Mol. Cells, Vol. 13, No. 3, pp. 413-418
M olecules
and
Cells
KSMCB 2002
Karyotype Analysis of a Korean Cucumber Cultivar (Cucumis
sativus L. cv. Winter Long) Using C-banding and Bicolor
Fluorescence in situ Hybridization
Dal-Hoe Koo, Yoonkang Hur, Dong-Chun Jin, and Jae-Wook Bang*
Department of Biology, Chungnam National University, Daejeon 305-764, Korea.
(Received December 27, 2001; Accepted February 18, 2002)
An intensive karyotype analysis of a Korean cucumber
cultivar (Cucumis sativus L. cv. Winter Long) was carried out with three different methods. These included
Feulgen staining, Giemsa C-banding, and fluorescence
in situ hybridization (FISH). The mitotic chromosomes
of the cucumber (2n = 2x = 14) were characterized,
based on the length and arm ratio values. A C-banding
analysis showed dark stains on the centromeric, telomeric, and intercalary regions of the chromosomes,
except that chromosome 2 had a heavy staining in the
long arm. Bicolor FISH, using 45S and 5S rDNA
probes, provided additional information to identify
cucumber chromosomes. The signals for 45S rDNA
were detected on the pericentromeric regions of
chromosomes 1, 2, and 4. The signals for 5S rDNA
were on the short arm of chromosome 5. Similar band
patterns (as the C-banding) were observed when the
chromosomes were counter-stained with 4′,6diamidino-2-phenyoindole (DAPI). The data implied
that the karyotype of the Korean cucumber cultivar is
peculiar and different from previous reports.
Keywords: Bicolor FISH; C-banding; Cucumis sativus
L.; Karyotype; rRNA Gene.
Introduction
Cucumbers (Cucumis sativus L., 2n = 2x = 14) are one of
the most important vegetable crops. They belong to one of
the top 10 worldwide vegetable species (FAO, 1993). The
karyotype of cucumber plants was analyzed using aceto
carmine, aceto orcein, and Feulgen staining (Ayyangar,
1967; Bhaduri and Bose, 1947; Chen et al., 1998; Hoshi-
Demo
* To whom correspondence should be addressed.
Tel: 82-42-821-5497; Fax: 82-42-822-9690
E-mail: [email protected]
et al., 1998; Ramachandran and Seshadri, 1986; Sing and
Roy, 1974; Trivedi and Roy, 1970). These studies provide
information on the overall nature of the chromosome
morphology and pairing, but their interpretations on
karyotype were inconsistent. This discrepancy on the cytological study of cucumbers may be due to the small size
of the mitotic chromosomes and poor stainability with the
conventional staining procedures (Dane, 1991; Ramachandran and Seshadri, 1986; Trivedi and Roy, 1970).
Nevertheless, chromosome identification of cucumbers
was achieved by using a C-banding method, even though
the banding patterns that were obtained were different
(Chen et al., 1998; Hoshi et al., 1998; Ramachandran and
Seshadri, 1986).
The fluorescence in situ hybridization (FISH) technique
that was developed by Welsh and McClelland (1990) as
well as Williams et al. (1990) is an excellent method for
the physical mapping of DNA sequences, because it is
capable of detecting several probes simultaneously on the
same metaphase chromosomal spread (Mukai et al., 1993).
The 18S, 5.8S, and 26S rRNA genes (45S rDNA) are organized in tandem arrays within the nucleolar orga-nizer
regions (NORs). Each rDNA repeat unit includes a nontranscribed region, a transcribed region that comprises the
externally transcribed spacer, and the coding regions for
the three rRNAs and two internally situated transcribed
spacers that flank the 5.8S rRNA gene (Gründler et al.,
1991; Wanzenböck et al., 1997). The 5S rRNA genes,
which are mapped outside the NORs, are again arranged
in highly repeated tandem arrays that consist of a 120 bp
coding region that is flanked by 100 to 700 bp long spacer
sequences (Danna et al., 1996; Sastri et al., 1992). Because two types of the arrays, the 5S and the 45S rDNA
loci, are often different in number but are not strictly
linked (Lapitan, 1992; Phillips et al., 1988),
Abbreviations: BSA, bovine serum albumin; dUTP, deoxyuridine triphosphate; SSC, standard sline citrate buffer.
414
Karyotype Analysis of Cucumber Using C-banding and FISH
they have been used as excellent markers for identifying
chromosomes (Fuchs et al., 1998; Schrader et al., 1997).
The physical mapping of the rRNA genes in cucumbers
was recently reported (Chen et al., 1999; Hoshi et al.,
1999). The results of FISH using the 45S rDNA probe
(Chen et al., 1999) observed four pairs of signals on
chromosomes 1, 2, 4, and 7; whereas, Hoshi et al. (1999)
observed three pairs of signals at the pericentromeric regions of chromosomes 1, 2, and 5. The FISH results, using either 5S rDNA or 5S and 45S rDNA probes together,
have not yet been reported in cucumber plants.
In this study, we report the karyotype of a Korean
cucumber cultivar and the physical mapping of 5S and
45S rDNA loci in the plant by the bi-color FISH method.
Materials and Methods
Plant materials Korean cucumber cultivar (Cucumis sativus L.
cv. Winter Long; 2n = 2x = 14) seeds were purchased from the
Dongbu Hannong Seeds Co., Korea. The seeds were germinated
in petri dishes at 25°C. The root tips were used for the chromosome analysis.
Feulgen staining Actively growing root tips were excised
when they were about 1 cm in length. They were pretreated with
distilled water at 4°C for 12 h. The treated root tips were then
fixed in an acetic acid: ethanol (1:3 v/v) solution for 1−2 d. The
samples were then hydrolyzed in 1 N HCl for 5 min at 60°C.
Afterward, squash mounts were prepared with Feulgen. For a
karyotype analysis, the chromosomes were arranged in order of
decreasing length, then classified according to the nomenclature
of Levan et al. (1964).
Chromosome preparation For the C-bandig and FISH analyses, the slides were prepared according to the previous method
(Kakeda et al., 1991) with minor modifications. Fixed root tips
were washed thoroughly in distilled water prior to the enzymatic
treatment. The meristematic portion of the root tips was treated
with an enzyme mixture (5% cellulase, 1% pectolyse, 1 mM
EDTA, pH 4.5) at 37°C for 1 h for degradation of the cell wall.
After rinsing, the root tips were tapped carefully with the forceps’ tip on a glass slide with a few drops of the fixative, then
air-dried.
Probe preparation The 5S rDNA probe was obtained from
PCR using total genomic DNA of C. sativus as the template
DNA. The reaction mixture of a total of 100 µl was composed
of 200 µM each of dATP, dGTP, and dCTP, 60 µM of dTTP, 140
µM of digoxigenin-11-dUTP (Boehringer Mannheim), 5 pM
each of the forward (5′-GATCCCATCAGAACTCC-3′) and
reverse (5′-GGTGCTTTAGTGCTGGTAT-3′) primers, 10 ng of
template DNA, and 2.5 units of Ex Taq Polymerase (TaKaRa).
Next, there was incubation at 94°C for 1 min, annealing at 55°C
for 1 min, and extension at 72°C for 2 min. Post elongation was
conducted at 72°C for 10 min.
To confirm the 5S rDNA that was obtained from PCR, the
PCR products were cloned and subjected to a sequence analysis.
After the PCR reaction, the DNA fragments were treated with a
Geneclean II Kit (Bio 101), ligated into a pGEM-T-Easy vector
(Promega), and transformed into E. coli DH5α (Sambrook et al.,
1989). Nucleotide sequences were determined using the automatic DNA sequencer (ABI377) at the Korea Research Institute
of Bioscience and Biotechnology. The DNA sequence data were
analyzed using the BLAST network service at the National Center for Biotechnology Information (NCBI). The 5S rDNA is 302
bp in size (data not shown), which is the first report for the cucumis species. The sequence homology with other crops is over
90%.
The probe labeling of the 45S rDNA was carried out by the
Nick translation method that is recommended for probes larger
than 1 kbp, according to the manufacturer’s protocol (GibcoBRL). Soybean 45S rDNA, 3.4 kbp, was obtained from Dr.
Hong-Gil Nam, Postech, Korea. The labeling mixture was composed of 5 µl of 0.2 mM dATP, dGTP and dCTP each, 1 µl of
biotin-16-dUTP or dioxigenin-11-dUTP instead of dTTP, 1 ng of
probe DNA, and 5 µl of DNaseI/DNA polymerase. The final
volume was adjusted to 50 µl with distilled water in a 1.5 ml
microfuge tube that was placed on ice, then mixed well. The
mixture was centrifuged for 5 s and incubated at 15°C for 90
min, sequentially at 65°C for 10 min, and placed at room temperature for 15 min. Then, 6.25 µl of 4 M ammonium acetate
and 5 µl of 100% EtOH were added to stop the reaction. The
mixture was centrifuged for 5 min at 12,000 rpm and the supernatant was discarded. The pellet was rehydrated in 20 µl of
100% formamide until all of the liquid evaporated.
C-banding analysis Sumner’s method (1972) was used for the
C-banding with minor modifications. The air-dried slides were
dehydrated in 100% ethanol at room temperature for 2 h. After
drying, the slides were immersed in 0.2 N HCl at 60°C for 2
min, rinsed with running water, and placed in 5% (w/v) barium
hydroxide at room temperature for 7 min. After washing, the
slides were incubated in 2× SSC at 60°C for 30 min. The slides
were then rinsed with distilled water and stained in 3% Giemsa
dilute with 1/15 M Sörensen phosphate buffer (pH 6.8) for 10
min.
Bicolor fluorescence in situ hybridization FISH was carried
out according to the previous method (Ohmido and Fukui, 1996)
with minor modifications. Chromosomal DNA on the slides was
denatured in 70% formamide at 70°C for 2 min, then dehydrated
in a 70, 85, 95, and 100% ethanol series at −20°C for 3 min each.
The probe mixture contained 50% formamide (v/v), 10% dextran sulfate (w/v), 5 ng/µl salmon sperm DNA, and 500 ng/ml of
each of the probe DNAs. The probe mixture was heated to 90°C
for 10 min and kept on ice for 5 min. Seventy microliters of the
probe mixture were applied to each denatured preparation and
covered with a glass cover-slip. The slides were then placed in a
humid chamber at 37°C for 18 h. After hybridization, the cover-
Dal-Hoe Koo et al.
slips were removed in 2× SSC. The slides were then washed in
2× SSC for 5 min, 50% formamide for 10 min at 37°C, 2× SSC
for 5 min, and 4× SSC/0.2% tween-20 for 5 min. They were
then incubated 10 min at room temperature in 600 µl of 5%
(w/v) BSA in 4× SSC/0.2% Tween-20 (blocking solution). The
cover-slips were removed and the blocking solution was drained.
The slides were covered with a 70 µl fluorescence staining mixture that was composed of rhodamine-conjugated antidigoxigenin and fluorescein isothiocyanate (FITC)-con-jugated avidin
(Boehringer Mannheim) that was dissolved in 5% BSA/4×
SSC/0.2% Tween-20 as a detection buffer. The slides were incubated at 37°C for 30 min. After fluorescence staining, a posthybridization washing was carried out to remove the unhybridized and nonspecifically bound probe. The slides were washed
in 4× SSC /0.2% Tween-20 three times for 10 min each at 37°C.
They were then incubated in the dark for 40 min in a McIlvaine’s buffer (aqueous 17.7 mM citric acid, 164.7 mM
Na2HPO4, pH 7.0) that contained 1 µg/µl DAPI (Sigma). The
slides were washed in distilled water and 20 µl of Vectashield
(Vector Laboratories) was added on each slide, then 24 × 32 mm
cover-slip was added. Cover-slips were sealed onto the slides
with fingernail polish. The signals were detected with a conforcal microscope (Carl Zeiss, LSM 510). Just DAPI counterstained image was taken with a Cooled CCD camera. The final
printed images were prepared with Adobe photoshop 6.0 programs.
415
Table 1. Analyses of somatic metaphase chromosomes of C.
sativus L.
Chromosome size (µm)
Chromosome
No.
1
2
3
4
5
6
7
Long
arm
Short
arm
Total
length
1.90
1.75
1.30
1.30
1.00
1.00
1.05
1.50
1.25
1.20
0.95
0.90
0.80
0.60
3.40
3.00
2.50
2.25
1.90
1.80
1.65
Arm
Centromeric
ratio
Index
(L/S)
1.27
1.40
1.08
1.37
1.11
1.25
1.75
M
M
M
M
M
M
SM
M, metacentric; SM, submetacentric.
A
B
Results and Discussion
Karyotype The chromosome complement of Cucumis
sativus L. cv. Winter Long by Fuelgen staining is shown
in Fig. 1A. The somatic chromosome number is 2n = 2x =
14. The length of the mitotic metaphase chromosomes
ranges from 1.65 to 3.40 µm with a total length of 33.00
µm. Both the values of the arm ratio and the total length
confer enough information to each chromosome pair (Table 1). The homologous chromosome complement is
composed of six metacentrics (chromosomes 1 to 6) and
one submetacentric (chromosome 7). Results of the cytogenetic studies on cucumbers have been inconsistent, depending on the researchers. Trivedi and Roy (1970) reported the karyotype of 14 submedian, while Ramachandran and Seshadri (1986) insisted that the haploid
complement is comprised of two median, four submedian,
and one subterminal chromosome. Hoshi et al. (1998)
reported five metacentric and two submetacentric chromosomes. However, Chen et al. (1998) described the haploid chromosome complement of cucumbers as six metacentrics and one submetacentric. This agreed with our
data. Different numbers of the secondary constrictions in
cucumber chromosomes have been reported and discussed.
Ayyangar (1967) found five pairs of chromosomes with
secondary constrictions in cucumbers, while Bhaduri and
Bose (1947) found four pairs. On the other hand,
Fig. 1. Somatic metaphase chromosome complement (2n = 14)
and the karyotype (A), and C-banded somatic metaphase chromosomes (B) of C. sativus. Bars = 5 µm.
Ramachandran and Seshadri (1986) described three pairs
of nucleolar organizers in the somatic chromosome as
well as in the pachytene chromosome complements. In
this study, the number of secondary constrictions were not
reliably observed with conventional staining. Our results
agreed with the results of Hoshi et al. (1998). Hoshi reported that different numbers of secondary constrictions
were counted in each metaphase cell because the chromosomes were too small.
C-banding All of the mitotic metaphase chromosomes
have clearly visible C-bands, especially the chromosome
pairs that were stained at the centromeric regions (Fig.
1B). Telomeric bands were observed on both arms of
chromosomes 1, 2, 3, 4, 5, and 7; whereas, they were observed only on the short arm of chromosome 6. In addition, chromosome 1 revealed intercalary bands only in its
short arm. A distinct C-banding marker was found on the
long arm of chromosome 2, where the entire region of the
long arm was heavily stained. However, this finding is
different from previous reports, which suggest that C-
416
Karyotype Analysis of Cucumber Using C-banding and FISH
Fig. 2. Somatic metaphase chromosome complements of C.
sativus counter-stained with DAPI. This represents the Cbanding-like patterns with major heterochromatin bands that
usually exhibit more intense fluorescence on the chromosomes 1
(B) and 2 ( → ).
bands were observed in the pericetromeric region (Hoshi
et al., 1998) and at both the centromeric and telomeric
regions (Chen et al., 1998). The reason why different cytogenetic results were observed in the cucumber varieties
may be due to the plant materials. This can be supported
by previous findings on the polymorphism of C-banding
disclosed varieties and cultivars in various plants, such as
barley (Kakeda et al., 1991; Linde-Saursen, 1978), onion
(Vosa, 1976), and rice (Hamoud et al., 1991). In addition,
the differences may come from the different methodology,
such as staining or growing conditions, slide preparation,
and C-banding technique (Chen et al., 1998; Hoshi et al.,
1998). The slide-preparation step is particularly critical in
the C-banding procedure. Our slide preparation, using
enzymatic maceration and air-drying, is a more effective
method for obtaining many qualitative metaphase chromosomes on a glass slide than the squash method (Kakeda
et al., 1991). We found that our C-banding pattern can be
confirmed by counter-staining with DAPI. The cucumber
chromosome, which was counter-stained with DAPI,
showed a C-banding-like pattern (Fig. 2). The heterochromatin bands were usually represented by more intense
fluorescence, particularly in chromosomes 1 and 2.
In situ hybridization A detailed morphological analysis
of cucumber chromosomes was difficult because of its
small size in chromosomes, poor staining, and the short
length of the satellite. Determination of the number of
secondary constrictions was also difficult, even though
well-spread mitotic metaphase chromosome preparations
were obtained. To solve this limitation, bicolor FISH,
using two different rDNA gene probes, was applied (5S
and 45S rDNAs). The signals for 45S rDNA were detected in the pericentromeric regions of chromosomes 1, 2,
and 4. The signal for 5S rDNA was detected in the short
arm of chromosome 5 (Fig. 3). The 45S rDNA multigene
families are located in a chromosomal nucleolus organizing region (NORs), which is cytologically visible
Fig. 3. Bicolor FISH pattern on the metaphase chromosomes of
C. sativus L. using both 5S and 45S rDNA probes. Digoxigenen-labeled 5S rDNA probe was detected with antidigoxigenin rhodamine conjugate (red). Biotin-labeled 45S rDNA
probe was detected with avidin-FITC conjugate (green).
Fig. 4. Ideogram of karyotype showing the physical location of
the 5S rDNA, 45S rDNA signals, and C-banded regions.
as a secondary constriction that is associated with a nucleolus and distal satellite. The FISH signals of 45S
rDNA were detected in the pericentromeric regions of
chromosomes 1, 2, and 4, which agrees with the previous
report (Hoshi et al., 1999). Chen et al. (1999) reported
four pairs of signals for 45S rDNA. This discrepancy may
be due to variability in rDNA loci, as found in the genus
Oryza (Fukui et al., 1994). They reported that varieties in
temperate regions have one rDNA locus, while those in
tropical and subtropical regions have two rDNA loci. It
appears that there has been selection pressure to reduce
the number of rDNA loci under adverse conditions, such
as the low temperature that is prevalent in temperate areas.
Since the 5S rDNA gene family consists of many repeating units, the probe preparation for detection by FISH was
very convenient. One 5S rDNA locus was detected in the
proximal region of the short arm of chromosome 5. FISH
patterns of 45S rDNA were different, depending on the
species, as follows: two-six in Arabidopsis and Brassica
Dal-Hoe Koo et al.
species (Maluszynska and Heslop-Harrison, 1993a;
1993b), two-four in Cucumis species (Chen et al., 1999),
one-two in Lathyrus species (Ali et al., 2000), and twofour in Sorghum species (Sang and Liang, 2000). In addition, the 5S rDNA patterns also showed a polymorphism
as follows: two pairs in Helianthus annuus (Schrader et
al., 1997), one pair in Capsicum species (Park et al.,
2000), and one-three pairs in Lathyrus species (Ali et al.,
2000). A FISH karyotype is a prerequisite for physical
mapping of the molecular markers on cucumber chromosomes. The illustrated cytogenetic markers in this study
(C-bands, 45S and 5S rDNAs) are highly valuable for
chromosome identification (Fig. 4). Our results demonstrate that simultaneous bicolor FISH in combination with
DAPI counter-staining is a powerful method, not only for
the physical mapping of DNA sequences, but also for
providing chromosomal landmarks and refined banded
karyotype on cucumber chromosomes.
Acknowledgments This work was supported by a grant from
the Korea Research Foundation (KRF-2000-DP0401: PI, YH) to
JWB.
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