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Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae
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Method paper
Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae ⁎
Laura Ghigliottia, , Chi-Hing C. Chengb, Catherine Ozouf-Costazc, Chantal Guidi-Rontanid, Marino Vacchia, Sara Federicie, Eva Pisanoa,e a
National Research Council of Italy (CNR), IAS, Genoa, Italy Department of Evolution, Ecology and Behavior, University of Illinois, Urbana-Champaign, IL 61801, USA Institute of Biology Paris Seine, Université Pierre et Marie Curie (CNRS, UMR 7138 « Evolution Paris-Seine»), Paris, France d Institut de Systematique, Evolution, Biodiversité (ISYEB) (CNRS UMR 7205), Museum National d'Histoire Naturelle, Paris, France e Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluorescence in situ hybridization Karyotype LINEs Notothenioidei Ribosomal genes
This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae. The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of twoarmed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes. Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.
1. Introduction The Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) is a pelagic notothenioid fish abundant and widely distributed around the coastal waters of the Antarctic continent (Duhamel et al., 2014). As a mid-trophic level plankton-feeder, it plays a pivotal role in the High Antarctic coastal marine ecosystem (reviews on the biology and ecology of the species in Vacchi et al., 2017). Despite its ancestral benthic origin, common to the Notothenioidei, the Antarctic silverfish occupies an ecological niche in the water column owing to extensive evolutionary changes that led to secondary pelagization (Voskoboinikova et al., 2017). The monotypic genus Pleuragramma has consistently been included in Nototheniidae, but the relation to its closest relatives is still unresolved. Based on morphological traits, Pleuragramma was initially
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included in the nototheniid subfamily Pleuragramminae, along with Aethotaxis (Andersen and Hureau, 1979). The subfamily name was later changed to Pleuragrammatinae, and the genus Gvozdarus was added (Balushkin, 1984). Following combined molecular and morphological analysis, the clade Pleuragrammatinae was recognized grouping all neutrally buoyant notothenioid species, including the two Dissostichus (Near et al., 2007). A classification of Notothenioidei based on the analysis of multiple mitochondrial/nuclear DNA markers (Dettai et al., 2012) assigned the monotypic genus Pleuragramma to the subfamily Pleuragramminae, whereas Dissostichus, Aethotaxis, and Gvozdarus were moved to Dissostichinae Nevertheless, the phylogenetic position of Dissostichus, Aetothaxis and Pleuragramma remained unresolved. A timecalibrated phylogeny (Colombo et al., 2015) still led uncertainty in the relationship among the pelagic taxa Pleuragramma, Dissostichus and Aethotaxis, and provided support to a very early divergence of
Corresponding author. E-mail address: [email protected] (L. Ghigliotti).
https://doi.org/10.1016/j.margen.2019.100737 Received 18 September 2019; Received in revised form 17 December 2019; Accepted 19 December 2019 1874-7787/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Laura Ghigliotti, et al., Marine Genomics, https://doi.org/10.1016/j.margen.2019.100737
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Pleuragramma in the process of Antarctic notothenioid fish adaptive radiation. The recent analysis by Near et al. (2018) does not resolve Pleuragrammatinae as a clade, with one poorly supported node separating P. antarctica from a monophyletic group containing A. mitopterix and the two Dissostichus. For its biological and ecological importance in the high Antarctic waters, and its persistent taxonomic ambiguities, P. antarctica is an interesting target for cytogenetic studies. Detailed characterization of the chromosomal features of P. antarctica can assist in resolving the evolutionary history of the lineage, and contribute to further understanding of changes in the genome architecture during notothenioid evolution and diversification (Pisano and Ozouf-Costaz, 2003). Despite its wide geographical distribution and abundance in the Antarctic coastal ecosystems, the Antarctic silverfish remained cytogenetically understudied due to difficulties of capturing live adult specimens, and of keeping them in healthy condition to undergo the treatments required by the classical direct chromosome preparation procedures. Only five adults (3 males and 2 females) from the Weddell Sea were analyzed prior to this study, yielding only basic information on the chromosomal number (2n = 48) and karyotype formula (8 m + 32sm + 8a) (OzoufCostaz et al., 1991). No cytogenetic data are available on P. antarctica from other Antarctic areas, including the Ross Sea, despite the historically intense cytogenetic surveys conducted on Ross Sea notothenioid fauna (reviewed in Ghigliotti et al., 2015). The discovery of an Antarctic silverfish nursery area at Terra Nova Bay (western Ross Sea), with thousands of eggs developing within the ice platelet layer under the sea-ice cover (Vacchi et al., 2012), provided a unique opportunity to develop alternative protocols for obtaining chromosomes from early larvae. Here we report on the cytogenetic characterization of the Antarctic silverfish from the western Ross Sea based on the analysis of chromosomes from early larvae of this cold adapted species. Conventional staining and fluorescence in situ hybridization (FISH) of repeated sequences were applied to obtain a refined picture of the Antarctic silverfish karyotype and chromosomal architecture. Given that Long Interspersed Nuclear Elements (LINEs) were found expanded in a number of Antarctic fishes, and are supposed to have contributed to the gene duplication and genome size enlargement in those species (Chen et al., 2008), the chromosomal organization of LINEs sequences in P. antarctica was explored. To the best of our knowledge, this is the first study on chromosomes from early life stages of Antarctic fishes.
Fig. 1. Near-hatching Antarctic silverfish eggs collected under the sea ice in the Ross Sea nursery area.
were used for chromosome preparation as follows. The young larvae were mechanically disrupted in 0.075 M KCl on a 350-μm cell strainer. The obtained cells were re-suspended in hypotonic 0.075 M KCl solution for 60–90 min, at 1–3 °C. Hypotonized cells were then fixed in Carnoy fixative (methanol/acetic acid 3:1 v/v) according to standard procedures for short term cell cultures in Antarctic fishes (Rey et al., 2015). Fixed cell suspensions were stored at −20 °C for later laboratory analyses. Reference cells preparations were deposited and cryopreserved at the National Antarctic Museum, Genoa. 2.2. Karyotyping Chromosome spreads from fixed cells dropped on microscope slides were treated for karyotyping and chromosome banding according to current protocols for notothenioid fishes (Ozouf-Costaz et al., 1997, 2015). Chromosomes were stained with DAPI (4,40,6-diamidino-2-phenylindole) and observed through an Olympus BX61 microscope. Multiple metaphases from each preparation (a minimum of 100 metaphases) were digitally imaged using Sensys (Photometrics) CCD camera. The digital images were processed either by the use of Genus Software (Applied Imaging) or by application of Adobe Photoshop image analysis. The characterization of chromosomal morphology followed the Levan et al. (1964). Based on the position of the centromere and on the arm ratio, chromosomes were assigned to the following categories: metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). Metacentric, submetacentric and subtelocentric chromosomes are two-armed, acrocentrics are one-armed. The long arm of twoarmed elements is referred to as q (queue), the short arm is referred as p (petit), according to the International system for human cytogenetic nomenclature (Mitelman, 1995). Chromosomes were arranged in the karyotype according to size. The karyotypic formula and the total number of chromosome arms (Fundamental Number, FN) were obtained from analyses of a minimum of 20 karyotypic sets from each of the 13 chromosome preparations.
2. Material and methods 2.1. Sampling and chromosome preparation Sampling was carried out on the 3rd and 8th of November 2012 at 74° 39.150S – 164° 41.302E, in the Antarctic silverfish nursery area at Terra Nova Bay, Ross Sea. Near term embryonated eggs, floating among icy sea water, were collected through holes drilled in the sea ice cover following the sampling procedure described in Vacchi et al. (2012). Samples, stored in insulated boxes, were immediately transferred to the aquaria at Mario Zucchelli Station. Pilot small-scale experiments were performed to set up and optimize a protocol for chromosome preparation. Three parameters were considered as critical and for those a range of values were tested. In particular, water temperature ranging 1–5 °C, duration of the colchicine treatment ranging 12–72 h, and hypotonic incubation time ranging 30–90 min, were tested. Eggs and larvae at various developmental stages, used individually or pooled in homogeneous batches, were tested. Based on the results of the smallscale trials, a protocol was finalized and applied to pools of embryos in large-scale experiments of chromosome extraction. Briefly, homogeneous near hatching eggs (Fig. 1) were reared in petri dishes at 3 °C in 0,005% colchicine in sea water. About half of the colchicined sea water was changed every 12 h. After 52 h, hatching was completed and 13 pools (10–20 specimens each) of just-hatched larvae
2.3. Fluorescence in situ hybridization (FISH) In situ mapping of repeated sequences, namely 28S ribosomal DNA and Long Interspersed Nuclear Elements (LINEs), was performed in order to gain a better resolution of the chromosomes structure. A clone obtained from the nototheniid Dissostichus mawsoni (GenBank accession number AY926497) was used as the probe for FISH of 28S rDNA according to the protocol described in Ghigliotti et al. (2007). To prepare the probe for FISH, a 551 nucleotides portion of the D. 2
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reported for adults (Ozouf-Costaz et al., 1991). Such a discrepancy is probably due to a better resolution of the chromosome morphology, leading to the detection of small arms in some chromosome pairs previously described as acrocentrics. The lack of any morphologically distinguishable heterochromosome in the large number of examined metaphases support the lack of a differentiated sex-chromosome system, as hypothesized from the examination of chromosomes of adult specimens (Ozouf-Costaz et al., 1991). A degree of variability was observed among separate metaphases for the chromosomes of pairs 20, 21, and 23. The p arms of those elements appeared heteromorphic in size and morphology due to the variable extension of a dim DAPI stained region differing between the homologues and between metaphases. Such a dim DAPI stained region is located at interstitial position on the p arms of pair 20, at sub-telomeric position in pair 21, and occupies the entire p arm in the homologous chromosomes pair 23 (Fig. 2 b). Whether the observed variability is intra-individual or inter-individual cannot be deduced from present analysis given that each chromosome preparation results from a pool of specimens. FISH mapping of 28S rDNA highlighted the location of major ribosomal genes in the same three chromosome pairs (20, 21, 23), corresponding to the dim DAPI stained regions (Fig. 2 c, boxed pairs). Variation in 28S rDNA signal patterns were detected, in agreement with the observed variability in the extent of the dim DAPI stained regions. The pattern most frequently observed was with the major ribosomal genes located on a large sub-telomeric region of the pair 21; occurrence of ribosomal genes at interstitial position on the p arms of pair 20, and on the entire p arms of the homologues of pair 23, were also found. The rDNA signals were never simultaneously observed on all the six chromosomes, possibly due to the variability in the number of gene copies at the single chromosome locations, sometimes falling under the resolution limits of the FISH technique. Based on current cytogenetic information on the Antarctic notothenioids (Arai, 2011; Ghigliotti et al., 2015), and according to the hypothesized karyotype rearrangements trajectory within the suborder (e.g. Ozouf-Costaz et al., 1999; Pisano and Ozouf-Costaz, 2003; Amores et al., 2017), the Antarctic silverfish chromosomal features are distinctively interesting. With 48 chromosomes and predominantly two-armed, the Antarctic silverfish karyotype differs substantially from the supposed ancestral karyotype for notothenioids, which comprises 48 simple one-armed chromosomes. Cytotaxonomically, the Antarctic silverfish karyotype features are different from those of the Pleuragrammatinae (sensu Near et al., 2007) Dissostichus species, which possess the ancestral diploid number of 48 primarily one-armed chromosomes (Ghigliotti et al., 2007). Intriguingly, the chromosomal changes leading to the distinct silverfish karyotype, seem different from the general trend recorded in the high-Antarctic lineages, where increase of two-armed chromosomes is paired with decrease of the diploid numbers (Ghigliotti et al., 2015). Furthermore, the pattern of major ribosomal genes of the Antarctic silverfish, unevenly distributed on up to three chromosome pairs, is unique among the Antarctic notothenioids, where the major ribosomal gene repeats map to a single chromosomal locus in most of the species, and to two chromosomal loci in a few species (Pisano and Ghigliotti, 2009). Among chromosome rearrangements, pericentric inversions and reciprocal translocations are generally most likely events that lead to such highly modified cytogenetic layouts. In such a highly dynamic karyotype restructuring scenario, where the number of chromosomal elements is conserved but the chromosomes are drastically reshaped, transposable elements (TE) have been hypothesized as promotors of structural alterations such as fusions (Auvinet et al., 2019). Among retrotransposable elements, seventeen LINE genes have been found to have undergone an expansion in a number of Antarctic fishes, and are supposed to have contributed to the gene duplication and genome size enlargement in those species (Chen et al., 2008). The Antarctic
mawsoni LINE EST sequence L042B01(GenBank accession number FE213337.1) was obtained by PCR amplification from D. mawsoni genomic DNA using the primers: L042B01-Fw (5′-GAT CAA CTG CTC CTG AAT GTG CCC-3′) and L042B01-Rev (5’-GCA CTA CGG CTT CCA CAG ACG AC-3′). The PCR product was cloned into pGem-T Easy vector (Promega) and sequenced. Probe template sequence was verified through clustal alignment to the reference sequence in Genbank, and labeled with biotin by nick translation, according to standard procedures. The labeled probe was purified by ethanol precipitation and dissolved in hybridization buffer (50% formamide/2× SSC, 40 mM KH2 PO4, 10% dextran sulfate) to yield final concentration of 2 ng/μl. Chromosomes on slides were denatured by heating at 70 °C for 1 min in 70% (v/v) formamide/2×SSC (pH 7), dehydrated in a cold ethanol series, and air-dried. The probe mixture was applied to chromosomal spreads (15 μl per slide) and incubated overnight in a moist chamber at 37 °C. Post-hybridization washes were performed at 45 °C: twice in 50% (v/v) formamide/2× SSC, twice in 2× SSC, and once in 4× SSC Tween-20, for 5 min each. Bound probe was detected by incubation with streptavidin-Cy3 (Amersham Biosciences). Chromosomes were counterstained in 0.3 μg/ml DAPI/2× SSC and mounted in a standard antifade solution (Vector). Images were captured and processed by the use of an Olympus BX61 microscope equipped with a Sensys (Photometrics) CCD camera. 3. Results and discussion Cytogenetic methods based on direct chromosome preparation from adult fish are widely used for taxonomic and evolutionary studies, as well as for ploidy determination and fish stock management (Pisano et al., 2007). In contrast, methods for direct chromosome preparation from early life stages of fishes have rarely been used, mostly due to difficulties in generating chromosome preparation that would provide a reasonable number of clear and identifiable metaphase spreads. Attempts to develop protocols to obtain high quality chromosome preparations have been performed in model species, such as zebrafish and tilapia, and in fish of commercial interest for aquaculture (e.g. Yamazaki et al., 1981; Ueda and Naoi, 1999; Shao et al., 2010; Ghigliotti et al., 2011; Pradeep et al., 2011; Karami et al., 2015). In most cases, the analysis of chromosomes from embryos and/or larvae was for testing genotoxicity or to check ploidy after chromosome manipulation. Standard karyotype analysis on chromosomes obtained from early life stages have been performed in very few fish species (Völker and Kullmann, 2006), and has never been attempted in Antarctic fish prior to this present work. The optimal parameters, determined in our experiments, for obtaining chromosomes from the Antarctic silverfish embryos differ from those reported for temperate-water species (reviewed in Völker and Ràb, 2015). We applied, with some modification, the protocol for the Atlantic cod Gadus morhua, a cold-temperate water species in which chromosome counting was used to check the ploidy after induction of meiotic gynogenesis (Ghigliotti et al., 2011). We found low temperature (1–3 °C), prolonged colchicine treatment (42 h in G. morhua, 36 h in P. antarctica), and long hypotonic incubation time (60–90 min), resulting in enhanced outcome in both species. Examination of multiple metaphase plates from each of the pooled larvae preparations led to confirm a diploid number (2n) = 48 for the Antarctic silverfish, as previously reported from adult specimens from the Weddell Sea (Ozouf-Costaz et al., 1991). The chromosome complement (Fig. 2 a, b) includes a single pair of one-armed elements (pair 23) but the majority of chromosomes are two-armed, leading to the karyotype formula (36 m/sm + 10st + 2a) and fundamental number (FN = 94). However, the chromosomes of pair 23 are not one-armed in all metaphases, and p arms of variable size have often been observed in one or both the homologues. The karyotype formula deduced from the examination of the larval chromosomes slightly differs from the one 3
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Fig. 2. Chromosomal features of the Antarctic silverfish from the Ross Sea. (a) DAPI-stained metaphase plate and (b) corresponding karyotype; (c) chromosomal architecture after physical mapping of a LINEs probe (red signals) and of a 28S rDNA probe (boxed chromosomes, green pseudo-color signals). Scale bar = 10 μm.
retrotransposons in the extensive reshaping of its karyotype. On the whole, the karyotype features of the Antarctic silverfish suggest a long independent cytogenetic and evolutionary history for the species. This is consistent with the available phylogenetic hypotheses for Antarctic notothenioids and with an early divergence of Pleuragramma in the notothenionid adaptive radiation. The chromosomal features might be consistent with the partitioning of Pleuragramma and Dissostichus to two different taxa, as suggested by Dettai et al. (2012). Nevertheless, based on the cytogenetic data, a
silverfish was not included in that study, however, the in situ mapping of LINEs presented here unambiguously indicated that extensive sweeps of these repeated sequences had occurred in the Antarctic silverfish genome, now present on the arms of all the chromosomes at interstitial position (Fig. 2 c). With widely acknowledged role of transposable elements in genome reshaping (reviewed in Blumenstiel, 2011; Belyayev, 2014) and chromosome evolution in fish (e.g. Chalopin et al., 2015), the abundance and pervasiveness of LINEs elements in the Antarctic silverfish genome strongly suggest involvement of these 4
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lineage-specific invasion of TEs, drastically modifying the chromosome features of Pleuragramma cannot be excluded. Cytotaxonomic comparisons between Pleuragramma and the remaining neutrally buoyant relatives Aethotaxis (A. mitopterix) and Gvozdarus (G. svetovidovi), will have to await sufficient cytogenetic information from the latter species, which are yet unavailable. Acknowledgments The work was carried out within the project IMAGES (PdR 2013/ AZ1.11) and POLICY (PNRA16_00281) funded by the Italian National Programme for Antarctic Research (PNRA), and contributes to the SCAR Scientific Research Program AntERA (Antarctic Thresholds − Ecosystems Resilience and Adaptation). We thank Jean-Pierre Coutanceau for the technical support to the analysis at the Institute of Biology Paris Seine. CHCC acknowledges support from US NSF award OPP-1645087. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Amores, A., Wilson, C.A., Allard, C.A., Detrich, H.W., Postlethwait, J.H., 2017. Cold fusion: massive karyotype evolution in the Antarctic bullhead Notothen Notothenia coriiceps. G3 (Bethesda) g3–117. https://doi.org/10.1534/g3.117.040063. Andersen, N.C., Hureau, J.-C., 1979. Proposition pour Une nouvelle classification des Nototheniinae (Pisces, Perciformes, Nototheniidae). Cybium 3, 47–53. Arai, R., 2011. Fish Karyotypes: A Check List. Springer, Tokyo. Auvinet, J., Graça, P., Ghigliotti, L., Pisano, E., Dettaï, A., Ozouf-Costaz, C., Higuet, D., 2019. Insertion hot spots of DIRS1 retrotransposon and chromosomal diversifications among the Antarctic Teleosts Nototheniidae. Int. J. Mol. Sci. 20, 701. Balushkin, A.V., 1984. Morphological Bases of the Systematics and Phylogeny of Nototheniid Fishes. Leningrad Zoological Institute of the Academy of Sciences of the USSR (In Russian). Belyayev, A., 2014. Bursts of transposable elements as an evolutionary driving force. J. Evol. Biol. 27, 2573–2584. https://doi.org/10.1111/jeb.12513. Blumenstiel, J.P., 2011. Evolutionary dynamics of transposable elements in a small RNA world. Trends Genet. 27, 23–31. https://doi.org/10.1016/j.tig.2010.10.003. Chalopin, D., Volff, J.N., Galiana, D., Anderson, J.L., Schartl, M., 2015. Transposable elements and early evolution of sex chromosomes in fish. Chromosom. Res. 23, 545–560. https://doi.org/10.1007/s10577-015-9490-8. Chen, Z., Cheng, C.H.C., Zhang, J., Cao, L., Chen, L., Zhou, L., et al., 2008. Transcriptomic and genomic evolution under constant cold in Antarctic notothenioid fish. Proc. Natl. Acad. Sci. 105, 12944–12949. https://doi.org/10.1073/pnas.0802432105. Colombo, M., Damerau, M., Hanel, R., Salzburger, W., Matschiner, M., 2015. Diversity and disparity through time in the adaptive radiation of Antarctic notothenioid fishes. J. Evol. Biol. 28, 376–394. https://doi.org/10.1111/jeb.12570. Dettai, A., Berkani, M., Lautredou, A.C., Couloux, A., Lecointre, G., Ozouf-Costaz, C., Gallut, C., 2012. Tracking the elusive monophyly of nototheniid fishes (Teleostei) with multiple mitochondrial and nuclear markers. Mar. Genomics 8, 49–58. https:// doi.org/10.1016/j.margen.2012.02.003. Duhamel, G., Hulley, P.A., Causse, R., Koubbi, P., Vacchi, M., Pruvost, P., et al., 2014. Biogeographic patterns of fish. Biogeographic Atlas Southern Ocean 7, 327–362. Ghigliotti, L., Mazzei, F., Ozouf-Costaz, C., Bonillo, C., Williams, R., Cheng, C.-H.C., Pisano, E., 2007. The two giant sister species of the Southern Ocean, Dissostichus eleginoides and Dissostichus mawsoni, differ in karyotype and chromosomal pattern of ribosomal RNA genes. Polar Biol. 30, 625–634. https://doi.org/10.1007/s00300006-0222-6. Ghigliotti, L., Bolla, S.L., Duc, M., Ottesen, O.H., Babiak, I., 2011. Induction of meiotic gynogenesis in Atlantic cod (Gadus morhua L) through pressure shock. Anim. Reprod. Sci. 127, 91–99. https://doi.org/10.1016/j.anireprosci.2011.07.011. Ghigliotti, L., Cheng, C.-H.C., Ozouf-Costaz, C., Vacchi, M., Pisano, E., 2015. Cytogenetic diversity of notothenioid fish from the Ross Sea: historical overview and updates. Hydrobiologia 761, 373–396. https://doi.org/10.1007/s10750-015-2355-5. Karami, A., Araghi, P.E., Syed, M.A., Wilson, S.P., 2015. Chromosome preparation in fish:
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Marine Genomics journal homepage: www.elsevier.com/locate/margen
Method paper
Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae ⁎
Laura Ghigliottia, , Chi-Hing C. Chengb, Catherine Ozouf-Costazc, Chantal Guidi-Rontanid, Marino Vacchia, Sara Federicie, Eva Pisanoa,e a
National Research Council of Italy (CNR), IAS, Genoa, Italy Department of Evolution, Ecology and Behavior, University of Illinois, Urbana-Champaign, IL 61801, USA Institute of Biology Paris Seine, Université Pierre et Marie Curie (CNRS, UMR 7138 « Evolution Paris-Seine»), Paris, France d Institut de Systematique, Evolution, Biodiversité (ISYEB) (CNRS UMR 7205), Museum National d'Histoire Naturelle, Paris, France e Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy b c
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A B S T R A C T
Keywords: Fluorescence in situ hybridization Karyotype LINEs Notothenioidei Ribosomal genes
This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae. The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of twoarmed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes. Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.
1. Introduction The Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) is a pelagic notothenioid fish abundant and widely distributed around the coastal waters of the Antarctic continent (Duhamel et al., 2014). As a mid-trophic level plankton-feeder, it plays a pivotal role in the High Antarctic coastal marine ecosystem (reviews on the biology and ecology of the species in Vacchi et al., 2017). Despite its ancestral benthic origin, common to the Notothenioidei, the Antarctic silverfish occupies an ecological niche in the water column owing to extensive evolutionary changes that led to secondary pelagization (Voskoboinikova et al., 2017). The monotypic genus Pleuragramma has consistently been included in Nototheniidae, but the relation to its closest relatives is still unresolved. Based on morphological traits, Pleuragramma was initially
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included in the nototheniid subfamily Pleuragramminae, along with Aethotaxis (Andersen and Hureau, 1979). The subfamily name was later changed to Pleuragrammatinae, and the genus Gvozdarus was added (Balushkin, 1984). Following combined molecular and morphological analysis, the clade Pleuragrammatinae was recognized grouping all neutrally buoyant notothenioid species, including the two Dissostichus (Near et al., 2007). A classification of Notothenioidei based on the analysis of multiple mitochondrial/nuclear DNA markers (Dettai et al., 2012) assigned the monotypic genus Pleuragramma to the subfamily Pleuragramminae, whereas Dissostichus, Aethotaxis, and Gvozdarus were moved to Dissostichinae Nevertheless, the phylogenetic position of Dissostichus, Aetothaxis and Pleuragramma remained unresolved. A timecalibrated phylogeny (Colombo et al., 2015) still led uncertainty in the relationship among the pelagic taxa Pleuragramma, Dissostichus and Aethotaxis, and provided support to a very early divergence of
Corresponding author. E-mail address: [email protected] (L. Ghigliotti).
https://doi.org/10.1016/j.margen.2019.100737 Received 18 September 2019; Received in revised form 17 December 2019; Accepted 19 December 2019 1874-7787/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Laura Ghigliotti, et al., Marine Genomics, https://doi.org/10.1016/j.margen.2019.100737
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Pleuragramma in the process of Antarctic notothenioid fish adaptive radiation. The recent analysis by Near et al. (2018) does not resolve Pleuragrammatinae as a clade, with one poorly supported node separating P. antarctica from a monophyletic group containing A. mitopterix and the two Dissostichus. For its biological and ecological importance in the high Antarctic waters, and its persistent taxonomic ambiguities, P. antarctica is an interesting target for cytogenetic studies. Detailed characterization of the chromosomal features of P. antarctica can assist in resolving the evolutionary history of the lineage, and contribute to further understanding of changes in the genome architecture during notothenioid evolution and diversification (Pisano and Ozouf-Costaz, 2003). Despite its wide geographical distribution and abundance in the Antarctic coastal ecosystems, the Antarctic silverfish remained cytogenetically understudied due to difficulties of capturing live adult specimens, and of keeping them in healthy condition to undergo the treatments required by the classical direct chromosome preparation procedures. Only five adults (3 males and 2 females) from the Weddell Sea were analyzed prior to this study, yielding only basic information on the chromosomal number (2n = 48) and karyotype formula (8 m + 32sm + 8a) (OzoufCostaz et al., 1991). No cytogenetic data are available on P. antarctica from other Antarctic areas, including the Ross Sea, despite the historically intense cytogenetic surveys conducted on Ross Sea notothenioid fauna (reviewed in Ghigliotti et al., 2015). The discovery of an Antarctic silverfish nursery area at Terra Nova Bay (western Ross Sea), with thousands of eggs developing within the ice platelet layer under the sea-ice cover (Vacchi et al., 2012), provided a unique opportunity to develop alternative protocols for obtaining chromosomes from early larvae. Here we report on the cytogenetic characterization of the Antarctic silverfish from the western Ross Sea based on the analysis of chromosomes from early larvae of this cold adapted species. Conventional staining and fluorescence in situ hybridization (FISH) of repeated sequences were applied to obtain a refined picture of the Antarctic silverfish karyotype and chromosomal architecture. Given that Long Interspersed Nuclear Elements (LINEs) were found expanded in a number of Antarctic fishes, and are supposed to have contributed to the gene duplication and genome size enlargement in those species (Chen et al., 2008), the chromosomal organization of LINEs sequences in P. antarctica was explored. To the best of our knowledge, this is the first study on chromosomes from early life stages of Antarctic fishes.
Fig. 1. Near-hatching Antarctic silverfish eggs collected under the sea ice in the Ross Sea nursery area.
were used for chromosome preparation as follows. The young larvae were mechanically disrupted in 0.075 M KCl on a 350-μm cell strainer. The obtained cells were re-suspended in hypotonic 0.075 M KCl solution for 60–90 min, at 1–3 °C. Hypotonized cells were then fixed in Carnoy fixative (methanol/acetic acid 3:1 v/v) according to standard procedures for short term cell cultures in Antarctic fishes (Rey et al., 2015). Fixed cell suspensions were stored at −20 °C for later laboratory analyses. Reference cells preparations were deposited and cryopreserved at the National Antarctic Museum, Genoa. 2.2. Karyotyping Chromosome spreads from fixed cells dropped on microscope slides were treated for karyotyping and chromosome banding according to current protocols for notothenioid fishes (Ozouf-Costaz et al., 1997, 2015). Chromosomes were stained with DAPI (4,40,6-diamidino-2-phenylindole) and observed through an Olympus BX61 microscope. Multiple metaphases from each preparation (a minimum of 100 metaphases) were digitally imaged using Sensys (Photometrics) CCD camera. The digital images were processed either by the use of Genus Software (Applied Imaging) or by application of Adobe Photoshop image analysis. The characterization of chromosomal morphology followed the Levan et al. (1964). Based on the position of the centromere and on the arm ratio, chromosomes were assigned to the following categories: metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). Metacentric, submetacentric and subtelocentric chromosomes are two-armed, acrocentrics are one-armed. The long arm of twoarmed elements is referred to as q (queue), the short arm is referred as p (petit), according to the International system for human cytogenetic nomenclature (Mitelman, 1995). Chromosomes were arranged in the karyotype according to size. The karyotypic formula and the total number of chromosome arms (Fundamental Number, FN) were obtained from analyses of a minimum of 20 karyotypic sets from each of the 13 chromosome preparations.
2. Material and methods 2.1. Sampling and chromosome preparation Sampling was carried out on the 3rd and 8th of November 2012 at 74° 39.150S – 164° 41.302E, in the Antarctic silverfish nursery area at Terra Nova Bay, Ross Sea. Near term embryonated eggs, floating among icy sea water, were collected through holes drilled in the sea ice cover following the sampling procedure described in Vacchi et al. (2012). Samples, stored in insulated boxes, were immediately transferred to the aquaria at Mario Zucchelli Station. Pilot small-scale experiments were performed to set up and optimize a protocol for chromosome preparation. Three parameters were considered as critical and for those a range of values were tested. In particular, water temperature ranging 1–5 °C, duration of the colchicine treatment ranging 12–72 h, and hypotonic incubation time ranging 30–90 min, were tested. Eggs and larvae at various developmental stages, used individually or pooled in homogeneous batches, were tested. Based on the results of the smallscale trials, a protocol was finalized and applied to pools of embryos in large-scale experiments of chromosome extraction. Briefly, homogeneous near hatching eggs (Fig. 1) were reared in petri dishes at 3 °C in 0,005% colchicine in sea water. About half of the colchicined sea water was changed every 12 h. After 52 h, hatching was completed and 13 pools (10–20 specimens each) of just-hatched larvae
2.3. Fluorescence in situ hybridization (FISH) In situ mapping of repeated sequences, namely 28S ribosomal DNA and Long Interspersed Nuclear Elements (LINEs), was performed in order to gain a better resolution of the chromosomes structure. A clone obtained from the nototheniid Dissostichus mawsoni (GenBank accession number AY926497) was used as the probe for FISH of 28S rDNA according to the protocol described in Ghigliotti et al. (2007). To prepare the probe for FISH, a 551 nucleotides portion of the D. 2
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reported for adults (Ozouf-Costaz et al., 1991). Such a discrepancy is probably due to a better resolution of the chromosome morphology, leading to the detection of small arms in some chromosome pairs previously described as acrocentrics. The lack of any morphologically distinguishable heterochromosome in the large number of examined metaphases support the lack of a differentiated sex-chromosome system, as hypothesized from the examination of chromosomes of adult specimens (Ozouf-Costaz et al., 1991). A degree of variability was observed among separate metaphases for the chromosomes of pairs 20, 21, and 23. The p arms of those elements appeared heteromorphic in size and morphology due to the variable extension of a dim DAPI stained region differing between the homologues and between metaphases. Such a dim DAPI stained region is located at interstitial position on the p arms of pair 20, at sub-telomeric position in pair 21, and occupies the entire p arm in the homologous chromosomes pair 23 (Fig. 2 b). Whether the observed variability is intra-individual or inter-individual cannot be deduced from present analysis given that each chromosome preparation results from a pool of specimens. FISH mapping of 28S rDNA highlighted the location of major ribosomal genes in the same three chromosome pairs (20, 21, 23), corresponding to the dim DAPI stained regions (Fig. 2 c, boxed pairs). Variation in 28S rDNA signal patterns were detected, in agreement with the observed variability in the extent of the dim DAPI stained regions. The pattern most frequently observed was with the major ribosomal genes located on a large sub-telomeric region of the pair 21; occurrence of ribosomal genes at interstitial position on the p arms of pair 20, and on the entire p arms of the homologues of pair 23, were also found. The rDNA signals were never simultaneously observed on all the six chromosomes, possibly due to the variability in the number of gene copies at the single chromosome locations, sometimes falling under the resolution limits of the FISH technique. Based on current cytogenetic information on the Antarctic notothenioids (Arai, 2011; Ghigliotti et al., 2015), and according to the hypothesized karyotype rearrangements trajectory within the suborder (e.g. Ozouf-Costaz et al., 1999; Pisano and Ozouf-Costaz, 2003; Amores et al., 2017), the Antarctic silverfish chromosomal features are distinctively interesting. With 48 chromosomes and predominantly two-armed, the Antarctic silverfish karyotype differs substantially from the supposed ancestral karyotype for notothenioids, which comprises 48 simple one-armed chromosomes. Cytotaxonomically, the Antarctic silverfish karyotype features are different from those of the Pleuragrammatinae (sensu Near et al., 2007) Dissostichus species, which possess the ancestral diploid number of 48 primarily one-armed chromosomes (Ghigliotti et al., 2007). Intriguingly, the chromosomal changes leading to the distinct silverfish karyotype, seem different from the general trend recorded in the high-Antarctic lineages, where increase of two-armed chromosomes is paired with decrease of the diploid numbers (Ghigliotti et al., 2015). Furthermore, the pattern of major ribosomal genes of the Antarctic silverfish, unevenly distributed on up to three chromosome pairs, is unique among the Antarctic notothenioids, where the major ribosomal gene repeats map to a single chromosomal locus in most of the species, and to two chromosomal loci in a few species (Pisano and Ghigliotti, 2009). Among chromosome rearrangements, pericentric inversions and reciprocal translocations are generally most likely events that lead to such highly modified cytogenetic layouts. In such a highly dynamic karyotype restructuring scenario, where the number of chromosomal elements is conserved but the chromosomes are drastically reshaped, transposable elements (TE) have been hypothesized as promotors of structural alterations such as fusions (Auvinet et al., 2019). Among retrotransposable elements, seventeen LINE genes have been found to have undergone an expansion in a number of Antarctic fishes, and are supposed to have contributed to the gene duplication and genome size enlargement in those species (Chen et al., 2008). The Antarctic
mawsoni LINE EST sequence L042B01(GenBank accession number FE213337.1) was obtained by PCR amplification from D. mawsoni genomic DNA using the primers: L042B01-Fw (5′-GAT CAA CTG CTC CTG AAT GTG CCC-3′) and L042B01-Rev (5’-GCA CTA CGG CTT CCA CAG ACG AC-3′). The PCR product was cloned into pGem-T Easy vector (Promega) and sequenced. Probe template sequence was verified through clustal alignment to the reference sequence in Genbank, and labeled with biotin by nick translation, according to standard procedures. The labeled probe was purified by ethanol precipitation and dissolved in hybridization buffer (50% formamide/2× SSC, 40 mM KH2 PO4, 10% dextran sulfate) to yield final concentration of 2 ng/μl. Chromosomes on slides were denatured by heating at 70 °C for 1 min in 70% (v/v) formamide/2×SSC (pH 7), dehydrated in a cold ethanol series, and air-dried. The probe mixture was applied to chromosomal spreads (15 μl per slide) and incubated overnight in a moist chamber at 37 °C. Post-hybridization washes were performed at 45 °C: twice in 50% (v/v) formamide/2× SSC, twice in 2× SSC, and once in 4× SSC Tween-20, for 5 min each. Bound probe was detected by incubation with streptavidin-Cy3 (Amersham Biosciences). Chromosomes were counterstained in 0.3 μg/ml DAPI/2× SSC and mounted in a standard antifade solution (Vector). Images were captured and processed by the use of an Olympus BX61 microscope equipped with a Sensys (Photometrics) CCD camera. 3. Results and discussion Cytogenetic methods based on direct chromosome preparation from adult fish are widely used for taxonomic and evolutionary studies, as well as for ploidy determination and fish stock management (Pisano et al., 2007). In contrast, methods for direct chromosome preparation from early life stages of fishes have rarely been used, mostly due to difficulties in generating chromosome preparation that would provide a reasonable number of clear and identifiable metaphase spreads. Attempts to develop protocols to obtain high quality chromosome preparations have been performed in model species, such as zebrafish and tilapia, and in fish of commercial interest for aquaculture (e.g. Yamazaki et al., 1981; Ueda and Naoi, 1999; Shao et al., 2010; Ghigliotti et al., 2011; Pradeep et al., 2011; Karami et al., 2015). In most cases, the analysis of chromosomes from embryos and/or larvae was for testing genotoxicity or to check ploidy after chromosome manipulation. Standard karyotype analysis on chromosomes obtained from early life stages have been performed in very few fish species (Völker and Kullmann, 2006), and has never been attempted in Antarctic fish prior to this present work. The optimal parameters, determined in our experiments, for obtaining chromosomes from the Antarctic silverfish embryos differ from those reported for temperate-water species (reviewed in Völker and Ràb, 2015). We applied, with some modification, the protocol for the Atlantic cod Gadus morhua, a cold-temperate water species in which chromosome counting was used to check the ploidy after induction of meiotic gynogenesis (Ghigliotti et al., 2011). We found low temperature (1–3 °C), prolonged colchicine treatment (42 h in G. morhua, 36 h in P. antarctica), and long hypotonic incubation time (60–90 min), resulting in enhanced outcome in both species. Examination of multiple metaphase plates from each of the pooled larvae preparations led to confirm a diploid number (2n) = 48 for the Antarctic silverfish, as previously reported from adult specimens from the Weddell Sea (Ozouf-Costaz et al., 1991). The chromosome complement (Fig. 2 a, b) includes a single pair of one-armed elements (pair 23) but the majority of chromosomes are two-armed, leading to the karyotype formula (36 m/sm + 10st + 2a) and fundamental number (FN = 94). However, the chromosomes of pair 23 are not one-armed in all metaphases, and p arms of variable size have often been observed in one or both the homologues. The karyotype formula deduced from the examination of the larval chromosomes slightly differs from the one 3
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Fig. 2. Chromosomal features of the Antarctic silverfish from the Ross Sea. (a) DAPI-stained metaphase plate and (b) corresponding karyotype; (c) chromosomal architecture after physical mapping of a LINEs probe (red signals) and of a 28S rDNA probe (boxed chromosomes, green pseudo-color signals). Scale bar = 10 μm.
retrotransposons in the extensive reshaping of its karyotype. On the whole, the karyotype features of the Antarctic silverfish suggest a long independent cytogenetic and evolutionary history for the species. This is consistent with the available phylogenetic hypotheses for Antarctic notothenioids and with an early divergence of Pleuragramma in the notothenionid adaptive radiation. The chromosomal features might be consistent with the partitioning of Pleuragramma and Dissostichus to two different taxa, as suggested by Dettai et al. (2012). Nevertheless, based on the cytogenetic data, a
silverfish was not included in that study, however, the in situ mapping of LINEs presented here unambiguously indicated that extensive sweeps of these repeated sequences had occurred in the Antarctic silverfish genome, now present on the arms of all the chromosomes at interstitial position (Fig. 2 c). With widely acknowledged role of transposable elements in genome reshaping (reviewed in Blumenstiel, 2011; Belyayev, 2014) and chromosome evolution in fish (e.g. Chalopin et al., 2015), the abundance and pervasiveness of LINEs elements in the Antarctic silverfish genome strongly suggest involvement of these 4
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lineage-specific invasion of TEs, drastically modifying the chromosome features of Pleuragramma cannot be excluded. Cytotaxonomic comparisons between Pleuragramma and the remaining neutrally buoyant relatives Aethotaxis (A. mitopterix) and Gvozdarus (G. svetovidovi), will have to await sufficient cytogenetic information from the latter species, which are yet unavailable. Acknowledgments The work was carried out within the project IMAGES (PdR 2013/ AZ1.11) and POLICY (PNRA16_00281) funded by the Italian National Programme for Antarctic Research (PNRA), and contributes to the SCAR Scientific Research Program AntERA (Antarctic Thresholds − Ecosystems Resilience and Adaptation). We thank Jean-Pierre Coutanceau for the technical support to the analysis at the Institute of Biology Paris Seine. CHCC acknowledges support from US NSF award OPP-1645087. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Amores, A., Wilson, C.A., Allard, C.A., Detrich, H.W., Postlethwait, J.H., 2017. Cold fusion: massive karyotype evolution in the Antarctic bullhead Notothen Notothenia coriiceps. G3 (Bethesda) g3–117. https://doi.org/10.1534/g3.117.040063. Andersen, N.C., Hureau, J.-C., 1979. Proposition pour Une nouvelle classification des Nototheniinae (Pisces, Perciformes, Nototheniidae). Cybium 3, 47–53. Arai, R., 2011. Fish Karyotypes: A Check List. Springer, Tokyo. Auvinet, J., Graça, P., Ghigliotti, L., Pisano, E., Dettaï, A., Ozouf-Costaz, C., Higuet, D., 2019. Insertion hot spots of DIRS1 retrotransposon and chromosomal diversifications among the Antarctic Teleosts Nototheniidae. Int. J. Mol. Sci. 20, 701. Balushkin, A.V., 1984. Morphological Bases of the Systematics and Phylogeny of Nototheniid Fishes. Leningrad Zoological Institute of the Academy of Sciences of the USSR (In Russian). Belyayev, A., 2014. Bursts of transposable elements as an evolutionary driving force. J. Evol. Biol. 27, 2573–2584. https://doi.org/10.1111/jeb.12513. Blumenstiel, J.P., 2011. Evolutionary dynamics of transposable elements in a small RNA world. Trends Genet. 27, 23–31. https://doi.org/10.1016/j.tig.2010.10.003. Chalopin, D., Volff, J.N., Galiana, D., Anderson, J.L., Schartl, M., 2015. Transposable elements and early evolution of sex chromosomes in fish. Chromosom. Res. 23, 545–560. https://doi.org/10.1007/s10577-015-9490-8. Chen, Z., Cheng, C.H.C., Zhang, J., Cao, L., Chen, L., Zhou, L., et al., 2008. Transcriptomic and genomic evolution under constant cold in Antarctic notothenioid fish. Proc. Natl. Acad. Sci. 105, 12944–12949. https://doi.org/10.1073/pnas.0802432105. Colombo, M., Damerau, M., Hanel, R., Salzburger, W., Matschiner, M., 2015. Diversity and disparity through time in the adaptive radiation of Antarctic notothenioid fishes. J. Evol. Biol. 28, 376–394. https://doi.org/10.1111/jeb.12570. Dettai, A., Berkani, M., Lautredou, A.C., Couloux, A., Lecointre, G., Ozouf-Costaz, C., Gallut, C., 2012. Tracking the elusive monophyly of nototheniid fishes (Teleostei) with multiple mitochondrial and nuclear markers. Mar. Genomics 8, 49–58. https:// doi.org/10.1016/j.margen.2012.02.003. Duhamel, G., Hulley, P.A., Causse, R., Koubbi, P., Vacchi, M., Pruvost, P., et al., 2014. Biogeographic patterns of fish. Biogeographic Atlas Southern Ocean 7, 327–362. Ghigliotti, L., Mazzei, F., Ozouf-Costaz, C., Bonillo, C., Williams, R., Cheng, C.-H.C., Pisano, E., 2007. The two giant sister species of the Southern Ocean, Dissostichus eleginoides and Dissostichus mawsoni, differ in karyotype and chromosomal pattern of ribosomal RNA genes. Polar Biol. 30, 625–634. https://doi.org/10.1007/s00300006-0222-6. Ghigliotti, L., Bolla, S.L., Duc, M., Ottesen, O.H., Babiak, I., 2011. Induction of meiotic gynogenesis in Atlantic cod (Gadus morhua L) through pressure shock. Anim. Reprod. Sci. 127, 91–99. https://doi.org/10.1016/j.anireprosci.2011.07.011. Ghigliotti, L., Cheng, C.-H.C., Ozouf-Costaz, C., Vacchi, M., Pisano, E., 2015. Cytogenetic diversity of notothenioid fish from the Ross Sea: historical overview and updates. Hydrobiologia 761, 373–396. https://doi.org/10.1007/s10750-015-2355-5. Karami, A., Araghi, P.E., Syed, M.A., Wilson, S.P., 2015. Chromosome preparation in fish:
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