Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.27 No.4 pp.242-253
DOI : https://doi.org/10.11623/frj.2019.27.4.02

Cytogenetic Studies of Chrysanthemum: A Review

Wang Yan1, Jae A Jung2, Ki-Byung Lim3, Raisa Aone M. Cabahug4, Yoon-Jung Hwang1*
1Department of Convergence Science, Sahmyook University, Seoul, 01795, Korea
2Floriculture Research Division, National Institute of Horticultural & Herbal Science, Wanju, 55365, Korea
3Department of Horticultural Science, Kyungpook National University, Daegu, 41566, Korea
4Chromosome Research Institute, Sahmyook University, Seoul, 01795, Korea
Corresponding author: Yoon-Jung Hwang Tel: +82-2-3399-1718 E-mail: hyj@syu.ac.kr
04/10/2019 18/11/2019 20/11/2019

Abstract


Chrysanthemum is a valuable ornamental plant worldwide, and several of its species are used as herbal tea, medicinal plants, and dietary supplements, among others. Commercial cultivars have been developed through interspecific hybridization and artificial selection to improve the characteristics, production quality, and environmental adaptation for enhancing ornamental value. To better understand the recent research in cytogenetic studies of Chrysanthemum, we examined studies concerning polyploidy, karyotyping, banding, fluorescence in situ hybridization (FISH) technique, and inter/intraspecific hybridization. Ploidy level is important in genomic characteristics and has a significant value to horticulturists and plant breeders. Studies have reported that flow cytometry analysis and single-dose molecular markers can be used to determine the Chrysanthemum ploidy level. As for karyotyping, a better understanding of karyomorphological relationships and evolution of Chrysanthemum and its closely related genera has already been gained. Moreover, karyotype parameters in Chrysanthemum studies play a critical role in cultivar identification, classification, and genetic analysis. The FISH technique in Chrysanthemum research provides more information on chromosome identification, sequences distribution, and evolution for expediting the development and improvement of plant species. The genomic in situ hybridization (GISH) technique can also be used to test hybridization in Chrysanthemum breeding. Hence, this review of molecular cytogenetic studies of Chrysanthemum will help us to have a better understanding and knowledge of the taxa breeding and the development and improvement of new cultivars.



초록


    Rural Development Administration
    PJ012804

    Introduction

    According to recent literature, Anthemideae tribe is a very large and widespread group of flowering plants consisting of 14 subtribes, 111 genera and 1800 species that are mainly inhabited in central Asia, the Mediterranean Basin, and southern Africa (Oberprieler et al. 2007;Qi et al. 2015). Many species in this tribe are important in herbal medicine and possesses desirable characteristics such as flowers varying greatly in shape, size and color, disease resistance, and other advantageous agronomical traits. These include species such as Artemisia, Chrysanthemum and Matricaria genus (El-Twab et al. 2008). The subtribes Artemisiinae, which belongs to Anthemideae, has currently 18 genera and 634 recognized species (Masuda et al. 2009;Qi et al. 2015). Chrysanthemum is a genus in this subtribe, which comprises about 41 species, and are largely distributed in East Asia, mainly in China, Korea, and Japan. China is thought to be the center of its species diversity (Hwang et al. 2013;Kim et al. 2003;Zhao et al. 2010).

    Chrysanthemum is a globally significant ornamental plant that is known for its commercial cultivars and commonly used as cut flowers, pot plants, and garden flowers (Hwang et al. 2013;Liu et al. 2012). It is the second largest species of cut flowers produced in Netherlands and United Kingdom, third in the world and the first in Japan and China, third in Germany in terms of cut flower trade (Datta and Janakiram 2015). In addition, it has been documented that several chrysanthemum species are herb medicinal plants in China (Li et al. 2011;Liu et al. 2018). Flowering head of some species is used as herbal tea and edible chrysanthemum has been reported as a dietary supplement in China and Korea and is also used as insecticide and parasiticide (Bhattacharya and da Silva 2006). In Korea, the chrysanthemum flower is also used as flavor for rice wine and is called as ‘Gukhwaju’ (Kushwah et al. 2018).

    Developing new commercial cultivars are often focused on improving the characteristics such as color, size, and shape of the flower, production quality, and adaptation to the environment to enhance the ornamental value (Rout and Das 1997). According to Wang et al. (2014), more than 20,000 cultivars have been developed with abundant morphological variation, powerful suitability, and wide distribution. Although some pseudo-self-incompatible plants have been identified, many scientists have proposed that chrysanthemum is a hybrid cultigen complex with a distinct mark of interspecific hybridization and artificial selection because of its long history of cultivation, natural selection, and artificial crossing (Khaing et al. 2013). Moreover, the characteristics of chrysanthemums are different from the wild donor species (Dai et al. 2005). In addition, many species are narrowly distributed and habitat-specific due to adaptation, hybridization, and polyploidy genotypes (Hwang et al. 2013;Liu et al. 2012).

    Because of the economic importance of the chrysanthemums, numerous articles and research studies has reported their breeding, cultivation, origins, and cytogenetics analysis (Liu et al. 2012). Several chrysanthemum species have been cytogenetically studied to develop a better understanding of their relationship and evolution progress among chrysanthemum and its related species (Table 1). Cytogenetic studies provide a powerful tool to study chromosome number and karyotyping the physical location of genes on chromosomes, and chromosomal behavior in cell division processes. It contributes very important knowledge on the patterns of genetic variation, phylogeny, taxonomy, and evolution of plants (Jara-Seguel and Urrutia 2012).

    The aim of this paper is to review the advances in cytogenetic studies of chrysanthemum, focusing on the analysis of polyploidy studies, karyotypes, banding, fluorescence in situ hybridization (FISH) technique, and inter/intraspecific hybridization.

    Polyploidy studies on chrysanthemum

    Chromosome number and ploidy level are one of the fundamental information to describe any of the species (Bourke et al. 2018). Ploidy level is defined as the number of set chromosomes in a cell and plays an important genomic characteristic in biodiversity (Xu et al. 2014). Chrysanthemum species have a complex ploidy ranging from d iploid ( 2n = 2x = 18) t o decaploid (2n = 2 x = 90) (El-Twab et al. 2008) and the polyploid genotype affects the growth of species under variable habitats (Hwang et al. 2013). A large-flowered cultivar of chrysanthemum has been reported to exhibit chromosome numbers ranging from 52 to 75 + B through cytogenetic studies, and most of these cultivars were found to be hexaploids (2n = 54) (Chang et al. 2009). Ploidy level of plants can modify the morphological, phonological, physiological and ecological character of the plants (Kushwah et al. 2018). Although the effects of the polyploidy on plant traits are significant to horticulturists and plant breeders, up until now there is still a lack of the fundamental ploidy information for most cultivars in chrysanthemum species (Guo et al. 2012) and only a few papers have been published on ploidy studies.

    Polyploidy can be induced through the use of chemical agents like colchicine (Bourke et al. 2018) for future breeding and propagation program. Studies demonstrated that using small cotton swabs soaked in aqueous colchicine solution (0.2%) placed over apical meristem of healthy young potted plants of Chrysanthemum carinatum proved to be an effective method for inducing polyploidy. Young flower buds were then used for cytological analysis to check their meiotic chromosome number. Furthermore, polyploidy is often related to some additional traits such as stronger stems, thicker leaves, larger flowers with thicker petals that tend to have longer blooming periods and postharvest life (Barlup 2002;Podwyszyńska et al. 2015).

    Traditionally, ploidy has been determined by chromosome counting on microscope slides and was proved in many different species (Ma et al. 2015;Wang et al. 2009;Xu et al. 2014). However, high level of ploidy with numerous chromosome and aneuploidy variation could cause lots of laborious work and inaccuracies in the end. Moreover, aneuploidy variation in chrysanthemum are widely present and inconsistent chromosome number could be generally observed in different root tip cells of the same individual (Guo et al. 2012). To solve these issues, studies have been reported that flow cytometry analysis could be used to identify the ploidy level of the chrysanthemum species. This technique has also been considered as a more accurate and effective method in studying the ploidy level of wild chrysanthemum species through Otto extraction buffer (Gao et al. 2012;Ma et al. 2015).

    Polyploidy level plays a significant role in plant evolution (Adams and Wendel 2005;El-Twab and Kondo 2007b) and developing desirable varieties of flower crops. However, it is difficult to classify the type of ploidy of a species because polyploidy genomes are highly dynamic (Klie et al. 2014). It is known that polyploidy is divided into two crucial categories namely: autopolyploid and allopolyploid. Allopolyploid is characterized by preferred paired chromosome or fixed heterozygosity, which is the result of a combination of dissimilative parent genomes, and disomic inheritance at each locus, whereas autopolyploid is formed by multiple chromosomes and polysomic inheritance at all chromosomes (Klie et al. 2014;van Geest et al. 2017).

    To determine whether a hexaploid chrysanthemum genome with 54 chromosomes was classified as an allopolyploid, Klie et al. (2014) combined cytological and molecular methods to identify allopolyploid from autopolyploid by using single-dose (SD) molecular marker. van Geest et al. (2017) suggested hexaploid cultivated chrysanthemum as a hexaploid with a polysomic inheritance based on conclusive evidence of 183 k SNP array through next-generation sequencing and high-throughput genotyping analysis. Bourke et al. (2018) also mentioned that most of the polyploidy genetics studies are depending on single nucleotide polymorphisms (SNPs) because of their great abundance over the genome.

    Although a variety of techniques have been designed to identify SNPs, these tools are not always suitable for polyploidy data set. Hence, we definitely need to establish appropriate methods to study polyploidy genotype and develop suitable tools to analyze SNP dosage from SNP array (van Geest et al. 2017). To develop a reliable method for investigating the type of ploidy of cultivated chrysanthemums, future studies may allow to explore this aspect.

    Karyotypes studies on chrysanthemum

    Chromosome karyotype analysis is necessary for the study of species origins, and are useful in taxonomy by measuring chromosome number, sizes, arm rations, and unique p atterns at t he s omatic metaphase s tage ( Nathewet et al. 2010). A karyotype is also a valuable tool to study chromosome variation such as the presence or absence of chromosome segments with different traits. Chromosome rearrangements have been observed through karyotype as well (Chung et al. 2018;Kim et al. 2008), and varying karyotypes in chrysanthemum are results of chromosome rearrangement. Furthermore, karyotype analysis has been used to define the genetic relationship between genera in Compositae (Qi et al. 2015). Information on chrysanthemum karyotype morphology studies has been carried out by several scientists (El-Twab et al. 2008;Li et al. 2008;Zhang et al. 2013).

    Most of the studies have claimed that karyomorphological analysis delivered a better understanding of karyomorphological relationships and evolution of Chrysanthemum and closely related genera (Chang et al. 2009;El-Twab et al. 2008;Li et al. 2011;Tatarenko et al. 2011). For example, a karyotype study between D. boreale and D. indicum showed a remarkable difference in somatic metaphase chromosome (Kim et al. 2003). Additionally, karyotype studies show a great potential to identify the heterogeneity in chrysanthemum (Hwang et al. 2013). The genome of the chrysanthemum cultivars usually has chromosome pairs with different morphology in karyotype study (Chang et al. 2009).

    According to karyotype data, it revealed that a conservative tendency to maintain the general structure of karyotypes characteristics among different groups in chrysanthemum genome (Hwang et al. 2013;Seijo and Fernández 2003). Rana (1965) has reported that there were no significant changes in karyotype symmetry either between the interchange heterozygotes or homozygotes. Most of the chrysanthemum cultivars showed metacentric or sub-metacentric chromosome (Hwang et al. 2013;Li et al. 2008). Karyotype evolution is suggested an isolating mechanism in speciation and has their own evolutionary tendencies such as the increasing number and acrocentric chromosome (El-Twab et al. 2008). It has been found that satellite chromosome might raise with increasing chromosome number and 2A and 2C karyotype also increased with increasing ploidy level of the cultivars by analysis of 40 Chinese chrysanthemum cultivars. Also, the karyotype asymmetry index and relative length of the chromosome are quite different among species. Therefore, karyotype parameters carry out great value for cultivar identification, classification, and genetic analysis in chrysanthemum (Zhang et al. 2013).

    Nowadays, molecular cytogenetic studies with specific markers offer a great opportunity to understand the phylogenetic relationship, improve taxonomic investigation, identify aneuploidy and assist in traits selection. Molecular cytogenetics methods have been widely used to directly observe and analyze plant chromosome during the last 30 years (Heslop‐Harrison 1992;Younis et al. 2015).

    Banding and FISH Technique

    Banding techniques are identified as part of chromosomes in metaphase can be clearly distinguished from its adjacent segments by staining with a suitable dye and visualized in the microscope. Most conventional cytogenetic analyses depend on the karyotyping of banded metaphase chromosomes, which including arm size, secondary constrictions, centromere position, and chromosome number (Younis et al. 2015). Earliest cytological papers in chrysanthemum studies were focused on morphological karyotype characteristic by using banding techniques (Chang et al. 2009;El-Twab et al. 2008;Hong-Bo et al. 2009;Tanaka and Shimotomai 1961;Tanaka and Shimotomai 1968). The development of fluorescent technique has greatly increased its application which now ranges from karyotype analysis to gene localization (Younis et al. 2015).

    Fluorescence in situ hybridization (FISH) has been developed as a powerful and consistent molecular cytogenetic tool to study repetitive elements and copy numbers of DNA sequences at different site, distribution of DNA sequences, and evolution changes in their physical genome organization (Harrison and Heslop-Harrison 1995;Zhmyleva and Kondo 2006;Qi et al. 2015;Younis et al. 2015). A pre-labeled probe with fluorescent dyes will bind specifically to the complementary site on the chromosome, which allows hybridization sites to be visualized directly (Heslop-Harrison et al. 1998;Waminal et al. 2018). For example, FISH signals of 5S (green signals) and 18S (red signals) rDNA loci on the mitotic metaphase chromosome of Chrysanthemum indicum from South Korea are showed in Fig. 1 (unpublished data).

    Fluorescence in situ hybridization (FISH) has been developed as a powerful and consistent molecular cytogenetic tool to study repetitive elements and copy numbers of DNA sequences at different site, distribution of DNA sequences, and evolution changes in their physical genome organization (Harrison and Heslop-Harrison 1995;Zhmyleva and Kondo 2006;Qi et al. 2015;Younis et al. 2015). A pre-labeled probe with fluorescent dyes will bind specifically to the complementary site on the chromosome, which allows hybridization sites to be visualized directly (Heslop-Harrison et al. 1998;Waminal et al. 2018). For example, FISH signals of 5S (green signals) and 18S (red signals) rDNA loci on the mitotic metaphase chromosome of Chrysanthemum indicum from South Korea are showed in Fig. 1 (unpublished data). Moreover, several probes can be simultaneously detected by different fluorochrome for determining physical order on the chromosome (Heslop-Harrison et al. 1998).

    FISH can be also used for chromosome identification and study of genome introgression (El-Twab et al. 2009;Jiang and Gill 1994). It has been applied to many plant species, including Allium species, Brassica species, Maize species, Oryza species, Triticum species, Solanum species, Lilium species, and so on (Hwang et al. 2010). To clarify the relationship between Japanese Chrysanthemum species, the FISH approach was used and was reported first by Kondo (1996). Since there are highly repetitive and conserved sequences in higher plants, ribosomal RNA genes (rDNAs) contain two different families which are the 5S rDNA and 45S rDNA (Zhang et al. 2016), it has been commonly used as a molecular marker for chromosome s tudies ( Hwang et al. 2010). Characteristics of 5S and 45S rDNA along its location on the chromosome provide useful information in chromosome analysis (Heslop-Harrison 2000). Therefore, 5S and 45S rDNA are generally used as FISH probes in molecular cytogenetics studies and most of the FISH analysis in Chrysanthemum species uses rDNA as probes.

    Karyotyping using FISH with 5S and 45S rDNA has been performed by several scientists to develop better understanding of connections among Chrysanthemum and related genera for expediting the development and improvement of plant species (Abd El-Twab and Kondo 2007a;Hwang et al. 2013;Matoba and Uchiyama 2009;Qi et al. 2015;Zhmyleva and Kondo 2006). To investigate the phylogenetic relationship of Dendranthema spp. and the origin of Chinese cultivated chrysanthemum, Dai et al. (2005) has performed FISH analysis using rDNA as probes. The physical map of the chromosomal distribution of 5S and 45S ribosomal RNA gene loci have been also studied in chrysanthemum species (Abd El-Twab and Kondo 2006a). Also, FISH with 5S and 45S rDNA has been applied to establish a karyotype analysis of the recently discovered tetraploid cytotype of Chrysanthemum zawadskii by Cuyacot et al. (2017).

    Heslop-Harrison (1991) also claimed that it provides more information on the nucleolar organizing region (NOR) when ribosomal DNA was used as a probe in the FISH analysis. NOR has been studied for understanding the events of divergence in plant species. For example, the rDNA probe of pTa71 has been used to mark the NORs of satellites for identification satellite chromosome in several Japanese and Chinese species of Chrysanthemum and other related genera (Abd El-Twab and Kondo 2006b). FISH analysis with 45S rDNA and telomeric DNA probes has been applied to investigate the structural relationship between 45S rDNA and telomere sequences as well and the evidence has shown some functional linkages between them (Li et al. 2012).

    Moreover, Cuyacot et al. (2016) used rDNA, telomeric repeats, and different types of C0t DNA fragments as FISH probes to visualize an overview of the genome characteristics and the distribution of major repetitive DNA sequences in C. boreale. Hence, molecular cytogenetic analysis of crops plays an important approach to understand their evolution, genetics, karyotype stability (Heslop-Harrison and Schwarzacher 1993), and the proportion of repetitive DNA sequences (Cuyacot et al. 2016).

    Inter and intraspecific hybridization

    Intraspecific, interspecific and intergeneric hybridization are well-known breeding strategies for developing various cultivars with d esirable t raits for ornamental f lowers ( Jiang et al. 2014). Hybrids between parents of the same species are called intraspecific hybridization which usually has a high cross-compatibility. Interspecific hybridization is the crossing of two different species, normally from the same genus (Chattopadhyay 2016). This allows the development of useful genes from wild, unimproved species for the benefit of cultivated species. For example, commercial lily with distinct characteristics has been developed by interspecific hybridization between distantly related species (Lim et al. 2008). Cultivated chrysanthemum (Dendranthema x grandiflora Tzvelv) are also considered as complex interspecific hybrids (Anderson 2007).

    Hybrids involving different genera are called intergeneric hybridization that provides an opportunity to improve the genetic diversity by introducing traits not found in the major genera of interest (Tang et al. 2010). Although hybridization becomes more difficulties due to gene pools diverge, hybridization in chrysanthemum continues to use for traits transfer (Anderson 2007). Most of the wide crosses failed because hybrid embryos were unable to develop during the earlier stage (Tang et al. 2010). Recently, research generally focused on the understanding of the phylogenetic relationships of Chrysanthemum and related genera using the FISH technique. For instance, Qi et al. (2015) have reported that Ajania species are closely related to chrysanthemum and these excellent traits such as drought, cold, salt, and insect resistances from Ajania can be transferred to chrysanthemum via an intergenic crossing.

    Nowadays, cytogenetic methods provide a direct visual analysis to distinguishing parental genomes as well as studying interspecific introgression and interspecific hybrids (Younis et al. 2015). Genomic in situ hybridization (GISH) technique is fast, sensitive, accurate, reproducible, and informative means to detect parental DNA in their F1 hybrids. GISH has been applied to discriminate parental genome and determine the incorporation of alien chromatin in recent days (Tang et al. 2010). One of the applications in breeding is to recognize parental genomes in interspecific and intergeneric hybrids (El-Twab and Kondo 2009). Tang et al. (2010) have reported that GISH can be used to prove the generation of a true hybrid between Dendranthema indica and Crossostephium chinense, and the taxonomic relationship between these two species.

    There are only few studies that have applied GISH technology to chrysanthemum and these were mostly performed by studies of El-Twab and Kondo. In their studies, GISH was used to investigate crossability, identify the parental genomic relationship, and genome composition in F1 hybrids. The interspecific crossability between Japanese and Chinese Chrysanthemum sensu lato species has been studied using GISH technique (El-Twab et al. (2012). Studies have also been carried out like intergeneric crossability between T. vulgare and Chrysanthemum horaimontanum (El-Twab and Kondo 2007a), an interspecific hybrid between C. boreale and C. vestitum ( Abd El-Twab and Kondo 2007b), and genomic relationships in hybrids between Chrysanthemum lavandulifolium x C. chanetiihas (El-Twab and Kondo 2008).

    Several combinations of artificial hybrids in Chrysanthemum species have been also analyzed through this technique (El-Twab and Kondo 2014) such as C. grandiflorum cv. ‘Happy Gold’ and Nipponanthemum nipponicum (El-Twab and Kondo 2009). Although GISH can be used to confirm allopolyploid species and identify intergenomic recombination, there are no studies that have been performed in chrysanthemum so far. In addition, most of the analysis was not studied by GISH alone but were combined with FISH techniques. For example, FISH and GISH were applied to characterize the parental chromosomes in the F1 hybrid between C. japonicum and N. nipponicum ( Abd El-Twab and Kondo 2006b). El-Twab and Kondo (2008) have clarified that using both FISH and GISH on different hybrids would likely add more information, and it will be helpful to understand the species relationship and dynamic genome changes in the polyploidy process within chrysanthemum.

    Conclusion

    Chrysanthemum is generally believed to be a result of complex hybridization events (Zhang et al. 2010) and exhibit natural variation in ploidy ranging from diploid to decaploid, and their polyploidy level leads to specification within its species. Morphological characteristics are used to differentiate species may not work well because several species like C. zawadskii varieties are almost the same (Cuyacot et al. 2017). However, cytogenetics technologies appear to bring many new opportunities in this field. With the development of new technology such as flow cytometry and molecular cytogenetic FISH and GISH techniques, studies in chrysanthemum will help us to have a better understanding what we can do in taxa breeding for new cultivar improvement. Nevertheless, techniques also have their own limitations. As for FISH, it can only be utilized for currently available probes. There is also a possibility of failure to detect signals and it requires fluorescence microscope and an image analysis system. Therefore, further investigations in chrysanthemum using multi-stage methodologies should be applied based on cytogenetic studies.

    Acknowledgements

    This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012804)” Rural Development Administration, Republic of Korea.

    Figure

    FRJ-27-4-242_F1.gif

    FISH signals of 5S (green signals) and 18S (red signals) rDNA loci on the mitotic metaphase chromosome of Chrysanthemum indicum from Republic of Korea: a) A7; b) A95; FISH karyotype of A7 from chromosome metaphase spread is arranged in a decreasing order according to the FISH signal, morphology, and length, scale bars = 10 μm.

    Table

    Application of cytogenetic techniques in chrysanthemum and related species.

    Reference

    1. Abd El-Twab MH , Kondo K (2006a) FISH physical mapping of 5S, 45S and Arabidopsis-type telomere sequence repeats in Chrysanthemum zawadskii showing intrachromosomal variation and complexity in nature. Chromosome Bot 1:1-5
    2. Abd El-Twab MH , Kondo K (2006b) Fluorescence in situ hybridization and genomic in situ hybridization to identify the parental genomes in the intergeneric hybrid between Chrysanthemum japonicum and Nipponanthemum nipponicum. Chromosome Bot 1:7-11
    3. Abd El-Twab MH , Kondo K (2007a) FISH physical mapping of 5S rDNA and telomere sequence repeats identified a peculiar chromosome mapping and mutation in Leucanthemella linearis and Nipponanthemum nipponicum in Chrysanthemum sensu lato. Chromosome Bot 2:11-17
    4. Abd El-Twab MH , Kondo K (2007b) Isolation of chromosomes and mutation in the interspecific hybrid between Chrysanthemum boreale and C. vestitum using fluorescence in situ hybridization and genomic in situ hybridization. Chromosome Bot 2:19-24
    5. Adams KL , Wendel JF (2005) Polyploidy and genome evolution in plants. Curr Opin Plant Biol 8:135-141
    6. Anderson NO (2007) Chrysanthemum. In: Anderson N.O. (eds) Flower Breeding and Genetics. Springer, Dordrecht
    7. Barlup J (2002) Let's talk hybridizing: Hybridizing with elepidote polyploid Rhododendrons. J Am Rhod Soc 76:75-77
    8. Bhattacharya A , da Silva JAT (2006) Molecular systematics in Chrysanthemum× grandiflorum (Ramat.) Kitamura. Sci Hortic 109:379-384
    9. Bourke PM , Voorrips RE , Visser RG , Maliepaard C (2018) Tools for genetic studies in experimental populations of polyploids. Front Plant Sci 9:513
    10. Chang L , Chen SM , Chen FD , Zhen L , Fang WM (2009) Karyomorphological studies on Chinese pot chrysanthemum cultivars with large inflorescences. Agric Sci China 8:793-802
    11. Chattopadhyay NR (2016) Induced fish breeding: A practical guide for hatcheries. Academic Press, London, UK
    12. Chung YS , Jun TH , Lee YG , Jung JA , Won SY , Hwang YJ , Silva RR , Choi SC , Kim C (2018) A genetic linkage map of wild chrysanthemum species indigenous to Korea and its challenges. Int J Agric Biol 20:2708-2716
    13. Cuyacot AR , Lim KB , Kim HH , Hwang YJ (2017) Chromosomal characterization based on repetitive DNA distribution in a tetraploid cytotype of Chrysanthemum zawadskii. Hortic Environ Biotechnol 58:488-494
    14. Cuyacot AR , Won SY , Park SK , Sohn SH , Lee J , Kim JS , Kim HH , Lim KB , Hwang YJ (2016) The chromosomal distribution of repetitive DNA sequences in Chrysanthemum boreale revealed a characterization in its genome. Sci Hortic 198:438-444
    15. Dai SL , Wang WK , Li MX , Xu YX (2005) Phylogenetic relationship of Dendranthema (DC.) Des Moul. revealed by fluorescent in situ hybridization. J Integr Plant Biol 47:783-791
    16. Datta S , Janakiram T (2015) Breeding and genetic diversity in Chrysanthemum morifolium in India: A review. Indian Journal of Agricultural Sciences 85:1379-1395
    17. El-Twab A , Hussein M , Kondo K (2012) Genome mutation revealed by artificial hybridization between Chrysanthemum yoshinaganthum and Chrysanthemum vestitum assessed by FISH and GISH. J Bot 2012:480310
    18. El-Twab MHA , Kondo K (2007a) Identification of parental chromosomes, intra-chromosomal changes and relationship of the artificial intergeneric hybrid between Chrysanthemum horaimontanum and Tanacetum vulgare by single color and simultaneous bicolor of FISH and GISH. Chromosome Bot 2:113-119
    19. El-Twab MHA , Kondo K (2007b) Rapid genome reshuffling induced by allopolyploidization in F1 hybrid in Chrysanthemum remotipinnum (formerly Ajania remotipinna) and Chrysanthemum chanetii (formerly Dendranthema chanetii). Chromosome Bot 2:1-9
    20. El-Twab MHA , Kondo K (2008) Visualization of genomic relationships in allotetraploid hybrids between Chrysanthemum lavandulifolium x C. chanetii by fluorescence in situ hybridization and genomic in situ hybridization. Chromosom Bot 3:19-25
    21. El-Twab MHA , Kondo K (2009) GISH identification of ancestor or closely related genome to Chrysanthemum grandiflorum cv. ‘Happy Gold’. Chromosome Bot 4:47-51
    22. El-Twab MHA , Kondo K (2014) Chrysanthemum latifolium x C. grandiflorum cv. ‘Red Betty’ crossed to induce new cultivars: Hybrid genome characterization and species relationship analyzed by FISH and GISH. Chromosome Bot 9:7-11
    23. El-Twab MHA , Mahmoud MAH , Helmey RK , Fadl-Allah EM (2009) Molecular cytogenetical effects of some mycotoxins to chromosomes of Triticum durum using RAPD-PCR fluorescence in situ hybridization. Chromosome Bot 4:13-18
    24. El-Twab MHA , Mekawy AM , El-Katatny MS (2008) Karyomorphological studies of some species of Chrysanthemum sensu lato in Egypt. Chromosome Bot 3:41-47
    25. Gao YD , Zhou SD , He XJ , Wan J (2012) Chromosome diversity and evolution in tribe Lilieae (Liliaceae) with emphasis on Chinese species. J Plant Res 125:55-69
    26. Guo X , Luo C , Wi Z , Zhang X , Cheng X , Huang C (2012) Polyploidy levels of Chinese large-flower chrysanthemum determined by flow cytometry. Afr J Biotechnol 11:7789-7794.
    27. Harrison G , Heslop-Harrison J (1995) Centromeric repetitive DNA sequences in the genus Brassica. Theor Appl Genet 90:157-165
    28. Heslop-Harrison J (1991) In situ hybridization with automated chromosome denaturation. Technique 3:109-116
    29. Heslop‐Harrison J (1992) Molecular cytogenetics, cytology and genomic comparisons in the Triticeae. Hereditas 116:93-99
    30. Heslop-Harrison J (2000) Comparative genome organization in plants: from sequence and markers to chromatin and chromosomes. Plant Cell 12:617-635
    31. Heslop-Harrison J , Schwarzacher T (1993) Molecular cytogenetics—biology and applications in plant breeding. Springer, Dordrecht
    32. Heslop-Harrison P , Osuji, J , Hull R , Harper G , D’Hont A , Carreel F (1998) Fluorescent in situ hybridization of plant chromosomes: illuminating the Musa genome. INIBAP Annual Report, pp 26-29
    33. Hong-Bo Z , Li C , Tang FP , Chen FD , Chen SM (2009) Chromosome numbers and morphology of eighteen Anthem i deae (Asteraceae) taxa f rom China a nd t heir systematic implications. Caryologia G Citol Citosistematica Citogenet 62:288-302
    34. Hwang YJ , Lee SN , Song KA , Ryu KB , Ryu KH , Kim HH (2010) Karyotype analyses of genetically modified (GM) and non-GM hot peppers by conventional staining and FISH method. Hortic Environ Biotechnol 51:525-530
    35. Hwang YJ , Younis A , Ryu KB , Lim KB , Eun CH , Lee J , Sohn SH , Kwon SJ (2013) Karyomorphological analysis of wild Chrysanthemum boreale collected from four natural habitats in Korea. Flower Res J 21:182-189
    36. Jara-Seguel P , Urrutia J (2012) Cytogenetics of Chilean angiosperms: Advances and prospects. Rev Chil Hist Nat 85:1-12
    37. Jiang J , Gill BS (1994) Different species-specific chromosome translocations in Triticum timopheevii and T. turgidum support the diphyletic origin of polyploid wheats. Chromosome Res 2:59-64
    38. Jiang L , Wang YW , Dunn BL (2014) Cross-compatibility in intraspecific and interspecific hybridization within Lychnis and intergeneric hybridization between Lychnis and Silene. HortScience 49:1136-1141
    39. Khaing A , Moe K , Hong W , Park C , Yeon K , Park H , Kim D , Choi B , Jung J , Chae S (2013) Phylogenetic relationships of chrysanthemums in Korea based on novel SSR markers. Genet Mol Res 12:5335-5347
    40. Kim JS , Oginuma K , Tobe H (2008) Analysis of meiotic chromosome behaviour in diploid individuals of Chrysanthemum zawadskii and related species (Asteraceae): evidence for chromosome rearrangements. Cytologia (Tokyo) 73:425-435
    41. Kim JS , Pak JH , Seo BB , Tobe H (2003) Karyotypes of metaphase chromosomes in diploid populations of Dendranthema zawadskii and related species (Asteraceae) from Korea: diversity and evolutionary implications. J Plant Res 116:47-54
    42. Klie M , Schie S , Linde M , Debener T (2014) The type of ploidy of chrysanthemum is not black or white: a comparison of a molecular approach to published cytological methods. Front Plant Sci 5:479
    43. Kondo K (1996) Chromosome marking in Dendranthema japonica var. wakasaense and its closely related species by fluorescence in situ hybridization using rDNA probe. La Kromosomo 81:2785-2791
    44. Kushwah K , Verma R , Patel S , Jain N (2018) Colchicine induced polyploidy in Chrysanthemum carinatum L. J Phylogenetics Evol Biol 6:193
    45. Li C , Chen S , Chen F , Li J , Fang W (2011) Cytogenetic study of three edible chrysanthemum cultivars. Russ J Genet 47:176-181
    46. Li J , Chen SM , Chen FD , Fang WM (2008) Karyotype and meiotic analyses of six species in the subtribe Chrysantheminae. Euphytica 164:293-301
    47. Li J , He S , Zhang L , Hu Y , Yang F , Ma L , Huang J , Li L (2012) Telomere and 45S rDNA sequences are structurally linked on the chromosomes in Chrysanthemum segetum L. Protoplasma 249:207-215
    48. Lim KB , Barba-Gonzalez R , Zhou S , Ramanna M , van Tuyl JM (2008) Interspecific hybridization in lily (Lilium): taxonomic and commercial aspects of using species hybrids in breeding. In: Floriculture, Ornamental and Plant Biotechnology, Global Books, UK, pp 138-145
    49. Liu PL , Wan Q , Guo YP , Yang J , Rao GY (2012) Phylogeny of the genus Chrysanthemum L.: Evidence from single-copy nuclear gene and chloroplast DNA sequences. PloS ONE 7:e48970
    50. Liu YH , Mou X , Zhou DY , Zhou DY , Shou CM (2018) Extraction of flavonoids from Chrysanthemum morifolium and antitumor activity in vitro. Exp Ther Med 15:1203-1210
    51. Ma YP , Wei JX , Yu ZY , Qin B , Dai SL (2015) Characterization of ploidy levels in Chrysanthemum L. by flow cytometry. J Forestry Res 26:771-775
    52. Masuda Y , Yukawa T , Kondo K (2009) Molecular phylogenetic analysis of members of Chrysanthemum and its related genera in the tribe Anthemideae, the Asteraceae in East Asia on the basis of the internal transcribed spacer (ITS) region and the external transcribed spacer (ETS) region of nrDNA. Chromosom Bot 4:25-36
    53. Matoba H , Uchiyama H (2009) Physical mapping of 5S rDNA, 18S rDNA a nd t elom ere sequences in t hree s pecies o f the genus Artemisia (Asteraceae) with distinct basic chromosome numbers. Cytologia (Tokyo) 74:115-123
    54. Nathewet P , Hummer KE , Yanagi T , Iwatsubo Y , Sone K (2010) Karyotype analysis in octoploid and decaploid wild strawberries in Fragaria (Rosaceae). Cytologia (Tokyo) 75:277-288
    55. Oberprieler C , Himmelreich S , Vogt R (2007) A new subtribal classification of the tribe Anthemideae (Compositae). Willdenowia 37:89-115
    56. Podwyszyńska M , Gabryszewska E , Dyki B , Stępowska A , Kowalski A , Jasiński A (2015) Phenotypic and genome size changes (variation) in synthetic tetraploids of daylily (Hemerocallis) in relation to their diploid counterparts. Euphytica 203:1-16
    57. Qi X , Zhang F , Guan Z , Wang H , Jiang J , Chen S , Chen F (2015) Localization of 45S and 5S rDNA sites and karyotype of Chrysanthemum and its related genera by fluorescent in situ hybridization. Biochem Syst Ecol 62:164-172
    58. Rana R (1965) Induced interchange heterozygosity in diploid Chrysanthemum. Chromosoma 16:477-485
    59. Rout G , Das P (1997) Recent trends in the biotechnology of Chrysanthemum: A critical review. Sci Hortic 69:239-257
    60. Seijo JG , Fernandez A (2003) Karyotype analysis and chromosome evolution in South American species of Lathyrus (Leguminosae). Am J Bot 90:980-987
    61. Tanaka R , Shimotomai N (1961) Karyotypes in four diploid species of Chrysanthemum. Cytologia (Tokyo) 26:309-319
    62. Tanaka R , Shimotomai N (1968) A Cytogenetic Study on the F1 Hybrid of Chrysanthemum makinoi × Ch. vulgare. Cytologia (Tokyo) 33:241-245
    63. Tang F , Chen F , Chen S , Wang XE , Zhao H (2010) Molecular cytogenetic identification and relationship of the artificial intergeneric hybrid between Dendranthema indica and Crossostephium chinense by GISH. Plant Sys Evol 289:91-99
    64. Tatarenko E , Kondo K , Smirnov SV , Kucev M , Yang QE , Hong DY , Ge S , Zhang DM , Zhou S , Damdinsuren O (2011) Chromosome relationships among the Chrysanthemum fruticulosum complex. Chromosome Bot 6:61-66
    65. van Geest G , Voorrips RE , Esselink D , Post A , Visser RG , Arens P (2017) Conclusive evidence for hexasomic inheritance in chrysanthemum based on analysis of a 183 k SNP array. BMC Genomics 18:585
    66. Waminal NE , Pellerin RJ , Kim NS , Jayakodi M , Park JY , Yang TJ , Kim HH (2018) Rapid and efficient FISH using pre-Labeled oligomer probes. Sci Rep 8:8224 (2018)
    67. Wang F , Zhang FJ , Chen FD , Fang WM , Teng NJ (2014) Identification of chrysanthemum (Chrysanthemum morifolium) self-incompatibility. The Scientific World Journal 2014: 625658
    68. Wang Y , Bigelow CA , Jiang Y (2009) Ploidy level and DNA content of perennial ryegrass germplasm as determined by flow cytometry. HortScience 44:2049-2052
    69. Xu H , Zhang W , Zhang T , Li J , Wu X , Dong L (2014) Determination of ploidy level and isolation of genes encoding acetyl-CoA carboxylase in Japanese foxtail (Alopecurus japonicus). PLoS ONE 9:e114712
    70. Younis A , Ramzan F , Hwang YJ , Lim KB (2015) FISH and GISH: molecular cytogenetic tools and their applications in ornamental plants. Plant Cell Rep 34:1477-1488
    71. Zhang F , Chen S , Chen F , Fang W , Li F (2010) A preliminary genetic linkage map of chrysanthemum (Chrysanthemum morifolium) cultivars using RAPD, ISSR and AFLP markers. Sci Hortic 125:422-428
    72. Zhang Y , Zhu ML , Dai SL (2013) Analysis of karyotype diversity of 40 Chinese chrysanthemum cultivars. J Syst Evol 51:335-352
    73. Zhang ZT , Yang SQ , Li ZA , Zhang YX , Wang YZ , Cheng CY , Li J , Chen JF , Lou QF (2016) Comparative chromosomal localization of 45S and 5S rDNAs and implications for genome evolution in Cucumis. Genome 59:449-457
    74. Zhao HB , Chen FD , Chen SM , Wu GS , Guo WM (2010) Molecular phylogeny of Chrysanthemum, Ajania and its allies (Anthemideae, Asteraceae) as inferred from nuclear ribosom al I TS a nd c hloroplast trnL-F IGS sequences. Plant Syst Evol 284:153-169
    75. Zhmyleva AP , Kondo K (2006) Comparison of somatic chromosomes in some species of Chrysanthemum sensu lato in Russia. Chromosome Bot 1:13-22