subunit in some species, which suggests that this microRNA family may be affected by DNA rearrangements. This study provides new data about the abundance, variability, and organization of the miR-430 family in fishes.

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.

Integration of Maps Enables a Cytogenomics Analysis of the Complete Karyotype in Solea senegalensis.

The Pleuronectiformes order, which includes several commercially-important species, has undergone extensive chromosome evolution. One of these species is Solea senegalensis, a flatfish with 2n = 42 chromosomes. In this study, a cytogenomics approach and integration with previous maps was applied to characterize the karyotype of the species. Synteny analysis of S. senegalensis was carried out using two flatfish as a reference: Cynoglossus semilaevis and Scophthalmus maximus. Most S. senegalensis chromosomes (or chromosome arms for metacentrics and submetacentrics) showed a one-to-one macrosyntenic pattern with the other two species. In addition, we studied how repetitive sequences could have played a role in the evolution of S. senegalensis bi-armed (3, and 5-9) and acrocentric (11, 12 and 16) chromosomes, which showed the highest rearrangements compared with the reference species. A higher abundance of TEs (Transposable Elements) and other repeated elements was observed adjacent to telomeric regions on chromosomes 3, 7, 9 and 16. However, on chromosome 11, a greater abundance of DNA transposons was detected in interstitial BACs. This chromosome is syntenic with several chromosomes of the other two flatfish species, suggesting rearrangements during its evolution. A similar situation was also found on chromosome 16 (for microsatellites and low complexity sequences), but not for TEs (retroelements and DNA transposons). These differences in the distribution and abundance of repetitive elements in chromosomes that have undergone remodeling processes during the course of evolution also suggest a possible role for simple repeat sequences in rearranged regions.

Detection and variability analyses of CRISPR-like loci in the H. pylori genome.

Helicobacter pylori is a human pathogenic bacterium with a high genomic plasticity. Although the functional CRISPR-Cas system has not been found in its genome, CRISPR-like loci have been recently identified. In this work, 53 genomes from different geographical areas are analyzed for the search and analysis of variability of this type of structure. We confirm the presence of a locus that was previously described in the VlpC gene in al lgenomes, and we characterize new CRISPR-like loci in other genomic locations. By studying the variability and gene location of these loci, the evolution and the possible roles of these sequences are discussed. Additionally, the usefulness of this type of sequences as a phylogenetic marker has been demonstrated, associating the different strains by geographical area.

Long-term monitoring of B-chromosome invasion and neutralization in a population of Prospero autumnale (Asparagaceae).

B chromosomes have been reported in about 15% of eukaryotes, but long-term dynamics of B chromosomes in a single natural population has rarely been analyzed. Prospero autumnale plants collected in 1981 and 1983 at Cuesta de La Palma population had shown the presence of B chromosomes. We analyze here seven additional samples collected between 1987 and 2015, and show that B frequency increased significantly during the 1980s and showed minor fluctuations between 2005 and 2015. A mother-offspring analysis of B chromosome transmission, at population level, showed significant drive on the male side (kB  = 0.65) and significant drag on the female side (kB  = 0.33), with average B transmission rate being very close to the Mendelian rate (0.5). No significant effects of B chromosomes were observed on a number of vigor and fertility-related traits. Within a parasite/host framework, these results suggest that B chromosomes' drive on the male side is the main pathway for B chromosome invasion, whereas B chromosome drag on the female side might be the main manifestation of host genome resistance in this species. Prospero autumnale thus illuminates a novel evolutionary pathway for B chromosome neutralization by means of a decrease in B transmission through the nondriving sex.

The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L.) Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1.

This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176-178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs) of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30) could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II) with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree.

Chromosomal manipulation in Senegalese sole (Solea senegalensis Kaup, 1858): induction of triploidy and gynogenesis.

In this study we have developed protocols for induced triploidy and gynogenesis of Senegalese sole (Solea senegalensis), a promising flatfish species for marine aquaculture, in order to: 1) identify the sex-determination mechanism; and 2) to improve its production by generating a) sterile fish, avoiding problems related with sexual maturation, and b) all-female stocks, of higher growth rate. Triploidy was induced by means of a cold shock. Gynogenesis was induced by activating eggs with UV-irradiated sperm, and to prompt diploid gynogenesis, a cold-shock step was also used. Ploidy of putative triploid larvae and gynogenetic embryos were determined by means of karyotyping and microsatellite analysis. Haploid gynogenetic embryos showed the typical "haploid syndrome". As expected, triploid and gynogenetic groups showed lower fertilization, hatching, and survival rates than in the diploid control group. Survival rate, calculated 49 days after hatching, for haploid and diploid gynogenetic groups was similar to those observed in other fish species (0% and 62.5%, respectively), whereas triploids showed worse values (45%). Sex was determined macroscopically and by histological procedures, revealing that all the diploid gynogenetic individuals were females. In conclusion, we have successfully applied chromosomal-manipulation techniques in the flatfish species Senegalese sole in order to produce triploid, haploid, and diploid gynogenetic progenies.

First haploid genetic map based on microsatellite markers in Senegalese sole (Solea senegalensis, Kaup 1858).

The Senegalese sole (Solea senegalensis, Kaup 1858) is a flatfish species of great value for aquaculture. In this study, we develop the first linkage map in this species based on microsatellite markers characterized from genomic DNA libraries and EST databases of Senegalese sole and from other flatfish species. Three reference gynogenetic families were obtained by chromosome-manipulation techniques: two haploid gynogenetics, used to assign and order microsatellites to linkage groups and another diploid gynogenetic family, used for estimating marker-centromere distances. The consensus map consists of 129 microsatellites distributed in 27 linkage groups (LG), with an average density of 4.7 markers per LG and comprising 1,004 centimorgans (cM). Additionally, 15 markers remained unlinked. Through half-tetrad analysis, we were able to estimate the centromere distance for 81 markers belonging to 24 LG, representing an average of 3 markers per LG. Comparative mapping was performed between flatfish species LG and model fish species chromosomes (stickleback, Tetraodon, medaka, fugu and zebrafish). The usefulness of microsatellite markers and the genetic map as tools for comparative mapping and evolution studies is discussed.

Identification of quantitative trait loci associated with resistance to viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus ): a comparison between bacterium, parasite and virus diseases.

One of the main objectives of genetic breeding programs in turbot industry is to reduce disease-related mortality. In the present study, a genome scan to detect quantitative trait loci (QTL) affecting resistance and survival to viral haemorrhagic septicaemia (VHS) was carried out. Three full-sib families with approximately 90 individuals each were genotyped and evaluated by linear regression and maximum likelihood approaches. In addition, a comparison between QTL detected for resistance and survival time to other important bacterial and parasite diseases affecting turbot (furunculosis and scuticociliatosis) was also carried out. Finally, the relationship between QTL affecting resistance/survival time to the virus and growth-related QTL was also evaluated. Several genomic regions controlling resistance and survival time to VHS were detected. Also significant associations between the evaluated traits and genotypes at particular markers were identified, explaining up to 14 % of the phenotypic variance. Several genomic regions controlling general and specific resistance to different diseases in turbot were detected. A preliminary gene mining approach identified candidate genes related to general or specific immunity. This information will be valuable to develop marker-assisted selection programs and to discover candidate genes related to disease resistance to improve turbot production.

Molecular characterization and evolution of an interspersed repetitive DNA family of oysters.

When genomic DNA from the European flat oyster Ostrea edulis L. was digested by BclI enzyme, a band of about 150 bp was observed in agarose gel. After cloning and sequencing this band and analysing their molecular characteristics and genomic organization by means of Southern blot, in situ hybridisation, and polymerase chain reaction (PCR) protocols, we concluded that this band is an interspersed highly repeated DNA element, which is related in sequence to the flanking regions of (CT)-microsatellite loci of the species O. edulis and Crassostrea gigas. Furthermore, we determined that this element forms part of a longer repetitive unit of 268 bp in length that, at least in some loci, is present in more than one copy. By Southern blot hybridisation and PCR amplifications-using primers designed for conserved regions of the 150-bp BclI clones of O. edulis-we determined that this repetitive DNA family is conserved in five other oyster species (O. stentina, C. angulata, C. gigas, C. ariakensis, and C. sikamea) while it is apparently absent in C. gasar. Finally, based on the analysis of the repetitive units in these oyster species, we discuss the slow degree of concerted evolution in this interspersed repetitive DNA family and its use for phylogenetic analysis.

Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain).

In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.

Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes.

The evolution of sex chromosomes in the genus Rumex (Polygonaceae): Identification of a new species with heteromorphic sex chromosomes.

The structural features and evolutionary state of the sex chromosomes of the XX/XY species of Rumex are unknown. Here, we report a study of the meiotic behaviour of the XY bivalent in Rumex acetosella and R. suffruticosus, a new species which we describe cytogenetically for the first time in this paper, and also that of the XY(1)Y(2) trivalent of R. acetosa by both conventional cytogenetic techniques and analysis of synaptonemal complex formation. Fluorescent in situ hybridization with satellite DNA and rDNA sequences as probes was used to analyse the degree of cytogenetic differentiation between the X and Y chromosomes in order to depict their evolutionary stage in the three species. Contrasting with the advanced state of genetic differentiation between the X and the Y chromosomes in R. acetosa, we have found that R. acetosella and R. suffruticosus represent an early stage of genetic differentiation between sex chromosomes. Our findings further demonstrate the usefulness of the genus Rumex as a model for analysing the evolution of sex chromosomes in plants, since within this genus it is now possible to study the different levels of genetic differentiation between the sex chromosomes and to analyse their evolutionary history from their origin.

The origin and evolution of the variability in a Y-specific satellite-DNA of Rumex acetosa and its relatives.

In this paper, we analyze a satellite-DNA family, the RAYSI family, which is specific of the Y chromosomes of Rumex acetosa, a dioecious plant species with a multiple sex-chromosome system in which the females are XX and the males are XY(1)Y(2). Here, we demonstrate that this satellite DNA is common to other relatives of R. acetosa, including Rumex papillaris, Rumex intermedius, Rumex thyrsoides and Rumex tuberosus that are also dioecious species with a multiple system of sex chromosomes. This satellite-DNA family is absent from the genomes of other dioecious Rumex species having an XX/XY sex-chromosome system. Our data confirm recent molecular phylogenies that support a unique origin for all dioecious species of Rumex and two separate lineages for species with single or complex sex-chromosome systems. Our data also support an accelerated degeneration of Y-chromosome in XX/XY(1)Y(2) species by the accumulation of satellite-DNA sequences. On the other hand, the particular non-recombining nature of the Y chromosomes of R. acetosa and their closest relatives lead to a particular mode of evolution of RAYSI sequences. Thus, mechanisms leading to the suppression of recombination between the Y chromosomes reduced the rate of concerted evolution and gave rise to the apparition of different RAYSI subfamilies. Thus, R. acetosa and R. intermedius have two subfamilies (the RAYSI-S and RAYSI-J subfamilies and the INT-A and INT-B subfamilies, respectively), while R. papillaris only has one, the RAYSI-J subfamily. The RAYSI-S and RAYSI-J subfamilies of R. acetosa differ in 83 fixed diagnostic sites and several diagnostic deletions while the INT-A and the INT-B of R. intermedius differ in 27 fixed diagnostic sites. Pairwise comparisons between RAYSI-S and RAYSI-J sequences or between INT-A and INT-B sequences revealed these sites to be shared mutations detectable in repeats of the same variant in same positions. Evolutionary comparisons suggest that the subfamily RAYSI-J has appeared in the common ancestor of R. acetosa and R. papillaris, in which RAYSI-J has replaced totally (R. papillaris) or almost totally the ancestral sequence (R. acetosa). This scenario assumes that RAYSI-S sequences should be considered ancestral sequences and that a secondary event of subfamily subdivision should be occurring in R. intermedius, with their RAYSI subfamilies more closely related to one another than with other RAYSI sequences. Our analysis suggests that the different subfamilies diverged by a gradual and cohesive way probably mediated by sister-chromatid interchanges while their expansion or contraction in number might be explained by alternating cycles of sudden mechanisms of amplification or elimination.

The evolution of reproductive systems and sex-determining mechanisms within rumex (polygonaceae) inferred from nuclear and chloroplastidial sequence data.

The genus Rumex includes hermaphroditic, polygamous, gynodioecious, monoecious, and dioecious species, with the dioecious species being represented by different sex-determining mechanisms and sex-chromosome systems. Therefore, this genus represents an exceptional case study to test several hypotheses concerning the evolution of both mating systems and the genetic control of sex determination in plants. Here, we compare nuclear intergenic transcribed spacers and chloroplast intergenic sequences of 31 species of Rumex. Our phylogenetic analysis supports a systematic classification of the genus, which differs from that currently accepted. In contrast to the current view, this new phylogeny suggests a common origin for all Eurasian and American dioecious species of Rumex, with gynodioecy as an intermediate state on the way to dioecy. Our results support the contention that sex determination based on the balance between the number of X chromosomes and the number of autosomes (X/A balance) has evolved secondarily from male-determining Y mechanisms and that multiple sex-chromosome systems, XX/XY1Y2, were derived twice from an XX/XY system. The resulting phylogeny is consistent with a classification of Rumex species according to their basic chromosome number, implying that the evolution of Rumex species might have followed a process of chromosomal reduction from x = 10 toward x = 7 through intermediate stages (x = 9 and x = 8).

The molecular phylogeny of oysters based on a satellite DNA related to transposons.

We have analysed a centromeric satellite DNA family that is conserved in several commercial and non-commercial oyster species (Ostrea edulis, O. stentina, Crassostrea angulata, C. gigas, C. gasar, C. ariakensis, C. virginica and C. sikamea). This satellite DNA family is composed of AT-rich repeat sequences of 166+/-2 bp and presents a 9-bp motif similar to the mammalian CENP-B box. The homology of oyster HindIII satellite DNA with satellite DNAs from other bivalves and its relation to a part of a mobile element suggest the existence of an ancient transposable element as a generating unit of satellite DNA in bivalve molluscs. Taking advantage of its degree of conservation in oyster species, we have used this element as a taxonomic marker. This marker clearly supports a high degree of differentiation between O. edulis and O. stentina, and, conversely, upholds the contention that C. gigas and C. angulata are the same species. Finally, we have used HindIII satellite DNA as a phylogenetic marker between these species, revealing two clades, one formed by Asiatic species (C. angulata, C. gigas and C. ariakensis) and another by the European, American and African species (O. edulis, C. virginica and C. gasar, respectively).

Evolution of ancient satellite DNAs in sturgeon genomes.

This study characterizes a repetitive DNA family of sequences in sturgeon, the PstI satellite DNA. We have found a high degree of preservation for these sequences, which are present in all 13 species analyzed, including within the genera Acipenser, Huso, and Scaphirhynchus of the family Acipenseridae. This is one of the most ancient satellite DNAs found to date, because it has been estimated to be more than 100 million years old. Alternatively, to the current view that most satellite DNAs are species-specific or preserved in a few closely related species, the PstI family and other previously characterized sturgeon satellite DNA, the HindIII, represent the most fascinating exceptions to the rapid sequence change usually undergone by satellite DNAs. Here, we compare the evolutionary pattern of these two satellite DNA families, PstI and HindIII, which differ markedly in length, sequence, and nucleotide composition. We have found that, in contrast to the situation in most other living beings, a high degree of preservation, a slow sequence change rate and slowed concerted evolution, appears to be a general rule for sturgeon satellite DNAs. The possible causes for all these features are discussed in the light of the evolutionary specifics found within these ancient organisms.

Angiotensin-converting enzyme and p22(phox) polymorphisms and the risk of coronary heart disease in a low-risk Spanish population.

OBJECTIVE: To evaluate the genetic contribution to myocardial infarction in a homogeneous Caucasian population (a Mediterranean Spanish population) with very low frequency of coronary heart disease (CHD). DESIGN: We analyzed a total of 210 subjects, younger than 55 years, considered to be a low-risk population (104 cases of myocardial infarction and 106 control), and genotyped them (using polymerase chain reaction and sequencing) for the angiotensin-converting enzyme (ACE) insertion/deletion (ACE I/D) and for the C242T polymorphism of NADPH oxidase p22(phox). Also, we sequenced 23 alleles of the ACE gene (9 D and 14 I) for the region that includes the end of the intron 16 and the exon 17. RESULTS: The ACE genotype-prevalence values for II, ID and DD were 4.81%, 28.85% and 66.34%, respectively, among the myocardial infarction patients, and 2.83%, 71.70% and 25.47% among controls. The statistical analysis comparing patients and controls revealed significant differences (chi(2)=25.09, P=0.00000055) between the two subpopulations. Also, we found a strong association between the genotype DD and the risk of suffering CHD (odds ratio (OR): 3.64; 95% CI: 2.37-8.07). The prevalence of the CC, TC and TT genotypes of p22(phox) gene among healthy controls proved to be 53.77%, 44.34% and 1.89%, while those of myocardial infarction were 58.65%, 39.42% and 1.93%, respectively. The association of C242T polymorphism of the p22(phox) gene with CHD was not statistically significant, (chi(2)=0.49, P=0.48). Logistic-regression analysis demonstrated that the independent risk factor for developing myocardial infarction was the DD genotype of ACE gene. Finally, our results indicate that alleles I and D of ACE gene are differentiated at three positions (nucleotide sites 14,480, 14,488 and 14,521) of which, the positions 14,480 and 14,488 were in absolute linkage disequilibrium. CONCLUSIONS: Among subjects of a Mediterranean population with low risk for CHD, the presence of DD ACE genotype could be a risk factor for myocardial infarction, and we confirm the linkage disequilibrium between two nucleotide positions of the ACE gene and the polymorphism for an Alu insertion.