0051-01-0021-0004-6.pdf1.75MB

Introduction

 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). Infraspecies 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 evolutionary pattern of Chrysanthemum has not been well explained although many researchers reported it their findings (Abd El-Twab and Kondo 2006b; Li and Shao 1990; Zhao et al. 2010).

 In chrysanthemum cytogenetic studies were carried out by several scientists (Li et al. 2011; Chang et al. 2009) and they observed that chromosome counts for large flowered chrysanthemum ranging from 52-75 + B (Endo and Inada 1992) and most of the cultivars having metacentric or sub-metacentric chromosomes (Chen et al. 2003; Li et al. 2008). Studies on Chinese and Japanese chrysanthemum species were conducted to understand the evolutionary mechanism and cytogenetic process (Abd El-Twab and Kondo 2003; Tanaka et al. 1989). 

 In higher plants, rDNAs are arranged in two different gene groups of major rDNA clusters that encode 45S rDNA, and minor rDNA clusters that encode 5S rDNA. It is reported that the minor rDNA are present in loci usually and they are separated from major rDNAs. It is also interesting to know that the minor rDNA has no involvement in the formation of nucleolus (Inafuku et al. 2000). Conventional staining approaches limitation reveals chromosome size, site of centromere and secondary constrictions existence or absence and it was difficult to elucidate individual chromosome of same size, form, and morphology (Song 1987). Fluorescence in situ hybridization (FISH), a molecular cytogenetic approach is employed for detecting specific sequences of chromosomes using ribosomal DNAs to detect nucleolar organizing regions (NORs) (Gasparini and Malazzi 2006; Harrison and Heslop- Harrison 1991). This method provides high resolution after detecting sequences of DNA and their copy number at different positions on chromosome to understand species divergence and to monitor the evolutionary variations in physical organization of any genome (Harrison and Heslop-Harrison, 1995). This technique was successfully used to identify the localization of chromosomes of 5S and 45S rDNA in several species of Chrysanthemum (Abd El-Twab and Kondo 2007a; Abd El-Twab and Kondo 2008; Khaung et al. 1997). In 1996, Kondo with other researchers conducted molecular cytogenetic analysis of Japanese chrysanthemum and they determine the relationship among species (Kondo et al. 1996). 

 In present study, Chrysanthemum boreale (syn. Dendranthema boreale) a perennial flowering plant that grows naturally in Korea showed variation in morphological characteristics in different locations, therefore it was necessary to detect the specific rearrangement of chromosomes and to examine the genome changes within species. We conducted FISH analysis of four genotypes of C. boreale collected from two locations in Korea to construct the physical map to identify the location of 5S and 45S rDNA and chromosomal position. 

Materials and Methods

Planting materials

 Four wildly grown genotypes of C. boreale were collected from two locations in Korea such as, 111016 and 111021 from Jangseong-gun, Jeollanam-do, whereas, 121001 and 121002 from Jeongeup-si, Jeollabuk-do, in year 2012. Detailed geographical information is presented in Table 1. Collected germplasm were grown in greenhouse at 25 ± 2℃ at NAAS, RDA, and Kyungpook National University, Daegu, Korea. 

Table 1. Information of geographical location in Korea where samples of wild C. boreale plants were collected

Chromosome preparation

 Root tips of actively growing cymbidium plants were pre-treated with 2 mM 8-hydroxyquinoline for 5 hrs at 20℃. Fixation was carried out in aceto-ethanol solution (v/v, 1:3) for 2 hrs at room temperature. The material was kept at -20℃ in 70% ethanol solution prior to use. For chromosome study, the root tips were washed thoroughly with distilled water and then treated with a mixture of enzymes (0.3% cellulase, 0.3% cytohelicase, 0.3% pectolyase in 150 mM of citrate buffer) for 1 hr at 37℃. Then root tips were squashed in a drop of 60% acetic acid and followed by air drying. 

Flow cytometric analysis

 20 mg of root tips per plantlet were sliced with a sharp blade in a petri dish having 500 mL of nuclei extraction ice-cool buffer (Partech, GmbH, Müster, Germany) to get a fine suspension (Lim et al. 2001). The sample was strained through a nylon mesh (30 μm). Then, 2.5 mL of staining buffer (Partech, GmbH, Müster, Germany) was added and the suspension was added with staining buffer subsequently, ploidy level from each sample was recorded using a flow cytometer (CyFlow, ploidy analyzer, Partech, GmbH, Müster, Germany). Leaf tissue from the R. sativus; genome size=554.2 Mbp 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 20 μg · mL^-1 of probe DNA was de-natured for 10 min at 70℃. The hybridization mixture was then transferred to slides and covered with cover slips. Each slide was de-natured for 5 min at 80℃ and incubated in a humid chamber for 1 hr at 37℃. After hybridization step, each slide was washed with 0.1x SSC for 30 min at 42℃, followed by detection of biotin and digoxygenin by using fluorescein iso-thiocyanate FITCconjugated 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 have leaves with separate lobed margins and possessed small compact flower with short petals (7.41 mm). It was found that environmental conditions of the habitats greatly influence the diversity of the species and as well as morphological characteristic of a particular species (Rodnikova 2012). Physiological and morphological characteristics in plants are interrelated with adaptive effects to environmental conditions (Dyer et al. 2006) and plants from more productive habitats having more conducive environmental conditions often have faster growth rates and it results into vigorous plant health with quality flowers (Grime et al. 1997). These signicant 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).

Table 2. The flower characteristics of C. boreale collected from wild habitats in Korea.

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.

 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 collected from Jeongeup-si, Jeollabuk-do, Korea were diploid and the chromosomes length in genotype 121001 ranges from 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. 

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

 Results regarding FISH using 5S rDNA and 45S rDNA as probes in four of Korean native C. boreale are presented in Fig. 2 and 3. Green fluorescence is showing 5S rDNA signals while red fluorescence indicating the 45S rDNA signals (Fig. 3). One pair of 5S rDNA was detected near to centromere region in long arm chromosome (Fig. 3B and 3C), while no signal of 5S rDNA displayed in 111016 and 121002. This may be due to the fact that naturally many closely related plants can cross to evolve new hybrids and polyploidy cultivars, which also attributed to perform its important role in chromosome evolution in different species. 5S rDNA signals were observed major 2 pairs and minor 1 pair in all materials. All of 45S rDNA signals located in terminal region of short arm chromosome. 

Fig. 2. Fluorescence in situ hybridization using 5S rDNA (green fluorescence) and 45S rDNA (red fluorescence) as probes in four of Korean native C. boreale. A, 111016; B, 111021; C, 121001; D, 121002 (size bar=10 μm).

Fig. 3. FISH karyotypes of four Korean native C. boreale. A, 111016; B. 111021; C, 121001; D, 121002.

 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).

Table 4. Karyomorphological data of four genotypes of C. boreale.

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.

 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 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.