Introduction

Rose is one of the most popular ornamental plants (Ding et al. 2016), accounting for 30% of the global floral market (Saint-Oyant et al. 2018). In addition to being a garden plant, it is used to produce cut flowers or manufacture fragrance products and has great cultural significance in many regions of the world (Khosh-Khui 2014;Kirov et al. 2015a;Saint-Oyant et al. 2018). Although rose cultivation is commercially important and great efforts have been made to develop new cultivars, little information is available on the inheritance of important traits in such cultivars (Debener and Mattiesch 1999). As a result of frequent natural and artificial hybridization, rose cultivars possess a complex genetic background. Modern rose cultivars are known as a tetraploid, and reports suggest that these formations are caused by autotetraploidy, allotetraploidy, or segmental allotetraploidy (Gar et al. 2011;Koning-Boucoiran et al. 2012;Rajapakse et al. 2001;Rusanov et al. 2009;Zhang et al. 2006).

In plants, the chromosome number is an important trait for genetic studies of species (Nair 2019). Different species possess a different number of chromosomes, while the same species typically possess an identical number of chromosomes (Verma 2011;Verma et al. 2018). Chromosome number variation may lead to the speciation or formation of geographic races (Bennett 1987;Epling and Dobzhansky 1942). Therefore, the knowledge of chromosome number is relevant to genetics, plant breeding, phytogeography, endemism, intraspecific variation, the origin of floral diversity, and evolution (Sharma and Sharma 1956;Simon et al. 2001;Verma et al. 2018).

Flow cytometry can rapidly determine the ploidy level of large samples and does not require the collection of buds for chromosome count during meiosis or roots for chromosome count during mitosis (Doležel et al. 2004;Yan et al. 2016); however, basic chromosome number must initially be determined using the conventional squash technique. Furthermore, flow cytometry has the limitation of detecting the specific chromosome number in aneuploids.

Although rose is an economically and a scientifically important floricultural crop, only a few cytogenetic studies have been performed on rose cultivars owing to their small genome size, small chromosomes, low root mitotic index, and high polyploid frequency (Kirov et al. 2015a;Kirov et al. 2016;Ma et al. 1996). Specifically, although chromosome information of some wild rose species is available (Fernández-Romero et al. 2001;Grossi and Jay 2002;Ma et al. 1997;Yu et al. 2014), there are only a few reports on rose cultivars (Ding et al. 2016;Hwang et al. 2012), which are the primary material for use in breeding programs to develop new rose cultivars. According to Hwang et al. (2010), chromosome ploidy level is a fundamental factor for rose breeding programs, as ploidy level affects pollen fertility, which in turn determines the efficiency and success rate of crossing.

To this end, in the present study, we explored the chromosome information of rose cultivars through conventional cytogenetic methods. Our data will serve as a basis for the development and breeding of new rose cultivars.

Materials and Methods

Plant Material

All seedlings from stem cuttings were provided by the National Institute of Horticultural and Herbal Science, Rural Development Administration, Korea.

Metaphase Chromosome Preparation

Young root tips of actively growing plants were collected, pre-treated with 2 mM 8-hydroxyquinoline for 5 h at 18°C, fixed in Carnoy’s solution [ethanol:glacial acetic acid (3:1, v/v)] for 24 h at room temperature, and finally stored in 70% ethanol at -20°C. To study the chromosomes, the prepared roots were thoroughly washed with distilled water and treated with an enzyme mixture [2% cellulose R-10 (Yakult) and 2% pectolyase Y-23] for 1.5 h at 37°C. Next, the root tips were washed again with distilled water to remove excess enzymes. Carnoy’s solution was pipetted into the meristematic root tissue, and the samples were vortexed for 15 s. The suspension was then centrifuged at 1,593 × g for 3 min, and the supernatant was carefully decanted. The remaining protoplasts were resuspended in acetic acid:ethanol (9:1, v/v) solution, dropped on a pre-warmed (70°C) glass slide in a humid chamber and air-dried at room temperature. Then, the slides were counterstained with a 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 fluorescence microscope (BX53, Olympus, Tokyo, Japan) equipped with a DFC365 FX CCD camera (Leica Microsystems, Wetzlar, Germany) and processed using Cytovision 7.2 (Leica Microsystems). Further image enhancements were made using Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA). The total chromosome length of each cell was measured using Image J from mitotic metaphase chromosome.

Results and Discussion

Images of somatic metaphase chromosomes of the 39 rose cultivars obtained using conventional cytogenetic methods (squashing technique) are shown in Fig. 1 and Fig. 2. Chromosome number, total chromosome length, and ploidy level of the 39 rose cultivars are presented in Table 1. Of the 39 rose cultivars studied, 36 (92.3%) were tetraploid (2n=4x=28). Meanwhile, ‘Rosada’ (Fig. 1A), ‘Rosemarin’ (Fig. 1B), and ‘Hanmaum’ (Fig. 2S) were diploid (2n=2x=14), triploid (2n=3x=21), and tetraploid-based aneuploid (2n=4x=28+2), respectively. All cultivars tested in the present study had the same basic chromosome number (x=7), which is consistent with previous reports (Kirov et al. 2015b;Rajapakse et al. 2001). The ploidy level of Rosa species typically ranges from diploid (2n=2x=14) to decaploid (2n=10x=70) (Jian et al. 2010;Kirov et al. 2016). In addition, most of the current rose cultivars have been reported to be tetraploid (2n=4x=28), evidently following the wild diploid progenitor species (Ma et al. 1997;Zhang et al. 2006). Currently, tetraploid roses, such as hybrid tea roses, represent the majority of the commercial varieties and remain the basis of rose breeding programs (Koning-Boucoiran et al. 2012).

In the present study, ‘Pearl Red’ was a tetraploid (2n=4x=28) but possessed 56 chromosomes (Fig. 2F) based on the chromosome image; this is because the mitotic chromosomes were in late metaphase when the replicated chromosomes are segregated to the daughter cells, as evidenced by two sets of chromosomes (Zhang and Nicklas 1996). ‘Rosemarin’ was a triploid (2n=3x=21). Generally, triploids exhibit reduced fertility, representing a genetic bottleneck during offspring development (Zlesak 2007). In a previous study examining pollen viability and cross-compatibility, when the triploid (2n=3x=21) rose cultivar ‘Mini Rosa’ was used as a maternal or paternal parent, the rate of seed set was substantially reduced and the seeds did not germinate (Hwang et al. 2010). Furthermore, ‘Hanmaum’ (2n=4x=28+2) was a tetraploid-based aneuploid. Aneuploids have been reported to be rare in the genus Rosa (Rowley 1960), which is consistent with our observation that only one of the 39 cultivars tested in the present study was aneuploid.Most modern roses have been bred by crossing tetraploid and diploid parents (Rajapakse et al. 2001), followed by backcrossing to derive lines with one or a few additional chromosomes (Zlesak 2007). This is because abnormal cell division, wherein chromosomes are lost or gained during mitosis or meiosis, leads to aneuploidy (Heslop-Harrison and Schwarzacher 2011).

In the present study, the total chromosome length ranged from 46.03±0.55 to 138.51±0.92 μm, and this length is slightly greater than the previously reported value (Lata 1981). This discrepancy may be attributed to the fact that total chromosome length is not stable, as metaphase chromosomes are usually measured on microscope slides and their length may vary depending on the degree of chromosome condensation (Mártonfiová 2013). Chromosome number and length have been studied over many decades, and these data have been confirmed to be useful in evolutionary and phylogenetic studies of plant species and families. In addition, chromosome length in a species is determined by chromosome number and genome size (Heslop-Harrison and Schwarzacher 2011). Chromosomes were metacentric and submetacentric in all tested cultivars in the present study, and metacentric chromosomes were longer than the rest of the chromosomes, which is consistent with previous reports (Lata 1981).

In addition to morphological and physiological characteristics, chromosomal information is one of the key factors for the taxonomic, phylogenetic, and evolutionary studies of plant species (Akasaka et al. 2003). In particular, chromosome data play a central role in breeding programs. In triploids or aneuploids, abnormal meiosis produces infertile pollen, which in turn reduces crossing efficiency (Zlesak 2007). The conventional pollen viability tests have the disadvantage that these can only be performed at certain flowering times when pollens are produced; however, ploidy level using root meristem can be confirmed year-round. Therefore, chromosome information is crucial for designing highly systematic breeding programs.

Chromosome numbers can be examined and shared through the following international databases: Chromosome Counts Database (CCDB, http://ccdb.tau.ac.il/home/), Chromosome Numbers of the Flora of Germany (http://chromosomes. senckenberg.de/index.php), and Index to Plant Chromosome Numbers (http://legacy.tropicos.org/projectwebportal.aspx? pagename=Home&projectid=9). In Korea, the National Institute of Biological Resources has published the “Chromosome Index of Korean Endangered and Native Plants” (Kim 2010, 2011, 2012, 2013;National Institute of Biological Resources 2011). However, this index is limited to endangered and native species. For domestic species, additional databases such as the Korean Plant Chromosome Database (www.plantchromosome. co.kr) are available, through which chromosome information is systematically updated and shared.

In conclusion, the chromosome information presented here will be useful for rose breeding to enhance breeding efficiency through germplasm analysis (Diaby and Casler 2003;Kim et al. 2011).