Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(4): 3-9, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-660 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: M. Kloch, A. Życzyński, W. Olech, Z. Nowak-Życzyńska (2020) Cytogenetic study of the Bison bonasus; I: Identification of heterochro- matic regions and NORs in European bison karyotype and comparison with domestic cattle (Bos taurus). Caryo- logia 73(4): 3-9. doi: 10.13128/caryolo- gia-660 Received: October 28, 2019 Accepted: October 02, 2020 Published: May 19, 2021 Copyright: © 2020 M. Kloch, A. Życzyński, W. Olech, Z. Nowak-Życzyńska. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Funding: This research was funded by the Forest Found (Poland), grant num- ber OR.271.3.10.20171. ORCID MK: 0000-0003-2569-5008 AŻ: 0000-0003-4128-9704 WO: 0000-0002-6166-3954 ZNŻ: 0000-0002-5907-9943 Cytogenetic study of the Bison bonasus; I: Identification of heterochromatic regions and NORs in European bison karyotype and comparison with domestic cattle (Bos taurus) Marta Kloch*, Andrzej Życzyński, Wanda Olech, Zuzanna Nowak- Życzyńska Department of Animal Genetics and Conservation, Institute of Animal Sciences, Warsaw University of Life Sciences, Warsaw, Poland *Corresponding author. E-mail: marta_kloch@sggw.edu.pl Abstract. The karyotypes of European bison Bison bonasus and domestic cattle Bos taurus are characterized by a diploid number of chromosomes, 2n = 60. Here, we char- acterized the European bison karyotype in terms of size and distribution of constitutive heterochromatin blocks (C-bands) and the location and number of nucleolar organizer regions (NORs), results were compared with those obtained for domestic cattle. For this purpose, staining for C and NOR bands was performed. In the chromosomes of both species, C-bands were located in the centromeric region of all chromosomes ana- lyzed, except for the X chromosome. Active NORs in European bison chromosomes were identified in the chromosomes from pairs 2, 3, 4, 25, and 28. In cattle, NORs were located in chromosomes from pairs 2, 3, 4, 11, and 25. The average number of NORs in the cell of European bison and cattle was 4.47 ± 1.74 and 4.56 ± 1.66, respectively. The obtained results shed new light on the European bison cytogenetics and confirmed the high similarity between studied species. Keywords: Bison bonasus, Bos taurus, heterochromatin, nucleolar organizer regions, nor, c-banding. INTRODUCTION The identification of homologous chromosomes is carried out on the basis of different banding techniques that reveal specific regions of the chromosome, for example constitutive heterochromatin (Sumner 1972) or Nucleolar Organizer Regions (NOR) (Goodpasture and Bloom 1975). The differentiated NOR sizes within a homology pair and the variability of con- stitutive heterochromatin blocks are treated as a chromosomal polymor- phism, and variants that differ in NOR or C-bands are considered as chro- mosomal markers. Constitutive heterochromatin is visible in the form of C-bands, is a late replicating fraction and contains a small number of genes (Lawce 2017). 4 Marta Kloch, Andrzej Życzyński, Wanda Olech, Zuzanna Nowak-Życzyńska Moreover, it was found that the DNA found in the con- stitutive heterochromatin blocks is a region that does not express genetic expression (Brown 1966). Methyla- tion within CpG islands affects the significant level of condensation of constitutive heterochromatin (Spector 2003). Heterochromatin is also involved in the transcrip- tion and segregation of chromosomes (Grewal and Jia 2007). Sipko et al. (2004) published a study in which they compared the karyotypes of Bison bonasus and Bos taurus by using the Sumner (1972) staining method of C-banding and SCE (sister chromatid exchange). They found no difference between the chromosomes of both species. The C-band method used by the authors showed that heterochromatic blocks were identified in all chro- mosomes except the X chromosome in both species. The nucleolar organizer regions usually form sec- ondary constrictions and are the localization site for genes encoding ribosomal nucleic acids (rRNA). NORs are organized as blocks of tandem repeating units whose distribution, i.e. the number of chromosomal loci and the number of genes in each locus, is a constant and characteristic for a given species (Weisenberger and Scheer 1995). Due to the affinity of NOR for heavy met- als, nucleolar organizer regions appear in the form of black silver stripes during the dyeing process. Silver staining does not allow revealing all areas containing rRNA genes, but only active areas for nucleus formation in interphase (Verma and Babu 1995). Graphodatsky et al. (1990) using classic staining method identified NORs on 2nd, 3rd, 4th and 28th chro- mosome pairs, while Gallagher et al. (1999) used in situ hybridization to identify NORs in Bison bonasus chro- mosomes and observed signals on chromosomes from pairs 2, 3, 11, 25, and 28. Because of the limited amount of available literature, there is still much scope to discover and describe the cytogenetics of the species Bison bonasus also from the standpoint of separately maintained two lines of Euro- pean bison: Lowland and Lowland-Caucasian (Pucek et al. 2004). The aim of the study was to characterize the Euro- pean bison karyotype in terms of size and distribution of constitutive heterochromatin blocks as well as the location and number of nucleolar organizer regions. It was decided to compare the karyotypes of the two stud- ied species and confirm their similarity. Furthermore, we decided to confirm the location of NORs in the Euro- pean bison. This is the first study in which the size of the heterochromatin area was measured in European bison. MATERIALS AND METHODS The experimental material consisted of 12 blood samples collected from Bison bonasus males from four localities (Figure 1, Table 1). The samples were stored in 9 ml heparinized tubes (Medlab Products) to pre- vent blood clotting and stored in cold until laboratory analysis. Blood from domestic cattle bull (Bos taurus) obtained in abattoir was used as a comparative material. Cell culture The cultures were carried out in 15 ml falcon tubes containing 8.5 ml of the culture medium RPMI 1640 Figure 1. Collection sites of European bison (Bison bonasus) sam- ples in Poland. The numbering of sampling localities corresponds to the data in the Table 1. Table 1. Collection sites of European bison (Bison bonasus) samples in Poland. The numbering of sampling localities corresponds to the Figure 1. SM – submetacentric, A – acrocentric. Locality Latitude, longitude No. of specimens 2n X Y Niepołomice 50°02’29.6”N, 20°21’54.1”E 3 60 SM A Gołuchów 51°51’35.2”N, 17°55’28.6”E 3 60 SM A Białowieża 52°42’20.0”N, 23°47’46.0”E 3 60 SM A Muczne 49°08’32.1”N, 22°42’50.5”E 3 60 SM A 5Cytogenetic study of the Bison bonasus (SIGMA) with addition of 10% fetal bovine serum (SIG- MA), pokeweed mitogen (SIGMA), and antibiotic (peni- cillin 100 µg/mL and streptomycin 100 µg/mL)(SIGMA). A thoroughly mixed blood sample was added to the final volume of 10 ml. The cultures were carried out in duplicate for each individual. The tubes were incubated at 38.5 °C for 72 hours and regularly mixed twice a day. The cultures were treated for 1 hour by colchicine (0.01 µg/mL) (SIGMA) (added at 71 hours). Subsequently, the cultures were treated with hypotonic solution (0.05 M KCl (POCH)) for 20 minutes at 38.5 °C, and then three times fixed in freshly prepared freezing cold Carnoy’s fixative (POCH). Microscope slide preparation Slides were prepared before the staining procedures. The mixture of the fixed precipitate was spotted on a microscope slide and then air dried. C-banding and AgNOR staining Initially, the slides were subjected to the standard staining procedure described by Sumner (1972). The results obtained in the form of poorly visible C-bands were unsatisfactory; hence, we decided to use the meth- odology described by Chaves et al. (2000). Silver staining was carried out according to the method described by Howell and Black (1980). Analysis of the slide preparations By using a Nikon Eclipse 90i microscope connected to the DS5-U1 digital camera (Nikon Corporation, Tokyo, Japan), and a Zeiss Axiophot fluorescence microscope and LUCIA software (Laboratory Imaging Ltd, Prague, Czech Republic), 25 clearly visible and well dispersed metaphase plates were photographed for each male of both species. The measurements were made in the IMAGEJ program by using the LEVAN plugin (Wayne Rasband, Research Ser- vices Branch, National Institute of Mental Health, Bethes- da, Maryland, USA, 2018). The obtained results were characterized statistically. RESULTS AND DISCUSSION The constitutive heterochromatin is a fraction which is most often located near the centromere of the chro- mosome, but it is also located in the distal parts of the chromosome arms, sometimes between the centromere and the telomeres or occupies the entire chromosomal arms (Lawce 2017). In our own research we measured the block size of constitutive heterochromatin in relation to the entire length of the arms of all analyzed chromo- somes. The used procedure allowed us to determine the position of C-bands on European bison and domestic cattle chromosomes, and thus to identify sex chromo- somes of both species (Figure 2A and 3A, respectively). The C-bands were located in the centromeric regions of all autosomes in both species and on Y chromosomes. In both species, no positive band was identified on X chro- mosomes. A positive, small, and dark band was found at the end of the short arm of the smallest chromosome of Bison bonasus, the Y chromosome. This chromosome is largely heterochromatic. Further, on the Y chromo- some of domestic cattle, a dark and distinct positive band located at the end of the shorter arm was visible. By comparing the image obtained for the Y chromo- some of European bison and domestic cattle, the differ- ence in their morphology can be seen. The Y chromo- some of domestic cattle is submetacentric, while that of the European bison is a small acrocentric as reported (Graphodatsky et al. 1990). Originally, the European bison’s Y chromosome was considered submetacentric (Fedyk and Sysa 1971). In both species, 59 blocks of con- stitutive heterochromatin were identified on the 30 pair of chromosomes (Figure 2B and 3B). In the analyzed acrocentric autosomes of European bison (Table 2) and domestic cattle (Table 3), a clear dif- ferentiation in the extent of heterochromatin areas was observed. In the measurement of constitutive heterochro- matin block size in relation to the entire length of the European bison chromosome, the first pair of chromo- somes showed the smallest mean value (14.52%), and the twelfth pair of chromosomes (26.40%) showed the high- est value. In cattle, the first pair of chromosomes showed the smallest mean value (14.06%), while the twenty-third pair showed the highest value (32.50%). There were no positive blocks of constitutive heterochromatin on the X chromosomes in both species. The heterochromatin con- tent of the Y chromosome of European bison and cattle was 39.15% and 28.95%, respectively. This indicates that the European bison’s Y chromosome is much more het- erochromatic. The variable length of heterochromatin blocks does not affect the phenotype, however it differ- entiates the morphology of the chromosomes of a given species. It has been found that the variable width of posi- tive C-banding blocks indicates the presence of non-iden- tical heterochromatin size (McFeely 1990). The number of active NORs and their location in chromosomes is a characteristic of each species. In the 6 Marta Kloch, Andrzej Życzyński, Wanda Olech, Zuzanna Nowak-Życzyńska metaphases of the same individual, there is an intercel- lular diversity of expression of NOR, which refers to the amount, size and intensity of silver staining (Weisen- berger and Scheer 1995). The AgNOR staining enabled to identify active nucleating regions (NORs) found on the chromosomes of European bison and domestic cattle Figure 2. Karyogram of European bison male (Bison bonasus), (a) C-banding, (b) AgNOR-banding. Heterochromatic blocks are mainly located in centromeric positions of all autosomes and in sex chromosomes. NORs are located in 2, 3, 4, 25 and 28 chromosome pairs (marked in squares). Figure 3. Karyogram of domestic cattle male (Bos taurus), (a) C-banding, (b) AgNOR-banding. Heterochromatic blocks are located in cen- tromeric positions of all autosomes and in sex chromosomes. NORs are located in 2, 3, 4, 11 and 25 chromosome pairs (marked in squares). 7Cytogenetic study of the Bison bonasus (Figure 2B and 3B, respectively). In European bison, the active nuclear regions were found in the terminal parts of chromosomes from pairs 2, 3, 4, 25, and 28 (Figure 2B). Similar conclusions were made by Gallagher et al. (1999) by using in situ hybridization for this purpose. In domestic cattle, visible silver grains were observed on chromosomes from pairs 2, 3, 4, 11, and 25 (Figure 3B). In a total of 325 metaphase plates analyzed (300 for European bison and 25 for domestic cattle) (Table 4), 1476 active nucleolar regions were observed (Table 5). There were visible differences in the intensity of NORs on chromosomes and the differences in the minimum and maximum number of active NORs in cells. At the cellular level, one to eight active NORs were observed in Bison bonasus species with an average value of 4.47 ± 1.75. In the Bos taurus species, it ranged from two to eight active nuclear regions with an average value of 4.56 ± 1.66. For comparison, in a similar experiment, the obtained active NORs average value for cattle was 6.06 (Mayr et al. 1987). The number of NORs in the cells of the animals varied and ranged from 107 to 115 within the individual. The most frequently observed cells had four active NORs, and the least frequently observed cells had eight. Our research allowed us to characterize the Euro- pean bison karyotype in terms of the C-band pattern and the number and location of active NORs. The fol- lowing conclusions were made on the basis of the ana- lyzes carried out. The number of active NORs per cell differed between the tested individuals and between the cells of the same individual. The method used in this study to identify C-bands can be successfully used to identify sex chromosomes. Constitutive heterochro- matin has been identified in all European bison (Bison bonasus) and domestic cattle (Bos taurus) chromosomes, except for the X chromosome in both species. Chro- mosomes of both species can be compared on the basis of different classical staining methods, but a thorough analysis aimed at comparison of the Y chromosome requires more advanced analysis. The Y chromosome of European bison was smaller than domestic cattle Y chromosome, and classified as acrocentric. Studies on the comparison of the Y chromosome structure of Bison and Bos species can bring very interesting results. Poten- tially identified differences within the Y-chromosome- linked sequences will allow to design a quick and easy- to-use test that unambiguously indicates father’s descent. In 1999, a genetic test was developed based on the sequence of the 16S rRNA subunit, which made it pos- sible to detect the presence of cattle-specific mitochon- drial sequences in individuals of the Bison genus (Ward et al. 1999). Unexpectedly, when performing the analysis Table 2. Size of constitutive heterochromatin blocks in European bison (Bison bonasus) chromosomes. Chromosomes 1-14; XY Average area of the constitutive heterochromatin x- ± S Chromosomes 15-29 Average area of the constitutive heterochromatin x- ± S 1 14.52 ± 5.23 15 20.64 ± 2.24 2 16.49 ± 2.29 16 23.64 ± 12.46 3 17.78 ± 4.17 17 22.40 ± 0.89 4 16.05 ± 5.86 18 23.48 ± 1.68 5 25.06 ± 4.08 19 22.47 ± 0.70 6 23.95 ± 1.66 20 20.38 ± 1.84 7 16.79 ± 1.52 21 19.22 ± 1.77 8 18.80 ± 4.22 22 22.64 ± 3.04 9 21.33 ± 3.20 23 22.01 ± 7.13 10 23.68 ± 2.25 24 23.93 ± 2.16 11 18.12 ± 0.80 25 25.55 ± 3.41 12 26.40 ± 0.26 26 18.04 ± 6.04 13 17.18 ± 1.89 27 21.77 ± 3.64 14 21.14 ± 3.82 28 21.92 ± 2.21 X - 29 21.11 ± 3.14 Y 39.15 ± 1.20 Table 3. Size of constitutive heterochromatin blocks in cattle (Bos taurus) chromosomes. Chromosomes 1-14; XY Average area of the constitutive heterochromatin x- ± S Chromosomes 15-29 Average area of the constitutive heterochromatin x- ± S 1 14.06 ± 1.60 15 19.95 ± 1.76 2 18.46 ± 3.15 16 18.39 ± 1.15 3 17.66 ± 1.40 17 23.11 ± 2.91 4 16.80 ± 2.51 18 25.19 ± 5.46 5 15.35 ± 1.87 19 25.51 ± 5.70 6 17.73 ± 3.63 20 26.69 ± 4.12 7 15.41 ± 4.47 21 32.15 ± 4.26 8 18.01 ± 1.13 22 30.98 ± 0.92 9 16.67 ± 2.53 23 32.50 ± 0.76 10 21.72 ± 4.86 24 21.47 ± 4.35 11 20.50 ± 3.35 25 27.57 ± 5.43 12 20.22 ± 2.44 26 25.54 ± 1.28 13 23.04 ± 3.21 27 25.77 ± 2.02 14 27.67 ± 2.80 28 24.56 ± 0.30 X - 29 27.78 ± 6.29 Y 28.95 ± 9.34 8 Marta Kloch, Andrzej Życzyński, Wanda Olech, Zuzanna Nowak-Życzyńska using this test in our laboratory, it turned out that over 75% of the studied European bison population showed the presence of sequences characteristic of the Bos genus (Nowak et al. 2008). 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Numbers of NOR Chromosome 2 x- ± S 3 x- ± S 4 x- ± S 11 x- ± S 25 x- ± S 28 x- ± S Niepołomice 1.22 ± 0.43 (81) 1.17 ± 0.52 (72) 1.17 ± 0.39 (42) - 1.59 ± 0.51 (81) 1.75 ± 0.45(84) Gołuchów 1.47 ± 0.51 (75) 1.36 ± 0.50 (57) 1.40 ± 0.51 (63) - 1.44 ± 0.51 (69) 1.47 ± 0.52 (66) Białowieża 1.50 ± 0.51 (81) 1.51 ± 0.52 (63) 1.38 ± 0.50 (66) - 1.63 ± 0.50 (78) 1.75 ± 0.45 (63) Muczne 1.39 ± 0.50 (75) 1.46 ± 0.52 (57) 1.22 ± 0.43 (66) - 1.40 ± 0.51 (63) 1.54 ± 0.52 (60) Bos taurus 1.20 ±0.41 (24) 1.36 ± 0.50 (19) 1.24 ± 0.44 (26) 1.05 ± 0.22 (22) 1.21 ± 0.42 (23) - Total 1.36 ± 0.47 (336) 1.37 ± 0.51 (268) 1.28 ± 0.10 (263) 1.05 ± 0.22 (22) 1.45 ± 0.49 (314) 1.63 ± 0.49 (273) Table 5. Number of NORs in the cells of the examined animals. 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