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.

The centromeric satellite of the wedge sole (Dicologoglossa cuneata, Pleuronectiformes) is composed mainly of a sequence motif conserved in other vertebrate centromeric DNAs.

Here, a new satellite-DNA family is isolated and characterized from wedge sole, Dicologoglossa cuneata Moreau, 1881 (Pleuronectiformes), a fish having a small genome. This satellite-DNA family of sequences was isolated by conventional cloning after digestion of genomic DNA with the DraI restriction enzyme. Repeat units are 171 bp in length with a high AT content (63%). Several runs of consecutive adenines and thymines were found, and concomitantly computer analyses revealed that these regions are prone to acquire stable sequence-directed curvature. Especially remarkable is that the DraI sequences are composed almost entirely of the repetition of up to fourteen 9-bp motifs (T/C)GTC(A/C)AAAA similar to other vertebrate centromeric satellite-DNA sequences. In fact, we demonstrate the origin of this satellite through duplication of this motif plus the addition of a stretch of cytosines. The centromeric location and the presence in this satellite-DNA sequence of not only different vertebrate motifs (CENP-B box, pJalpha) but also others such as the CDEIII motif of Saccharomyces cerevisiae reveal a possible role in centromere function. All these characteristics provide important information on the origin, function, and the evolution of the centromeric satellite DNAs in 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.

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.

A heterochromatic satellite DNA is highly amplified in a single chromosome of Muscari (Hyacinthaceae).

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.

Chromosomal location and evolution of a satellite DNA family in seven sturgeon species.

The Hind III satellite DNA family, isolated from the Acipenser naccarii genome, was used as a probe for fluorescent in-situ hybridization (FISH) on the karyotype of seven sturgeon species, six belonging to the genus Acipenser and one to Huso. All species except one (A. sturio) exhibit from 8 to 80 chromosome hybridization signals, mainly localized at the pericentromeric regions. Eight chromosomes with weak hybridization signals are present in H. huso and A. ruthenus, which are characterized by a karyotype with about 120 chromosomes. The species with 240-260 chromosomes, A. transmontanus, A. naccarii, A. gueldenstaedtii, and A. baerii, show from 50 to 80 signals, prevalently localized around centromeres. Moreover, A. transmontanus and A. gueldenstaedtii show from 4 to 8 chromosomes with a double signal. The phylogenetic and evolutionary relationships among sturgeon species are discussed on the basis of number and morphology of signal-bearing chromosomes and on the localization of signals.

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.

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.

Evolution of centromeric satellite DNA and its use in phylogenetic studies of the Sparidae family (Pisces, Perciformes).

In this paper, we use the EcoRI centromeric satellite DNA family conserved in Sparidae as a taxonomic and a phylogenetic marker. The analyses of 56 monomeric units (187 bp in size) obtained by means of cloning and PCR from 10 sparid species indicate that this repetitive DNA evolves by concerted evolution. Different phylogenetic inference methods, such as neighbor-joining and UPGMA, group the 56 repeats by taxonomic affinity and support the existence of at least two monophyletic groups within the Sparidae family. These results reinforce the recent taxonomic revision of the genera Sparus and Pagrus and contradict previous classifications of the Sparidae family.

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris.

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a B-specific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.

The EcoRI centromeric satellite DNA of the Sparidae family (Pisces, Perciformes) contains a sequence motive common to other vertebrate centromeric satellite DNAs.

By means of cloning, sequencing, and fluorescence in situ hybridization, we have determined that the EcoRI satellite DNA family is conserved in the 10 sparid species analyzed here. Its conservation, its chromosomal location at the centromere of each chromosome, and its structural features could make this satellite DNA family an important structural and/or functional element of the centromeres of these species. Monomeric units of this satellite DNA have a consensus length of 187 bp. Its sequence is characterized by a high AT content and the presence of short runs of consecutive AT base pairs. These monomeric EcoRI repeats also contain three to four copies, depending on the species, of a short sequence reflecting the repetitive duplication and subsequent divergence of an ancestral 9-bp sequence in this family. This sequence motive is conserved in some parts of the monomeric units of the different species studied at the same positions, and, precisely, surrounding the area in which the curvature of the monomeric molecule is greatest. The 9-bp sequence motive is similar to other direct-repeat sequences of the centromeric satellite DNAs of other vertebrates, including those of amphibians and mammals.

Cytogenetic analysis of gilthead seabream Sparus aurata (Pisces, Perciformes), a deletion affecting the NOR in a hatchery stock.

We have cytogenetically characterized a hatchery stock of gilthead seabream, Sparus aurata. The study included larvae, juveniles and adults. In S. aurata (diploid chromosome number 2n = 48), a pair of NORs is located at the ends of the short arms of the first submetacentric pair of chromosomes. In this stock we discovered a polymorphism which affects the NORs, and, by means of several cytogenetic and molecular techniques, we demonstrate that this polymorphism is due to the complete deletion of one of the two NORs in a high number of individuals. The significance of these cytogenetic characteristics for this species are discussed since they may be the source of aquaculture problems.

A molecular analysis of the origin of the Crepis capillaris B chromosome.

The origin of the B chromosome of Crepis capillaris has been studied by using in situ hybridization with different DNA probes. Genomic in situ hybridization (GISH) with DNA from plants with and without Bs as probes indicates that the B chromosome has many DNA sequences in common with A chromosomes, showing no region rich in B-specific sequences. Six additional DNA probes were used to test the possible origin of this B from the standard NOR chromosome (chromosome 3). In the short arm of the NOR chromosome, we detected not only 18 S + 25 S rDNA, but also 5 S rDNA and a specific repetitive sequence from the NOR chromosome (pCcH32); in the heterochromatic bands of the long arm, we found two different repetitive sequences (pCcE9 and pCcD29). In the B chromosome, however, only the 18 S + 25 S rDNA and the telomeric sequences from Arabidopsis thaliana were observed. Our in situ hybridization data with telomeric repeats indicate that the two telomeres of the B are larger than those of the A chromosomes, confirming the isochromosomal nature of this B. Hybridizations of 18 S + 25 S rDNA and telomeric repeats to blots of DNA from plants with and without Bs reveal a high homology between A and B 18 S + 25 S rDNA genes, but some sequence dissimilarities between A and B telomeres. Taken as a whole, these data indicate that the entire B of C. capillaris, although possibly having originated from the standard genome, did not derive directly from the NOR chromosome.

rDNA site number polymorphism and NOR inactivation in natural populations of Allium schoenoprasum.

Nucleolar-organiser activity has been studied by silver staining and by in situ hybridization with an rDNA probe in two populations of Allium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution of A. schoenoprasum.

Cloning and characterization of a fish centromeric satellite DNA.

A highly repetitive DNA sequence family from the genome of Sparus aurata has been cloned and characterized. The family is composed of repeat units of 186 bp in length, and it accounts for 2% of the fish genome. Data from Southern blots and in situ hybridization demonstrate that repeating units are tandemly arranged at the centromeres of all the chromosomes in this species. The repetitive sequence is AT rich (67%) and is characterized by short stretches of consecutive AT base pairs and by short direct and inverted repeats. Sequence analysis of six cloned monomers of the family reveals some variation among clones at random positions and also distinguishes two subfamilies of repeats that differ in a highly divergent block of 31 bp. These two subfamilies do not seem to be located in separate domains but occur together in the centromere of each chromosome pair. The presence of this repeat family in the genome of other Sparidae species, some of which are relatively distant from S. aurata, indicates that this repetitive sequence could be an important component of the centromere in this fish family.

Repetitive DNA sequence families in Crepis capillaris.

Three families of tandemly repetitive DNA from Crepis capillaris were cloned and characterized. Data obtained from in situ hybridization indicate that these families are located mainly in the heterochromatic C-bands. The pCcH32 family hybridizes at the paracentromeric C-band of the NOR (nucleolus-organized region) chromosome and along most of the long arm of the same chromosome. The pCcD29 family is located in all the remaining C-bands of the karyotype, while the third family, pCcE9, is restricted to the more proximal C-bands. Nucleotide sequence comparisons between one cloned repeating unit from each DNA family showed some significant regions of homology between the families. We discuss the sequence relationships between the three DNA families and the significance of our data in relation to models of heterochromatin evolution, emphasizing the concepts of equilocality and the differentiation of the NOR-bearing chromosome. We also examine the possible role that chromosome disposition, in either mitotic or meiotic nuclei, plays in the distribution and homogenization of heterochromatic DNA sequences.

A method for increasing the number of mitoses available for cytogenetic analysis in rainbow trout.

A new method for the cytogenetic analysis of fishes with special interest for the rainbow trout (Salmo gairdneri Rich.) is described. This reliable method that includes treatment with a yeast solution provides high quality spreads of a great number of metaphases.