Francisca Robles

Genetic Identification of Western Mediterranean Sturgeons and its Implication for Conservation

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.

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.

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

Abstract. 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.

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 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.

Analysis of Mitochondrial and Nuclear DNA Markers in Old Museum Sturgeons Yield Insights About the Species Existing in Western Europe: A. sturio, A. naccarii and A. oxyrinchus

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.

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, pJα) 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.

Identification of Sturgeon Caviar Using DNA Markers

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.

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.