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Int'l Journal of Marine Science 2011, Vol.1, No.2, 2-5
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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
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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.
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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
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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.
References
Cross I., Vega L., and Rebordinos L., 2003, Nucleolar organizing regions in
Crassostrea angulata: chromosomal location and polymorphism,
Genetica, 119: 65-74
http://dx.doi.org/10.1023/A:1024478407781 PMid:12903748
He M., Jiang W., and Huang L., 2004, Studies on aneuploid pearl oyster
(Pinctada martensii Dunker) produced by crossing triploid females and
a diploid male following the inhibition of PB1., Aquaculture, 230:
117-124
http://dx.doi.org/10.1016/j.aquaculture.2003.10.002
Katherine Lam and Brian Morton, 2004, The oysters of Hong Kong
(Bivalvia: Ostreidae and Gryphaeidae), The Raffles Bulletin of
Zoology, 52(1): 11-28
Levan A., Fredga D., and Sandberg A.A., 1964, Nomenclature for
centromeric position on chromosomes, Hereditas, 52: 201-220
http://dx.doi.org/10.1111/j.1601-5223.1964.tb01953.x
Shi Y.H., Gui J.F., Wang Y., Wang A.M., and Qu Y.B., 2007, Studies on the
genetic diversity of three cultured populations of Pinctada maxima,
Shuisheng Shengwu Xuebao (Acta Hydrobiologica Sinica), 31(1):
131-134
Wang Y., and Guo X., 2004, Chromosomal rearrangement in pectinidae
revealed by rRNA loci and implications for bivalve evolution, Biol
Bull, 207: 247-256
http://dx.doi.org/10.2307/1543213 PMid:15616355
Wang Y., Xu Z., and Guo X., 2004, Differences in the rDNA-Bearing
Chromosome Divide the Asian-Pacific and Atlantic Species of
Crassostrea (Bivalvia, Mollusca), Biol Bull, 206: 46-54
http://dx.doi.org/10.2307/1543197 PMid:14977729
Wang Y., Xu Z., Pierce J., and Guo X., 2005, Characterization of Eastern
Oyster (Crassostrea virginica Gmelin) Chromosomes by Fluorescence
In Situ Hybridization with Bacteriophage P1 Clones, Marine
Biotechnology, 7: 207-214
http://dx.doi.org/10.1007/s10126-004-0051-y PMid:15933900
Xu Z., Guo X., Gaffney P.M., and Pierce J.C., 2001, Chromosomal location
of the major ribosomal RNA genes in Crassostrea virginica and
Crassostrea gigas, Veliger, 44: 79-83
Zhang Q.Y., Yu G., Cooper R.K., and Tiersch T.R., 1999, Chromosomal
location by fluorescence in situ hybridization of the 28S ribosomal
RNA gene of the eastern oyster, J. 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.
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