Chromosome and ploidy analysis of winter hardy Hibiscus species by FISH and flow cytometry

Determination of nuclear DNA content, genome size, and ploidy level and, information on cytogenetic characteristics are all prerequisite of modern plant breeding. However, identification of individual chromosomes of Hibiscus species is extremely difficult due to high number, small size and similar shape of mitotic chromosomes. The goal of the study was to ascertain the chromosome number, karyomorphology, distribution of 5S and 18S rDNA signals, chromosome length, and centromere positions as well as the ploidy level, genome sizes, 2C - DNA content of winter-hardy Hibiscus (H. syriacus ‘Saejamyung’, H. sinosyriacus ‘Seobong’, H. moscheutos ‘Luna Red’ and, H. paramutabilis). 5S rDNA and 18S rDNA signals were detected by fluorescence in situ hybridization (FISH). According to the FISH results, there are two 5S rDNA signals (green) in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA signals in H. paramutabilis. The range in length of somatic chromosomes in H. syriacus, H. sinosyriacus, H. moscheutos, and H. paramutabilis is 2.66–7.06, 3.18–7.31, 2.91–5.23, and 4.75–7.60, respectively. The 2C - DNA content of H. syriacus, H. sinosyriacus, and H. paramutabilis are very similar, the amount was 4.06, 4.11, and 4.18 pg, respectively whereas, H. moscheutos has nearly half and that amount was 2.06 pg. These findings will contribute to the detailed cytogenetic assessment of Hibiscus and thus benefit plant breeding in this genus.


Introduction
Hibiscus a polymorphic genus of the Malvaceae family include approximately 300 species of trees, shrubs, and herbs growing in tropical, subtropical, and temperate regions around the world (Mohammad et al. 2020). There are as many as 350 varieties of the shrub Hibiscus syriacus also known as Rose of Sharon or Althea, which has a flowering period from 60 to 120 days usually blooming starts from last of June to mid of October (Lee 2013). They are categorized as single, semi double or double flowers depending on the number and arrangement of the petals. H. sinosyriacus is a deciduous, perennial, winter hardy shrub that is very similar to H. syriacus. The H. sinosyriacus cultivar 'Malmauve' cultivated in France in Abstract Determination of nuclear DNA content, genome size, and ploidy level and, information on cytogenetic characteristics are all prerequisite of modern plant breeding. However, identification of individual chromosomes of Hibiscus species is extremely difficult due to high number, small size and similar shape of mitotic chromosomes. The goal of the study was to ascertain the chromosome number, karyomorphology, distribution of 5S and 18S rDNA signals, chromosome length, and centromere positions as well as the ploidy level, genome sizes, 2C -DNA content of winter-hardy Hibiscus (H. syriacus 'Saejamyung', H. sinosyriacus 'Seobong', H. moscheutos 'Luna Red' and, H. paramutabilis). 5S rDNA and 18S rDNA signals were detected by fluorescence in situ hybridization (FISH). According to the FISH results, there are two 5S rDNA signals (green) in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA signals in H. paramutabilis. The range 2001, was introduced in Korea in 2003 and named as 'Seobong' (Jo et al. 2019). Hibiscus moscheutos L., often called swamp rose-mallow, is winter-hardy herbaceous, plant native to wetland areas in eastern North America. It has long been bred for its compact stature and large flowers in various in colors and shapes (Li and Ruter 2017). Hibiscus paramutabilis is a hardy, semi-woody, deciduous shrubs; compare with H. syriacus and H. sinosyriacus, it has larger, round shape leaves (Bates 1965).
In order to carry out an efficient plant breeding system, it is important to know beforehand the chromosome number, ploidy level, and DNA contents of the species (Sakhanokho et al. 2020). However, contained in the Hibiscus genus are a wide variety of plants with complex interspecific relationships (Li et al. 2015). Basic chromosome number is one of the most important features of the taxonomic genus. Notwithstanding, Hibiscus species with varied basic chromosome numbers (7 -44) have a wide range of ploidy levels, from diploid to sixteen-ploidy and total chromosome number from 22 to 180 (Fryxell 1988). Therefore, the basic chromosome number, ploidy levels, and total chromosome numbers of Hibiscus species are still in the disputes. Fluorescence in situ hybridization (FISH) and karyotyping are efficient techniques for cytogenetic studies (Anamthawat-Jónsson 2004;Liang and Chen 2015). FISH results can provide basic information on the ploidy level, and chromosome characteristics of each species. Moreover, the location of ribosomal DNA of 5S, 18S, 25S, and 45S loci has been widely used to study the genetic relationships among various plant species (Sakhanokho et al. 2020). Using FISH, oligos specific to a repetitive sequence or to a particular genomic region can be visualized . Karyotype analysis of plants with a high number of chromosomes and small chromosomes is labor-intensive and difficult due to the differences in the physical features of the homologues and similarities of chromosomes in a complement . Moreover, genome size is a significant character of living organisms and estimates of genome size have been useful in systematic and evolutionary studies (Knight et al. 2005). In most plant species, complete and accurate genetic information is shared with the DNA content changed by ploidy level, while the change in DNA consistency and ploidy degree are positively correlated. In case of H. syriacus, DNA content was 4.63 pg/2C for tetraploid cultivar 'Blue Bird' whereas, it was 7.05 pg/2C for the hexaploid cutivar 'Aprodite' (Lattier et al. 2019). Determination of genome size can be helpful in elucidating the relationship between the Hibiscus species, although very few studies of genome size or nuclear DNA contents of Hibiscus have been published. We therefore aimed to determine chromosome number, karyomorphology, and 5 and 18S rDNA loci mapping, nuclear DNA content, genome size, and ploidy level, of H. syriacus 'Saejamyung', H. sinosyriacus 'Seobong', H. moscheutos 'Luna red' and, H. paramutabilis.

Plant materials
Hibiscus syriacus 'Saejamyung', H. sinosyriacus 'Seobong', H. moscheutos 'Luna Red' and H. paramutabilis were used for this experiment. These plants are grown in greenhouse and field conditions at Kyungpook National University, Daegu, Republic of Korea.

Root collection and chromosome preparation
Stem of H. syriacus 'Saejamyung', H. sinosyriacus 'Seobong', H. moscheutos 'Luna Red' and H. paramutabilis were cut keeping minimum two buds and planted in rockwool media using rooting hormone (Rhizopone AA). Cutting stems were nurtured in a greenhouse at a temperature of 25 °C during the day and 20 °C at night, with a humidity of 60%. After two and half months regular watering and observation, media allowed to spread roots. In early in the morning, healthy roots tips were collected and treated with 2m 8-hydroxyquinoline solution for 3 -4 h at room temperature. The root tips were then washed with double-distilled water and transferred in an acetoethanol (1:3, v/v) solution to fix the roots overnight. After the roots were rinsed with distilled water, they were preserved in 70% ethanol at − 20 °C until further use. For slide preparation, the root tips were washed with distilled water to remove the ethanol solution and incubated with an enzyme mixture consisting of 0.3% pectolyase (Duchefa, Haarlem, The Netherlands), 0.3% cellulose (Duchefa), and 0.3% cytohelicase (Sigma, St. Louis, MO, USA) at 37 °C Page 3 of 12 81 Vol.: (0123456789) for 40 min. The digested root tips were then transferred to a clean slide one by one and only milky part of roots were kept and other debris were removed with the help of needles. 20 µL acetic acid (60%) was added per slide to spread the cells, and the slides were air-dried. Finally, slides were checked and location of well spread chromosomes were recorded in top parts of slides.
Fluorescence in situ hybridization (FISH) FISH analysis was conducted according to Lim et al. (2001) with a few modifications. Briefly, slides were pre-treated with 100 µg mL −1 RNase A at 37 °C for 50 min. After rinsing with 2× SSC and fixing with 4% paraformaldehyde for 10 min, the slides were rinsed again with 2× SSC and dehydrated in an ethanol series (70, 90, and 100%), followed by air-drying. The hybridization mixture contained formamide, 50% dextran sulfate, 20× SSC, 10% sodium dodecyl sulfate, herring sperm DNA, and rDNA probes. The mixture was placed in a water bath for DNA denaturation at 70 °C for 5 min and then placed on ice for 15 min for fixation. Forty microliters of the mixture were added to each slide, and a cover slip was placed on the slide, while ensuring there were no bubbles. The slides were placed in a water bath for hybridization at 80 °C for 5 min and then placed in a container with wet tissue paper and incubated in a humid chamber at 37 °C for 16 h. The slides were then washed with 2× SSC buffer for 5 min followed by 0.1× SSC buffer for 30 min at 42 °C with shaking and 2× SSC buffer for 5 min. The slides were then submerged in 1× detection buffer for 5 min. Streptavidin CY3 (Invitrogen, Carlsbad, CA, USA) and anti-digoxigenin fluorescein (Roche, Basel, Switzerland) were used to detect the labeled chromosomes. The slides with cover-slips were placed in a humid chamber to incubate at 37 °C for 50 min. Three jars containing 1× detection buffer was incubated in the dark in a water bath at 37 °C for 5 min followed by dehydration an ethanol series (70, 90, and 100%). The slides were then air-dried in the dark and counterstained with 4′6-diamidino-2-phenylindole (DAPI) and Vectashield at a 2:100 ratio (Vector Laboratories, Burlingame, CA, USA). The prepared slides were examined with a model Nikon BX 61 fluorescence microscope (Tokyo, Japan). Probe signals were analyzed using ultraviolet excitation filters. Cytovision and imaging software were used to acquire images of the chromosomes with 5S rDNA and 18S rDNA signals.

Karyotype analysis
For the karyotype analysis, five cells showing wellspread metaphase chromosomes were selected. The length of each chromosome was measured using software (Cytovision), and chromosomes were ordered and numbers assigned based on short arm length (Lim et al. 2001). Chromosome types were classified according to the ratio of the short arm to the long arm (Levan, 1964). The images of the chromosomes were captured with and 600× and 1000× magnification.
Flow cytometry analysis DNA content and genome sizes were measured using flow cytometry (Partec PA, Ploidy Analyzer, Sysmex, Kobe, Japan) with five randomly collected recently expanded leaves. Leaves were cut to an area approximately 1 cm 2 and chopped in 500 µL of extraction buffer solution (Sysmex) in a petri dish and shaken for 30 s. The resulting solution was poured through a 30-µm nylon mesh filter into a 3-mL tube and 2 µL DAPI containing staining buffer was added. Finally, the nuclei suspension was injected into the flow cytometry analyzer. 2C genome sizes were calculated as 2C = DNA content of standard × mean fluorescence value of sample / mean fluorescence value of standard (Greilhuber et al. 2005). Raphanus sativus cv Tetra Ilowiecka (2C = 2.00 pg).

Chromosome counting
We performed photomicrographs of 45 well spread cells from each species to confirm the chromosome number. We clearly detected 88 chromosomes in H. syriacus 'Saejamyung' while H. sinosyriacus 'Seobong' has 80 chromosomes ( Figs. 1 and 2). Among the four species, H. moscheutos 'Luna Red' had the least number of chromosomes 38 whereas, 82 chromosomes are found in H. paramutabilis (Fig. 2). Chromosomal localization of 5S rDNA and 18S rDNA sites Two 5S rDNA loci (green fluorescence) were found in H. syriacus, H. sinosyriacus, and H. moscheutos, and four 5S rDNA were detected in H. paramutabilis ( Fig. 3; Table 1). Six 18S rDNA loci (red fluorescence) were found in H. sinosyriacus and H. moscheutos, and four and ten loci were noticed in H. syriacus and H. paramutabilis respectively ( Fig. 3; Table 1). The distribution pattern of 5S rDNA loci for H. syriacus, H. sinosyriacus, H. moscheutos and H. paramutabilis were in the long arm of chromosome number #19 ( Fig. 4a; Table 1), the long arm of chromosome #6 (Fig. 4b; Table 1), the short arm of chromosome number #10 ( Fig. 4c; Table 1) and, the long arm of chromosome number (Fig. 4d; Table 1) respectively. In contrast, 18S rDNA loci were distributed in different locations of the chromosomes. Among four rDNA signals of H. syriacus, we found two signals in the long arm in chromosomes number #8 and two were found in the short arm in chromosomes number #18 ( Fig. 4a; Table 1). In H. sinosyriacus, we detected four 18S signals in long arms of chromosome number #6, #8 and two signals were in the short arm of chromosome number #11 ( Fig. 4b; Table 1). In the case of H. moscheutos, all 18S rDNA signals were in the long arm of the chromosome numbers #1, #7, and #13 ( Fig. 4c; Table 1). The highest number of 18 rDNA signals was found in H. paramuatabilis; among ten signals, eight were in the short arm of chromosomes #5 and #13 and two are in the centromere position in the chromosome numbers #3 ( Fig. 4d; Table 1).

Karyomorphological analysis
The chromosomes were arranged according to decreasing order of short arm lengths. An ideogram of the four species is presented in Fig. 4. The number of chromosomes complements of are H. syriacus 88 (Fig. 4a), and H. sinosyriacus 80 (Fig. 4b), with metaphase chromosome lengths ranging from 2.66 ± 0.02 to 7.06 ± 0.30 μm and from 3.18 ± 0.29 to 7.31 ± 0.25 μm, respectively. Among the four species, H. moscheutos 2n = 38 has the shortest range of chromosome length (2.91-5.23 μm) whereas, H. paramutabilis (Fig. 4d) has the longest-range chromosome length, from 4.75 ± 0.55 to 7.60 ± 0.47 μm. Moreover, we found that the last two chromosomes were in a pair and placed in chromosome #21 (Fig. 4d). Depending on the centromere positions, the homologous chromosomes are composed of metacentric and sub metacentric pairs, no telocentric or sub telocentric chromosome were detected except H. moscheutos. 12 pairs of metacentric, 6 pairs of sub metacentric and one pair of sub telocentric chromosomes were detected in H. moscheutos. In H. syriacus and H. sinosyriacus, the majority of chromosome pairs are metacentric, with numbers were 13 and 12, respectively, while 9 and 8 were sub metacentric accordingly. In contrast, the chromosomes of H. paramutabilis were composed of 14 sub metacentric and 7 of metacentric chromosomes ( Fig. 4; Table 2).   (Table 3).

Chromosome counts
Basic chromosome number is one of the key features of cytogenetic characteristics of any genus. However, Hibiscus species have a high number of small chromosomes and the basic number of chromosomes of most species are not yet determined. The range in the basic chromosome number is wide 7/8/9/10/11/12/13/ 14/15/16/17/18/19/20/21/23/24/39/40/44. Ploidy levels in this genus from diploid to sixteen-ploidy and total chromosome number from 22 to 180 (Fryxell 1988;Lattier et al. 2019). In our experiment, we found 88 chromosomes of H. syriacus 'Saejamyung' and 80 chromosomes of H. sinosyriacus 'Seobong', which is consistent with a previous result for H. sinosyriacus (80 chromosome) (Skovsted 1941;Jo et al. 2019) however, not for H. syriacus. We noticed that there are over 150 cultivars of H. syriacus, with chromosomal numbers and ploidy levels that are highly varied (unpublished data). There is a lot of variation in Old World lupins (Lupinus), not only for chromosomal counts (2n = 32 -52), but also for the basic chromosome number (x = 5 -9, 13) (Susek et al. 2019). In the present study we report the chromosome count result of 'Saejamyung' for the first time. Pedigree of that cultivar is not determined. Its ploidy may term as hypertetraploid. It may happen for unreduced gamete or failure of chromosome pairing during meiosis (Karlov et al. 1999;Lee et al. 2020). H. moscheutos and, H. paramutabilis were found to have a chromosome number of 38 and 82 respectively which is consistent with reports in the Chromosomal localization of 5S rDNA and 18S rDNA loci FISH is a molecular cytogenetic technique in which fluorescent-label probes are used to classify complementary DNA sequence in nuclei (Speicher and Carter 2005); the technique has been shown to be an efficient technique for cytogenetic study of woody angiosperms (Prado et al., 1996). The use of rDNA signals, combined with flow cytometry, has proved meaningful for confirmation of ploidy levels in Hibiscus a genus characterized by numerous small chromosomes and tolerance of high level of polyploids (Lattier et al. 2019). In our results, two 5S rDNA loci were in each of H. syriacus 'Saejamuyng' H. sinosyriacus 'Seobong' and H. moscheutos 'Luna Red', whereas four 5S rDNA loci were seen in H. paramutabilis. A study of 5S rDNA in cotton plants (close relatives to Hibiscus) revealed that most diploid had two 5S rDNA signals and all allotetraploid species had four 5S rDNA signals (Gan et al. 2013); the same result was found in woody species of genus Rubus (Wang et al. 2015). A recent FISH analysis of tetraploid H. sabdaiffa var. 'sabdariffa' (Mohammad et al. 2020) resulted in the identification of four 5S rDNA loci. The study of only two 5S rDNA loci in tetraploid H. syriacus and H. sinosyriacus could provide evidence being diploid however, recent genome analysis confirmed their polyploidy status after multiple occurrence of whole-genome duplication followed by polyploidization after speciation (Kim et al. 2017). In addition, among-ploidy level variation in rDNA loci, it is possible to have variation within the same ploidy for a species. The reduced copy number and interstitial of 5S rDNA often observed in flowering plants (Nguyen et al. 2021). Species with the same chromosome number have been found to have up to a five-fold difference in rDNA loci in a comparative analysis of species in Brassicaceae (Hasterok et al. 2006

Karyomorphology of chromosomes
Karyotyping is a useful tool for detecting chromosome variation and is also helpful to interpret in constructing of genetic map (Chung et al. 2018). Karyotype is considered to be the prime cytological trait employed in empirical and theoretical research (Liang and Chen 2015). In karyotype analysis, the difference within each species or cultivar may indicate changes in chromosomes arrangement. According to the finding in the present study, the average chromosomes length of Hibiscus spp. is 2 to 8 μm. The chromosome length of H. mutabilis f. mutabilis were recorded 1.24 to 10.89 μm which were slightly higher than our studies (Li et al. 2015). Moreover, small chromosome studies such as chromosomes length of the investigated chrysanthemum species were 9.70 to 12.24 μm, and 9.02 to 13.37 μm for Chrysanthemum boreale, C. makinoi respectively (Hoang et al. 2020). The karyotype studies of Populus species (woody plants having small chromosomes) found that relative chromosome length of P. trichocarpa is 4.30 to 11.02 and P. euphratica is 4.10 to 10.28 (Xin et al. 2020). Depending upon the position of the centromere, the chromosomes  (Li et al. 2015).
Nuclear DNA content analysis Flow cytometry offers a quick, precise and simple way to determine nuclear DNA content (C-value) of plants (Galbraith, 2009). The technique facilitates the characterization of plants species in natural and agricultural settings, allows easy identification of ploidy level, and is responsive to the environment in term of evolutionary fitness. Despite, the feasibility of flow cytometry methods, C-values have been calculated for only approximately 2% of the described angiosperm species (Galbraith 2009