Introduction

The Asteraceae is one of the largest angiosperm families in the herbaceous plants throughout the world. It contains 1600 genera, including genus Chrysanthemum and genus Aster (Atri et al. 2009). The genus Chrysanthemum contains about 41 species that are mainly distributed in East Asia and chiefly in China, Korea, and Japan (Hwang et al. 2013;Liu et al. 2012;Masuda et al. 2009;Zhao et al. 2010). In Korea, there are 17 known species of wild chrysanthemums that grow in their respective habitats. Among them, Chrysanthemum boreale and C. indicum are used for landscaping and medicinal purposes. Many Aster species are serving as garden plants due to their attractive and colorful flowers.

One of the most widely marketed cut-flower in the world, chrysanthemum is famous for its wide array of flower variations in size, color, and shape as well as its long vase life, early production and photoperiod responses (Junqueira and Peetz 2014;Zandonadi et al. 2018). Due to these desirable traits, chrysanthemums rank among the top three highest cut flowers imported annually by Netherlands (2.7 billion pieces) and Taiwan (7.1 Million bunches) (Hanks 2018).

The increasing awareness of the importance of wild germplasm recognizes that wild species play a key role as progenitors in many plant improvement programs. Therefore, it is necessary to use desirable genetic traits from wild chrysanthemums that have not been exploited in these breeding programs (Datta and Janakiram 2015). Take for example the white rust disease, caused by the fungus Puccinia horiana, which caused economical loses in chrysanthemum (Park and Kim 1993). In the study of Park et al. (2014), it was reported that among the wild chrysanthemum infected by Puccinia horiana, C. boreale and C. zawadskii were found to be resistant, and C. indicum and Aster spathulifolius were highly susceptible. Studies like these can contribute to the improvement of genetic diversity in chrysanthemum.

However, breeding chrysanthemum proves to be difficult because they have various ploidy level, self-incompatibility, and genetic heterozygosity. Therefore, chromosomal information in the chrysanthemum breeding program is essential and crucial. The basic chromosome number of chrysanthemum is x = 9 and shows a variety of their ploidy levels ranging from diploid (2n = 2x = 18) to decaploid (2n = 10x = 90). In particular, chrysanthemum cultivars (C. morifolium) are mostly known as allohexaploid (2n = 6x = 54). It was reported that polyploidization is caused by natural cross-hybrid between C. indicum, C. japonense, C. lavandulifolium, C. makinoi, C. sinense, C. ornatum, C. vesticum, and C. zawadskii etc. (Dai et al. 2005;Ma et al. 2016) and possessing various aneuploidy (2n = 45-71) (Chang et al. 2009;Zhu et al. 2011). The formation of abnormal pairs of chromosomes during meiosis caused by the aneuploidy affects pollen viability. Therefore, identification of chrysanthemum species using cytological investigation is essential for an efficient breeding program.

Hence, this paper investigated the chromosome number and ploidy level information of Korean wild Asteraceae species collected from 17 different areas within Republic of Korea by performing cytogenetic analysis. The results of this study provide basic information of Asteraceae genetic diversity in Korea and can be used for breeding schemes as basic data for breeder.

Materials and methods

Plant materials

Twenty-three individuals of Asteraceae species were collected from 17 different areas in Republic of Korea (Table 1). Plant samples were provided by Sejong University, Seoul, Korea and being cultivated in the green-house of Sahmyook University, Seoul, Korea.

Ploidy level determination

Investigation of ploidy level was determined by chromosome counting method. Young root tips of actively growing plants were harvested and pre-treated with 2 mM 8-hydroxyquinoline for 5 h at 18°C, and then fixed in Carnoy’s solution [3:1 = ethanol : glacial acetic acid (v/v)] for 24 h at room temperature. The material was stored at -20°C in 70% ethanol solution. For chromosome analysis study, pretreated roots were washed thoroughly with distilled water and the root tips were treated with a mixture of enzymes (0.3% cellulase, 0.3% cytohelicase, 0.3% pectolyase in 150 mM of citrate buffer) for 1.5 h at 37°C. Carnoy’s solution was pipetted into meristematic tissue, and vortexed for 15 sec. The suspension was then centrifuged at 5,000 rpm for 5 min, and the supernatant was decanted carefully. The protoplast was re-suspended in acetic acid:ethanol (9:1 v/v) solution. The final suspension was spread on an 80°C pre-warmed glass slide in a humid chamber and followed by air dried at room temperature. The slides were counterstained with premixed 4′,6-diamidino-2-phenylindole (DAPI) solution (1 μg･ml^-1; DAPI in Vectashield, Vector Laboratories, Burlingame, CA, USA).

Image acquisition

Images were captured under a model BX53 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a DFC365 FS CCD camera (Leica Microsystems, Wetzlar, Germany) and processed using Cytovision ver. 7.2 (Leica Microsystems). Further image enhancements were performed with Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA).

Results and discussion

Of the twenty-three individuals across genus Chrysanthemum and Aster have been examined, and the chromosome results revealed that chromosome number are listed in Table 1. All the investigated plants are the first time to report except A05 and B06 under the species of C. boreale and B27 under the species of Aster spathulifolius. Extensive variation in ploidy levels was found in wild Asteraceae species and ploidy level varies from diploid (2x) to hexaploid (6x) with a basic chromosome number of 9.

Chrysanthemum boreale (2n = 18)

C. boreale is a perennial flowering plant that is often well-known by its yellow flower. In Korea, wild C. boreale can be found in various habitats, such as Gyeongsangbuk-do, Gangwon-do, Gyeonggi-do, Gyeongsangnam-do, and Jeollabuk-do (Table 1). All the investigated plant showed diploid with chromosome number of 2n = 18 (Fig. 1), which is consistent with the previously report (Cuyacot et al. 2016;Dowrick 1952;Hwang et al. 2013;Kim 1999;Kim 2015;Kim et al. 2008;Kim et al. 2015;Liu et al. 2012).

Chrysanthemum indicum (2n = 18 / 36)

C. indicum is flowering plant with small capitula and yellow ray florets (Lim 2012) and commonly called Indian chrysanthemum and Yejuhua (China). It is used to treat infectious diseases and hypertension disorders in Korea, China and Japan (Lograda et al. 2013). As an antiphlogistic used for the treatment of carbuncle, furuncle, conjunctivitis, headache, and vertigo in traditional Chinese medicine (Tang and Eisenbrand 1992). The species are wild to Korea and being extensively distributed at Jeollabuk-do, Chungcheongbuk-do, Jeju-do, Gyeongsangbuk-do, and Incheon (Table 1). It was reported that C. indicum was diploid (2n = 18) and tetraploid (2n = 36) by Kim (1999), Kim et al. (2008), Hong-Bo et al. (2009), Liu et al. (2012), Li et al. (2013). The current study also revealed that C. indicum has chromosome number of 18 (diploid) and 36 (tetraploid) within the infraspecies. The species is also known to have hexaploid with 2n = 54 (Kim et al. 2015;Liu et al. 2012). Fig. 2

Chrysanthemum zawadskii (2n = 18 / 36 / 45 / 54)

C. zawadskii has a white-purple ray floret with large captitula (Kim 2015). C. zawadskii is the major and most popular wild plant in Korea (Lim 2012). Diploid (2n = 18), tetraploid (2n = 36), hexaploid (2n = 54), and octaploid (2n = 72) species from Korea have been reported by Kim et al. (2003), Kim et al. (2008), Kim et al. (2015), Stuessy and Ono (2007). Tetraploid (2n = 36) and hexaploid (2n = 54) were also reported in China and Japan (Liu et al. 2012;Suzuki et al. 2014) Besides, the present study adds a new cytotype of pentaploid (5X) to this species (Fig. 3D).

Chrysanthemum zawadskii spp. lucidum (2n = 36)

C. zawadskii spp. lucidum is a Chrysanthemum subspecies from Ullengdo, Korea. Chromosome number of C. zawadskii spp. lucidum were showed tetraploid (2n = 4x = 36), which are same as the earlier study (Cuyacot et al. 2017;Kim 1999;Kim et al. 2015;Kim 2015;Lee 1975). Fig. 4

Aster spathulifolius (2n = 18)

A. spathulifolius, a perennial herb of the genus Aster within the family Asteraceae, are native to the coastal regions of Korea and Japan (Maki and Morita 1998;Yoon et al. 2005). It grows on seashores due to its short height and thick leaves. This species has vivid white or light blue flowers (Maki and Morita 1998). The plants used for studies were collected from Ulleungdo, Busan, and Jeju-do (Table 1) and all showed diploid with chromosome number of 2n = 18. The number of chromosome is same with previous report (Huziwara 1957;Maki and Morita 1998;Li and Zhang 2004;Yoon et al. 2005). Moreover, all three plants from different native areas had a pair of satellite chromosome (Fig. 5), which is consistent with the study of Yoon et al. (2005).

Variations in ploidy level

Chromosome counts reveal that subspecies variation were highly occurred except C. boreale. For example, C. indicum was found to have 2x (A06, A13, and A16), 4x (A01, A96, and B25), and C. zawadskii was found to have 2x (B75), 4x (A57 and B30), 5x (A99), and 6x (A25) populations. The existence of a regular succession of chromosome number within a species is an evidence for polyploidy (Heslop‐ Harrison and Schwarzacher 2011) and this probably due to plants from different location is examined (Gutiérrez-Flores et al. 2018). Variation in chromosome numbers may be the result of habitat adaptation, which explains their widely distribution (El-Twab and Kondo 2006).

However, both of the C. boreale and A. spathulifolius are diploid with a chromosome number of 2n = 18, but their distribution patter is opposite. C. boreale is widely distributed around the country (Table 1), while A. spathulifolius are narrowly distributed and habitat-specific in the coastline. According to Liu et al. (2012), this can be explained that the diploid species appear to be endemic or narrowly distributed, and polyploidy are usually widely distributed in most instances. All this information suggests that speciation is related to polyploidy and ecological tolerances.

Polyploidy is one of the major biological events in plant evolution process that often leading to complex patterns of genetic diversity, reproductive isolation, and discrepancy in breeding systems (Gutiérrez-Flores et al. 2018). It is often the result of climatic change, human, and other disturbance (Li et al. 2013). Studies of chromosome numbers could provide information for a better understanding of evolutionary relationships between species (Peng and Hsu 1978). Thus, studies that focus on chromosomal number can be used to determine the distribution of different cytotypes and reproductive variation at both intra- and inter-population levels. As well as being of basic research, understanding and exploiting evolutionary mechanisms in plant is likely to be a key for crop development.