Research Article |
Corresponding author: Teresa Capriglione ( teresa.capriglione@unina.it ) Academic editor: Nazar Shapoval
© 2015 Ennio Cocca, Agnese Petraccioli, Maria Alessandra Morescalchi, Gaetano Odierna, Teresa Capriglione.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Cocca E, Petraccioli A, Morescalchi MA, Odierna G, Capriglione T (2015) Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodraco hamatus (Notothenioidei, Channichthyidae). Comparative Cytogenetics 9(1): 1-15. https://doi.org/10.3897/CompCytogen.v9i1.8731
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Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodraco hamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Ch. hamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Ch. hamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.
Sex chromosomes, Antarctic fish, CHD1 gene
The chromosome microdissection and microcloning technique (
While sex-determining genes have been identified and mapped on the heteromorphic sex chromosomes of mammals and birds (
Fish sex chromosomes often appear undifferentiated, sharing a similar morphology and a comparable gene composition (
Some fishes present multiple sex-chromosome systems, in which the initial stage of differentiation seems to be independent of heterochromatin accumulation in chromosomes and to be related to chromosome rearrangement, whereas simple sex-chromosome systems show progressive heterochromatin accumulation (
The variety of sex-chromosome systems and the broad range of sex-determination mechanisms typical of fishes make it an interesting group in which to explore sex-chromosome evolution. The present study was designed to seek correlations between cytologically recognizable fish X and Y chromosomes and to establish whether they are related to different genetic make-up or simply to fusion events, perhaps trigged by differential amplification of repetitive DNA fractions.
To shed light on the genome composition of a teleost sex chromosome, laser microdissection and pressure catapulting (LMPC) was used to isolate the Y chromosome from standard mitotic plates of the Antarctic icefish Chionodraco hamatus (Lönnberg, 1905) belonging to the Notothenioidei, the dominant endemic component of the Southern Ocean fauna. The physiological and molecular adaptations that have enabled their evolution have made this Antarctic clade unique among vertebrates. The karyotype of most Notothenioidei shows 2n = 48 mostly acrocentric chromosomes, which is considered basic for Perciformes, and nucleolus organizer regions (NORs) localized on the telomeres of a single chromosome pair (
Ch. hamatus Y chromosomes were isolated and amplified by degenerate oligonucleotide primed PCR (DOP-PCR) as described by
Ch. hamatus specimens were collected near Mario Zucchelli Station, on the coast of Terra Nova Bay (74°42'S, 164°07'E), during Italian Antarctic expeditions in 2000 and 2005. They were killed by severing the spinal cord according to animal welfare guidelines.
Mitotic chromosome plates were prepared from a Ch. hamatus male as described in
Y chromosomes (20/slide) were outlined and excised using a low-energy laser beam and catapulted by a single laser pulse directly into the cap of an Eppendorf tube, to minimize contamination.
DOP-PCR reactions were carried out without chromosome pre-treatment (
The first amplification was carried out by RAMP-PCR: 94 °C for 5 min; 12 cycles at low stringency (94 °C for 90 s, 32 °C for 2 min, increasing by 0.2 °C/s to 72 °C, then 72 °C for 2 min), followed by 35 cycles at high stringency (94 °C for 90 s, 52 °C for 90 s and 72 °C for 90 s). 10 μl of the DOP-PCR product was run on 1% agarose gel.
The species specificity of the probe was checked by dot-blot hybridization under stringent conditions using digoxigenin-labelled Y chromosome material as a probe on human and fish total genomic DNA spotted on a nitrocellulose membrane. Dot-blot analysis confirmed the absence of contamination, showing a strong hybridization signal only to Ch. hamatus genomic DNA (data not shown).
The DOP-PCR-amplified chromosome material was re-amplified and labelled with 16-dUTP-biotin (Roche). The PCR mix comprised 1 x Taq polymerase buffer (2 mM MgCl2), 40 μM dATP, dGTP and dCTP, 28 μM dTTP, 12 μM 16-dUTP biotin, 2 μM DOP primer, and 0.05 U/μl Taq polymerase under the following conditions: (1×) 94 °C for 5 min; (35×) 90 °C for 1 min and 30 s, 52 °C for 1 min and 30 s, 72 °C for 1 min and 30 s, and (1×) 72 °C for 5 min. The FISH procedure was performed under high stringency conditions (50% formamide, 2 × SSC, 10% dextran sulphate). Metaphase spreads for FISH were prepared on ethanol-cleaned slides, air-dried and incubated overnight at 37 °C. Chromosomes were denatured in 70% deionized formamide 2 × SSC at 72 °C for 7 min and dehydrated in an ethanol series.
A DNA mixture of approximately 400 ng of the chromosome painting probe and 10 μg of the Cot-1 fraction of Ch. hamatus (
Post-hybridization washes were in 0.4 × SSC/0.1% Triton X-100 for 5 min at 42 °C and in 2 × SSC/0.1% Triton X-100 for 15 min at room temperature. Signal detection was accomplished using AlexaFluor-Streptavidin 488 (Molecular Probes) according to
DOP-PCR fragments were purified with the NucleoSpin Extract II Kit (Macherey-Nagel). Three μl aliquots were cloned into the TOPO TA Cloning Kit (Invitrogen Life Technologies). Transformation and clone analysis were conducted according to the manufacturer’s instructions. Recombinant clones were isolated and PCR amplification was used to establish insert presence and size. Sequencing was performed by Primm Biotech (Milano, Italy). The nucleotide sequences, ranging from 200 to 800 bp, were used for homology searches in four databases (National Center for Biotechnology Information, NCBI: http://www.ncbi.nlm.nih.gov; DNA DataBank of Japan, DDBJ: http://www.ddbj.nig.ac.jp; Nucleotide EST Database; and RepBase: http://www.girinst.org/repbase) using BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov), FASTA (http://www.ebi.ac.uk/Tools/sss/fasta), and RepeatMasker (http://www.repeatmasker.org) sequence alignment programs.
The results of FISH, performed on metaphase plates of Ch. hamatus males and females using the newly generated Y-chromosome paint, are shown in Fig.
Mitotic chromosomes of Ch. hamatus. FISH with the Y chromosome paint on Ch. hamatus a male (2n = 47 and X1X2Y sex-chromosome system) and b female (2n = 48) metaphase spreads.
The identification of syntenic regions may have been hampered by sequence length and by the lack of whole sequenced genomes of Antarctic fishes. Most clones were found to contain redundant repetitive sequences; moreover, several clones did not show significant homologies to any genomic or translated sequence contained in the GenBank database, suggesting that they may correspond to untranslated or non-conserved regions of genes already known from other species.
FASTX analysis of clone C11 (251 bp) against library ‘B170.0 vertebrates’ yielded the highest similarity score for fish genome fragments. Interestingly, its uninterrupted amino acid translation was found to be homologous to an N-terminal portion of a Chromodomain-helicase-DNA-binding protein 1-like (CHD1) (The European nucleotide archive: at http://www.ebi.ac.uk/ena/data/view/LM999957), a conserved protein with a putative role in chromatin architecture (Fig.
Alignment of the N-terminal portion of a Chromodomain-helicase-DNA-binding protein 1-like (CHD1) with the homologous CHD1 sequences of fish: Danio rerio, Tetraodon nigroviridis, and Gasterosteus aculeatus.
RepeatMasker analysis allowed identification in clone CHS4 of a microsatellite strand whose sequence is conserved in other teleost species. FASTA and BLAST analysis showed that our sequence aligned with several loci in the genome of Plecoglossus altivelis (Temminck & Schlegel, 1846), Gasterosteus aculeatus (Linnaeus, 1758), Scaphirhynchus platorynchus (Rafinesque, 1820), and Eptatretus burgeri (Girard, 1855). Notably, in E. burgeri the sequence is found in Sox9 mRNA for Sry-box protein 9 (
Repetitive sequence of clone CHS4 aligned with several loci of Plecoglossus altivelis, Gasterosteus aculeatus, Scaphirhynchus platorynchus and Eptatretus burgeri. Notably, in E. burgeri the sequence is found in Sox9.
In two other clones, C3 (825 bp) and C5 (244 bp), putative ancient LINE (Long Interspersed Element) fragments (non-LTR retrotransposon of the L1/CIN4 family) were found.
In clone C3 the partial LINE (position 342–503 bp) is bordered by repetitive regions both upstream (232–327 bp) and downstream (521–639 bp) (Fig.
Structure of clone C3: the partial LINE (position 341–504 bp) is bordered by microsatellite regions both upstream (232–325 bp) and downstream (520–640 bp).
In situ hybridization of the C3 probe (CIN1/L4) to the Ch. hamatus male karyotype: the probe clearly maps interstitially on the q arm of the Y chromosome. Insert: a C-banded and chromomycin-stained Y chromosome of Ch. hamatus and b FISH performed using the Tc1-like DNA transposon as probe (modified from fig. 5 of
Chromosome microdissection provides valuable chromosome information on lower vertebrate, fish, amphibian, and reptilian genomes, in which chromosome identification is hampered by difficulties in obtaining specific chromosome markers, and detection of syntenic regions is prevented by the lack of sequenced genomes.
Ch. hamatus Y-chromosome paint hybridized strongly to a single acrocentric chromosome pair, perhaps X2X2, in the female karyotype. The signal localization on the pericentromeric region suggests that, at least in this species, sequences, which are still found on the Y chromosome, might have been accumulated on the female X2X2 pair after sex chromosome emergence.
Because the heteromorphic Y is probably the result of a tandem fusion of autosomes and morphologically undifferentiated sex chromosomes, this event may have been followed by a reduction in recombination and by formation of new linkage groups between genes that were originally found on different chromosomes, including sexually antagonistic ones (
The interstitial chromomycin-positive heterochromatic region of the q arm, corresponding to the region involved in the tandem fusion event (
In a previous paper we mapped a mobile element belonging to the Tc1/mariner DNA transposon family on the q arm of the Y chromosome (
Repetitive sequences preserved in teleost species have helped to identify conserved genetic synteny and measure the degree and patterns of sex-chromosome differentiation in comparative genomic studies of fish (
The microsatellite sequence isolated from the C. hamatus Y chromosome in our study appears to be conserved. In addition, it aligned with several loci in the genome of ancient and modern fishes, including T. newnesi, an Antarctic notothenioid.
The presence of Y chromosome-specific microsatellites is well established in literature, mainly in mammals and the variability of their loci has been widely exploited to generate haplotypes for phylogenetic and forensic analysis (
In fish, comparative genomic studies have shown that some of these microsatellite sequences, preserved in different teleost species, can help to examine conserved genetic sinteny, to clarify phylogeny as well as to measure the degree and patterns of sex chromosome differentiation (
Finally, although the present data do not allow any hypothesis to be advanced, it is intriguing that in E. burgeri this sequence is found in Sox9 mRNA for Sry-box protein 9, which together with steroidogenic factor 1 regulates transcription of the anti-Muellerian hormone (AMH) gene (
With regard to the partial CHD1 gene sequences found in one of our Y clones, two CHD1 gene homologues have been described in birds, which are characterized by ZW female heterogamety. In birds these homologues are associated with the heteromorphic sex-chromosome pair, one being W-linked and the other Z-linked (
Even though the SDR has been identified in some teleost species (
The data seem to confirm that sex chromosomes arose independently in distant taxa several times during fish radiation (
This work was supported by the Italian M. U. R. S. T. (Ministry of the University and Scientific and Technological Research)-PRIN 2005.