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Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes. We sequenced the genomes of Pistacia vera cultivar (cv.) Siirt, the female parent, and P.vera cv. Bagyolu, the male parent. Two chromosome-level reference genomes of pistachio were generated, and Z and W chromosomes were assembled. The ZW chromosomes originated from an autosome following the first inversion, which occurred approximately 8.18 Mya. Three inversion events in the W chromosome led to the formation of a 12.7-Mb (22.8% of the W chromosome) non-recombining region. These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio. The W-specific genes, including defA, defA-like, DYT1, two PTEN1, and two tandem duplications of six VPS13A paralogs, are strong candidates for sex determination or differentiation. Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago, dating the domestication event close to the archeological record of pistachio domestication in Iran. We identified 390, 211, and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality, grafting success, flowering time shift, and drought tolerance. These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Infectious diseases represent one of the most pressing threats to modern aquaculture at a global scale [...]

The striped soldier shrimp, Plesionika edwardsii (Brandt, 1851) is a pandalid with economic value in the Mediterranean region. We have sequenced and assembled its complete mitochondrial genome, which is 15,956 bp in length and contains the same 37 genes found in most metazoan mitochondrial genomes. Its gene order and nucleotide content are similar to most of the caridean mitochondrial genomes. In the comparative analysis, however, we detected in other species changes in the gene order that could be mediated by the recombination of transfer RNA genes, as well as AT skew shifts that could indicate changes in the origins of replication. All protein-coding genes of the mitochondrial genome of P. edwardsii seem to be under purifying selection, although the differences in Ka:Ks ratios suggest a disparity in the mutational constraints of some genes. This genome also presents a 1,118 bp-long non-coding sequence that encompass the control region. We have been able to find a previously described conserved sequence block in this region and assess that it forms a stem-loop structure in different species of Pandalidae, which is a shared feature with the conserved sequence blocks described in the family Alvinocarididae. We also detected microsatellites in the control region of P. edwardsii and in other species of Pandalidae and minisatellites in Lysmata vittata (Stimpson, 1860) that can account for around 20% of the additional non-coding region of this species. The phylogenetic relationships of P. edwardsii with other pandalids were assessed by two analyses: one based on the complete mitochondrial sequences and another based only on the protein-coding genes. Our study, thus, contributes to the genomic resources available for P. edwardsii and expands the current biological knowledge about the mitochondrial genomes of other caridean species.

The Pleuronectiformes order, which includes several commercially-important species, has undergone extensive chromosome evolution. One of these species is

This study was supported by the Spanish Ministry of Economy and Competitiveness, FEDER Grants (RTI2018-096847-B-C21 and RTI2018-096847-B-C22), Junta de Andalucía-FEDER Grant (P20-00938)., 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., RTI2018-096847B-C21 AND RTI2018-096847-B-C22 SPANISH MINISTRY OF ECONOMY AND COMPETITIVENESS -FEDER

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.

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.

a b s t r a c t A new Pistacia variety, VIGROS, has been characterized here using nuclear and chloroplastidial DNA sequences, which are suitable tools for genetic improvement and cultivar identification. The nuclear DNA sequences (ribosomal ITS1, 5.8S, ITS2) determined that VIGROS is an interspecific hybrid between Pistacia vera L. and Pistacia atlantica Desf. The chloroplastidial DNA sequences (trnC-D, trnL-F regions) demonstrated that VIGROS bears a haplotype 100% coincident with that of P. vera, which would then be regarded as the female parent, while P. atlantica would be the male one. Due to its vigorous phenotype VIGROS has the potential to be used as a rootstock. This characterization may benefit producers and contribute to improved grafting of pistachios. Also, this paper demonstrates the validity of this set of molecular markers to characterize interspecific Pistacia hybrids.

The bud–rootstock compatibility is decisive in the success of grafting pistachios. Climate conditions and appropriate rootstock choice are the two key factors influencing grafting success. Despite producers’ efforts, fluctuations in these factors result in frequent differences in annual yield. To minimize production losses by increasing the percentage of successful grafting would greatly benefit the introduction of this crop in new areas. Here, we analyze the viability of Pistacia terebinthus L. differentially treated with mycorrhizae or phytohormones and used as rootstock for Pistacia vera buds. Our results, on an experimental plot of 12,905 plants, demonstrate that mycorrhiza-treated plants reached ∼80% positive grafts, while the phytohormone-treated plants and controls had 32.3% and 38.4% success, respectively. The increase in grafting success could be explained by more efficient nutrient uptake in mycorrhiza-treated plants. An analysis of chemical element accumulation and assimilation in leaves reveals that mycorrhiza-treated plants selectively accumulated Ca, Fe, Mg, N, Al, S, Sr, Ti, V, Mn, and Tl, but lacked K with respect to the rest of the plants. Mycorrhiza-treated plants were shorter, but no significant differences were found in trunk diameter and circumference. We propose the use of mycorrhizae to increase bud–rootstock compatibility for this tandem of species.

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.

Two new centromeric satellite DNAs in flatfish (Order Pleuronectiformes) have been characterized. The SacI-family from Hippoglossus hippoglossus, restricted to this species, had a monomeric size of 334 base pair (bp) and was located in most of the centromeres of its karyotype. The PvuII-family, with a monomeric size of 177 bp, was initially isolated from the genome of Solea senegalensis, and fluorescent in situ hybridization (FISH) localized the repeat to centromeres of most of the chromosomes. This family could only be amplified in 2 other species of the genus Solea (Solea solea and Solea lascaris). Molecular features and chromosomal location indicated a possible structural and/or functional role of these sequence repeats. The presence of species-specific satellite-DNA families in the centromeres and their possible role in the speciation processes in this group of fishes is discussed.

To clarify the species status of sturgeon from rivers of the Iberian Peninsula, eight molecular markers (4 nuclear and 4 mitochondrial) have been analysed in different specimens from historical museum samples and prehistoric samples from archaeological sites. These analyses indicate that one of these specimens (UGP captured in the Guadalquivir River in the 19th century) is A. sturio, based on all the eight molecular markers, four of them used from the first time in this study. In previous analyses based on 5 genetic markers, our group assigned two specimens captured in this river in the 1970-80s (EBD8173 and EBD8401) to the species A. naccarii, suggesting the presence of this species in the Iberian Peninsula. In this work, this conclusion is drawn after successfully obtaining a mitochondrial marker in a very old scute from a prehistoric site (Acinipo, about 1500 BC, from the Guadalquivir River basin). On the other hand, in the specimen EBD8174 captured in the Guadalquivir in 1975, we have obtained two new mitochondrial markers confirming that it can be considered A. sturio for all the mitochondrial markers, but nuclear ones identify it as A. naccarii. Finally, two very old samples (Nerja E-VI and Nerja N/62-63) were not successfully characterized by any molecular markers. Some aspects and consequences of our results are discussed, such as the origin of the “mosaic” specimen EBD8174 and, above all, the native status of A. naccarii in historic and prehistoric times in the southern Iberian Peninsula.

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.

From a battery of microsatellite markers (100 loci), recently identified by our group, we have selected eight for parentage assignment in Senegal sole (Solea senegalensis). This tool is based on microsatellite loci obtained from four genomic DNA libraries and one cDNA library. Within the eight loci (six from anonymous genomic DNA sequences and two located in expressed sequence tags of known genes), we have found, in an analysis of a reproductive broodstock, between nine and 16 alleles. The expected heterozygosity was between 0.616 and 0.860. In addition, we have optimized the polymerase chain reaction (PCR) conditions to amplify all loci simultaneously in a single multiplex PCR reaction, and we have tested three lots of male and female (five to six individuals) and three offspring (50–60 larvae each). The use of the eight microsatellite loci, the possibility of amplifying them in a single PCR reaction and the high value of the exclusion probability (0.9992) make this multiplex PCR method a unique tool for parentage assignment. Finally, analysing one meiotic gynogenetic progeny, we have determined the relative distance of six of these loci to the centromere, and we have also found that all of them are unlinked. All these characteristics confer this tool with a high accuracy for parentage studies and genetic population analyses of Senegal sole.

Currently, three species ofMuscari subg. Botryanthus are recognized in the Iberian Peninsula: two diploids (2n = 18), M. atlanticum and M. cazorlanum, and one morphologically variable species with three different ploidy levels, M. neglectum (2n = 36, 45, 54). We have made a comparative study of numerous Iberian populations to clarify the taxonomy and evolution of this group. To this end we carried out morphological and cytogenetic analyses, and phylogenetic analysis of the internal transcribed spacers of nuclear ribosomal DNA. Comparative and UPGMA analyses of the morphological characteristics show that the different ploidy levels of M. neglectum represent different species. We describe the pentaploid and hexaploid levels as two new species, M. olivetorum (2n = 45) and M. baeticum (2n = 54), each with an exclusive combination of morphological characters and a characteristic ecological behavior pattern. Phylogenetic study of ITS shows that the two new species are not autopolyploids from M. neglectum but allopolyploids. These findings are supported by the additivity of the three ITS variants found in M. olivetorum with the ITS of M. neglectum and M. baeticum, and also by morphology. Possible parents for both new species are proposed. Absence of homogenization between homeologous M olivetorum nrDNA loci is explained by the absence of sexual reproduction and by nucleolar dominance, indicating that this is a recent species.

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 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/XY 1 Y 2 , 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).

Marteilia refringens is a paramyxean parasite which infects the flat oyster Ostrea edulis and mussels (Mytilus galloprovincialis), where it has been attributed to a separate species, Marteilia maurini, by several authors. Doubts persist though as to the existence or not of two species of Marteilia in Europe. We have devised a molecular method for the diagnosis of M. refringens based on 358 bp nested-PCR of the rDNA intergene spacer (rDNA IGS) which is capable of detecting 0.5 fg of M. refringens DNA. Molecular characterization of this spacer indicates that the Marteilia parasites which infect oysters and mussels are two different strains of the same species.

To date, the only native sturgeon species in Western Europe was believed to be Acipenser sturio. However, this species is currently restricted to the Gironde River (Southern France), and it poses serious difficulties for rearing in captivity and for using in recovery programme. Furthermore, it has been questioned whether A. sturio is in fact the only species within the rivers of Western Europe, as A. naccarii, a species previously considered endemic of the Adriatic region, has been reported from the Iberian Peninsula in recent years. Here, we have used forensic techniques to obtain DNA from several museum specimens of sturgeons caught in the Spanish Guadalquivir River and in other European rivers. We analysed DNA sequences from these specimens for five genetic markers (three nuclear and two mitochondrial regions), which were subsequently compared with sequences obtained from A. sturioand A. naccarii. Our study demonstrates that A. naccarii coexists withA. sturio, from the Adriatic Sea to the Iberian Peninsula, a finding that could be taken into account in future sturgeon recovery programmes in Western Europe.

To solve problems concerning the status of the taxa described in the genus Sarcocapnos, we have conducted a study using morphological, pollen morphology (light microscopy), cytogenetic and molecular techniques. Focusing on the last technique, we have sequenced ITS-1 and ITS-2 of nuclear rDNA. The species differ basically according to 5 morphological traits (leaf shape, flower spur, corolla colour, corolla size, and crest of the stigmatic surface). The cytogenetic analyses indicated n=16 to be the standard chromosome number. The ITS analyses showed that the genus is monophyletic, defining two main well-supported clades, one containing S. saetabensis and S. enneaphylla, and one containing the rest of the species. In this second clade, S. speciosa, S. pulcherrima, and S. baetica subsp. ardalii are related, as are S. integrifolia, S. crassifolia subsp. crassifolia, and S. crassifolia subsp. atlantis; S. baetica subsp. baetica forms a trichotomy with the foregoing groups. S. speciosa is shown to be a species separate from S. crassifolia subsp. crassifolia, as in the case of S. baetica with respect to S. integrifolia. Palynologically, the parameters used enabled us to establish clear differences between the taxa, often corroborating the macromorphological and genetic data. The flower spur has been reduced several times in different groups of the genus, for which the classifications established on the basis of this trait are paraphyletic.

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.

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. © 2013 Springer Science+Business Media New York.

In this study, the phylogenetic relationships and which the taxonomic status of the species belonging to the Sparidae family (Pisces: Perciformes) are analysed and revised. This study includes species of this family that are distributed by the North-eastern Atlantic and Mediterranean coasts, is based on the analysis of two satellite DNA families. While one satellite DNA, the centromeric EcoRI family, extends to all the species analysed, the other, the subtelomeric DraI family, is restricted to only six of the 16 species studied. Based on phylogenetic use of these two markers, we conclude that the Sparidae family is composed by two major lineages: one comprising the species of the genera Sparus, Diplodus, Lithognathus, Boops, Sarpa and Spondyliosoma, and one species of Pagellus (P. bogaraveo); and the other lineage is comprised of the species of Pagrus and Dentex, and one species of Pagellus (P. erythrinus). This classification is consistent across the two markers used and clearly contradicts previous morphological phylogenies based mainly on dentition. In addition, the current status and the phylogenetic position of some of the species analysed (i.e. species of Pagrus, Dentex and Pagellus) are not supported by our analyses. Finally, we discuss the value of the morphological characters used until now for the classification of this group of fish.

This paper reports on the isolation and localization of the subtelomeric DraI satellite DNA in the Sparidae family. Gene cloning determined that the DraI satellite DNA is present in only 3 species (Pagrus pagrus, P. auriga, and Pagellus erythrinus) of the 10 Sparidae species analyzed. The results were confirmed by PCR amplification. This satellite DNA is located in a subtelomeric position in all 48 acrocentric chromosomes of these species. However, interstitial loci are also observed. Sequence analysis of monomers of this repetitive family indicates that the satellite DNA is associated with telomeric sequences, (TTAGGG)n, in at least one species, P. erythrinus. This is the first direct demonstration of the existence of the consensus telomere sequences of vertebrates in fish. Likewise, this report also demonstrates that the ends of fish chromosomes have a structure similar to those of most eukaryote chromosomes, viz., telomere sequences and subtelomeric sequences associated by a boundary in which both types of sequences are interspersed. The recent origin of the DraI satellite DNA and its use as a phylogenetic marker is discussed.

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.

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.

The meat and, above all, the caviar from sturgeons have been gastronomic emblems of delicacy from time immemorial. When sturgeons were abundant, caviar was marketed primarily from three species (Huso huso, Acipenser gueldenstaedtii and A. stellatus) mostly from the Caspian Sea. However, these are not the only species of sturgeons used traditionally to produce quality caviar, nor is the Caspian its only geographic origin. For example, caviar from sturgeons belonging to the species A. sturio or A. naccarii captured in the Guadalquivir River (southern Spain) gained renown in the 1960s, winning prizes at world fairs. Now, when sturgeons are almost disappearing all over the world, the caviar trade has become far more complex. In fact, practically all sturgeon species can produce quality caviar, not only in the wild (in steady decline) but also in fish farms. This scenario makes it necessary to monitor the sale of caviar at a worldwide level, and for this the application of molecular techniques based on DNA, together with others, can be of great use. In this chapter, we present two types of DNA markers for this purpose: some, especially nuclear-DNA markers, indicate qualitative differences between the different species (presence/absence of these markers); and others, particularly mitochondrial-DNA, indicate differences in certain bases of nucleotide sequences. In addition, sturgeons produce interspecific hybrids. Consequently, nuclear markers along with mitochondrial ones are needed for accurately identifying the species responsible for a given caviar.

Today, with all the sturgeon species almost disappearing all over the world, it is necessary to undertake their recovery under the programs for the conservation of genetic resources. The complete absence of these fish from most rivers increases the difficulties in carrying out such programs, hampering the genetic identification of specimens and their correct species assignment. However, with the development of new and reliable molecular genetic techniques, many studies such as this are yielding insights concerning the sturgeon species that inhabited European rivers in the past. In the last few years, our group has developed forensic techniques to isolate DNA from ancient sturgeon specimens preserved in museums. These DNA samples have been the subject of various analyses conducted on several nuclear and mitochondrial DNA markers. The combined use of both types of markers has provided accurate genetic identification of these specimens and has overcome the problem of misinterpretation caused by hybridization and introgression. Here, we show that, in addition to Acipenser sturio (the only species previously believed to inhabit the rivers of Western Europe), two other species, Acipenser naccarii and Acipenser oxyrinchus lived in these rivers. Thus, we have found evidence for the presence of A. naccarii in the Guadalquivir river in the Iberian Peninsula and in some rivers in Italy from the Tyrrhenian/Ligurian side, as well as for the presence of A. oxyrinchus in the Ebro river in Spain. Our studies clarify the distribution of sturgeon species in the Western Mediterranean and open new perspectives for recovery plans.

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.

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.

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.

Ribosomal DNA in sturgeon is informative when analyzed at the molecular level because it bears unique characteristics that are, to a certain extent, ancestral within vertebrates. In this paper, we examine the structure and the molecular evolution of the 5S ribosomal DNA (rDNA) region in 13 sturgeon species, comparing both the 5S ribosomal RNA (rRNA) genes and the non-transcribed spacer (NTS) sequences between the coding regions. We have found that different NTS and 5S gene variants are intermixed in the 5S rDNA arrays of the different sturgeon species and that all variants are ancestral, having been maintained over many millions of years. Using predictive models, we have found similar levels of sequence diversity in the coding regions, as well as in the non-coding region, but fixed interspecific differences are underrepresented for 5S genes. However, contrary to the expectations, we have not found fixed differences between NTS sequences when comparing many pairs of species. Specifically, when they belong to the same phylogeographic clade of the four into which the sturgeon is divided, but fixation of mutations and divergence is found between species belonging to different phylogeographic clades. Our results suggest that the evolution of the two parts of the 5S rDNA region cannot be explained exclusively as the outcome of a balance between mutational, homogenizing (i.e., gene conversion as a predominant force in sturgeon), and selective forces. Rather, they suggest that other factors (i.e., hybridization) might be superimposed over those forces and thus could to some extent be masking their effects.Key words: sturgeon, 5S rDNA, NTS sequence, 5S gene, concerted evolution, sequence homogenization, gene conversion, hybridization.

One characteristic of sex chromosomes is the accumulation of a set of different types of repetitive DNA sequences in the Y chromosomes. However, little is known about how this occurs or about how the absence of recombination affects the subsequent evolutionary fate of the repetitive sequences in the Y chromosome. Here we compare the evolutionary pathways leading to the appearance of three different families of satellite-DNA sequences within the genomes of Rumex acetosa and R. papillaris, two dioecious plant species with a complex XX/XY(1)Y(2) sex-chromosome system. We have found that two of these families, one autosomic (the RAE730 family) and one Y-linked (the RAYSI family), arose independently from the ancestral duplication of the same 120-bp repeat unit. Conversely, a comparative analysis of the three satellite-DNA families reveals no evolutionary relationships between these two and the third, RAE180, also located in the Y chromosomes. However, we have demonstrated that, regardless of the mechanisms that gave rise to these families, satellite-DNA sequences have different evolutionary fates according to their location in different types of chromosomes. Specifically, those in the Y chromosomes have evolved at half the rate of those in the autosomes, our results supporting the hypothesis that satellite DNAs in nonrecombining Y chromosomes undergo lower rates of sequence evolution and homogenization than do satellite DNAs in autosomes.

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 p22phox. 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 (χ2=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 p22phox 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 p22phox gene with CHD was not statistically significant, (χ2=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. This work has been financed by Spanish grants of Fondo de Investigaciones Sanitarias (FIS 94/0553) and by Plan Andaluz de Investigación (Research Group CVI0200).

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).

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.

We examined the composition and evolution of a large heterochromatic region present in the genomes of certain species of the genus Muscari (Hyacinthaceae). We found that in Muscari comosum this heterochromatic region is composed mainly of a satellite DNA family, which we named MCSAT. Molecular analyses and in situ hybridization revealed that, through the evolution of Muscari species, the MCSAT sequences have been progressively amplified in several species of the genus, such as M. matritensis and M. dionysicum, attaining enormous amplification in the genome of M. comosum. We discuss the characteristics of this satellite DNA family, which, being exclusively amplified in one chromosome pair of M. comosum, constitute the major exception to the equilocal model of satellite DNA and heterochromatin distribution. Also, we discuss the possibility that the amplification of these sequences in a single chromosome could have contributed to a progressive increase in the asymmetry of the karyotypes in Muscari species.