R. de la Herrán

A satellite DNA of the Sparidae family (Pisces, perciformes) associated with telomeric sequences

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.

The molecular diagnosis of Marteilia refringens and differentiation between Marteilia strains infecting oysters and mussels based on the rDNA IGS sequence.

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.

Molecular characterization of the ribosomal RNA gene region of Perkinsus atlanticus: its use in phylogenetic analysis and as a target for a molecular diagnosis.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S-5S-18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.

Organization of Repetitive DNA Sequences at Pachytene Chromosomes of Gilthead Seabream Sparus aurata (Pisces, Perciformes)

A method of preparing two-dimensional surface spreads of fish synaptonemal complexes (SCs) associated with fluorescent in-situ hybridization is described. This technique permits a novel approach to the analysis of chromatin organization and the construction of physical maps at meiosis, since surface-spread pachytene chromosomes are several times the length of metaphase chromosomes and the decondensed chromatin loops are attached to the lateral elements of the SC. We have applied this technique to analyze the location and organization of three different repetitive DNA sequences, rDNA, an EcoRI satellite DNA of the Sparidae family and telomere DNA in the gilthead seabream Sparus aurata. Our observations indicate that, depending on the type of sequence, the chromatin has different properties with regard to anchorage to the SC.

Characterization of Vibrio tapetis strains isolated from diseased cultured Wedge sole (Dicologoglossa cuneata Moreau)

The first isolation of Vibrio tapetis from Wedge sole (Dicologoglossa cuneata) is reported. The bacterium was recovered from ulcers of ailing cultured fish, from two different outbreaks occurred in spring 2005. The four isolates found (a200, a201, a204 and a255) were biochemically, genetically and serologically characterized and diagnosis was confirmed by PCR V. tapetis specific primers and multilocus sequencing analysis (MLSA). The isolates constituted a homogeneous phenotypic and genotypic group, being distinct to the already serological and genetic groups defined within the species. A virulence evaluation of the isolate a255 was also carried out; however this strain was unable to induce disease in fry and juvenile Wedge sole.

The controversial telomeres of lily plants

The molecular structure of the exceptional telomeres of six plant species belonging to the order Asparagales and two species of the order Liliales was analyzed using Southern blot and fluorescence in situ hybridization. Three different situations were found, namely: i) In the two Liliales species, Tulipa australis (Liliaceae) and Merendera montana (Colchicaceae), the chromosome ends display hybridization signals with oligonucleotides resembling telomere repeats of both plants (TTTAGGG)n and vertebrates (TTAGGG)n. ii) Asparagales species such as Phormium tenax (Hemerocallidaceae), Muscari comosum (Hyacinthaceae), Narcissus jonquilla (Amaryllidaceae) and Allium sativum (Alliaceae) lack both the plant telomere repeats and the vertebrate telomere repeats. iii) Two other Asparagales species, Aloe vera (Asphodelaceae) and an Iris hybrid (Iridaceae), display positive hybridization with the vertebrate telomere repeats but not with the plant telomere repeats. Southern blot hybridization revealed concurring results. On this basis, the composition of the telomere structure in this plant group is discussed.

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

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.

Exploitation of a turbot (Scophthalmus maximus L.) immune‐related expressed sequence tag (EST) database for microsatellite screening and validation

In this study, we identified and characterized 160 microsatellite loci from an expressed sequence tag (EST) database generated from immune‐related organs of turbot (Scophthalmus maximus). A final set of 83 new polymorphic microsatellites were validated after the analysis of 40 individuals of Atlantic origin including both wild and farmed individuals. The allele number and the expected heterozygosity ranged from 2 to 18 and from 0.021 to 0.951, respectively. Evidences of null alleles at moderate–high frequencies were detected at six loci using population data. None of the analysed loci showed deviations from Mendelian segregation after the analysis of five full‐sib families including approximately 92 individuals/family. The markers are used to consolidate the turbot genetic map, and because they are mostly EST‐derived, they will be very useful for comparative genomic studies within flatfishes and with model fish species. Using an in silico approach, we detected significant homologies of microsatellite sequences with the EST databases of the flatfish species with highest genomic resources (Senegalese sole, Atlantic halibut, bastard halibut) in 31% of these turbot markers. The conservation of these microsatellites within Pleuronectiformes will pave the way for anchoring genetic maps of different species and identifying genomic regions related to productive traits.

First isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata (Moreau).

The first isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata, is reported. The pathogen was recovered from ulcers of cultured fish, from three different outbreaks. The six isolates obtained were biochemically and serologically characterized and diagnosis was confirmed by polymerase chain reaction using specific primers and partial 16S rRNA gene sequencing. The isolates constituted a homogeneous phenotypic group; however, they belong to two of the different serotypes described within this species. A virulence evaluation of the isolates using Wedge sole fry was also performed.

Inheritance and fitness effects of a pericentric inversion and a supernumerary chromosome segment in Muscari comosum (Liliaceae)

The inheritance and the effects on fitness of a pericentric inversion and a supernumerary chromosome segment (SCS) involving the second chromosome pair of Muscari comosum, have been analysed in an experimental population. Whereas the inversion is inherited in a Mendelian fashion through the female side, the SCS shows a powerful accumulation mechanism (k=0.796). Cases of nonrandom pollination were observed in homozygous bulbs for the standard chromosomes (++) and also in those homozygous for the inverted chromosomes (ii), which may be caused by a certain rate of self-pollination. The different karyotypes for both the inversion and the SCS showed similar values for several life-history traits related to female fitness, such as bulb weight, number of fruits, number of seeds and seed weight. However, plants carrying the inversion had heavier bulbs than those lacking it. The significance of these observations is discussed in relation to the maintenance of the two types of polymorphism in natural populations.