MAPPING OF RIBOSOMAL DNA AND (TTAGGG) n TELOMERIC
SEQUENCE BY FISH IN THE BIVALVE PATINOPECTEN YESSOENSIS
(JAY, 1857)
XIAOTING HUANG, XIAOLI HU, JINGJIE HU, LINGLING ZHANG, SHI WANG,
WEI LU AND ZHENMIN BAO
Laboratory of Marine Genetics and Breeding (MGB), Division of Life Science and Technology, Ocean University of China, Qingdao 266003, China
(Received 4 January 2007; accepted 1 September 2007)
ABSTRACT
The chromosome set of Patinopecten yessoensis (Jay, 1857) was characterized using Giemsa staining, DAPI
staining and fluorescence in situ hybridization (FISH) with three repetitive DNA probes [18S– 28S
rDNA, 5S rDNA and telomeric (TTAGGG)n]. DAPI staining showed that AT-rich regions were
located on the centromere of almost all chromosomes and interstitial banding was not observed.
FISH showed that 18S– 28S rDNA spread over the short arms of two subtelocentric chromosome
pairs and 5S rDNA was located on the long arm of one subtelocentric chromosome pair. Sequential
FISH demonstrated that 18S– 28S and 5S rDNA were located on different chromosomes. FISH also
showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S– 28S rDNA and (TTAGGG)n FISH indicated that repeated units of the two multicopy families were closely associated on the same chromosome
pair.
INTRODUCTION
The Yesso scallop, Patinopecten yessoensis (Jay, 1857), family
Pectinidae, is a cold-tolerant species that inhabits the coastal
areas of cold temperate zones. It is one of the most extensively
cultivated bivalve molluscs and among the twelve most important species in global aquaculture (Saavedra & Bachère, 2006). A
more detailed genetic characterization of P. yessoensis may help
to improve conservation and restocking plans, and offers a considerable advantage for environmental protection of the species.
Unfortunately, the genome of P. yessoensis is poorly understood.
The chromosome number of P. yessoensis is 2n ¼ 38. Its chromosome complement consists of two pairs of metacentric (m), one
pair of metacentric – submetacentric (m/sm), four pairs of submetacentric (sm), six pairs of submetacentric – subtelocentric
(sm/st), three pairs of subtelocentric (st) and three pairs of telocentric (t) chromosomes (Komaru & Wada, 1985). Little other
cytogenetic information is known about P. yessoensis.
The family Pectinidae includes more than 300 living species.
Phylogenetic analyses using mitochondrial genes and morphological characters have been used to infer the basic evolutionary
relationships in Pectinidae (Barucca et al., 2004; Waller, 1993).
Chromosomal studies may provide a new perspective on this
evolution. Moreover, the chromosome number and karyotype
vary considerably in Pectinidae. However, a detailed analysis
of chromosome changes in Pectinidae has been prevented by
the inability to identify individual chromosomes, and because
banding techniques were not clear and stable enough. The fluorescence in situ hybridization (FISH) technique is a useful tool for
reconstruction of chromosome rearrangement. Recently, this
technique has been used for comparative cytogenetic investigations in Pectinidae. Wang & Guo (2004), for example,
applied FISH to study the patterning of ribosomal DNA in
Chlamys farreri (Jones & Preston, 1904) and Argopecten irradians
irradians (Lamarck, 1819) and postulated that chromosomal
translocation and duplication may play a dominant role in the
Correspondence: Z. Bao; e-mail: [email protected]
karyotypic evolution of Pectinidae. In addition, the histone H3
gene has been developed as a new FISH marker and the
results suggested that gene duplication/diminution as well as
chromosome rearrangements by inversion and translocation
might play important roles in the genomic evolution of Pectinidae (Zhang et al., 2007). With regard to Pectinidae, FISH has
also been used to map highly repetitive ribosomal DNA (18S –
28S rDNA and 5S rDNA) on the chromosomes of seven scallops
(Table 1). These studies have revealed that ribosomal DNA is
useful for examining the genome constitution at the molecular
level and for investigating chromosomal changes during evolution. However, little is known about the ribosomal DNA
location in P. yessoensis.
The other repetitive sequence used frequently by FISH is
(TTAGGG)n, which occurs in the telomere of all vertebrate
chromosomes, whereas in invertebrates it is present in some
species (Colomba et al., 2002) but not in others (Vitturi et al.,
2000). In bivalve molluscs, this vertebrate telomeric sequence
has been mapped using FISH in oysters (Guo & Allen, 1997;
Wang & Guo, 2001), clams (Wang & Guo, 2001) and mussels
(Plohl et al., 2002). Nevertheless, telomeric sequences in Pectinidae are still unknown.
In the present study, both advanced and conventional cytogenetic techniques were applied to gain more detailed cytogenetic
knowledge of the P. yessoensis chromosome complement. In
addition to DAPI banding, chromosomal mapping of two
rDNA families (28S and 5S) and the vertebrate telomeric
sequence (TTAGGG)n was carried out using the FISH technique. Moreover, we used sequential FISH to examine the physical relationships between 18S– 28S rDNA, 5S rDNA and
(TTAGGG)n telomeric repeats.
MATERIAL AND METHODS
Specimens and chromosome preparations
Sexually mature scallop Patinopecten yessoensis (10 females and 3
males) were obtained from a hatchery in Shandong Province,
Journal of Molluscan Studies (2007) 73: 393–398. Advance Access Publication: 6 November 2007
# The Author 2007. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.
doi:10.1093/mollus/eym036
X. HUANG ET AL.
Table 1. Summary of the location of ribosomal DNA in Pectinidae.
Species
Ribosomal DNA
Location†
No.
Type‡
Reference
Pectininae
Aequipecten opercularis
Argopecten purpuratus
18S –28S rDNA
1
Telomeric q
t
Insua et al., 1998
5S rDNA
2
Interstitial q
m
Insua et al., 1998
Interstitial q
m
18S –28S rDNA
3
Telomeric q
Unknown
Gajardo et al., 2002
Wang & Guo, 2004
Pericentromeric
Pericentromeric
Argopecten irradians irradians
Pecten maximus
5S rDNA
Unknown
18S –28S rDNA
2
Telomeric p
st
Telomeric p
st
5S rDNA
1
Interstitial q
t
Wang & Guo, 2004
18S –28S rDNA
1
Telomeric
m/sm
Insua et al., 2006
5S rDNA
1
Interstitial q
t
Insua et al., 2006
18S –28S rDNA
1
Paracentromeric p
st
Odierna et al., 2006
5S rDNA
Unknown
18S –28S rDNA
2
This study
Adamusslinae
Adamussium colbecki
Chlamidinae
Patinopecten yessoensis
Chlamys farreri
Hinnites distortus
st
st
5S rDNA
1
Interstitial q
st
This study
18S –28S rDNA
1
Telomeric p
sm/st
Wang & Guo, 2004
1
Telomeric p
st
Huang et al., 2006
1
Interstitial q
sm/st
Wang & Guo, 2004
5S rDNA
Mimachlamys varia
Telomeric p
Telomeric p
18S –28S rDNA
1
Telomeric p
sm/st
Insua et al., 2006
5S rDNA
1
Interstitial q
st
Insua et al., 2006
18S –28S rDNA
2
López-Piñón et al., 2005
5S rDNA
1
Centromeric
st
Centromeric
st
Pericentromeric q
st
López-Piñón et al., 2005
The species assignation to these subfamilies based on the classification data from Barucca et al. (2004), where phylogenetic relationships within family Pectinidae
have been explored using molecular analysis techniques based on the sequences of 12S and 16S rRNA gene.
China. Eggs and sperm were collected from mature scallops. Eggs
were fertilized by adding sperm suspension. After fertilization,
excessive sperm were removed by rinsing with seawater on a 20mm screen. The progeny individuals were sampled at the swimming trochophore larvae stage and used for analysis. Briefly, the
larvae were treated with a colchicine solution (10 mg/ml) at
room temperature for 2 h and KCl (0.075 M) for about 30 min,
and then fixed three times (15 min each) in Carnoy’s fixative
(methanol: glacial acetic acid ¼ 3:1 v/v). The fixed larvae were
dissociated into a cell suspension in 50% acetic acid and then
dropped on hot wet glass slides and air-dried.
Conventional karyotype was made from slides stained with
10% Giemsa for 20 min. For karyotype analysis, the chromosomes were paired according to their morphology, and then
short and long arms were measured using the Photoshop 7.0
program for calculation of relative length (RL) and centrometric index (CI), and classified according to criteria defined
by Levan, Fredga & Sandberg (1964).
Fluorescence in situ hybridization
The telomeric probes (TTAGGG)7 were synthesized and 50 -end
labelled with biotin (Invitrogen). Both 18S – 28S rDNA and 5S
rDNA probes were obtained by PCR amplification as described
by Wang & Guo (2004).
FISH was carried out according to Bi et al. (2005). Chromosomes were pretreated with pepsin, denatured in a mixture containing 75% formamide and 2 SSC for 2 – 3 min at 728C,
dehydrated through an ice-cold ethanol series (70, 90, 100%,
5 min each) and air-dried. The probe hybridization mixture
containing 5 ng/ml PCR-biotinylated probe DNA, 50% (v/v)
formamide, 10% (v/v) dextran sulphate and 2 SSC was
denatured at 808C for 5 min and rapidly cooled by putting it
on ice for at least 10 min. Denatured probes were applied to
the slides and DNA – DNA in situ hybridization was carried out
at 378C for 12 – 16 h. After hybridization, the slides were
washed by placing them in 2 SSC at 428C for 5 min, 50% formamide in 2 SSC at 428C for 10 min, 2 SSC at 428C for
10 min and finally 2 SSC at room temperature for 10 min.
Biotinylated probes were detected with avidin-FITC (Vector).
Digoxigenin-labelled probes were detected with Rhodaminelabelled anti-digoxigenin antibody (Vector). Chromosomes
were counterstained with PI (1.5 mg/ml, Vector) or DAPI
(20 ng/ml, Vector) at room temperature for 10 min. Slides
were visualized with a Nikon epifluorescence microscope
(Eclipse E-600) equipped with a CCD camera and analysed a
with Lucia-FISH Image System.
Sequential hybridization using 18S– 28S rDNA and the 5S
rDNA or (TTAGGG)n probes was performed as follows: after
Banding technique
For fluorescent pattern analysis, chromosomes were stained with
DAPI (4, 6-diamidino-2-phenylindole, Vector). Chromosome
slides aged 1–30 days at 2208C were baked and then stained
with DAPI for 5–10 min and mounted with Canada balsam
(Heng and Tsui, 1993). The number of DAPI bands was determined in 16 well-banded metaphase plates prepared from the trochophore stage. Chromosomes were measured for RL and CI,
and classified according to criteria defined by Levan, Fredga &
Sandberg (1964).
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CYTOGENETICS IN PATINOPECTEN YESSOENSIS
Figure 1. Karyotype of P. yessoensis after conventional Giemsa staining (A) and DAPI staining (B).
located on chromosome 15 (Fig. 2C). Sequential hybridization
using 18S – 28S rDNA and 5S rDNA probes on the same metaphase showed that the two ribosomal DNA clusters were
located on different chromosomes. After karyotype analysis,
the result was consistent with that of FISH using a single
probe, i.e. 18S– 28S rDNA was located on chromosomes 11
and 13 and 5S rDNA was located on chromosome 15 (Fig. 2).
Telomeric repeats, detected by FISH, were located at both
ends of each chromosome, although the signal intensity varied
among different chromosomes (Fig. 3B). No hybridization
signal was observed at any interstitial chromosomal site. Sequential hybridization using the 18S– 28S rDNA and the
(TTAGGG)n probes on the same metaphase showed that the
two repetitive sequences were located adjacent to each other
(Fig. 3A, B).
the first round of probing and image acquisition, the slides were
soaked in 1 PBS solution at 428C to remove the coverslips.
The slides were then dehydrated in an ethanol series (70, 90
and 100%, 5 min each), denatured again in 70% formamide
at 728C for 2 – 3 min, dehydrated in a second ethanol series
and incubated using a different FISH probe.
RESULTS
The diploid chromosome number, counted under Giemsa-staining,
was 2n ¼ 38 in 81% of the Patinopecten yessoensis metaphases. To
characterize the chromosomes, homologous chromosomes from
10 metaphases were paired and measured. The karyotype is
composed of three pairs of metacentric, five pairs of submetacentric, eight pairs of subtelocentric and three pairs of telocentric
chromosomes (Fig. 1A).
Staining with fluorochrome DAPI showed the existence of
centromeric AT-bands on chromosomes of P. yessoensis
(Fig. 1B). Centromeric DAPI-positive bands were clearly
visible on almost all chromosomes. Interstitial DAPI-positive
bands were not observed.
FISH using the 18S– 28S rDNA probe alone showed that four
hybridization signals were located on the telomeric region of the
short arms of two pairs of homologous chromosomes 11 and 13
(Figs 2A, C, 3A). FISH using the 5S rDNA probe revealed
that two hybridization signals were located on the interstitial
region of the long arm of one pair of homologous chromosomes
(Fig. 2B). Karyotype analysis showed that FISH signals were
DISCUSSION
Komaru & Wada (1985) reported that the chromosome number
of Patinopecten yessoensis was 2n ¼ 38 and the karyotype was
2 m þ 1 m/sm þ 4sm þ 6sm/st þ 3st þ 3t. The present study
confirmed these results, although the number of chromosomes
assigned to each chromosome group was slightly different.
These slight differences may result from the condensation
state of chromosomes and due to the difficulty in distinguishing similar metacentric/submetacentric or submetacentric/
subtelocentric chromosomes. A chromosome number of 38 is
the most frequent among scallop species (Insua et al., 2006).
395
X. HUANG ET AL.
Figure 2. FISH signals and chromosomal location of the 18S–28S rDNA (A) and 5S rDNA (B) on the same metaphase in P. yessoensis. (C) Karyotype
of P. yessoensis. Scale bar ¼ 5 mm.
the close relationship between the two species, which has been
demonstrated by a molecular phylogeny based on mitochondrial
16S and 12S rRNA genes (Barucca et al., 2004) and by morphological characteristics (Waller, 1993).
Staining with DAPI revealed that AT-rich regions were only
on the centromeric region of most P. yessoensis chromosomes. In
The karyotype of P. yessoensis studied here was similar to that of
Chlamys farreri, which was 3 m þ 5sm þ 11st (Huang et al., 2006),
and the only difference between the two species was that P. yessoensis had three pairs of telocentric chromosome while C. farreri
did not. Both P. yessoensis and C. farreri live in the coastal water of
the northwestern Pacific. Their similar karyotype may reflect
Figure 3. FISH signals and chromosomal location of the 18S–28S rDNA (A) and telomeric (TTAGGG)n sequence (B) on the same metaphase in
P. yessoensis. Scale bar ¼ 5 mm.
396
CYTOGENETICS IN PATINOPECTEN YESSOENSIS
histone H3 suggested that gene duplication/diminution as well
as chromosome rearrangements by inversion and translocation
might play important roles in the genomic evolution of Pectinidae (Zhang et al., 2007). We assume that the slight difference of
5S rDNA loci on the homologous chromosome of P. yessoensis
may be a result of chromosomal inversion.
In the present study, we used a (TTAGGG)n probe to test for
the presence of vertebrate telomeric sequences in P. yessoensis
chromosomes. The results demonstrated that the telomeric
hexanucleotide occurred at the terminus of all chromosomes in
P. yessoensis, without the production of any internal signals.
Among bivalve molluscs, this sequence has also been located
on the terminus of the chromosomes of clams (Wang & Guo,
2001; Plohl et al., 2002) and oysters (Wang & Guo, 2001). In
the mussel Mytilus galloprovincialis (Lamarck, 1819), the chromosomes have also shown some interstitial positions in addition to a
telomeric position (Plohl et al., 2002).
Sequential hybridization using biotin-labelled 5S rDNA and
digoxigenin-labelled 18S– 28S rDNA showed that major
(18S– 28S rDNA) and minor (5S rDNA) ribosomal clusters
were on different chromosome pairs. The same results have
been reported in A. opercularis (Insua et al., 1998), A. i. irradians
(Wang & Guo (2004)), H. distortus (López-Piñón et al., 2005),
P. maximus and M. varia (Insua et al., 2006). Although 18S–
28S and 5S rDNA were located on the same chromosome pair
in C. farreri, (Wang & Guo (2004)), the most frequent situation
is that the two types of rDNA are located on different chromosomes (Insua et al., 2006). A single chromosome with rRNA
genes is rare in vertebrates and is considered to be the ancestral
state within taxa. In addition, the same technique with biotinlabelled (TTAGGG)n and digoxigenin-labelled 18S– 28S
rDNA probes revealed that ribosomal and telomeric signals
were adjacent. A similar configuration has been observed in a
periwinkle (Colomba et al., 2002) and a slug (Vitturi et al.,
2004). Among vertebrates, an adjacent disposition between telomere and NORs has been reported to be an unusual finding
(Liu & Fredga, 1999). However, this condition occurs more frequently in invertebrates than in vertebrates (Colomba et al.,
2002). Nevertheless, the significance of the telomere within
NORs is unclear. One of the functions of telomeres is nuclear
organization (Liu & Fredga, 1999). Therefore, telomeric
sequences near NORs in P. yessoensis may play a role in nucleolus
organization.
In conclusion, we have reported the first successful application
of FISH to locate ribosomal DNA and vertebrate telomeric
sequence in P. yessoensis. The results obtained here should help
to identify the chromosomes of P. yessoensis and will be useful
for assessing the evolutionary relationships within Pectinidae.
Pectinidae, only Hinnites distortus (Da Costa, 1778) has been
studied by DAPI staining and identical DAPI banding patterns
were observed (López-Piñón, Insua & Méndez, 2005). In contrast, centromeric, terminal and interstitial DAPI bandings
were observed in Argopecten irradians irradians in our other experiment (Huang et al., 2007). Gajardo, Parraguez & Colihueque
(2002) reported that the Hoechst/Actinomycin D counterstain
revealed a fluorescent heterochromatic block in the centromeric
area of few chromosomes in Argopecten purpuratus (Lamarck,
1819). Heng & Tsui (1993) obtained a full C-banding pattern
in human chromosomes using a method based on one step
DA/DAPI staining protocol: chromosome slides aged 1– 30
days at 2208C are baked and then stained in DA/DAPI solution
for 5 – 10 min; slides are washed twice in 2 SSC and mounted
in McIlcaine buffer and glycerol. Thus the DAPI banding
obtained here may reflect constitutive heterochromatin regions
in P. yessoensis. In Pectinidae, constitutive heterochromatin was
observed by C-banding on centromeric, intercalary or subterminal positions in Aequipecten opercularis (Lamarck, 1758) (Insua,
López-Piñón & Méndez, 1998), and on pericentromeric, telomeric or interstitial positions in Nodipecten nodosus (Linnaeus,
1758) (Pauls & Affonso, 2000). To explain this difference in the
distribution of heterochromatin, it has been proposed that the
centromeres are the starting points for the transfer of heterochromatin through the telomeres, hence, a karyotype with higher
telomeric heterochromatin must have a relatively basal phylogenetic status (Martı́nez-Lage, González-Tizón & Méndez, 1995).
As a result, in Pectinidae, A. opercularis, N. nodosus and
A. i. irradians may have an older phylogenetic status because
they show considerable telocentric heterochromatin; in contrast,
P. yessoensis and H. distortus may have a more recent origin
because they have more centromeric heterochromatin.
This report described the first application of FISH to identify
the location of repetitive DNAs [rDNA and (TTAGGG)n
sequence] on chromosomes of P. yessoensis. 18S– 28S rDNA
spread over the short arm of subtelocentric chromosomes 11
and 13. In Pectinidae (Table 1), most species show one NORbearing chromosome pair; A. i. irradians and H. distortus
show NORs on two chromosome pairs; only in one scallop
A. purpuratus, was the NOR located on three chromosomes
pairs. The more than one NOR-bearing chromosome pairs
may have evolved from one NOR-bearing chromosome pair
through chromosomal translocation or duplication. The variation observed in the distribution of 18S– 28S rDNA in scallops
is similar to that reported in other bivalve families. Clams,
mussels and oysters show one to four 18S– 28S rDNA sites,
mostly located on the telomere of chromosomes (Martı́nez
et al., 2002; Insua et al., 2006). In addition, we also noticed
that FISH signals were often considerably stronger on one of
the homologous chromosomes than on the other (Figs 2A, 3A).
A similar situation has also been observed in oyster and
abalone, which has explained by random variation in FISH or
the differences (loss or gain) in rDNA sequences (Xu et al.,
2001; Gallardo-Escárate et al., 2005). We speculate that this
difference in intensity may result from different numbers of
repetitive rDNA units between homologous chromosomes. 5S
rDNA was located on the long arm of chromosome 15 in P. yessoensis. For the family Pectinidae, one interstitial 5S rDNA locus
has mostly been described. Only A. opercularis has shown two
adjacent hybridization sites on the long arm (Insua et al.,
1998). In A. i. irradians, C. farreri, H. distortus, Pecten maximus
(Linnaeus, 1758) and Mimachlamys varia (Linnaeus, 1758), 5S
rDNA was located on the interstitial parts of the long arms of
one chromosome pair (Table 1). Furthermore, the loci of 5S
rDNA show a slight difference on the homologous chromosomes
in this study (Fig. 2B), which was observed only in one metaphase of our analysed 30 metaphases. Previous reports on
chromosome mapping of major and minor rRNA genes, and
ACKNOWLEDGEMENTS
We thank Prof. Guanpin Yang for his assistance with the use of
English. This work was supported by the National High Technology Research and Development Programme of China
(2006AA10A408 and 2006AA10A402), Specialized Research
Fund for the Doctoral Programme of Higher Education
(20060423015) and a Grant for Agricultural Technique Application Project of Ministry of Science and Technology of China
(2006GB23600451).
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