Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5x genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and < 5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.

Molecular cytogenetic detection of paternal chromosome fragments in allogynogenetic gibel carp, Carassius auratus gibelio Bloch

In gynogenesis, sperm from related species activates egg and embryonic development, but normally does not contribute genetically to the offspring. In gibel carp, Carassius auratus gibelio Bloch, however, gynogenetic offspring often show some phenotypes apparently derived from the heterologous sperm donor. This paternal effect of allogynogenesis is outstanding in an artificial clone F produced by cold treatment of clone E eggs after insemination with blunt-nose black bream (Megaloabrama amblycephala Yin) sperm. Karyotype analysis revealed 5-15 supernumerary microchromosomes in different individuals of clone F in addition to 156 normal chromosomes inherited from the maternal clone E. A painting probe was prepared from the microdissected microchromosomes, and used to investigate the origin of these microchromosomes. Strong positive signals were detected on each microchromosomes of clone F and on 4 pairs of chromosomes in blunt-nose black bream, whereas no signals were detected on the chromosomes of clone E. This result indicates that some paternal chromosome fragments of blunt-nose black bream have been incorporated into the artificial clone F. Therefore, the manipulation of allogynogenesis may provide a unique method to transfer DNA between diverse species for fish breeding.

Cytogenetic and histological studies of the brook trout (Salvelinus fontinalis) x arctic charr (Salvelinus alpinus) hybrids

In aquaculture, application of fish hybrids has increased. This technique permits improvement of the fish production by providing specimens showing better growth rate when compared to the parental species. Indeed, sterile individuals are highly demanded because quite frequently parental fish mature before they reach the market size, which impairs their growth and decrease their economic value. Throughout the last years, the commercial and scientific interest in salmonids has increased rapidly, among them, the brook trout (Salvelinus fontinalis), Arctic charr (Salvelinus alpinus) are species that can be crossed to produce hybrids that might by cultured in the fish farms. In the present thesis, we have assessed chromosome numbers and evaluate gonadal sex in the brook trout X Arctic charr hybrid progenies. In our populations, the karyotype of the brook trout comprises 84 chromosomes: 16 bi-armed chromosomes (meta-submetacentric) and 68 one-armed chromosomes (telo-acrocentrics) and the chromosome arm number, NF= 100. Arctic charr karyotype shows variation related to the chromosome number (2n= 81-82) and stable chromosome arm number (NF= 100). 2n= 81 chromosomes consisted of 19 bi-armed and 62 one-armed chromosomes, while 2n= 82 karyotype was organized into 18 meta-submetacentric and 64 acrocentrics. The cytogenetic and histological analysis of the brook trout X Arctic charr hybrids (sparctics) was carried out to asses chromosome and chromosome arm number and gonadal sex of the studied specimens. Diploid chromosome number in the hybrids varied from 81 to 84 and individuals with 83 and 84 chromosomes were predominant. Most of the fish had chromosome arm number equal to 100. Robertsonian fusion in the Arctic charr and chromosome behaviour in the hybrid fish cells might lead to the observed variation in chromosome numbers in the hybrids. Among studied fish, 12 were males, 3 were females and 9 had intersex gonads. No correlation between chromosome number and disturbances in the gonadal development was found. This might suggest that intersex gonads might have been developed as a consequence of disturbances in the genetic sex determination process. Genetic sex determination acts properly in the parental species but in the hybrids this may not be as efficient.

Polysomy 8 defines a clinico-cytogenetic entity representing a subset of myeloid hematologic malignancies associated with a poor prognosis: report on a cohort of 12 patients and review of 105 published cases.

Tetrasomy, pentasomy, and hexasomy 8 (polysomy 8) are relatively rare compared to trisomy 8. Here we report on a series of 12 patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or myeloproliferative disorder (MPD) associated with polysomy 8 as detected by conventional cytogenetics and fluorescence in situ hybridization (FISH). In an attempt to better characterize the clinical and hematological profile of this cytogenetic entity, our data were combined with those of 105 published patients. Tetrasomy 8 was the most common presentation of polysomy 8. In 60.7% of patients, polysomy 8 occurred as part of complex changes (16.2% with 11q23 rearrangements). No cryptic MLL rearrangements were found in cases in which polysomy 8 was the only karyotypic change. Our study demonstrates the existence of a polysomy 8 syndrome, which represents a subtype of AML, MDS, and MPD characterized by a high incidence of secondary diseases, myelomonocytic or monocytic involvement in AML and poor overall survival (6 months). Age significantly reduced median survival, but associated cytogenetic abnormalities did not modify it. Cytogenetic results further demonstrate an in vitro preferential growth of the cells with a high level of aneuploidy suggesting a selective advantage for polysomy 8 cells.

Cytogenetic characterization of Metynnis maculatus (Teleostei; Characiformes): the description in Serrasalminae of a small B chromosome bearing inactive NOR-like sequences

Mitotic chromosomes of Metynnis maculatus (KNER 1860) (Teleostei, Characiformes), a fish species that occurs in the Amazon and Parana-Paraguay river basins, were analyzed for the first time by Giemsa and Ag-NOR staining, C-banding and fluorescence in situ hybridization (FISH) with 18S and 5S rDNA sequences. The basic chromosome number of the species is 2n=62 (32M+22SM+4ST+4A) and, in addition to the 62 regular chromosomes, one small acrocentric supernumerary B chromosome was found in part of the specimens analyzed. Four active NORs were present, and constitutive heterochromatin blocks were found in the pericentromeric region of several chromosomes. A heterochromatic block was also present in the interstitial portion of the submetacentric NOR-bearing pair and the B chromosome was entirely heterochromatic. FISH using an 18S rDNA probe confirmed the results obtained with AgNO(3) staining, and an additional signal was also present on the B chromosomes. 5S rDNA sequences mapped only to the largest acrocentric pair. This is the first description of supernumerary B chromosomes in Serrasalminae, and this karyotype characterization may be useful in further studies about chromosome evolution in this fish group.

Supernumerary chromosomes in the pufferfish Sphoeroides spengleri: first occurrence in marine Teleostean Tetraodontiformes fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytogenetic characterization of the strongly electric Amazonian eel, Electrophorus electricus (Teleostei, Gymnotiformes), from the Brazilian rivers Amazon and Araguaia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytogenetic characterization of six species of flatfishes with comments to karyotype differentiation patterns in Pleuronectiformes (Teleostei)

The karyotypes and cytogenetic characteristics of flatfishes species Paralichthys orbignyanus, Paralichthys patagonicus, Citarichthys spilopterus and Etropus crossotus (Paralichthyidae), Bothus ocellatus (Bothidae) and Symphurus tessellatus (Cynoglossidae) were investigated by conventional [Giemsa staining, C-banding, Ag- and chromomycin (CMA(3))-stainings] and molecular [in situ hybridization (ISH)] cytogenetic techniques. The results showed 2n = 46 and FN = 48 (2msm + 46sta) in P. orbignyanus, 2n = 46 and FN = 46 (46sta) in P. patagonicus, 2n = 26 and FN = 44 (18msm + 8sta) in C. spilopterus, 2n = 38 and FN = 64 (26msm + 12sta) in E. crossotus, 2n = 32 and FN = 50 (18msm + 14sta) in B. ocellatus, and 2n = 46 and FN = 62 (46msm + 62sta) in S. tessellatus. All species exhibited weak C-band positive segments in terminal and centromeric positions of some chromosome pairs. Silver staining of the nucleolus organizer regions (Ag-NOR) technique showed a single Ag-NOR-bearing chromosome pair in all species except E. crossotus. All these sites were CMA(3) positive and showed clear ISH signals after probing with a 18S rRNA probe. Etropus crossotus presented until seven chromosomes with Ag-NORs and CMA(3) positively stained segments in five chromosome pairs. Conversely only one chromosome pair was identified with the ISH experiments in this species. The available results show that the fishes of the order Pleuronectiformes experienced a marked chromosome evolution that included reduction in diploid number, mainly due to Robertsonian rearrangements, and several chromosome inversions. (c) 2007 the Authors Journal compilation (c) 2007 the Fisheries Society of the British Isles.

Classical and molecular cytogenetic analysis in head and neck squamous cell carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular cytogenetic analysis of heterochromatin in the chromosomes of tilapia, Oreochromis niloticus (Teleostei : Cichlidae)

The structure of the heterochromatic bands in mitotic chromosomes of the important tropical aquaculture species of tilapia, Oreochromis niloticus, was investigated by the combination of the C-banding technique, chromosomal digestion with two restriction endonucleases and fluorescence in situ hybridization (FISH) of two satellite DNAs (SATA and SATB). The tilapia chromosomes presented heterochromatic bands in the centromeres and in the short arms of almost all chromosomes that were differentially digested by the restriction endonucleases HaeIII and EcoRI. FISH of SATA showed that the satellite sequence is distributed in the centromeric region of all chromosomes of tilapia. FISH also revealed an intense hybridization signal for SATB in only one chromosome pair, but less intense signals were also present in several other pairs. The digestion of tilapia chromosomes by HaeIII and EcoRI was positively correlated with the position of SATA and SATB in chromosomes as revealed by FISH. The results obtained may be useful in future molecular and genetic studies of tilapias.

Chromosome evolution in fish: sex chromosome variability in Eigenmannia virescens (Gymnotiformes : Sternopygidae)

New data are presented on the sex chromosomes of the fish species Eigenmannia virescens (Gymnotiformes, Sternopygidae). A new finding, involving the occurrence of ZZ/ ZW sex chromosomes, is described in specimens sampled from the Sao Francisco and Amazon river basins in Brazil. All individuals had a chromosome number of 2n = 38. The homologs of the sex chromosome pair from the Sao Francisco river basin sample differed only in their morphology, while those from the Amazonian sample differed both in morphology and heterochromatin pattern. A possible model for the evolution of the sex chromosomes in E virescens is proposed, including data from populations from the Parana (Brazil) river basin, in which male heterogamety has already been described. The occurrence of different sex chromosome systems in species and populations of the neotropical freshwater fish fauna is discussed. Copyright (C) 2002 S. Karger AG, Basel.

Cytogenetic analysis of A- and B-chromosomes of Prochilodus lineatus (Teleostei, Prochilodontidae) using different restriction enzyme banding and staining methods

Different cytogenetic techniques were used to analyse the chromosomes of Prochilodus lineatus with the main objective of comparing the base composition of A- and B-chromosomes. The results of digestion of chromosomes with 10 different restriction endonucleases (REs), silver staining, CMA(3) staining and C-banding indicated the existence of different classes of highly repetitive DNA in the A-set and also suggested the existence of compositional differences between the chromatin of A- and B-chromosomes. The 5-BrdU incorporation technique showed a late replicating pattern in all B-chromosomes and in some heterochromatic pericentromeric regions of A-chromosomes. The cleavage with RE BamHI produced a band pattern in all chromosomes of P. lineatus which permitted the tentative pairing of homologues in the karyotype of this species. We concluded that the combined use of the above techniques can contribute to the correct identification of chromosomes and the karyotypic analysis in fishes. on the basis of the results, some aspects of chromosome structure and the origin of the B-chromosomes in P. lineatus are discussed.

Cytogenetic studies of Mastacembelus armatus (Pisces, Mastacembelidae)

The mitotic chromosomes, nucleolus organizer regions (NORs), C-banding pattern and nuclear DNA content of Mastacembelus armatus were studied. The karyotype (2n = 48; 10m + 6sm + 4st + 28a) was characterized by the presence of one chromosome pair with NORs and a small quantity of heterochromatin. The DNA content observed in erythrocyte nuclei of M. armatus was 1.39 +/- 0.08 pg. Comparison of this karyotype with those of other Synbranchiformes revealed a strong similarity, suggesting that small chromosome rearrangements may have been maintained during its evolutionary history.

Cytogenetic characterization of a supernumerary chromosome segment and of B-chromosomes in Astyanax scabripinnis (Teleostei, Characidae)

The chromosome complement of a local population of Astyanax scabripinnis in Brazil was investigated with emphasis on the study of the heterochromatin attached to the A-chromosomes and present in the macro B-chromosome. Analysis after C-banding, silver and CMA(3) staining, incorporation of 5-bromo-2'-deoxyuridine and chromosome digestion with nine restriction endonucleases revealed that the heterochromatin in the B-chromosomes was different from that found in the A-chromosomes. A polymorphism due to the presence of a supernumerary heterochromatic chromosome segment was observed in the population investigated. Some aspects related to the origin of the heterochromatin polymorphism in Astyanax scabripinnis are discussed.