﻿Karyotypic analysis and isolation of four DNA markers of the scleractinian coral Favitespentagona (Esper, 1795) (Scleractinia, Anthozoa, Cnidaria)

﻿Abstract We performed conventional and molecular cytogenetic studies on the Favitespentagona Esper, 1795, a scleractinian coral mostly found along the west coast of Japan. Karyotype analysis of F.pentagona by G-banding revealed a karyogram containing a homogenously staining region (HSR) on chromosome 10 in more than 50% of the examined metaphase spreads. This HSR consisted of sequences from 18S ribosomal RNA (rRNA) genes, as demonstrated by fluorescence in situ hybridization (FISH) and DNA sequencing. We highlighted the development of four chromosomal FISH markers from repetitive genes such as U2 small nuclear RNA linked to 5S rRNA sequence (U2 snRNA-5S), 18S rRNA, histone H3, and uncharacterized gene FP-9X. The chromosomal locations of the U2 snRNA-5S and 18S RNA were on the terminal end of long arm of chromosomes 2 and 10, respectively, while the histone H3 and the uncharacterized gene were located near the centromeres of chromosomes 1 and 9, respectively. These FISH markers will improve the karyotyping of F.pentagona from mitotic preparations which helps in widening our understanding of coral genetic structure and chromosome organization. In addition, these improvements in karyotyping will provide the basis in constructing of chromosome-level genome assembly for F.pentagona.


Introduction
Cytogenetic information from karyotypic analysis gives us a deeper understanding the way genetic material is packaged inside a nucleus of a cell and how certain genetic diseases are associated with chromosome defects and aberrations (Testa 1990;Super 1991). Cytogenetic information is also valuable in understanding the genome structure and organization of an individual which may vary greatly within and across species (Sullivan 2020). These variations in chromosome characteristics will give insight into their evolutionary process as they reflect genome shuffling, translocation, and chromosomal duplication/deletion (Guo et al. 2018). This information might be valuable for stony corals, in which taxonomic classification poses a great challenge as the integration of its morphological and molecular characteristics often reveal conflicting results (Fukami et al. 2004). This difficulty might be caused, in part, by hybridization of closely related species and by morphological plasticity. Confusion about the stony coral taxonomy necessitates the search for new coral characters such as cytogenetic information to understand coral systematics and evolution. However, the cytogenetic information of stony corals, such as karyotypes and gene maps, is limited. To date, molecular cytogenetic information on stony corals has only been reported for five species from three different families (2 species from Acroporidae, 2 species from Merulinidae, and 1 species from Lobophyliidae). These are Acropora solitaryensis Veron et Wallace,, 1984 and Acropora pruinosa Brook, 1893 (Acroporidae), Coelastrea aspera Verrill, 1866 and Platygyra contorta Veron, 1990 (Merulinidae), and Echinophyllia aspera Ellis et Solander, 1786 (Lobophylliidae) (Taguchi et al. 2013(Taguchi et al. , 2014(Taguchi et al. , 2016(Taguchi et al. , 2017(Taguchi et al. , 2020Vacarizas et al. 2021). In those studies, new cytogenetic evidence was presented, including information regarding chromosome numbers, rRNA gene loci, the presence of a homogenously staining regions (HSR), and some repeated sequences shared with human satellite DNA.
In this study, we reported the detailed molecular cytogenetic analysis of stony coral Favites pentagona Esper, 1758, from family Merulinidae. F. pentagona is commonly observed along the west coast of Japan (Veron, 2000). Colonies of F. pentagona range from massive, encrusting to columnar forms. The valleys with colonies are usually long and relatively straight at colony margins, becoming increasingly short, sinuous, and contorted towards the colony center; septa have thin walls and are highly irregular (Veron, 2000).
Cytogenetic analysis of F. pentagona was conducted using conventional and molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH). We identified a homogenously staining region (HSR) on F. pentagona chromosome 10 using G-banding and 4',6-diamidino-2-phenylindole (DAPI) staining, followed by karyotyping. Furthermore, the chromosomal locations of four tandemly repetitive genes were identified for F. pentagona. The development of four FISH markers was described, and the FISH signals of each gene were characterized showing the effectivity of these markers to identify specific chromosomes from mitotic cells.

Coral collection
F. pentagona gametes were collected from spawning colonies at Nishidomari (32°46'N, 132°43'E), Kochi Prefecture, Japan (Fig. 1). The release of gamete bundles was observed between 8:00 pm and 9:30 pm on July 24, 2019. Coral bundles were collected using plastic cups placed over the colonies during spawning. After collection, eggs and sperm from the spawned bundles were separated. The separated gametes were then transferred to a new container to allow fertilization. Successful cell divisions were observed under the light microscope. Embryos were then rinsed in 0.2 µm filtered seawater (ADVANTEC cartridge filter; Advantec Toyo Corp., Tokyo, Japan) to remove external contaminants.

Chromosome preparations and G-banding
Coral chromosome preparations were conducted according to the method described by Taguchi et al. (2013). About 10-14 hours after artificial fertilization, embryos were treated in filtered seawater supplemented with 0.01% (v/v) colchicine (Sigma, St. Louis, MO, USA) for 1 h, followed by treatment with hypotonic solution (0.5 × sea water; diluted in distilled water) to spread the chromosomes. Embryos were fixed using a freshly prepared fixative containing absolute methanol and glacial acetic acid (3:1). Fixed embryos were soaked in diethyl ether overnight to remove intracellular lipids and incubated again in the fixative. Approximately 20 to 50 embryos were isolated using a fine needle to tear the embryos apart into their constituent cells. Suspensions containing embryo cells were transferred into a 1.5 ml tube filled with the fixative. The tube was centrifuged at 2000 g for 2 min and the pellet was re-suspended in 0.5 ml of fresh fixative. A drop containing separated cells was placed on a clean slide and then air-or flame-dried to spread the chromosomes. For G-banding, slides were treated with 0.025% trypsin solution at room temperature (approximately 25 °C) for 1 min (Seabright, 1973) and then stained with 5% Giemsa.

DNA extraction
Genomic DNA was extracted from F. pentagona sperm (approximately 0.1 ml) using a Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions.

FISH analysis
FISH analysis was performed as previously reported (Taguchi et al. 1993), with slight modifications. Metaphase preparations were denatured in 70% formamide/2x Salinesodium citrate (SSC) at 70 °C for 2 min; 0.8 µl of the prepared probe was mixed with 10 µl of hybridization solution (H7782, Sigma, Japan) and denatured at 82 °C for 10 min. Hybridization was performed at 37 °C in a CO 2 incubator for 12-15 h. After hybridization, samples were washed twice in 2x SSC and 1x Phosphate buffered detergent (PBD; 0.05% Tween20/4xSSC). Chromosomes were counterstained with DAPI.

Image acquisition and processing
FISH slides were examined under a fluorescence microscope (Olympus BX-50, Tokyo, Japan) equipped with a cooled charge-coupled device. Images of suitable metaphase spreads were acquired using an Olympus DP70 workstation and the FISH analysis software. The mirror units used for each fluorescence light (FITC, Cy-3, and DAPI) were U-NIBA, U-MWU, and U-MWIB (Olympus), respectively.

Diploid karyotypes in F. pentagona
Chromosomes in metaphase cells were karyotyped by conventional trypsin G-banding, and an HSR in terminal end of one of the chromosomes was observed in approximately 50% of the observed metaphase spreads (Fig. 2). HSRs were shown in Gbanded karyograms as long and lightly stained region of the chromosomes. The HSRs were also revealed using the inverted images of DAPI fluorescent staining showing the similar characteristics (Fig. 2, inset). Chromosomes were arranged in decreasing order  of chromosome length from 1 to 14, which revealed that the chromosome with HSR is chromosome 10 (Fig. 2). The length and arm ratio of chromosomes were measured using eight metaphase spreads, as summarized in Table 2. The modal number of chromosomes per metaphase spread, which determined from 100 examined F. pentagona cells, was 28 (2n = 28). The percentage of metaphase spreads with an HSR from all cells was greater than 50% (43/80). Based on the arm ratio (Levan et al. 1964), this karyogram consisted of six submetacentric (1, 2, 3, 4, 5 and 7) and eight metacentric (6,8,9,10,11,12,13, and 14) chromosomes (Table 2).

Physical mapping of four FISH markers
FISH signals for the 18S rRNA gene locus were identified at the terminal ends of the long arm of chromosome 10 (Fig. 3a, c). The HSR, previously observed on chromosome 10 by G-banding, is hybridized by 18S rRNA gene probe, exhibiting a broad and intense hybridization signal ( Fig. 3a and c, green signals indicated by arrows). The HSR was also recognized as a pale part of chromosome 10 in DAPI staining metaphase (Fig. 3b, arrow). FISH probe from the uncharacterized gene FP-9X was mapped on the centromeres of chromosome 9 (Fig. 3a, c, red signals indicated by arrowheads,). The histone H3 gene, on the other hand, was mapped near the centromere of the long arm of chromosome 1 (Fig. 4a, c), whereas the U2 snRNA-5S gene locus was mapped on the terminal ends of the long arm of chromosome 2 (Fig. 5a, c).

Discussion
To solve the difficulties in taxonomically classifying stony corals (Fukami et al. 2004) and promote research on coral genetics, we have accumulated molecular cytogenetic data on several coral species. At this point, we have published six molecular cytogenetic reports with both conventional and molecular cytogenetic analyses of scleractinian corals, such as Acropora solitaryensis, Acropora pruinosa, Echinophyllia aspera, Coelastrea aspera, and Platygyra contorta (Taguchi et al. 2013(Taguchi et al. , 2014(Taguchi et al. , 2016(Taguchi et al. , 2017(Taguchi et al. , 2020Vacarizas 2021), which are commonly found on the western coast of Japan (Wallace, 1999). The present study focuses on F. pentagona, belonging to the genus Favites and the family Merulinidae.
We carried out conventional G-bandings to establish the karyotype of F. pentagona embryos. In general, obtaining high-quality G-banding in invertebrate chromosomes is difficult because of the relatively small chromosome size and the weak effect of trypsin on G-banding. Karyotyping of F. pentagona revealed the three chromosome groups that cannot be precisely identified because of their similar lengths. Furthermore, differences in chromosome condensation depending on the stage of the cell cycle at which cells were fixed sometimes made it difficult to measure the precise lengths of chromosomes. To develop a coral chromosome study, it is necessary to identify each chromosome precisely using a painting probe (Gokhman et al. 2019). Nonetheless, G-banded chromosomes revealed the presence of HSRs. HSRs are created by the amplification Chromosomes were karyotyped according to size and centromere positions showing the hybridization signal on chromosome 1 (c, above alignment). DAPI-only channel revealing the HSR region pointed by the arrow (b) and its karyogram (c, below alignment). Scale bar: 2 µm. and accumulation of certain DNA region within chromosomes. The amplification can be detected by conventional G-banding and FISH using locus-specific probes, which show intense hybridization signals on a single chromosome, as opposed to two copies with normal-homologous chromosomes (Biedler and Spencer 1976). In this study, we demonstrated that the HSR in the F. pentagona chromosomes is composed mainly of sequences from 18S rRNA gene. Generally, genetic amplification at the chromosomal level is manifested in the form of HSRs in tumor cells (Takaoka et 1 (a and c). DAPI-only channel without HSR region (b) and its karyotype (c, below alignment). Scale bar: 2 µm. al. 2012). We arranged each chromosome in decreasing order of their lengths and located the long chromosome 10 with an HSR. In our previous studies (Taguchi et al. 2013(Taguchi et al. , 2016(Taguchi et al. , 2017, using G-banding and FISH analyses, we revealed the presence of HSRs in chromosomes 11, 12, and 13 in each of the following three corals, C. aspera, E. aspera, and P. contorta. Interestingly, based on our recent studies on five stony coral species, including F. pentagona, HSRs are commonly found in the coral chromosomes of non-Acropora species. This indicates that the presence of an HSR is a cytogenetic characteristic of certain taxa of stony corals (Taguchi et al. 2013(Taguchi et al. , 2014(Taguchi et al. , 2016(Taguchi et al. , 2017(Taguchi et al. , 2020Vacarizas et al. 2021).
The FISH marker for histone H3 gene was observed at the centromeric region of chromosome 1. The core histone genes are highly conserved and repetitive, and their loci can thus be detected using FISH probes containing the sequence of a single array composed of tandem repeats (Huang et al. 2011). Previous molecular cytogenetic study on stony coral Acropora pruinosa has shown that the core histone gene locus which contains the H2a and H2b sequences were on the centromeric region of chromosome 8 (Vacarizas et al. 2021). These results imply that the chromosomal location of a highly conserved core histone varies across different family (Acroporidae and Merulinidae) of order Scleractinia (stony corals).
Information on 5S rDNA gene among stony corals is very limited (Taguchi et al. 2017;Taguchi et al. 2020;Vacarizas et al. 2021). We therefore developed the 5S rRNA gene primers reported not only in scleractinian corals, but also from other distant taxa such as hydra, starfish, and jellyfish (Hori et al. 1982;Walker and Doolittle 1983;Hendriks et al. 1987;Stover and Steele 2001) in which cytogenetic studies have identify specific FISH markers for 5S rRNA gene. The 5S rRNA gene primer from Hydra utilized by Stover and Steele (2001) were used to amplify suitable FISH probe sequences for this coral F. pentagona. Surprisingly, the sequence contain not only sequence of 5S rRNA gene but also the complete region of the U2-snRNA. The 5S rRNA gene and the U2-snRNA gene sequence were homologous to those of C. aspera (LC120341) and A. pruinosa (LC 557013), respectively (Fig. 6). In this study, we showed that chromosomal location of this U2 snRNA-5S of F. pentagona was near the telomere region of chromosome 2. This is different from the chromosomal location of 5S-snRNA in C. aspera (Taguchi et al. 2017) and A. pruinosa (Vacarizas et al. 2021) chromosomes. Based on the alignments (red rectangle in Fig. 7), several insert nucleotide sequences were found only in the U2 snRNA gene of F. pentagona. This suggests that U2 snRNA and 5S rRNA genes seemed to be fused, as cloning-sequence analysis and the sequence of the cloned insert is a part of the pseudogene.
Each of the FISH markers derived from the four different amplicons (FP-18S, FP-H3, FP-U2-5S, and FP-X9) were observed at a single site (locus), even in uncontracted prometaphase spreads with elongated chromosomes. As the specificity of the markers is very high due to the highly repetitive and conserved nature of these genes, these markers will be useful for karyotyping and identifying specific chromosomes containing similarity of sequences within closely related species.

Conclusion
The series of cytogenetic studies on stony corals including the development of specific FISH markers may contribute to understand changes in chromosomal structures across taxa thereby gain insight into its evolutionary processes (Guo et al. 2018). Thus, these results might shed light on coral diversity and improve the classification of corals. However, it is important to develop more suitable FISH markers which can identify specific gene loci. In this study, we isolated FISH markers that provided distinct and bright hybridization signals on chromosomes that suitable for routine observation.
We highlighted the development of four chromosomal FISH markers for U2 snRNA-5S, 18S rRNA, histone H3, and uncharacterized FP-9X genes, which were found to be located on chromosomes 2, 10, 1, and 9, respectively. The loci of the gene histone H3 were mapped in the chromosomes of stony corals for the first time. The isolation of four FISH markers for F. pentagona will also promote gene mapping and understand genome structure and organization for this species. This cytogenetic information on stony coral along with morphological and molecular characteristics may contribute to understand its evolutionary processes and resolve taxonomic problems in stony coral taxonomy.