Marcin Kucinski

Pericentromeric location of the telomeric DNA sequences on the European grayling chromosomes

AbstractThe chromosomal characteristics, locations and variations of the C-band positive heterochromatin and telomeric DNA sequences were studied in the European grayling karyotype (Thymallus thymallus, Salmonidae) using conventional C-banding, endonucleases digestion banding, silver nitrate (AgNO3), chromomycin A3 and 4′,6-diamidino-2-phenylindole staining techniques as well as fluorescence in situ hybridization (FISH) and primed in situ labelling. Original data on the chromosomal distribution of segments resistant to AluI restriction endonuclease and identification of the C-banded heterochromatin presented here have been used to characterize the grayling karyotype polymorphism. Structural and length polymorphism of the chromosome 21 showing a conspicuous heterochromatin block adjacent to the centromere seems to be the result of the deletion and inversion. Two pairs of nuclear organizer regions (NOR)-bearing chromosomes were found to be polymorphic in size and displaying several distinct forms. FISH with telomeric peptide nucleic acid probe enabled recognition of the conservative telomeric DNA sequences. The karyotype of the thymallid fish is thought to experienced numerous pericentric inversions and internal telomeric sites (ITSs) observed at the pericentromeric regions of the six European grayling metacentric chromosomes are likely relics of the these rearrangements. None of the ITS sites matched either chromosome 21 or NOR bearing chromosomes.

Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814)

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3–6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Disturbances in the ploidy level in the gynogenetic sterlet Acipenser ruthenus

Artificial mitotic gynogenesis, a chromosome set manipulation, is applied to provide the homozygous progeny with only maternal inheritance. Here, gynogenetic development was induced in the sterlet Acipenser ruthenus L. (Acipenseridae) by activation of the eggs originating from albino females with the UV-irradiated spermatozoa from wild-coloured males, followed by the heat shock applied to suppress the first mitotic division in the haploid zygotes. All experimentally obtained gynogenetic offspring possessed recessive albino coloration. Moreover, the genetic verification, based on three microsatellite DNA markers, confirmed the only maternal inheritance in the albino progeny. Cytogenetic screening enabled identification of the aneuploids, haploids, diploids, triploids, tetraploids and mosaic individuals among the gynogenetic larvae that hatched from the eggs subjected to the heat shock. Furthermore, 40% of the larvae from the haploid variants of the research that were not exposed to the temperature shock showed the diploid chromosome number. A variation of the ploidy level observed in the gynogenetic sterlets may be the consequence of the spontaneous polyploidisation that occurred in the haploid zygotes. Moreover, observation during embryogenesis showed varied stages of eggs development and the asynchronous cell cleavages that may have resulted in the chromosomal disturbances observed in the gynogenetic sterlets here.

Genetic Variability in Farmed Brood Stocks of the Siberian Sturgeon in Poland

The Siberian sturgeon Acipenser baerii is one of the most common and most important sturgeon species cultured in Europe, being the main source of sturgeon meat and black caviar produced by large fish farms and international aquaculture companies. The present paper describes the genetic characteristics of Siberian sturgeon Acipenser baerii brood stock from the Polish fish farm. The genetic analysis, based on six polymorphic microsatellite DNA analysis, revealed a high level of genetic diversity in studied broodstock of Siberian sturgeon (PIC = 0.504–0.837 and I = 1.036–2.150). The observed values of allelic richness (Ar) varied from 6.000 to 13.500 in examined fish group. Observed (Ho) and expected (He) heterozygosity across the studied loci showed values from 0.723 to 1.000 and from 0.586 to 0.857, respectively. Overall, the examined broodstock were not in Hardy-Weinberg equilibrium (H-WE), where five of the six microsatellite loci deviated from H-WE. The estimated effective population size (Ne) values by the linkage disequilibrium and the molecular coancestry methods were at the level of 47.3 (95% CI = 39.6–57.2) and 41.3 (95% CI = 3.0–128.7), respectively. A total number of 38 rare alleles within investigated microsatellite loci were found, which consisted 51% of qualitative composition of all detected alleles. All the analyzed genetic indicators suggested the good genetic condition and high genetic value of studied Siberian sturgeon farmed broodstock.

Heterochromatin organization and chromosome mapping of rRNA genes and telomeric DNA sequences in the burbot Lota lota (Linnaeus, 1758) (Teleostei: Gadiformes: Lotidae)

Abstract The knowledge about the genome of the burbot Lota lota (Linnaeus, 1758), the only freshwater gadiform fish (Gadidae, Gadiformes), is limited to chromosome number (2n = 48, NF = 78). In this work analysis of burbot chromosomes by C-banding, DAPI staining and restriction endonuclease digestion proved both heterogeneity of the heterochromatin and interchromosomal differences in the vulnerability to the endonuclease treatment. Silver nitrate and chromomycine A3 staining and fluorescence in situ hybridization using 28S rDNA probes showed that nucleolus organizer regions appear in one pair of burbot chromosomes. There is also a single locus for 5S rDNA repeats exhibited in burbot; however, major and minor rRNA gene families are not linked. The single and separated location of major and minor rDNA families suggests that the burbot karyotype still retains some characteristics of the putative ancestral karyotype. Although the increased number of the chromosome arms in relation to the ancestral karyotype suggests that burbot karyotype experienced several inversions, we did not find any intercalary telomeric DNA sequences in their chromosomes.

The morphological, histological and cytogenetic characteristics of goldfish Carassius auratus (L.) × common carp Cyprinus carpio (L.) hybrids

Morphology of hybrids between the goldfish (Carassius auratus) and common carp (Cyprinus carpio) are studied by biometric and meristic methods. Additionally, histological analyses concerning gonads as well as genetic characteristics of chromosomes are performed. Studied hybrids were morphologically very similar to a goldfish but possessed one pair of reduced posterior barbels. Most meristic and biometric indices had intermediate values when compared to parental species. Histological study showed that investigated hybrids produce gonads with germ cells. All studied males had gonads at the same stage of maturity (III stage). By contrast, female gonads were in various stages of development – from sexually immature to post-spawning stage. Karyotype characteristics were assessed in terms of chromosomes number, fundamental arm number and location of nucleolus organizer regions (NOR). The data obtained were compared with baseline karyotypes of species used for hybridization, based on information from the literature. The diploid chromosome number of studied hybrids equaled 2n = 100, NF = 152. Ag-NORs were localized on the short arms of two to three submetacentric chromosomes. Additionally, one to two supernumerary (B) chromosomes were also found, which is a typical phenomenon for interspecific hybrids, especially between cyprinid fish species.

Example of the application the microsatellite DNA fragments in the study of farmed European catfish (Silurus glanis, L.) broodstock

Abstract European catfish, Silurus glanis L., is the second largest freshwater fish in Europe. The species is very popular among the farmers, as it is one of the most promising European aquaculture species. Despite the growing importance of European catfish in freshwater aquaculture, the genetic data available on this species are still limited. The main purpose of the present study was to develop a reliable, feasible genetic protocol for future studies on European catfish populations and broodstocks in Poland. The genetic characteristics of the tested fish group were based on genetic parameters such as the polymorphism information content (PIC), the effective population size (Ne), the inbreeding coefficient (Fis), and the Garza-Williamson index (M), among others. Additionally, the potential effects of a genetic bottleneck on the genetic variation of the broodstock were examined. The genetic analysis protocol described in this study can be used to establish genetic-based records for European catfish broodstocks, including for sperm cryobanking. This approach will be useful for elaborating the selection procedures that allow for optimal assemblages of spawning pairs in artificial reproduction. The application of the genetic analysis protocol in practice will permit maintaining high quality in European catfish broodstocks.