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.
