Physical Map of FISH 5S rDNA and (AG3T3)3 Signals Displays Chimonanthus campanulatus R.H. Chang & C.S. Ding Chromosomes, Reproduces its Metaphase Dynamics and Distinguishes Its Chromosomes

Chimonanthus campanulatus R.H. Chang & C.S. Ding is a good horticultural tree because of its beautiful yellow flowers and evergreen leaves. In this study, fluorescence in situ hybridization (FISH) was used to analyse mitotic metaphase chromosomes of Ch. campanulatus with 5S rDNA and (AG3T3)3 oligonucleotides. Twenty-two small chromosomes were observed. Weak 5S rDNA signals were observed only in proximal regions of two chromosomes, which were adjacent to the (AG3T3)3 proximal signals. Weak (AG3T3)3 signals were observed on both chromosome ends, which enabled accurate chromosome counts. A pair of satellite bodies was observed. (AG3T3)3 signals displayed quite high diversity, changing in intensity from weak to very strong as follows: far away from the chromosome ends (satellites), ends, subtelomeric regions, and proximal regions. Ten high-quality spreads revealed metaphase dynamics from the beginning to the end and the transition to anaphase. Chromosomes gradually grew larger and thicker into linked chromatids, which grew more significantly in width than in length. Based on the combination of 5S rDNA and (AG3T3)3 signal patterns, ten chromosomes were exclusively distinguished, and the remaining twelve chromosomes were divided into two distinct groups. Our physical map, which can reproduce dynamic metaphase progression and distinguish chromosomes, will powerfully guide cytogenetic research on Chimonanthus and other trees.

Chen [10] studied the biosystematics of species in the genus Chimonanthus and inferred a close relationship between Ch. campanulatus and Ch. praecox. Dai [11] analysed the phylogeography, phylogeny and genetic diversity of species in the genus Chimonanthus by inter-simple sequence repeats (ISSRs), chloroplast DNA (trnL-F, trnS-G, and trnH-psbA), and the internal transcribed spacer (ITS) and speculated that current populations evolved independently in their respective refuges, e.g., Ch. campanulatus in Yunnan. Shu et al. [12] reviewed the non-volatile components and pharmacology of species in the genus Chimonanthus and found no reports on Ch. campanulatus. Similarly, other types of studies on Ch. campanulatus are also quite scarce.
Since the number of studies on Ch. campanulatus is currently quite small, in this study, fluorescence in situ hybridization (FISH) was used to analyse mitotic metaphase chromosomes of Ch. campanulatus with the oligonucleotides 5S rDNA and (AG 3 T 3 ) 3 . The aim was to construct a physical map and distinguish the chromosomes of Ch. campanulatus, which will aid in molecular genetic map construction and pharmacological studies in Ch. campanulatus.

Chromosomes and Probe Preparation
Seeds of Ch. campanulatus R.H. Chang & C.S. Ding were collected from Chengdu Botanical Garden and germinated in wet sand at room temperature (15-25 • C). When the roots reached a length of 2 cm, the root tips were excised and immediately treated with nitrous oxide for four hours. Later, the root tips were transferred to 100% acetic acid for 5 min. Next, the meristems of the root tips were treated with cellulase and pectinase (1 mL buffer + 0.04 g cellulase + 0.02 g pectinase, the buffer 50 mL was included 0.5707 g trisodium citrate + 0.4324 g citric acid), which were produced by Yakult Pharmaceutical Ind. Co., Ltd. (Tokyo, Japan) and Kyowa Chemical Products Co., Ltd. (Osaka, Japan), and then placed into suspension for dropping onto slides. Finally, air-dried slides were examined using an Olympus CX23 microscope (Olympus, Japan), and high-quality spreads were further used in a follow-up experiment.
Two oligonucleotides, namely, 5S rDNA [62] and (AG 3 T 3 ) 3 [63], were used in this study. The probes were synthesized by Sangon Biotechnology Limited Corporation (Shanghai, China), and their 5 ends were labelled by carboxyfluorescein (FAM) or carboxytetramethylrhodamine (TAMRA). The synthetic probes were dissolved in ddH 2 O and maintained at a concentration of 10 µM.
The slides were examined by an Olympus BX63 fluorescence microscope equipped with a Photometric SenSys Olympus DP70 CCD camera (Olympus, Japan). Approximately 60 metaphases from ten slides of ten Ch. campanulatus root tips were observed in this study. Greater than 30 metaphases in which the chromosomes were well separated were selected to count the chromosome number. Ten better spreads were used for karyotype analysis. Single chromosomes were isolated using Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA, USA), and each spread was measured three times to provide consistent karyotype data. Chromosomes in the physical maps were aligned based on length and signal patterns. Karyotype idiograms were constructed in Excel 2019 and PowerPoint 2019 based on the relative chromosome lengths.

S rDNA and (AG 3 T 3 ) 3 Enabled Visualization of Ch. Campanulatus Chromosomes
Ten high-quality spreads of mitotic metaphase chromosomes of Ch. campanulatus after FISH are illustrated in Figure 1 Figure 4g-i), which exhibited dynamic metaphase progression from the preliminary stage to the final metaphase and confirmed the repeatability and stability of our FISH results. In total, twenty-two chromosomes were counted in each spread except the last spread, as shown in Figure 4g-i (twenty-four chromosomes). In the last spread, chromosomes were at the end of metaphase to the beginning of anaphase, with twenty chromosomes were preparing to split (chromatids) and two chromosomes had already split into four chromosomes (yellow arrows in Figure 4g Figure 5 also shows the lengths of the longest (1) and shortest chromosomes (22) in the ten Ch. campanulatus spreads originally shown in Figure 1a,d,g, Figure 2a,d, Figure 3a,d, and Figure 4a,d,g, with the lengths ranging from 1. 66- 1.07 µM, 1.83-1.21 µM, 1.84-1.21 µM, 1.90-1.21 µM, 1.93-1.34 µM, 2.14-1.36 µM, 2.21-1.49 µM, 2.41-1.53 µM, 2.24-1.65 µM, and 2.21-1.41 µM, respectively. There was a very significant difference in chromosome length among these ten spreads (p = 0.00019), revealing dynamic chromosome growth in terms of length from the preliminary stage to the end of metaphase. The longest chromosome was 2.41 µM long, i.e., still less than 3 µM; hence, all of the chromosomes were small chromosomes. Due to the small size and unclear centromere locations of the chromosomes, it was difficult to determine the long arms and short arms, and karyotype analysis was not performed further. Figure 5 further displays a conserved 5S rDNA signal distribution and a diverse (AG 3 T 3 ) 3 signal distribution for Ch. campanulatus. Weak 5S rDNA signals were observed only in proximal regions of two chromosomes (7/8) (red colour in Figure 1a,c,d,f,g,i, Figure 2a,c,d,f, Figure 3a,c,d,f, Figure 4a,c,d,f,g,i, and Figure 5), which were adjacent to the (AG 3 T 3 ) 3 proximal signals on these two chromosomes. Weak (AG 3 T 3 ) 3 signals were observed on both chromosome ends (green colour in Figure 1b

Physical Map Reproduced Metaphase Dynamics and Distinguished Chromosomes
To better describe the chromosomes of Ch. campanulatus, the chromosome hybridizations with 5S rDNA and (AG 3 T 3 ) 3 shown in Figure 5 were separated to create Figure 6. The metaphase dynamic idiograms on the right were constructed based on the metaphase dynamics of chromosomes on the left. Although this dynamic metaphase progression is not a novel mitotic phenomenon, it still provides the first visualization of the dynamic metaphase progression of Ch. campanulatus, from the preliminary stage to the end of metaphase and from single-chromosome duplication to the linkage or splitting of chromosomes. In addition, obvious satellite bodies gradually moved from far away from the arm ends to close to the arm ends from the beginning to the end of metaphase.
Furthermore, idiograms of the seven types of 5S rDNA and (AG 3 T 3 ) 3 signal patterns observed for Ch. campanulatus chromosomes (top of Figure 6) were constructed: Type I: Weak (AG 3 T 3 ) 3 signals on both chromosome ends were observed for six chromosomes (11/12/17/18/19/20). Type II: Weak (AG 3 T 3 ) 3 signals on both chromosome ends and in the proximal regions were observed for two chromosomes (1/2). Type III: Weak (AG 3 T 3 ) 3 signals on both chromosome ends and strong (AG 3 T 3 ) 3 signals in the distal regions were observed for two chromosomes (15/16). A peculiar phenomenon was observed for these two chromosomes: Satellite bodies were shown by upper-end signals located far away from the designated chromosome ends. Type IV: Weak (AG 3 T 3 ) 3 signals on both chromosome ends and very strong (AG 3 T 3 ) 3 signals in the subtelomeric regions (adjacent to the end signals) were observed for two chromosomes (13/14). Type V: Weak (AG 3 T 3 ) 3 signals on both chromosome ends and very strong (AG 3 T 3 ) 3 signals in the subtelomeric regions (adjacent to the end signals) were observed for the two shortest chromosomes (21/22). Type VI: Weak (AG 3 T 3 ) 3 signals on both chromosome ends and very strong (AG 3 T 3 ) 3 signals in the proximal regions were observed for six chromosomes (3/4/5/6/9/10). Type VII: Weak (AG 3 T 3 ) 3 signals on both chromosome ends and very strong (AG 3 T 3 ) 3 signals in the proximal regions, as well as 5S rDNA signals adjacent to proximal (AG 3 T 3 ) 3 signals, were observed for two chromosomes (7/8). In contrast to types I and VI, which included six chromosomes, types II, III, IV, V, and VII each included only two chromosomes. Therefore, 5S rDNA and (AG 3 T 3 ) 3 signal patterns exclusively distinguished ten chromosomes, namely, 1/2/7/8/13/14/15/16/21/22, and divided the other twelve chromosomes into two obvious groups, namely, 3/4/5/6/9/10 and 11/12/17/18/19/20. The physical map of Ch. campanulatus convincingly demonstrated that (AG 3 T 3 ) 3 not only labelled chromosome ends (including satellite bodies), which are used to count chromosome numbers, but also labelled chromosome proximal regions and subtelomeric regions with obviously different signal intensities, which are used as effective FISH markers for cytogenetic analysis.   In summary, Figure 7 shows the refined metaphase dynamics and signal patterns of Ch. campanulatus chromosomes isolated from Figure 6. Chromosome 20, represented by ten spreads (visualized in Figure 1c,f,i, Figure 2c,f, Figure 3c,f, Figure 4c,f,i), presented dynamic metaphase progression from the beginning to the end. Chromosomes gradually grew larger and wider to form linked chromatids, which grew more significantly in width than in length ( Figure 7A). The chromosomes shown in Figure 1c exhibited seven types of signal patterns. The intensity of (AG 3 T 3 ) 3 signals showed quite high diversity, ranging from weak to very strong as follows: far away from the ends (satellite bodies), ends, subtelomeric regions, and proximal regions ( Figure 7B). Based on a combination of 5S rDNA and (AG 3 T 3 ) 3 signal patterns, ten chromosomes were exclusively distinguished, and the remaining twelve chromosomes were divided into two obvious groups. In the future, we will explore more oligonucleotides to discern the twelve chromosomes categorized as types I and VII.

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The length of the longest chromosome in Ch. campanulatus was approximately equal to that in Z. Figure 7. Dynamic metaphase progression and signal patterns of Chimonanthus campanulatus chromosomes isolated from Figure 6. The left panel ( Figure 7A) shows chromosome 20 in ten spreads to present metaphase dynamics (Figure 1c,f,i, Figure 2c,f, Figure 3c,f, Figure 4c,f,i). The right panel ( Figure 7B) uses the 22 chromosomes in Figure 1c to demonstrate signal patterns. The signal intensity of the distal repeat probe (AG 3 T 3 ) 3 was variable, ranging from weak on the chromosome ends to strong in the proximal regions.

Karyotype Analysis and Dynamic Metaphase Progression
Ten high-quality spreads displayed a very significant difference in chromosome length, which revealed chromosome dynamic growth in terms of length from the preliminary stage to the end of metaphase and transition to anaphase. The corresponding physical map of chromosomes reproduced the dynamic mitotic metaphase progression in Ch. campanulatus for the first time. Similarly, Matsui [64] examined dynamic changes in proteins during mitotic metaphase, anaphase, and telophase in human epithelial HeLa S3 cell populations to reproduce dynamic mitotic progression. The length of Ch. campanulatus chromosomes was 1.07-2.41 µM in this study. In previous works, chromosome lengths were reported to be 1.05-1.81 µM in Ligustrum lucidum Lindl. [ [70], and 9-13 µM in Triticum aestivum L. 'Chinese Spring' [71]. In our unpublished works, chromosome lengths were recorded to be 0.97-2. In this study, twenty-two chromosomes were observed in Ch. campanulatus, which was in agreement with the numbers reported in previous works [2,3]. Similar to the results from Liu et al. [3], one pair of satellite chromosomes was clearly observed in this study. Satellite bodies, as hereditary features, may be used to distinguish species [72,73]. The other five species in the genus Chimonanthus (Ch. grammatus, Ch. nitens, Ch. nitens var. salicifolius, Ch. praecox, and Ch. zhejiangensis) do not possess satellite bodies, based on the findings of previous works [3][4][5], revealing relatively distant relationships between Ch. campanulatus and the other five Chimonanthus species. However, in the same family (Calycanthaceae) but a different genus (Calycanthus), the species Ca. chinensis and Ca. occidentalis possess one pair of satellite chromosomes and twenty-two chromosomes [6,7], indicating moderately close relationships between these two species and Ch. campanulatus.

Distinguishing Ch. campanulatus chromosomes
No FISH technology has been previously applied in Chimonanthus species. Here, (AG 3 T 3 ) 3 and 5S rDNA were tested in Ch. campanulatus for the first time. In contrast to 5S rDNA, (AG 3 T 3 ) 3 generally labels only chromosome ends, rendering it a less effective FISH marker for distinguishing chromosomes [65,69,74]. However, in this study, (AG 3 T 3 ) 3 was an excellent marker because it distinguished Ch. campanulatus chromosomes based on its location: far away from chromosome ends (satellite bodies), ends, subtelomeric regions and proximal regions; in this order, its intensity ranged from weak to strong and very strong. Such high diversity in (AG 3 T 3 ) 3 signal intensity has rarely been found in other species, but this oligonucleotide was found to locate to the proximal region of chromosomes in Zea mays L. [63], Podocarpus L. Her. ex Persoon species [75], and Philodendron Schott species [76].
In this study, (AG 3 T 3 ) 3 labelling of satellite bodies in Ch. campanulatus was reported for the first time. Satellite bodies of B. diaphana are a quarter of the chromosome size (~0.6 µM) and are labelled at the ends by (AG 3 T 3 ) 3 [69]. Satellite bodies are often connected to the main body of the chromosome by very lightly staining strands [72]. These bodies vary in size according to the position of the secondary constriction. If the secondary constriction is very close to an end of the chromosome, the satellite may be a barely perceptible dot [73]. In this study, the satellite bodies were small, barely perceptible dots (~0.1 µM) located far away from their designated ends and were obvious based on their green (AG 3 T 3 ) 3 signals. Hence, (AG 3 T 3 ) 3 may aid in the visualization of such unobservable satellite bodies.

Conclusions
In this study, based on the combination of (AG 3 T 3 ) 3 and 5S rDNA signal patterns, only ten chromosomes were exclusively discerned among the twenty-two chromosomes of Ch. campanulatus. The remaining twelve chromosomes were divided into two obvious groups. It is necessary to explore more oligonucleotide probes to further distinguish Ch. campanulatus chromosomes and establish detailed physical maps. writing-review and editing, X.L.; visualization, J.C.; supervision, X.L.; project administration, X.L.; funding acquisition, X.L. All authors consented to this submission.
Funding: This research was funded by the Natural Science Foundation of China (grant number 31500993).