Dorota Fopp-Bayat

Genetic characterization of Polish cultured brook trout, Salvelinus fontinalis (Mitchill), based on microsatellite DNA analysis

Genetic characterization of Polish cultured brook trout, Salvelinus fontinalis (Mitchill), based on microsatellite DNA analysis The genetic variability of two farmed strains of brook trout, Salvelinus fontinalis (Mitchill), was examined using six microsatellite DNA loci. The objective of the present study was to evaluate locus-specific genetic markers in brook trout strains cultured in Poland and to assess their levels of genetic diversity. The average number of alleles at all loci in the studied stocks differed between the two groups: 2.20 in the Rutki strain and 7.20 in the Canada strain. For the microsatellites examined, the number of alleles per locus ranged from 1 (locus Ssa-171 in the Canada strain) to 24 (locus Sfo-292 in the Canada strain). The results suggested that the Canada strain had higher gene diversity than the Rutki strain. The molecular analysis described in the present study will be useful for monitoring long-term genetic variation and for the identification of suitable parents for the development of stocks with suitable gene diversity in cultured Polish brook trout. Charakterystyka genetyczna pstrąga źródlanego (Salvelinus fontinalis Mitchill) hodowanego w Polsce przy zastosowaniu mikrosatelitarnych fragmentów DNA Przedmiotem opisywanych badań była analiza genetyczna dwóch stad pstrąga źródlanego Salvelinus fontinalis. Głównym celem badań było zidentyfikowanie alleli w wybranych loci mikrosatelitarnego DNA, charakterystycznych dla dwóch stad pstrąga źródlanego, pochodzących z Zakładu Hodowli Ryb Łososiowatych w Rutkach Instytutu Rybactwa Śródlądowego w Olsztynie. Średnia liczba alleli w analizowanych loci u badanych ryb wynosiła od 2,20 (stado Rutki) do 7,20 (stado Canada). Liczba alleli w badanych loci wynosiła od 1 (locus Ssa-171 dla stada Canada) do 24 (locus Sfo-292 dla stada Canada). W wyniku przeprowadzonych analiz stwierdzono, iż stado Canada charakteryzowało się wyższą zmiennością genetyczną w porównaniu do stada Rutki. Opisaną metodę przy zastosowaniu markerów mikrosatelitarnego DNA można wykorzystać do monitoringu genetycznego w trakcie prowadzenia prac hodowlanych.

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl-Lav-8, Cocl-Lav-18, Cocl-Lav-28, Cocl-Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5 × 10⁶ which is less than 0.1% of spermatozoa in standard 250 μL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.

Microsatellite DNA variation in the Siberian sturgeon, Acipenser baeri (Actinopterygii, Acipenseriformes, Acipenseridae), cultured in a Polish fish farm

Background. The Siberian sturgeon, Acipenser baeri, is one of the most important sturgeon species cultured in Poland. The effective management of aquaculture production of this species requires contemporary knowledge of broodstock structure, mating patterns, and genetic diversity of broodstock. The aim of the present study was the application of microsatellite DNA analysis for estimation of gene diversity in the S iberian sturgeon farmed at a Polish fish farm. Materials and Methods. Fin clips were randomly sampled from 94 specimens of Siberian sturgeon broodstock reared at the Wąsosze Fish Farm near Konin, Poland. The analysed broodstock has been kept there since 1996, with new specimens being introduced annually. The fish were studied in 2007-2008. Genomic DNA for ampli- fication of microsatellite loci was extracted using Chelex 100. Six microsatellite loci (Afu-19, Afu-39, A fu-68, AfuB-68, Spl-163, and Spl-168) were amplified for examination of the genetic variability of the studied fish. Results. Within 94 individuals of the Siberian sturgeon, a total of 74 alleles were detected in six polymorphic microsatellite loci. The number of alleles per locus ranged from 8 to 18, with an average allele number being 12. The genetic diversity of six microsatellite loci varied from 0.686 to 0.811. Conclusion. This technology has great potential for u se in aquaculture of sturgeon fish, especially when levels of genetic variation could be monitored and inbreeding controlled in commercial breeding programs.

Genetic Characteristics of Polish Whitefish ( Coregonus lavaretus Maraena ) Broodstocks-Recommendations for the Conservation Management

European whitefi sh Coregonus lavaretus maraena is an important fi sh species in Poland. Unfor - tunately, most of the whitefi sh populations are currently threatened with extinction, generally due to water pollution and over fi shing. Because the whitefi sh populations have decreased drastically in Poland, a protec - tion plan has been developed, which includes the creation of whitefi sh broodstocks in aquaculture condi - tions. During its implementation, genetic analysis of these valuable fi sh populations should be performed. This manuscript describes a study, in which microsatellite DNA analysis was applied to investigations into the genetic structure of two whitefi sh broodstocks maintained in Poland (Pomorska Bay and Galadus) and two natural populations from Lebsko Lake and Mamry Lake, northern Poland. Genetic differentiation was detected between the analyzed populations through the pairwise genetic differentiation index ( F ST ). The D A measure of genetic distance between pairs of populations indicated that the shortest distance was between the Pomorska Bay broodstock and the Lebsko Lake population (0.062), while the longest one was between the Lebsko Lake and the Galadus populations (0.172). The present results reveal genetic characterization of important popula - tions of whitefi sh in Poland and provide the first information about the genetic condition of these fi sh stocks.

Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes)

Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI‐negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50–61, 2017. ©© 2016 Wiley Periodicals,Inc.

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.

Genome Manipulation and Sex Control in the Siberian Sturgeon: An Updated Synthesis with Regard to Objectives, Constraints and Findings

The genome manipulations, such as gynogenesis or androgenesis, are widely applied in fish for artificial modification of chromosome set and allow the production of monosex stocks. Moreover, such manipulations enable rapid production of inbred populations that can be applied in crossbreeding programs. The application of the gynogenesis in sturgeons seems to be very important in creation of all-female stocks for caviar production. The Siberian sturgeon Acipenser baerii is the species most frequently cultured in European fish farms for black caviar. The production and all-female stocks of this species are highly desirable and commercially reasonable. Unfortunately, in Siberian sturgeon, the available sex identification methods are not effective for fish younger than age 3 years because these fish have no morphological sex specific features and no sex chromosomes were identified in this species. Therefore the direct production of all-female stock is very important. Although genome manipulations were successfully applied in the production of monosex populations of some fish species, especially with the XY sex-determination system, in Siberian sturgeon, such manipulations were characterized by low efficiency due to the low hatching rate. The present review provides a summary of genome manipulations in the Siberian sturgeon.

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.

Ovarian nests in cultured females of the Siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes)

Ovaries of Acipenser baerii are of an alimentary type and probably are meroistic. They contain ovarian nests, individual follicles, inner germinal ovarian epithelium, and fat tissue. Nests comprise cystoblasts, germline cysts, numerous early previtellogenic oocytes, and somatic cells. Cysts are composed of cystocytes, which are connected by intercellular bridges and are in the pachytene stage of the first meiotic prophase. They contain bivalents, finely granular, medium electron dense material, and nucleoli in the nucleoplasm. Many cystocytes degenerate. Oocytes differ in size and structure. Most oocytes are in the pachytene and early diplotene stages and are referred to as the PACH oocytes. Oocytes in more advanced diplotene stage are referred to as the DIP oocytes. Nuclei in the PACH oocytes contain bivalents and irregularly shaped accumulation of DNA (DNA‐body), most probably corresponding to the rDNA‐body. The DNA‐body is composed of loose, fine granular material, and comprises multiple nucleoli. At peripheries, it is fragmented into blocks that remain in contact with the inner nuclear membrane. In the ooplasm, there is the rough endoplasmic reticulum, Golgi complexes, free ribosomes, complexes of mitochondria with cement, fine fibrillar material containing granules, and lipid droplets. The organelles and material of nuclear origin form a distinct accumulation (a granular ooplasm) in the vicinity of the nucleus. Some of the PACH oocytes are surrounded by flat somatic cells. There are lampbrush chromosomes and multiple nucleoli present (early diplotene stage) in the nucleoplasm. These PACH oocytes and neighboring somatic cells have initiated the formation of ovarian follicles. The remaining PACH oocytes transform to the DIP oocytes. The DIP oocytes contain lampbrush chromosomes and a DNA‐body is absent in nuclei. Multiple nucleoli are numerous in the nucleoplasm and granular ooplasm is present at the vegetal region of the oocyte.

Presence of gynogenetic males suggests a female heterogamety in sterlet Acipenser ruthenus L.

Investigation of the heterogametic sex in sterlet Acipenser ruthenus L. was performed using meiotic gynogenesis and gonadal histology. Eggs from the albino females were activated by UV irradiated sperm of wild colored males and exposed to a heat shock. The resultant fish were all albino and exhibited exclusively maternal inheritance of the microsatellite DNA markers. Cytogenetic analysis indicated that gynogenetic progeny were diploids with 120 chromosomes. Based on the histological analysis, more than 86% of the gynogenetic individuals were found to be females. Moreover, some males (7%), sterile speciemens (3.5%) and fish with unidentified gonads (3.5%) were observed among the gynogenetic fish. Presence of both females and males in the gynogenetic offspring is indicative that the heterogametic sex in sterlets is female.