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Karyomorphological Analysis of Wild Chrysanthemum boreale Collected from

Four Natural Habitats in Korea

Article · December 2013

DOI: 10.11623/frj.2013.21.4.34

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Flower Res. J. (2013) 21(4):182-189 ISSN 1225-5009(Print)
DOI http://dx.doi.org/10.11623/frj.2013.21.4.34 ISSN 2287-772X(Online)

O RIG IN AL AR TIC LE

Karyomorphological Analysis of Wild Chrysanthemum boreale

Collected from Four Natural Habitats in Korea
Yoon-Jung Hwang1, Adnan Younis2,3, Kwang Bok Ryu2, Ki-Byung Lim2, Chang-Ho Eun4, Jungho Lee5,
Seong-Han Sohn4, and Soo-Jin Kwon4*
1
Department of Life Science, Sahmyook University, Seoul 139-742, Korea
2
Department of Horticultural Science, Kyungpook National University, Daegu 702-701, Korea
3
Institute of Horticultural Sciences, University of Agriculture, Faisalabad, 38040, Pakistan
4
Dep. of Agricultural Biotechnology, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea
5
Green Plant Institute, Suwon 441-853, Korea

Received 10 November 2013; Revised 29 November 2013; Accepted 24 December 2013

© The Korean Society for Floricultural Science 2013

Abstract Chrysanthemum is one of the most popular and Introduction

economically important ornamental plants due to its huge
diversity in growing habits, wide range of colors, and different
patterns of flower. In the present study, we conducted the
karyotype analysis in four naturally occurring genotypes of
Chrysanthemum boreale. Karyotype studies based on the
fluorescence in situ hybridization (FISH) using 5S and
45S rDNAs. Four chrysanthemum genoteyps showed an
aneuploid chromosome number of 2n = 18+2 (111016 and
111021) or a diploid of 2n=18 (121001 and 121002). All
the genoteyps had the same karyotype formula of 14
metacentrics and 4 submetacentrics. In 111016, the
chromosome length during somatic metaphase ranged from
3.11 ± 0.26 µm (shortest) to 3.94 ± 0.20 µm (longest), with a
total length of 32.94 µm. The chromosome length at the
mitotic metaphase ranged from 3.11 to 6.46 µm, with a total
length of 32.94 µm in 111016 and 51.05, 32.81, and
46.00 µm in 111021, 121001, and 121002, respectively. The
5S rDNA and 45S rDNA signals recorded different in all four
wildly grown genotypes of C. boreale. This information can
be useful in cultivar improvement, as well as in elucidation
of the evolution of the chrysanthemum.
Additional key words: Asteraceae, cytogenetics, FISH flow
cytometry, ploidy level, rDNA
*Corresponding author: Soo-Jin kwon
Tel: 031-299-1949
E-mail: sjkwon67@korea.kr
www.ijfs.org
Chrysanthemums (Asteraceae) are important ornamental
plants that are well known for its commercial valuable cultivars
for cut flower production, potted plant, and garden flowering
plant (Bhattacharya and Teixerra 2006; Maoxue et al. 1983).
Chrysanthemum genus comprises about 41 species that are
mostly well-distributed in East Asia and the species diversity
is thought to be centered in China (Bremer and Humphries
1993; Oberprieler et al. 2006).
Chrysanthemum is considered as a hybrid cultigen complex
due clear evidence of artificial selection and inter-specific
hybridization in its origin process (Dai et al. 2002). The
morphology and ploidy level are quite variable among species
in this genus such as C. rhombifolium is diploid (2n= 2x=18),
C. hypargyrum is tetraploid (2n=4x=36) while C. vestitum is
hexaploids (2n=6x =54) (Chen et al. 2008; Dowrick 1953; Li
et al. 2013). The occurrence of polyploid genotypes has been
attributed to the prospective of polyploids species to grow
under variable habitats and survives better under unfavorable
climatic conditions (Abd El-Twab and Kondo 2006a). Infra-
species and even sometimes within population differences
in ploidy level have been observed (Yang et al. 2006).
Moreover, in Chrysanthemum many species are narrowly
distributed and some of them are habitat specific (Kim et al
2003; Kondo et al 2003; Tsukaya 2002). This reflects that
speciation in Chrysanthemum is related with habitat
adaptation, hybridization, and polyploidy. The changes in
chromosome and genome structure during the species
evolution can affect the botanical and morphological
characteristics of plants which fascinate the plant researchers
to explore the genome evolution (Heslop-Harrison 2000). The
Flower Res. J. (2013) 21(4):182-189 183

evolutionary pattern of Chrysanthemum has not been well Table 1. Information of geographical location in Korea where samples
of wild C. boreale plants were collected.
explained although many researchers reported it their findings
(Abd El-Twab and Kondo 2006b; Li and Shao 1990; Zhao et Genotype Location GPSz Elevation (m)
al. 2010). Jangseong-gun, N 35° 25' 00''
111016 95
Jeollanam-do E126° 50' 00''
In chrysanthemum cytogenetic studies were carried out
Jangseong-gun, N 35° 25' 00''
by several scientists (Li et al. 2011; Chang et al. 2009) and 111021 95
Jeollanam-do E126° 50' 00''
they observed that chromosome counts for large flowered
Jeongeup-si, N 35° 29' 00''
chrysanthemum ranging from 52-75 + B (Endo and Inada 121001 98
Jeollanam-do E 126° 48' 00''
1992) and most of the cultivars having metacentric or Jeongeup-si, N 35° 29' 00''
121002 98
sub-metacentric chromosomes (Chen et al. 2003; Li et al. Jeollanam-do E 126° 48' 00''
2008). Studies on Chinese and Japanese chrysanthemum z
Global positioning system.
species were conducted to understand the evolutionary
mechanism and cytogenetic process (Abd El-Twab and locations in Korea to construct the physical map to identify the
Kondo 2003; Tanaka et al. 1989). location of 5S and 45S rDNA and chromosomal position.
In higher plants, rDNAs are arranged in two different
gene groups of major rDNA clusters that encode 45S rDNA, Materials and Methods
and minor rDNA clusters that encode 5S rDNA. It is reported
that the minor rDNA are present in loci usually and they are Planting materials
separated from major rDNAs. It is also interesting to know that Four wildly grown genotypes of C. boreale were collected
the minor rDNA has no involvement in the formation of from two locations in Korea such as, 111016 and 111021 from
nucleolus (Inafuku et al. 2000). Conventional staining Jangseong-gun, Jeollanam-do, whereas, 121001 and 121002
approaches limitation reveals chromosome size, site of from Jeongeup-si, Jeollabuk-do, in year 2012. Detailed
centromere and secondary constrictions existence or absence geographical information is presented in Table 1. Collected
and it was difficult to elucidate individual chromosome of germplasm were grown in greenhouse at 25 ± 2oC at NAAS,
same size, form, and morphology (Song 1987). Fluorescence RDA, and Kyungpook National University, Daegu, Korea.
in situ hybridization (FISH), a molecular cytogenetic approach
is employed for detecting specific sequences of chromosomes Chromosome preparation
using ribosomal DNAs to detect nucleolar organizing regions Root tips of actively growing cymbidium plants were
(NORs) (Gasparini and Malazzi 2006; Harrison and Heslop- pre-treated with 2 mM 8-hydroxyquinoline for 5 hrs at 20oC.
Harrison 1991). This method provides high resolution after Fixation was carried out in aceto-ethanol solution (v/v, 1:3) for
detecting sequences of DNA and their copy number at 2 hrs at room temperature. The material was kept at -20oC in
different positions on chromosome to understand species 70% ethanol solution prior to use. For chromosome study, the
divergence and to monitor the evolutionary variations in root tips were washed thoroughly with distilled water and then
physical organization of any genome (Harrison and treated with a mixture of enzymes (0.3% cellulase, 0.3%
Heslop-Harrison, 1995). This technique was successfully cytohelicase, 0.3% pectolyase in 150 mM of citrate buffer) for
used to identify the localization of chromosomes of 5S 1 hr at 37oC. Then root tips were squashed in a drop of 60%
and 45S rDNA in several species of Chrysanthemum (Abd acetic acid and followed by air drying.
El-Twab and Kondo 2007a; Abd El-Twab and Kondo 2008;
Khaung et al. 1997). In 1996, Kondo with other researchers Flow cytometric analysis
conducted molecular cytogenetic analysis of Japanese 20 mg of root tips per plantlet were sliced with a sharp
chrysanthemum and they determine the relationship among blade in a petri dish having 500 mL of nuclei extraction
species (Kondo et al. 1996). ice-cool buffer (Partech, GmbH, Müster, Germany) to get a
In present study, Chrysanthemum boreale (syn. Dendranthema fine suspension (Lim et al. 2001). The sample was strained
boreale) a perennial flowering plant that grows naturally in through a nylon mesh (30 µm). Then, 2.5 mL of staining
Korea showed variation in morphological characteristics in buffer (Partech, GmbH, Müster, Germany) was added and the
different locations, therefore it was necessary to detect the suspension was added with staining buffer subsequently, ploidy
specific rearrangement of chromosomes and to examine level from each sample was recorded using a flow cytometer
the genome changes within species. We conducted FISH (CyFlow, ploidy analyzer, Partech, GmbH, Müster, Germany).
analysis of four genotypes of C. boreale collected from two Leaf tissue from the R. sativus; genome size=554.2 Mbp
184 Flower Res. J. (2013) 21(4):182-189

Table 2. The flower characteristics of C. boreale collected from wild habitats in Korea.
Ray flower Disk flower Pedicel
Genotype Length Width Length Color Diameter Color Number Pollen color
Number Length
(mm) (mm) /Width (RHSz) (mm) (RHS) of branch (RHS)
111016 11.05 ± 0.27 1.94 ± 0.05 5.7 4A 18.4 6.10 ± 0.30 145B 29.20 ± 0.66 4.9 12A
111021 09.14 ± 0.32 2.00 ± 0.05 4.6 4A 15.7 5.26 ± 0.26 145B 27.04 ± 1.17 4.1 12A
121001 07.62 ± 0.19 2.47 ± 0.08 3.1 4A 17.6 6.50 ± 0.19 145B 24.09 ± 1.85 6.1 12A
121002 07.41 ± 0.26 2.15 ± 0.02 3.4 4A 19.7 6.95 ± 0.20 145B 23.38 ± 1.07 5.8 12A
z
RHS denotes Royal Horticultural Society color chart.

was used as a diploid reference to adjust the flow cytometer.

All the experiments were replicated for three times. Data
were presented as histograms to indicate the relative DNA
contents of each sample. The peak area in the histogram
is showing the number of nuclei for each ploidy level.

Fluorescence in situ hybridization (FISH)

FISH was carried out as adopted by Lim et al. (2007). The
slides were pretreated with RNase A in 2x SSC
(100 µL • mL-1, DNase-free) for 1 hr at 37°C. The slides were
washed in 2x SSC three times and then post-fixed in
paraformaldehyde solution (4%) for 10 min. 5S and 45S rDNA
were tagged with digoxygenin-11-dUTP and biotin-16-dUTP
by nick translation mix (Roche, Mannheim, Germany),
respectively. The hybridization mixture containing deionized
formamide (50%), dextran sulfate (10%), 2x SSC and
Fig. 1. Phenotypic characteristics of Chrysanthemum boreale plants
collected from four natural habitats in Korea A. 111016 with joint-lobed
leaf margin (left) and large flower size with cushion-like dick (right); B.
I111021 with separate-lobed leaf margin (left) and medium flower size
with open dick florets (right); C. 121001 with light green color leaf, with
separate lobed margins (left) and medium compact flower with
broad petals (right); D. 121002 with leaf with separate-lobed margins
(left) and small compact flower with short petals (right). Bar=1 cm.

20 µg • mL-1 of probe DNA was de-natured for 10 min at 70oC.

The hybridization mixture was then transferred to slides and
covered with cover slips. Each slide was de-natured for 5 min
at 80oC and incubated in a humid chamber for 1 hr at 37oC.
After hybridization step, each slide was washed with 0.1x
SSC for 30 min at 42oC, followed by detection of biotin and
digoxygenin by using fluorescein iso-thiocyanate FITC-
conjugated anti-digoxygenin antibody (Roche, Mannheim,
Germany) and streptavidin Cy3 (Zymed Laboratoties Inc.,
California, USA). The chromosomes were counter-stained
with 2 µL • mL-1 of 4’, 6-diamidino-2-phenylindole (DAPI) in
vecta-shield (Vector Laboratories Inc., California, USA) and
observed under the fluorescent microscope (Nikon BX 61,
Newyork, USA). Images were taken through charge coupled
device (CCD) and then images processing were done
through the Genus FISH imaging system (Applied Imaging
Corporation, Genus version 3.8 program, USA). Confirmation
of putative homologous chromosomes was done on the
basis of their morphological characteristics and FISH results.
Karyotype analysis
Minimum 10 cells with metaphase spreads chromosomes
were used for karyotype analysis. The length of individual
chromosome was measured through computer software and
chromosome numbers were determined on the basis of short
arm length order. Chromosome types were then observed
based on arm ratio value (Levan et al. 1964).
Results and Discussion
Morphological analysis of four genotypes collected from two
different locations was carried out in this study. From the
results it was revealed that there were variations in leaves and
flowering characteristics of all four genotypes collected from
two separate locations (Table 2). Phenotypic characteristics of
one of the genotypes (111016) showed dark green color leaf
with joint lobed leaf margin having large flower size with
cushion like dick compared with other genotypes under
study (Fig. 1). Maximum 11.05 mm length of ray floret was
recorded in plant of (111016) with 6.10 mm diameter of disk
florets (Table 2). The plants of 111021 have separate lobed
leaf margin with medium sized flowers (Fig. 1) having open
dick florets of 5.26 mm diameter. Whereas, plants of 121002
Flower Res. J. (2013) 21(4):182-189 185

have leaves with separate lobed margins and possessed signals while red fluorescence indicating the 45S rDNA
small compact flower with short petals (7.41 mm). It was signals (Fig. 3). One pair of 5S rDNA was detected near to
found that environmental conditions of the habitats greatly centromere region in long arm chromosome (Fig. 3B and
influence the diversity of the species and as well as 3C), while no signal of 5S rDNA displayed in 111016 and
morphological characteristic of a particular species (Rodnikova 121002. This may be due to the fact that naturally many
2012). Physiological and morphological characteristics in closely related plants can cross to evolve new hybrids and
plants are interrelated with adaptive effects to environmental polyploidy cultivars, which also attributed to perform its
conditions (Dyer et al. 2006) and plants from more productive important role in chromosome evolution in different species.
habitats having more conducive environmental conditions 5S rDNA signals were observed major 2 pairs and minor 1
often have faster growth rates and it results into vigorous plant pair in all materials. All of 45S rDNA signals located in
health with quality flowers (Grime et al. 1997). These signicant terminal region of short arm chromosome.
variations in different morphological characteristics among
different populations might be due to differences in habitats,
and this phenotypic plasticity facilitate any plant to alter its
growing pattern as it comes under different stresses (Guo et
al. 2007; Jugrana et al. 2013).
Four chrys-anthemum materials showed aneuploid
chromosome number of 2n=18+2 (111016 and 111021) or
diploid of 2n=18 (121001 and 121002). All materials had same
karyotype formula, 14 metacentrics and 4 submetacentrics. In
111016, the chromosomes length during somatic metaphase
ranges from 3.11 ± 0.26 µm (shortest) to 3.94 ± 0.20 µm
(longest), with a total length of 32.94 µm. In case of 111021
the chromosomes length ranges from 4.63 ± 0.19 µm to
6.46 ± 0.30 µm, with a total of 51.05 µm. Plants of C. boreale Fig. 2. Fluorescence in situ hybridization using 5S rDNA (green flu-
orescence) and 45S rDNA (red fluorescence) as probes in four of
collected from Jeongeup-si, Jeollabuk-do, Korea were diploid Korean native C. boreale. A, 111016; B, 111021; C, 121001; D, 121002
and the chromosomes length in genotype 121001 ranges from (size bar=10 µm).

3.21 ± 0.27 µm to 3.70 ± 0.07 µm, with a total length of

32.81 µm having two unpaired chromosomes of 3.28 µm and
3.17 µm in each chromosome length, respectively (Table 3).
In genotype 121002, total length of 46 µm was recorded with
no unpaired chromosomes. Some chromosomes were difficult
to classify from its homologous counterpart due to resemblance
in the short arm length of chromosomes. However all identified
chromosomes had variable morphological characteristics
having different short arm and long arm lengths.
Results regarding FISH using 5S rDNA and 45S rDNA as
probes in four of Korean native C. boreale are presented in Fig. 3. FISH karyotypes of four Korean native C. boreale. A, 111016; B.
Fig. 2 and 3. Green fluorescence is showing 5S rDNA 111021; C, 121001; D, 121002.

Fig. 4. Histograms of DNA content obtained after analyses of nuclei isolated from leaf tissue of four Korean native C. boreale. A, 111016; B,
111021; C, 121001; D, 121002.
186 Flower Res. J. (2013) 21(4):182-189

Table 3. Cytogenetic characteristics of C. boreale habitats in Korea.

Genotype Chr. no. Short arm (µm) Long arm (µm) Total (µm) Karyotype formula
a 1.81 ± 0.01 2.13 ± 0.19 3.94 ± 0.20
b 1.89 ± 0.01 1.99 ± 0.01 3.88 ± 0.03
c 1.76 ± 0.18 2.12 ± 0.17 3.88 ± 0.01
d 1.61 ± 0.05 2.22 ± 0.12 3.83 ± 0.07
111016 e 1.64 ± 0.04 2.17 ± 0.04 3.81 ± 0.01 14 mz + 4 smy
f 1.77 ± 0.01 1.93 ± 0.06 3.70 ± 0.05
g 1.53 ± 0.07 2.05 ± 0.25 3.58 ± 0.17
h 1.08 ± 0.12 2.12 ± 0.10 3.21 ± 0.02
i 1.10 ± 0.28 2.01 ± 0.02 3.11 ± 0.26
total length 14.19 18.74 32.94
a 2.93 ± 0.08 3.58 ± 0.23 6.46 ± 0.30
b 2.91 ± 0.18 3.13 ± 0.22 6.04 ± 0.39
c 2.44 ± 0.15 3.51 ± 0.34 5.95 ± 0.20
d 2.84 ± 0.11 3.08 ± 0.01 5.92 ± 0.12
111021 e 2.69 ± 0.01 3.15 ± 0.04 5.84 ± 0.05 14 m + 4 sm
f 2.55 ± 0.07 3.26 ± 0.09 5.81 ± 0.02
g 2.23 ± 0.12 3.49 ± 0.07 5.72 ± 0.19
h 1.70 ± 0.08 2.98 ± 0.18 4.68 ± 0.26
i 1.30 ± 0.02 3.33 ± 0.07 4.63 ± 0.19
total length 21.59 29.51 51.05
a 1.63 ± 0.12 2.07 ± 0.05 3.70 ± 0.07
b 1.57 ± 0.08 1.96 ± 0.03 3.54 ± 0.06
c 1.47 ± 0.06 1.89 ± 0.14 3.35 ± 0.07
d 1.37 ± 0.07 1.98 ± 0.05 3.35 ± 0.12
e 1.38 ± 0.08 1.84 ± 0.01 3.22 ± 0.09
121001 14 m + 4 sm
f 1.56 ± 0.10 1.66 ± 0.08 3.22 ± 0.19
g 0.82 ± 0.13 1.95 ± 0.12 2.76 ± 0.01
h 1.28 ± 0.23 1.92 ± 0.04 3.21 ± 0.27
i-Ix 1.53 1.75 3.28
w
i-II 1.09 2.09 3.17
total length 13.7 19.11 32.81
a 2.48 ± 0.10 3.08 ± 0.21 5.56 ± 0.11
b 2.68 ± 0.02 2.84 ± 0.04 5.51 ± 0.06
c 2.26 ± 0.12 3.22 ± 0.03 5.48 ± 0.15
d 2.04 ± 0.09 3.30 ± 0.27 5.34 ± 0.18
e 2.48 ± 0.02 2.82 ± 0.01 5.31 ± 0.03 14 m + 4 sm
121002
f 2.06 ± 0.05 2.96 ± 0.34 5.02 ± 0.09
g 2.07 ± 0.04 2.81 ± 0.12 4.87 ± 0.16
h 1.58 ± 0.17 2.92 ± 0.12 4.50 ± 0.29
i 1.16 ± 0.04 3.24 ± 0.08 4.41 ± 0.13
total length 18.81 27.19 46.00
z
m; metacentric.
y
sm; submetacentric.
x
i-I and wi-II; chromosome composition of chromosome i in 121001 observed different centromere position.
Flower Res. J. (2013) 21(4):182-189
Table 4. Karyomorphological data of four genotypes of C. boreale.
Genotype 2n Ploidy
111016 2x=18+2 Aneuploid
111021 2x=18+2 Aneuploid
121001 2x=18 Diploid
121002 2x=18 Diploid
Compared with the basic chromosomes number (2n=18),
estimation of chromosomes count and ploidy were carried out
in four genotypes of C. boreale (Table 4). Present study
results showed that among four wild genotypes, two were
aneuplouid (2n=2x=18+2) and the remaining two were diploid
(2n =2x =18). It is important to note that plants (111016 and
111021) collected from Jangseong-gun showed aneuploidy
behavior whereas, 121001 and 121002 which were collected
from Jeongeup-si, Jeollabuk-do were diploid in nature. The
nuclear contents and genome size of four genotypes of
chrysanthemum are presented in Table 4. The 1C nuclear
contents were ranged from ~ 3.96 to ~ 5.49 pg in selected
chrysanthemum genotypes whereas, the genome size ranged
from 1,938 to 2,684 Mbp (Table 4). DNA contents of nuclei
isolated from the root tips of chrysanthemum wild genotypes
were compared with the standard genotype R. sativus
(genome size=554.2 Mbp) and results are presented in
histograms (Fig. 4).
In this study, two genotypes appeared as aneuploidy, while
in a previous report showed that most of the Japanese
chrysanthemum cultivars were euhexaploid (Endo 1969) but
American genotypes were also aneuploidy (Dowrick 1953). It
was well-established that rDNA’s loci can change their relative
position on chromosome (Dubcovsky and Dvorak 1995), this
phenomenon was also observed in chrysanthemum hybrids
(Abd El-Twab and Kondo 2007b). Moreover, the rDNA loci
have the ability to exchange its genetic makeup with rest of
the genome (Dubcovsky and Dvorak 1995; Lim et al. 2004).
Conclusions
Present results provide useful information about organization
and physical location of 5S rDNA and 45S rDNA gene loci
and their multiple copies which can be used for constructing
chromosomes physical maps which proved a valuable
method for identification of genome and it will open new
breeding avenues in Chrysanthemum cultivar improvement.
Further, karyotype analysis offers important information to
construct physical maps of plant species and also for plant
classication (Maria et al. 2010). The data from karyotype
187
Total chromosome Putative genome
length (µm) Size (pg) Mbp
32.94 3.96 1,938
51.05 4.69 2,297
32.81 5.49 2,684
46.00 4.41 2,156
studies has the potential to identify the heterogeneity and
to indicate genetic relationships of plants (Diao 2004).
Chrysanthemum genome generally contains chromosome
pairs with variable morphology and allopolyploidization has an
important role in genesis of chrysanthemum (Dai et al. 2005).
Present study results indicated that karyotype characteristics
are comparatively more stable in chrysanthemum genome
and possess a divergent significance for cytogenetic studies.
Acknowledgments
This study was carried out with the support of “Research
Program for Dept. of Agricultural Biotechnology (Project No.
PJ008725)”, National Academy of Agricultural Science, Rural
Development Administration, Republic of Korea.
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