Research Article |
Corresponding author: Takahiro Taguchi ( ttaguchi@kochi-u.ac.jp ) Academic editor: Vladimir Lukhtanov
© 2016 Takahiro Taguchi, Satoshi Kubota, Takuma Mezaki, Erika Tagami, Satoko Sekida, Shu Nakachi, Kazuo Okuda, Akira Tominaga.
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:
Taguchi T, Kubota S, Mezaki T, Tagami E, Sekida S, Nakachi S, Okuda K, Tominaga A (2016) Identification of homogeneously staining regions by G-banding and chromosome microdissection, and FISH marker selection using human Alu sequence primers in a scleractinian coral Coelastrea aspera Verrill, 1866 (Cnidaria). Comparative Cytogenetics 10(1): 61-75. https://doi.org/10.3897/CompCytogen.v10i1.5699
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Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30–35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of C. aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this C. aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution.
Coral, karyotype, hsr, FISH, chromosome microdissection, Alu repeats
Species of the genus Coelastrea Verrill, 1866 belong to the family Merulinidae (
In scleractinian corals, the available chromosomal data, including their karyotypes and gene loci on chromosomes, has been limited, although many studies of other aspects of their biology such as ecology and physiology have been reported. Chromosomal evolution in corals occurs in closely related taxa within and at the species level (
In humans, a karyotype is usually established using the G-banding pattern shown on chromosomes (
Obtaining FISH markers is not only profitable in helping to establish karyotypes but also in comparing syntenic relations among coral species. In our previous study (
In this study, we analyzed C. aspera chromosomes using not only a conventional G-banding method but also molecular cytogenetic techniques. We successfully identified the hsr of C. aspera chromosome 11 using trypsin-treated G-banding, followed by karyotyping. Following that, we conducted chromosome microdissection (CMD) to regenerate DNA sequences consisting of the hsr to see if this involved not only rDNA but also some other specific sequences. Then, we applied CMD technique. Microdissected DNA was amplified by PCR and used as a painting probe of hsr. Moreover, to see the nature of the rRNA gene on the chromosomes of C. aspera, we performed silver-stained nucleolus organizer region (Ag-NOR) analysis (
C. aspera is hermaphroditic; that is during spawning, eggs and sperm packed together into discreet bundles are released from the mouths of fertile polyps. The gametes of C. aspera were collected at Nishidomari (32°46'N; 132°43'E), in Kochi Prefecture, Japan (Fig.
We have previously reported the method for making coral chromosome preparations (
Metaphase spreads on a slide were stained with silver solution (50% AgNO3 solution at 37 °C for 1–5 h), which binds to the nucleolus organizing regions (NOR), i.e., the secondary constrictions (stalks) of acrocentric chromosomes in the case of humans (
Coral DNA from C. aspera embryos (approximately 200–300) or sperm (approximately 0.1 mL) was extracted using a Wizard genomic DNA purification kit purchased from Promega corporation (Madison, WI, USA) according to the manufacturer’s instructions.
Five hsrs were scraped using a chromosome microdissection (CMD) technique as previously described (
The probes for the rRNA genes and Alu-derived DNAs were obtained from PCR products using the primers described by
A human digoxigenin-labeled telomere probe was purchased from Appligene Oncor (Lifescreen, Watford, UK). Random prime labeling of the probe DNA from PCR products was performed with fluorescein-12-dUTP (F-dUTP) or cyanine-3-dUTP (Cy3-dUTP) in accordance with the kit protocol (Invitrogen, Tokyo, Japan). FISH was conducted as previously reported (
The slides were examined with an Olympus BX-50 fluorescence microscope. Images of suitable metaphase spreads and interphase nuclei were acquired on an Olympus DP70 microscope workstation equipped with a cooled charge-coupled device and FISH analysis software. The miller units used for each fluorescence light (FITC, Cy-3 and DAPI) were U-NIBA, U-MWU, and U-MWIB (Olympus), respectively.
A survey of 50 metaphase plates of C. aspera identified a complement of 28 chromosomes. Then we tried to make a conventional trypsin G-banding (Fig.
Localizations of the rRNA genes by FISH are shown in Figure
A dual color FISH image of C. aspera obtained by a probe generated by PCR using rRNA gene primers for 28S (green) and human telomere probe (red). Arrowheads indicate rRNA genes loci on one homologous chromosome 11 (one has an hsr with a long and large green signal). Scale bar: 5 μm.
Ag-NOR staining. A Interphase nuclei and metaphases of C. aspera. Note that five to seven dark domains on interphase nuclei were seen but did not seem to appear on two metaphases B The human metaphase spread with black dots on the seven acrocentric chromosomes (arrowheads) C Human interphase nuclei. Scale bar: 10 μm.
Five hsr portions were scraped from five chromosome 11 by a glass needle (Fig.
FISH signal by the probe from the microdissection-generated DNA probe of C. aspera’s hsr. A Metaphase cells with a distinct signal on the hsr (an arrowhead). Note that the hsr-derived probe hybridized not only with rRNA gene (arrowheads) but also with terminal ends of other chromosomes (arrows) in both full (2n=28, upper) and partial (lower) metaphases B The same cells were stained with DAPI. Chromosome microdissection of the hsr in chromosome 11 C Before microdissection of the hsrD After scraping of the hsr (an arrow). Scale bar: 5 μm.
Figure
Dual FISH image with human Alu-derived and rRNA gene probes. A Red FISH signals by human Alu-repetitious DNA-derived probe were seen on two homologous chromosomes (arrowheads). Green signals by 28S rRNA genes (arrows). In this cell, no hsr was seen B The same chromosomes stained with DAPI. Scale bar: 5 μm.
There are more than 800 species of scleractinian corals in the world (
We carried out G-banding to establish the C. aspera karyotype using embryos. It is difficult in general to obtain a high quality G-banding in invertebrate chromosomes due to the relatively small chromosome sizes and insufficient digestion by trypsin. In this coral, we could produce a G-banding pattern on chromosomes by trypsin-treatment, although it was not fully satisfactory (Fig.
To verify the nature of an hsr composer of rDNA cytogenetically, we tried to visualize NOR proteins using Ag-NOR staining (
To elucidate if the hsr of C. aspera is composed only of rDNA or includes some other sequences together with rDNA, we carried out CMD, followed by FISH. We successfully regenerated probes made from the microdissected DNA by DOP-PCR. The FISH experiment confirmed that these probes hybridized on rRNA gene loci and on the terminal end of another chromosome. This suggested that the hsr consists not only of rRNA genes but also some other repeated sequences.
In the previous study (
In conclusion, we have successfully performed chromosome analysis by using conventional banding and molecular cytogenetic techniques, such as G-banding, CMD and FISH. The telomere sequences and rRNA genes could be mapped on the C. aspera chromosomes. Our research suggests that the telomere sequence of C. aspera may be identical to the human telomere sequence. We demonstrated through G-banding that C. aspera has an hsr on chromosome 11. CMD-FISH revealed that the elongated segment of chromosome 11 was an hsr and was occupied by rDNAs and some other sequences. Metaphase spreads with an hsr were seen in more than 50% of cells observed. These new findings would lead to surveys of other scleractinian coral species to find clues in solving difficulties in taxonomy.
This work was supported by grants from the Kuroshio fund (Kuroshio-kou) of the Graduate School of Kuroshio Science, Kochi University (to T.T.), Kurita Water and Environment Foundation (13B094: to S.K.) and the Japan Society for Promotion of Science (26292107 and 15K14789: to T.T.).