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Int'l Journal of Marine Science 2011, Vol.1, No.2, 2-5 http://ijms.sophiapublisher.com A letter Open Access Chromosomal Mapping of Ribosomal rRNA Genes in the Small Rock Oyster, Saccostrea mordax (Gould 1850) Hu Lu * , Xuzhen Huang * , Jintian Zhu , Yan Wang Key Laboratory of Tropical Biological Resources of Ministry of Education, Marine Biology Experiment Teaching Demonstration Center, Ocean College, Hainan University, Haikou, Hainan 570228, China Corresponding author email: [email protected]; Authors * The authors who contribute equally International Journal of Marine Science, 2011, Vol.1, No.2 doi: 10.5376/ijms.2011.01.0002 Received: 02 Nov., 2011 Accepted: 18 Dec., 2011 Published: 20 Dec., 2011 Copyright: © 2011 Lu et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Lu et al., 2011, Chromosomal Mapping of Ribosomal rRNA Genes in the Small Rock Oyster (Saccostrea mordax, Gould 1850), International Journal of Marine Science, Vol.1, No.2 2-5 (doi: 10.5376/ijms. 2011.01.0002) Abstract Chromosomal location of minor (5S) and major (18-28S) ribosomal RNA genes (rDNA) were studied in the small rock oyster (Saccostrea mordax) using sequential fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from gill tissue of adult oysters. Both minor and major rDNA probes were obtained by PCR amplifications, and labeled by PCR incorporation of Biotin-11-dUTP and detected with fluorescein-labeled anti-digoxigenin antibodies. The results showed that small rock oyster had a haploid number of 10 chromosomes with a karyotype of ten metacentric chromosomes (10 M), which was similar to most species in genus Crassostrea. FISH with metaphase chromosomes detected a single locus for both 5S and 18-28S rDNA in the small rock oyster. 5S rDNA was mapped on the long arm of chromosome 2. And 18-28S rDNA was located on the telomeric regions of the short arms of chromosome 3. This study is the first report of karyotype and chromosomal assignment of the minor and major rRNA genes in S. mordax. Keywords Small rock oyster; Saccostrea mordax; Chromosome; FISH; rRNA gene Introduction marine mollusk belonging to family Ostreidae, which occurs only on oceanic, exposed rocky shores and is widely distributed in the Indo-West Pacific, e.g. Japan, Korea, Taiwan, Hong Kong, South China Sea, Peninsula Malaysia, Singapore, Indonesia, New Hebrides and Australia (Katherine and Brian, 2004). Because the small rock oyster is small and is not of economic importance, few research works were undertaking about it and little was known about its molecular and cytogenetic characteristics. In this article we study the karyotype and chromosomal assignment of the RNA genes in S. mordax using fluorescence in situ hybridization (FISH). Chromosomes are basic units of genome organization. Chromosomes and karyotype are inheritable characteristics, and their variation among species is a measure of evolutionary changes(Wang and Guo, 2004). The identification and characterization of chromosomes are also essential for studies on aneuploidy and chromosomal organization of genes (He et al., 2004; Wang et al., 2005). Studies on chromosomes of marine mollusks are more significant, especially for those as oysters, whose shell coloration and morphology are highly variable and sensitive to environmental influence, and anatomy of soft tissue is difficult, resulting in classification and phylogenetic analysis problematic (Wang et al., 2004). So phylogenetic analyses of oysters may have to rely on a multidiscipline approach using morphological, molecular, and cytogenetic characteristics. 1 Result and Discussions 1.1 Karyotype The majority (121 of 135) of metaphases screened had 20 chromosomes, conforming the haploid number is n=10. To characterize the chromosomes, homologous chromosomes from fifteen metaphases The small rock oyster (S. mordax, Gould 1850) is a 2 Int'l Journal of Marine Science 2011, Vol.1, No.2, 2-5 http://ijms.sophiapublisher.com were paired (Figure 1B) and measured (Table 1). The karyotype is composed of 10 pairs of metacentric chromosomes (n=10 M), as previously reported in most other oysters (Wang et al., 2004). The loss of one or two chromosomes was occasionally seen in a few metaphases that appeared to be overly spread (Figure 1A). Figure 2 Products of PCR amplification of probes. Note: M: Marker DL2000; A. Products of PCR amplification of 18S-28S sequence (Lane 1) and Biotin-16-dUTP labeled sequence (Lane 2); B. Products of PCR amplification of 5S sequence (Lane 1) and Biotin-16-dUTP labeled sequence (Lane 2). Figure 1 The ideogram (A) and Karyotypes (B) of small rock oyster S. mordax. 1.3 Chromosomal location of rRNA genes Table 1 Chromosome measurements and classification of S. mordax Chromosome Relative length no. (mean±SD) Arm ratio Type 1 5.41±0.03 1.15±0.03 m 2 5.40±0.04 1.15±0.04 m 3 5.38±0.02 1.14±0.03 m 4 5.38±0.02 1.14±0.03 m 5 5.39±0.02 1.15±0.03 m 6 5.33±0.02 1.16±0.03 m 7 5.34±0.03 1.13±0.03 m 8 5.36±0.02 1.15±0.03 m 9 5.27±0.07 1.15±0.03 m 10 5.28±0.04 1.15±0.03 m FISH with both 18-28S and 5S probes were successful. The counterstain dye PI stained the entire chromosome red and allowed karyotypic analysis of chromosomes. FISH signals detected by fluorescein appeared as greenish yellow dots or clusters of dots over a red background. FISH with 5S probe to the metaphase detected a single locus (four signals) (Figure 3A), while 18-28S probe to the interphase nuclei also produced four signals (1 locus) per nucleus (Figure 3B). The number and intensity of FISH signals varied depending on the stringency of post-hybridization wash. Intensity apparently differed among signals at different sites, with one to two signals were often stronger (Figure 3). 1.2 PCR results PCR with both 18-28S and 5S primer sets were successful. PCR with 18-28S primers produced a single fragment of about 1200 bp (Figure 2A), and 5S primers produced a single fragment of about 100 bp (Figure 2B). Incorporation of digoxigenin-11-dUTP significantly reduced the mobility of both fragments, suggesting that the labeling was effective. After labeling, the 18-28S fragment shifted to about 1500 bp (Figure 2A), and the 5S fragment shifted to about 150 bp (Figure 2B). Figure 3 FISH signals and chromosomal location of 5S and 18-28 S rDNA sequence on the same metaphase of S. mordax. Note: A. Metaphase of S. mordax after FISH with 5S rDNA; B. Metaphase of S. mordax after FISH with 18S-28S rDNA. 3 Int'l Journal of Marine Science 2011, Vol.1, No.2, 2-5 http://ijms.sophiapublisher.com and then with 0.005% colchicine for 8 h. Gill tissue were sample alive and were cut into little piece in 0.075 M KCl, and the the tissue suspension were transferred to a 10 mL centrifugal tube. The KCl treatment lasted for 30 min. The suspension were then fixed with 1:3 (v:v) acetic acid and methanol and stored in 4℃ overnight. Karyotypic analysis showed that for 18S-28S rDNA the specific FISH signals were located on telomeric region of the short arms of Chromosome 3 and for 5S rDNA, it is located on the long arm of chromosome 2 (Figure 3). The major ribosomal RNA genes (rDNA), which correspond to NORs, have also been mapped by FISH in three Crassostrea species of oysters (Zhang et al., 1999; Xu et al., 2001; Cross et al., 2003). FISH analysis of major rDNA provided validation for Ag-NOR staining and eliminated any uncertainty and intraspecific variations. The previous study suggests that differences in the 18-28S rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea oysters (Wang et al., 2004). As for Asian-Pacific S. mordax, the characters of major rDNA-bearing chromosome are apparently different from species of Crassostrea in the same region. Further effort should focus on the systematical study on the chromosomal divergence within and between various genus of small oysters, eg. Saccostrea and Dendostrea (Zhao et al., 2012), so that we can discover the mechanism of reproductive isolation and speciation resulted from chromosomal divergence. Chromosome metaphases were made by dropping cell suspension onto clean glass slides and subsequently flooded with 2~3 drops of 1:1 methanol and acetic acid. Then the slides were air-dried and stored at -20℃ until FISH analysis. Chromosomes were measured and classified according to Levan et al (1964). 2.2 Probe construction Genomic DNA of small rock oyster was extracted from adductor muscle according to Shi et al (2007) using proteinase K digestion. The major and minor rDNA were amplified and used as FISH probes. PCR primers were designed using conserved regions of sequences from several bivalve species. PCR primer sequences were 5’-GTTTCTGTAGGTGAACCTG C-3’ and 5’-CTCGTCTGATCTGA GGTCG-3’ for 18-28S rDNA, and 5’-GTCTACGACCATATCACGT TGAAAA-3’, 5’-TGTCTACAACACCCGGTATTCC C-3’ for 5S rDNA. Primers were synthesized by Sangong Technologies (China). Probes were labeled with digoxigenin-11-dUTP (alkali-stable) by PCR incorporation. PCR were performed in a 100 μL solution containing 1×PCR buffer with 1.5 mM of MgCl2, 0.2 mM each of dATP, dCTP, and dGTP, 0.13 mM of dTTP, 0.07 mM of Digoxigenin-11-dUTP, 2.5 U of Taq DNA polymerase, 1 μM of each primer, and 1 μg of oyster genomic DNA. The optimized thermal cycling parameters were 30 cycles of 1 min at 95℃, 1 min at 50℃, and 1 min at 72℃. Amplified products were visualized on 2% agarose gels. DIG-labeled PCR products were purified using G-50 columns and used as FISH probes. This study provides the first chromosomal assignment by FISH of the rRNA genes in S. mordax. Results of this study show that FISH is a powerful tool for cytogenetic analysis, especially in species where chromosome identification by traditional methods is challenging. Cytogenetic analysis in most marine invertebrates has been limited primarily due to difficulties of chromosome identification. The application of FISH techniques and development of chromosome-specific probes may enable chromosome identification and phylogenetic comparisons of mollusks and other marine invertebrates. 2 Materials and Methods 2.3 Fluorescence in situ hybridization Sequential FISH was performed according to Wang and Guo (2004) with minor modifications. Briefly, after adding 100 μL RNase, the metaphase slides were incubated for 0.5~2 h, and dehydrated for 5 min each in 2.1 Chromosome preparation and measurement The small rock oysters (S. mordax) used in this study were sampled from West Island Port, Sanya city, Hainan province, China. Three females and three males oysters were bathed with 0.01% PHA for 24 h 4 Int'l Journal of Marine Science 2011, Vol.1, No.2, 2-5 http://ijms.sophiapublisher.com 70%, 80% and 95% ethanol, and airdried. Metaphases were then denatured in 2×SSC containing 50% deionized formamide and 10% dextran sulfate at 80℃ for 10 min. Denatured FISH probe (12~15 μL) was added to the slide carrying denatured metaphases and covered with coverslip. After sealing the perimeter of the coverslip with rubber cement, the slides were incubated overnight at 37℃ in a humidified chamber. Acknowledgements This study was funded by the National Natural Science Foundation of China (#31060354,40866003), and the Natural Science Foundation of Hainan Province (#311024). And we thank Prof. Yingya Cai for her effort in indentifying the samples. 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Shellfish Res., 18: 431-435 Zhao W.X., Zhang N., Wang Y., Shi Y.H., Gu Z.F., and Wang A.M., 2012, Karyotypes and Chromosomal Mapping of Major Ribosomal rRNA Genes in the Leaf Oyster (Dendostrea folium), Genomics and Applied Biology, 31: 20-25 Coverslips were then removed by rinsing slides in solution A (2×SSC, containing 50% formamide) and B (2×SSC) for 5 min, 3 times. The slides were washed with 40 μL stringencies (5% BSA 3 μL, 0.1% Tween-20 5 μL, 20×SSC, 1 μL) and incubated at 37℃ for 20 min. 30 μL 10 μg/mL Fluorescein-labeled anti-digoxigenin antibody Avidin-FITC (Roche) was added to each slide, covered with a plastic coverslip, and incubated at 37℃ for 15 min in a humidified chamber. The slides were then washed in 1×PBD at room temperature three times for 2 min each. Twenty microlites propidium iodide (PI)/antifade (0.5 μg/mL) was added to each metaphase spread and covered with glass coverslips. Hybridization signals were analyzed and documented using a Nikon epi-fluorescence microscope equipped with a 3CCD camera and the Image-Pro Plus software. For sequential hybridiaztion using 18-28S rDNA and 5S rDNA probes, the methods were as follows: after the first round of probing and image taking, the sildes were soaked in 1 × PBS solution to remove the coversilips. The slides were then dehydrated and denatured again as descrised before, and then incubated using a different FISH probe. Authors’ contributions HL and XZH are the executor of experimental research in this study, they have the same contribution to the paper. YW and JTZ make the experimental design, data analysis, paper writing and revising. 5