Next Article in Journal
Genome-Wide Identification and Comprehensive Analysis of the AP2/ERF Gene Family in Pomegranate Fruit Development and Postharvest Preservation
Previous Article in Journal
New Insights into the Neuromyogenic Spectrum of a Gain of Function Mutation in SPTLC1
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 13
Issue 5
10.3390/genes13050894
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions

by
Jun He
,
Yong Zhao
,
Shuangshuang Zhang
,
Yanze He
,
Jiafu Jiang
,
Sumei Chen
,
Weimin Fang
,
Zhiyong Guan
,
Yuan Liao
,
Zhenxing Wang
,
Fadi Chen
and
Haibin Wang
*
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Genes 2022, 13(5), 894; https://doi.org/10.3390/genes13050894
Submission received: 16 March 2022 / Revised: 30 April 2022 / Accepted: 6 May 2022 / Published: 17 May 2022
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
Review Reports Versions Notes

Abstract

:
Ribosomal DNA (rDNA) is an excellent cytogenetic marker owing to its tandem arrangement and high copy numbers. However, comparative studies have focused more on the number of rDNA site variations within the Chrysanthemum genus, and studies on the types of rDNA sites with the same experimental procedures at the species levels are lacking. To further explore the number and types of rDNA site variations, we combined related data to draw ideograms of the rDNA sites of Chrysanthemum accessions using oligonucleotide fluorescence in situ hybridization (Oligo-FISH). Latent variations (such as polymorphisms of 45S rDNA sites and co-localized 5S-45S rDNA) also occurred among the investigated accessions. Meanwhile, a significant correlation was observed between the number of 5S rDNA sites and chromosome number. Additionally, the clumped and concentrated geographical distribution of different ploidy Chrysanthemum accessions may significantly promote the karyotype evolution. Based on the results above, we identified the formation mechanism of rDNA variations. Furthermore, these findings may provide a reliable method to examine the sites and number of rDNA variations among Chrysanthemum and its related accessions and allow researchers to further understand the evolutionary and phylogenetic relationships of the Chrysanthemum genus.

1. Introduction

Asteraceae (Compositae) is one of the most prominent angiosperm families in herbaceous plants worldwide, containing 1600 genera [1]. Significantly, the Anthemideae tribe belonging to Asteraceae is a large and widespread group of flowering plants mainly distributed in Central Asia, the Mediterranean Basin, and Southern Africa [2]. Especially the cultivated chrysanthemums among the Chrysanthemum genus (Asteraceae, Anthemideae tribe), as some of the most widely marketed cut flowers globally, are popular owing to their wide variety of flower variations in size, color, and shape; long vase-life; early production; and photoperiod responses [3]. In addition, other species in the Chrysanthemum genus, primarily native to Asia, are essential germplasm resources for cultivated chrysanthemum (Chrysanthemum morifolium) and contribute to increasing its aesthetic value [4,5]. Consequently, promoting studies on the origin and molecular cytogenetics of wild Chrysanthemum accessions may further aid the understanding of phylogenetic and species relationships [6].
Repetitive DNA occupies a significant fraction of the nuclear genome in higher eukaryotes and contributes significantly to the plant chromosome structure [7,8]. Turnover of this repetitive DNA occurs across the genome with repetitive DNA variations, such as amplification, deletion, transposition, and mutation. This may be associated with population divergence and speciation processes. Ribosomal DNA (rDNA), a type of repetitive DNA, is highly conserved and ubiquitously present in plants under natural selection pressure [9]. The rDNA has two distinct gene classes in higher eukaryotes: the primary 45S rDNA cluster encoding 28S (26S), 5.8S, and 18S rRNA and the minor 5S rDNA cluster encoding 5S rRNA [4,10,11,12,13].
Consequently, 5S rDNA and 45S rDNA have been shown to be good cytogenetic markers by tandemly arranging and presenting in high copy numbers (up to several thousand copies) with different chromosomal distributions [14,15]. In addition, 5S rDNA and 45S rDNA are regarded as vital markers for fluorescence in situ hybridization (FISH) analysis in molecular phylogenetics and evolution. They are widely used in cytogenetic research, not only for essential crops [16,17], but also for horticultural plants, such as strawberry [18], cucumber [19], sweet potato [20], and chrysanthemum [4,11,21]. For example, the rDNA-FISH results for Taraxacum (Asteraceae) suggest a highly dynamic evolution of karyotypes among the genera owing to its polyploid apomictic events [22]. In the Rosa genus, rDNA probes were used to observe meiotic chromosome behavior and further the retention of substantial ancient rDNA sequences in genomes resonates with drastic allelic heterozygosity encountered in previous studies [23].
Compared with traditional FISH analysis, the novel oligonucleotide FISH (Oligo-FISH) technology is extensively utilized in related cytogenetic research because of its cost-effectiveness [24,25,26]. Moreover, based on high-throughput DNA sequencing data, oligonucleotide probes have been developed and successfully applied to many plants [27]. Therefore, examining the rDNA sites by Oligo-FISH would efficiently contribute to further cytogenetic research of the Chrysanthemum genus.
In a previous study, the amplification or deletion of rDNA arrays during the evolutionary process was observed, which represented the inter-individual fluctuations in several plant groups [28,29]. Studies have assessed the extent of rDNA variation at the sequence, site, and copy number levels in some accessions of the Chrysanthemum genus [2,3,4,6,21]. In addition, it has been previously reported that 45S rDNA sites may display intra-specific variations in size and number, and most variations are restricted to artificial hybrids, polyploid systems, and crop species [30,31]. These findings further illustrate the possibility for rDNA variations. However, comparative studies have focused more on rDNA site number variations among the Chrysanthemum genus [4,12,15,21,32,33] and explored the types of rDNA sites with the same experimental procedures at accession levels. Crucially, the 5S rDNA and pTa71 (45S rDNA) probes in the conventional FISH analysis contain some heterologous fragments, and non-specific sequences may result in background noise and interfere with the FISH results. In a previous study, prelabeled oligomer probes of 5S and 45S rDNA were designed based on the reference genome data of Chrysanthemum and its allied genera in the Asteraceae, and more credible FISH results were obtained [5].
In this study, we aimed to further explore the 5S and 45S rDNA sites by conducting the Oligo-FISH analysis among ten wild Chrysanthemum plants ranging from 2n = 18 (2x) to 2n = 54 (6x). Consequently, our specific aims were to (i) clarify the number and different types of 5S and 45S rDNA sites, (ii) analyze the number of rDNA sites associated with geographic distribution and ploidy, and (iii) identify the possible underlying mechanisms for the variation in rDNA sites.

2. Materials and Methods

2.1. Plant Materials

Chrysanthemum plants from diploid (2n = 2x = 18) to hexaploid (2n = 6x = 54) lines were maintained at the Chrysanthemum Germplasm Resource Preserving Center, Nanjing Agricultural University, China (32°05′ N, 118°8′ E, 58 m elevation) (Table 1). Ten investigated plant materials in this study were identified using cytogenetics and morphological analysis. Morphological identification of leaves and inflorescences (ray flowers and tubular flowers) was conducted separately during the periods of vegetative growth and blossoming based on the morphological descriptions in Flora of China. All plants were propagated through cuttings, and the medium contained a 2:2:1 (v/v) mixture of perlite, vermiculite, and leaf mold. Rooted seedlings were grown in a greenhouse at 22 °C during the day and at least 15 °C at night, with a relative humidity range of 70–75% under natural light.

2.2. Cytological Preparation and Multiplex Oligo-FISH

The chromosome spread preparations and Oligo-FISH analysis were performed as previously described [34,35,36] with minor modifications in chromosome preparations. The root tips from cutting seedlings measuring about 2 cm were taken, pretreated in 0.2 μmol/L amiprophos-methyl solutions (APM A0185, DuchefaBiochemie, The Netherlands) for three hours, and washed thrice using tap water. Next, the roots were excised, placed in 1.5 mL Eppendorf tubes, and treated with N2O at 1.0 MPa for one hour. The root tips were transferred to 90% acetic acid for 10 min on an icebox and then stored in 70% ethanol at −20 °C until their use in chromosome preparations. After cell spreads were dried on slides, then they were subjected to UV-crosslinked treatment (total energy, 120 mJ/cm2). Then, at the center of the cell spreads, 10.0 μL hybridization solution per spread containing 1.0 μL 5S rDNA probe (0.55 ng/μL), 1.0 μL 45S rDNA probe (0.55 ng/μL), and 8.0 μL buffer (equal amount of 1 × TE and 2 × SSC) was dropped. After the application of a plastic coverslip, the slide preparation was denatured by being placed on a wet paper towel in an aluminum tray floating in boiling water (100 °C) for 5 min in dark conditions. Finally, the slides were incubated at 55 °C overnight in a humidity chamber containing 2 × SSC soaked paper toweling. The next day, slides were washed in 2 × SSC for 5 min at room temperature. After drying, the slides were mounted with DAPI mounting medium (H-1200, Vector Laboratories, Burlingame, CA, USA) while we waited to make observations.
The oligonucleotide probes of 5S and 45S rDNA were developed according to the method described by Waminal et al. [37] and then synthesized by General Biosystems (Chuzhou, Anhui province, China), and the sequences were as previously described by He et al. [5]. Finally, the chromosomes were visualized with an Olympus BX60 microscope (Olympus, Tokyo, Japan). All synthetic oligonucleotide probes for 5S rDNA and 45S rDNA emerged as distinct fluorescent signals at a 0.55 ng/μL concentration.

2.3. Karyotyping Analysis

Images were captured using a SPOT CCD camera (SPOT Cooled Color Digital, Olympus DP72, Tokyo, Japan). Then, multi-color component images were merged using Cellsens Dimension software (version 1.6). For karyotyping, 3–5 cells from each accession were observed, and karyotypes were generally obtained from a single cell. Otherwise, they were sampled from 1 to 4 cells because of overlapping chromosomes.
Furthermore, chromosome measurements based on Oligo-FISH results and idiograms were obtained using the computer software KaryoMeasure [38]. Finally, the idiograms of the investigated materials were integrated using Photoshop (2021) (Adobe, San Jose, CA, USA).

3. Results

3.1. Chromosome Numbers and Karyotype Features

Ten wild Chrysanthemum plants were analyzed in this study. We identified ten wild Chrysanthemum plants by conducting a morphological analysis and cytological observations (Figure 1). These results suggest that the chromosome numbers ranged from 2n = 18 (2x) to 2n = 54 (6x), following previous reports. In addition, the karyotypes of Chrysanthemum accessions mostly comprised metacentric and submetacentric chromosomes of similar sizes, and the B chromosome rarely emerged in C. indicum (NAU079, Taibaishan, Shaanxi, China) (Figure 1h).

3.2. Geographic Distribution of Wild Chrysanthemum Genus Plants

The geographic distribution plays a pivotal role in the karyotype evolution of the Chrysanthemum genus. We visualized the sources of related wild Chrysanthemum plants listed in Table 1 using RStudio (Version 1.2.5033) (Figure 2).
From the result of the geographic distribution analysis, most Chrysanthemum accessions were located in the mid-latitude area of China (Figure 2a). In addition, the diploid and tetraploid Chrysanthemum accessions were distributed along the Yangtze River and Yellow River, which may make the dispersion of seeds and pollens by water possible (Figure 2b). Moreover, the detailed topographic amplitude map equally supports the spreading rules of seeds and pollens (Figure 2c). With the spread of seeds and pollens, different ploidy Chrysanthemum accessions simultaneously gathered together to form diverse populations, extensively promoting karyotype evolution among the Chrysanthemum genus.

3.3. Identification of 5S and 45S rDNA Sites in Chrysanthemum Accessions Using Oligo-FISH

The double-color Oligo-FISH localization of the 5S and 45S rDNA loci on mitotic metaphase chromosomes was assessed for the wild Chrysanthemum accessions listed in Table 1 (Figure 3). Overall, the numbers and positions of 5S rDNA sites were relatively conserved among all examined Chrysanthemum accessions, unlike that of 45S rDNA, which varied considerably in the number and parts of its sites.
For 45S rDNA, the number of sites ranged from 3 (C. lavandulifolium var. aromaticum (NAU010) and C. mongolicum (NAU011)) to 17 in an autotetraploid C. nankingense (NAU172) (Figure 3). Additionally, the Oligo-FISH results indicated that 45S rDNA sites were mainly located at chromosomal termini and minor sites were located at the interstitial region of chromosomes. Simultaneously, the signal intensity levels of the Chrysanthemum accessions showed substantial differences.
Compared to 45S rDNA, the number of 5S rDNA sites was conserved across all ten Chrysanthemum accessions and may be related to its ploidy, except for the diploid plants C. indicum (Wuhan, Hubei) (NAU031) (Figure 3a) and C. mongolicum (NAU011) (Figure 3d). Compared to hexaploid wild Chrysanthemum accessions, the diploid and tetraploid wild Chrysanthemum accessions account for a large proportion of the wild Chrysanthemum germplasm. Consequently, we conducted the t-test analysis mainly based on the diploid and tetraploid wild Chrysanthemum accessions. As shown in Table 1, we further found that a significant correlation existed between the number of 5S rDNA sites and chromosomes (p < 0.001), but not between 45S rDNA sites and chromosome numbers (p > 0.05) (Figure 4). In addition, we found that the 5S rDNA signals of ten Chrysanthemum accessions were mainly located in subterminal chromosome regions (interstitial regions near centromeres). However, 5S rDNA signals emerged at the chromosomal termini and interstitial areas near the terminal in diploid C. mongolicum (NAU011) and C. indicum (Wuhan, Hubei) (NAU031), respectively. Consequently, we hypothesized potential chromosomal variations in the two accessions.
Significantly, the 5S rDNA and 45S rDNA signals were colocalized on the same chromosome in diploid C. indicum (Wuhan, Hubei) (NAU031) (Figure 3a) and tetraploid C. indicum (Tianzhushan, Shaanxi) (NAU047) (Figure 3f).

3.4. Patterns of rDNA Sites

Consistently, by integrating the previous related data (Table 1), we constructed the standard karyotype and painted the idiograms using the computer software KaryoMeasure, then the rDNA sites were marked (Figure 5). Based on the results shown above, we divided the patterns of 5S rDNA and 45S rDNA sites into three types.
For 5S rDNA sites, there were three basic types: type I (terminal), type II (interstitial on the short arm), and type III (interstitial on the long arm). Statistically, the 5S rDNA sites were more likely to be located at the subterminal chromosome regions (type I and type II) rather than at chromosomal termini (Type III). Likewise, statistical data further demonstrated that 5S rDNA sites were conserved across Chrysanthemum accessions (Table 1).
The patterns found in the 45S rDNA sites were divided into three types: type I (terminal on the short arm), type II (terminal on the long arm), and type III (interstitial). They indicated that most 45S rDNA sites were detected on chromosomal termini (type I and type II), which coincided with previously reported results. However, interstitial areas (type III) of 45S rDNA rarely appeared in C. indicum (Botanical Garden, Beijing) (NAU077) and C. nankingense (Nanjing, Jiangsu) (NAU172). Briefly, all of the above findings proved that unequal crossing-over or ectopic recombination might emerge in 45S rDNA sites, leading to a highly variable number of 45S rDNA sites.
Furthermore, 5S rDNA and 45S rDNA signals were colocalized on the same chromosome of three accessions, C. indicum (Shennongjia, Hubei) (NAU030), C. indicum (Wuhan, Hubei) (NAU031), and C. indicum (Tianzhushan, Shaanxi) (NAU047), which has rarely been reported in previous studies. Hence, we inferred that these three accessions were highly heterogeneous and exhibited potential chromosomal variation among the 18 investigated accessions.

4. Discussion

4.1. Distribution Patterns of rDNA Sites in Wild Chrysanthemum Genus Plants

Typically, in the prevailing views, the highly repetitive sequences constitute a significant fraction of the highest plant genomes (from 20% to 90%) and are arranged hundreds or thousands of times over in separate arrays [8,39,40]. However, the specific functions of these repetitive sequences in the genomes are not yet completely understood. With the improvement of high-throughput DNA sequencing techniques, repetitive DNA sequences have been identified using repetitive sequence analysis software. These repetitive sequences seem to be associated with the organization, evolution, and behavior of plant genomes [41].
The rDNA has been shown to be an excellent cytogenetic marker in related studies [42]. In the genomes of higher eukaryotes, rDNA comprises 45S and 5S rDNA. The 45S rDNA cluster is located in the nucleolar organizer region and encodes the 28S (26S), 5.8S, and 18S rRNAs [16,43]. Nevertheless, the 5S rDNA cluster embodies a highly conserved coding region of approximately 120 bp, even among unrelated species, and a non-transcribed spacer region that varies between 100 and 900 bp [20]. Based on the sequences of 5S and 45S rDNA, genome restructuring [44], intra-species genetic diversity [31], and re-discovery of the status of certain species [45] can be easily visualized using FISH analysis. In previous studies, traditional plasmid DNA probes of rDNA (such as pTa71, consisting of a 9-kb EcoRI fragment of rDNA derived from Triticum aestivum) were used to observe the distribution of rDNA sites in the Chrysanthemum genus and its allied genera in the Asteraceae [11,32,33]. With the development of DNA synthesis technology, novel Oligo-FISH technology has been widely utilized instead of conventional FISH technology [37]. Consequently, oligo probes of rDNA have been employed in cytogenetic studies of the Chrysanthemum genus [2,3,4,6,21]. Nonetheless, the results of the FISH analysis were limited to karyotype and statistical investigations of sites, and the patterns of rDNA sites with the same experimental procedures at accession levels are lacking. Therefore, examining the differences in the number and distribution patterns of rDNA sites can help to further analyze the chromosomal behavior of different species within the genera.
In this study, we integrated the previously reported data listed in Table 1 to paint idiograms of the rDNA sites of Chrysanthemum mitotic metaphase chromosomes and divided the patterns of 5S rDNA and 45S rDNA sites into three equal types (Figure 5). For 5S rDNA sites, there are three basic types: type I (terminal), type II (interstitial on the short arm), and type III (interstitial on the long arm). These results further demonstrated that 5S rDNA sites were conserved across Chrysanthemum accessions. The patterns of the 45S rDNA sites were divided into three types: type I (terminal on the short arm), type II (terminal on the long arm), and type III (interstitial). Coinciding with the results previously reported, a highly variable number of 45S rDNA sites was observed.
Generally, 5S and 45S rDNA sites tend to be distributed independently on chromosomes owing to physical distance [46]. Although the probability of 5S-45S rDNA colocalization is relatively low, this phenomenon does exist in some genera, such as Brassica [47], Cucumis [48], and Artemisia [49]. As reported for another genus, we also found that the 5S rDNA and 45S rDNA signals were colocalized on the same chromosome of three accessions of the Chrysanthemum genus: C. indicum (Shennongjia, Hubei, China) (NAU030), C. indicum (Wuhan, Hubei, China) (NAU031), and C. indicum (Tianzhushan, Shaanxi, China) (NAU047) (Figure 3 and Figure 4). Consequently, the composition of the polyploid Chrysanthemum genome is considered very complex now. Although the potential chromosomal variations, such as translocation or inversion, may not involve a loss or addition of chromosome materials in the Chrysanthemum genus, these variations frequently become associated with differences, duplications, and unbalanced combinations of genetic units [50]. These results further indicate that potential chromosomal variation might occur not only in polyploid Chrysanthemum species and F1 hybrids, but also in diploid species. Finally, these variations might also be facilitated by transposable elements and important ornamental and resistance characters during the evolution of the species in Chrysanthemum sensu lato.
Furthermore, geographical factors (such as topographic features and river systems) may be involved in hybridization, thereby influencing the distribution patterns of rDNA sites through chromosomal rearrangement (such as breakage inversion, translocation, and recombination) owing to rapid genome changes in the F1 hybrid. First, the plant germplasm (such as the seeds and pollen) is dispersed by the wind and the water. Then, the plant forms the new natural populations or speciation through intra- and interspecies hybridization. For Isoetes in East Asia, the spatiotemporal pattern and environmental gradient play essential roles in the speciation and evolution of different ploidy Isoetes species from China [51,52]. Similarly, geographical and ecological factors are vital in differentiating and specifying Chrysanthemum and its related accessions [53,54]. Moreover, in the Chrysanthemum genus, the different ploidy species generally gathered to form hybrids. In the previous studies, the FISH results of the artificial hybridization F1 of C. yoshinaganthum (2n = 36) × C. vestitum (2n = 54), C. indicum (2n = 36) × C. vestitum (2n = 54), and C. remotipinum (2n = 18) × C. chanetii (2n = 36) showed the site and number of rDNA variations, which also supported the hypothesis above [50]. Combined with the geographic distribution (Figure 2) and distribution patterns of rDNA sites (Figure 3 and Figure 5), we infer that the Chrysanthemum accessions are seen as chromosome donors and are located upstream (C. rhombifolium (NAU004) and C. lavandulifolium var. aromaticum (NAU010)). They may have spread through the Yangtze River and may be involved in the karyotype evolution of the downstream accessions C. indicum (Wuhan, Hubei) (NAU031) during the evolution process.

4.2. Relationship between Chromosome Number and Number of rDNA Sites in Wild Chrysanthemum Genus Plants

Over the past few decades, researchers have found that the number of 5S rDNA sites is related to the ploidy level (chromosome number) in some genera such as cultivated Cichorium [55] and Passiflora [56]. Conversely, the numbers of 45S rDNA sites show high variability at the same ploidy level (chromosome number) in many different plants [43,57,58]. Significantly, high variability in 45S rDNA sites was also demonstrated in Anacyclus (Asteraceae) from other geographical areas at the population level [59].
In the present study, extensive variations in ploidy levels were found in wild Chrysanthemum accessions, and ploidy levels varied from diploid (2x) to hexaploid (6x), with a basic chromosome number of 9 (Figure 1 and Figure 3). Compared to the results in previous studies, there were slight differences in the statistical data for rDNA site numbers, especially for the same wild Chrysanthemum accessions, such as C. nankingense (NAU001) and C. lavandulifolium (NAU007). More 45S rDNA sites were discovered in the FISH results by using pTa71(45S rDNA) derived from T. aestivum [11]. Consequently, the non-homologous fragments in rDNA probes may cause the signal noise and interfere with the FISH results. Simultaneously, the 5S rDNA sequences differ in monocotyledon and dicotyledon, and even 5S rDNA is highly conservative in plants. Equally, the FISH results for 5SrDNA sites using homologous probes showed more reliable sites than those using the probes derived from non-homologous species [11,32,33]. From the results of Oligo-FISH analysis, we found that the numbers of 5S rDNA sites were conserved across all ten Chrysanthemum accessions and may be related to their ploidy, except for two diploid plants, C. indicum (Wuhan, Hubei) (NAU031) (Figure 3a) and C. mongolicum (NAU011) (Figure 3d). Furthermore, based on the statistical data of rDNA site number, a significant correlation (p < 0.001) existed between the number of 5S rDNA sites and chromosome number, but not between the number of 45S rDNA sites and chromosome number (Figure 4). Similarly, the number of 5S rDNA sites in other genera in the Asteraceae also showed a substantial significant correlation with ploidy (chromosome number), such as Achyrocline [60], Lactuca [61], and Centaurea [62]. In polyploids, the number of duplicated rDNA sites tends to reduce owing to diploidization mechanisms such as multiple chromosomal variations and gene silencing [56]. Furthermore, these mechanisms may further lead to rapid homogenization of rDNA sequences and induce copy number variation, blurring the hybridogenic signatures of allopolyploids [5]. Our findings strongly support the hypothesis above. Consequently, these results provided the possibility of achieving quick ploidy identification based on the number of 5S rDNA sites combined with flow cytometry results.

4.3. Potential Mechanism of rDNA Sites Variation

According to previous studies, rDNA sites are the predominant targets of repeated recombination events [63]. Related studies have shown that rDNA arrays and adjacent regions are frequent targets for mobile element insertions [64]. These transposition events might promote changes in the number of rDNA sites. The number and location of 5S rDNA sites are highly conserved in different plants. In this study, we discovered variations in the numbers and locations of 5S rDNA sites in three Chrysanthemum accessions: C. rhombifolium (NAU004), C. mongolicum (NAU011), and C. indicum (Wuhan, Hubei, China) (NAU031). These variations may lead to the rapid homogenization of rDNA sequences and induce copy number variation, thereby blurring the hybridogenic signatures of allopolyploids [5]. In addition, the results of other studies indicated that the variation in the number of 5S rDNA sites might be caused by amplification of the covert rDNA copy number during crossing over or transposition events [65]. In contrast, translocation of 5S rDNA sites to other chromosomes without rDNA sites or fusion with different DNA sequences may lead to the disappearance of these sites [20].
Compared with 5S rDNA, 45S rDNA repeats are fragile sites associated with epigenetic alterations and DNA damage under stress responses [16,66]. In addition, high variability in the number of 45S rDNA sites has been observed in many plants [22,67]. Furthermore, we also found that the number and location of 45S rDNA sites varied in some Chrysanthemum accessions with the same ploidy (Figure 3 and Figure 5). Therefore, various cytogenetic and molecular mechanisms may underlie the dynamics of the 45 rDNA sites.
First, unequal crossover and transposition events are considered to be the predominant factors that lead to the variation in rDNA sites. Significantly, the entire 45S rDNA repeat sequence in the chromosomes of Allium and its subgenus can be transferred from one location to another, suggesting that 45S rDNA may move freely or along with neighboring transposable elements [68]. Regarding conserved 5S rDNA, the different types of 5S rDNA sites that emerged in C. indicum (Wuhan, Hubei) (NAU031) may arise from the unequal crossover in homologous chromosomes carrying the same loci.
Additionally, potential variations in rDNA sites may occur during polyploidy changes to different degrees [69]. Significantly, the different ploidy species within the genera distributed in clusters would contribute to hybridization between intra- and interspecies. These variations may also emerge in the targeted chromosome carrying the rDNA site along with karyotype evolution among the genera, further forming new populations or species. Furthermore, the silencing of rDNA loci in chromosomes would cause the disappearance of the secondary constrictions [70]. Correspondingly, variations in 5SrDNA and 45SrDNA sites were also found between autopolyploid C. nankingense (NAU172) and C. nankingense (NAU001) (Figure 5). Especially for the variations in 45rDNA sites in autopolyploid C. nankingense (NAU172), the potential DNA damage and repair mechanism during the autopolyploid process may cause the 45SrDNA sites of type III to change from long-arm to short-arm. Furthermore, the amplification of Ty1-copia and Ty3-gypsy elements in long terminal repeat (LTR) retrotransposons along the chromosomes of autopolyploid C. nankingense (NAU172) also suggests that the rDNA sites may transfer to another site by these transposition events during autopolyploidization [36]. Repetitive sequences (inverted or tandem) can give rise to the production of dsRNA, an important trigger for RNA silencing as well as heterochromatin formation, and may further lead to diverse phenotypes during the polyploidization [71]. Consequently, the variations in different types of repetitive sequences, such as rDNA sequences, may cause gene expression changes and further lead to abundant phenotypic diversity in the Chrysanthemum genus.
Finally, the variation in rDNA sites could also cause high-frequency chromosomal structural fractures and rearrangement, especially for the fragile 45S rDNA sites. Based on the results of previous studies, 45S rDNA is mainly located at chromosomal termini and on satellite chromosomes, which may contribute to the variable number of 45S rDNA sites [66]. Furthermore, the colocalization of 5S and 45S rDNA sites in C. indicum (Shennongjia, Hubei) (NAU030), C. indicum (Wuhan, Hubei) (NAU031), and C. indicum (Tianzhushan, Shaanxi) (NAU047) also strongly supports this potential mechanism of rDNA site variations.

5. Conclusions

We studied the molecular cytogenetics of ten wild Chrysanthemum accessions using rDNA Oligo-FISH. Based on the Oligo-FISH results, we divided the patterns of 5S and 45S rDNA sites into three types separately and drew idiograms of the rDNA sites of Chrysanthemum mitotic metaphase chromosomes. Overall, the 45S rDNA sites showed numerical variations in location and number, whereas the 5S rDNA sites were conserved. In addition, we speculated that clustered groups might greatly promote karyotype evolution in the genus Chrysanthemum. Our work may provide a reliable method of examining the sites and number of rDNA variations among Chrysanthemum and its related accessions and allow researchers to further understand the evolutionary and phylogenetic relationships of the Chrysanthemum genus.

Author Contributions

H.W., F.C., Z.W., J.J., S.C., W.F. and Z.G. planned and designed the research. J.H., Y.Z., S.Z. and Y.H. performed experiments. H.W., Z.G., Y.Z. and Y.L. analyzed the data. J.H. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 32172613), Natural Science Foundation of Jiangsu Province (BK20211215), and the Fundamental Research Funds for the Central Universities, a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article. The figures and tables presented in this study are available here.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Atri, M.; Chehregani, A.; Jalali, F.; Yousefi, S. New chromosome counts in some species of the genus Artemisia L. (Asteraceae) from Iran. Cytologia 2009, 74, 443–448. [Google Scholar] [CrossRef] [Green Version]
  2. Wang, Y.; Lim, J.H.; Jung, J.A.; Kim, W.H.; Lim, K.-B.; Cabahug, R.A.M.; Hwang, Y.-J. Analysis of ploidy levels of Korean wild Asteraceae species using chromosome counting. Flower Res. J. 2019, 27, 278–284. [Google Scholar]
  3. Zandonadi, A.S.; Maia, C.; Barbosa, J.G.; Finger, F.L.; Grossi, J.A.S. Influence of long days on the production of cut chrysanthemum cultivars. Hortic. Bras. 2018, 36, 33–39. [Google Scholar] [CrossRef] [Green Version]
  4. Hoang, T.K.; Wang, Y.; Hwang, Y.J.; Lim, J.H. Analysis of the morphological characteristics and karyomorphology of wild Chrysanthemum species in Korea. Hortic. Environ. Biotechnol. 2020, 61, 359–369. [Google Scholar] [CrossRef]
  5. He, J.; Lin, S.; Yu, Z.; Song, A.; Guan, Z.; Fang, W.; Chen, S.; Zhang, F.; Jiang, J.; Chen, F.; et al. Identification of 5S and 45S rDNA sites in Chrysanthemum species by using oligonucleotide fluorescence in situ hybridization (Oligo-FISH). Mol. Biol. Rep. 2021, 48, 21–31. [Google Scholar] [CrossRef] [PubMed]
  6. Wang, Y.; Jung, J.A.; Lim, K.-B.; Cabahug, R.A.M.; Hwang, Y.-J. Cytogenetic studies of Chrysanthemum: A Review. Flower Res. J. 2019, 27, 242–253. [Google Scholar]
  7. Flavell, R.B. Repetitive DNA and chromosome evolution in plants. Philos. Trans. R Soc. Lond. B Biol. Sci. 1986, 312, 227–242. [Google Scholar]
  8. Biscotti, M.A.; Olmo, E.; Heslop-Harrison, J.S. Repetitive DNA in eukaryotic genomes. Chromosome Res. 2015, 23, 415–420. [Google Scholar] [CrossRef]
  9. Garcia, S.; Kovarik, A.; Leitch, A.R.; Garnatje, T. Cytogenetic features of rRNA genes across land plants: Analysis of the Plant rDNA database. Plant J. 2017, 89, 1020–1030. [Google Scholar] [CrossRef] [Green Version]
  10. She, C.W.; Jiang, X.H. Karyotype analysis of Lablab purpureus (L.) Sweet using fluorochrome banding and fluorescence in situ hybridization with rDNA probes. Czech J. Genet. Plant 2015, 51, 110–116. [Google Scholar] [CrossRef] [Green Version]
  11. Qi, X.Y.; Zhang, F.; Guan, Z.Y.; Wang, H.B.; Jiang, J.F.; Chen, S.M.; Chen, F.D. Localization of 45S and 5S rDNA sites and karyotype of Chrysanthemum and its related genera by fluorescent in situ hybridization. Biochem. Syst. Ecol. 2015, 62, 164–172. [Google Scholar] [CrossRef]
  12. Abd El-Twab, M.H.; Kondo, K. Physical mapping of 5S and 45S rDNA in Chrysanthemum and related genera of the Anthemideae by FISH, and species relationships. J. Genet. 2012, 91, 245–249. [Google Scholar] [CrossRef] [PubMed]
  13. Cuyacot, A.R.; Lim, K.B.; Kim, H.H.; Hwang, Y.J. Chromosomal characterization based on repetitive DNA distribution in a tetraploid cytotype of Chrysanthemum zawadskii. Hortic. Environ. Biotechnol. 2017, 58, 488–494. [Google Scholar] [CrossRef]
  14. Heslop-Harrison, J.S. Comparative genome organization in plants: From sequence and markers to chromatin and chromosomes. Plant Cell 2000, 12, 617–636. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Abd El-Twab, M.H.; Kondo, K. FISH physical mapping of 5S, 45S and Arabidopsis-type telomere sequence repeats in Chrysanthemum zawadskii showing intra-chromosomal variation and complexity in nature. Chromosome Bot. 2006, 1, 1–5. [Google Scholar] [CrossRef] [Green Version]
  16. Yue, M.; Gautam, M.; Chen, Z.; Hou, J.; Zheng, X.; Hou, H.; Li, L. Histone acetylation of 45S rDNA correlates with disrupted nucleolar organization during heat stress response in Zea mays L. Physiol. Plant 2021, 172, 2079–2089. [Google Scholar] [CrossRef]
  17. Xu, C.M.; Bie, T.D.; Wang, C.M.; Zhou, B.; Chen, P.D. Distribution of 45S rDNA sequence on chromosomes of Triticum aestivum and its relative species. Yi Chuan 2007, 29, 1126–1130. [Google Scholar] [CrossRef]
  18. Liu, B.; Davis, T.M. Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae). BMC Plant Biol. 2011, 11, 157. [Google Scholar] [CrossRef] [Green Version]
  19. Wibowo, A.; Setiawan, A.; Purwantoro, A.; Kikuchi, S.; Koba, T. Cytological variation of rRNA genes and subtelomeric repeat sequences in Indonesian and Japanese cucumber accessions. Chromosome Sci. 2018, 21, 81–87. [Google Scholar]
  20. Su, D.; Chen, L.; Sun, J.; Zhang, L.; Gao, R.; Li, Q.; Han, Y.; Li, Z. Comparative chromosomal localization of 45S and 5S rDNA sites in 76 purple-fleshed sweet potato cultivars. Plants 2020, 9, 865. [Google Scholar] [CrossRef]
  21. Wang, Y.; Jung, J.A.; Kim, W.H.; Lim, K.B.; Hwang, Y.J. Morphological and rDNA fluorescence in situ hybridization analyses of Chrysanthemum cultivars from Korea. Hortic. Environ. Biotechnol. 2021, 62, 917–925. [Google Scholar] [CrossRef]
  22. Machackova, P.; Majesky, L.; Hrones, M.; Bilkova, L.; Hribova, E.; Vasut, R.J. New insights into ribosomal DNA variation in apomictic and sexual Taraxacum (Asteraceae). Bot. J. Linn. Soc. 2021, boab094. [Google Scholar] [CrossRef]
  23. Vozarova, R.; Herklotz, V.; Kovarik, A.; Tynkevich, Y.O.; Volkov, R.A.; Ritz, C.M.; Lunerova, J. Ancient origin of two 5S rDNA families dominating in the genus Rosa and their behavior in the Canina-type meiosis. Front. Plant Sci. 2021, 12, 643548. [Google Scholar] [CrossRef] [PubMed]
  24. Tang, S.; Qiu, L.; Xiao, Z.; Fu, S.; Tang, Z. New oligonucleotide probes for ND-FISH analysis to identify barley chromosomes and to investigate polymorphisms of wheat chromosomes. Genes 2016, 7, 118. [Google Scholar] [CrossRef] [Green Version]
  25. Beliveau, B.J.; Joyce, E.F.; Apostolopoulos, N.; Yilmaz, F.; Fonseka, C.Y.; McCole, R.B.; Chang, Y.; Li, J.B.; Senaratne, T.N.; Williams, B.R.; et al. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl. Acad. Sci. USA 2012, 109, 21301–21306. [Google Scholar] [CrossRef] [Green Version]
  26. Luo, X.; Liu, J.; He, Z. Oligo-FISH can identify chromosomes and distinguish Hippophae rhamnoides L. Taxa. Genes 2022, 13, 195. [Google Scholar] [CrossRef]
  27. Jiang, J. Fluorescence in situ hybridization in plants: Recent developments and future applications. Chromosome Res. 2019, 27, 153–165. [Google Scholar] [CrossRef]
  28. Roa, F.; Guerra, M. Distribution of 45S rDNA sites in chromosomes of plants: Structural and evolutionary implications. BMC Evol. Biol. 2012, 12, 225. [Google Scholar] [CrossRef] [Green Version]
  29. Garcia, S.; Galvez, F.; Gras, A.; Kovarik, A.; Garnatje, T. Plant rDNA database: Update and new features. Database 2014, 2014, bau063. [Google Scholar] [CrossRef] [Green Version]
  30. Kovarik, A.; Dadejova, M.; Lim, Y.K.; Chase, M.W.; Clarkson, J.J.; Knapp, S.; Leitch, A.R. Evolution of rDNA in Nicotiana allopolyploids: A potential link between rDNA homogenization and epigenetics. Ann. Bot. 2008, 101, 815–823. [Google Scholar] [CrossRef] [Green Version]
  31. Pedrosa-Harand, A.; de Almeida, C.C.; Mosiolek, M.; Blair, M.W.; Schweizer, D.; Guerra, M. Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution. Appl. Genet. 2006, 112, 924–933. [Google Scholar] [CrossRef] [PubMed]
  32. El-Twab, M.H.; Kondo, K. Physical mapping of 5S rDNA in chromosomes of Dendranthema by fluorescence in situ hybridization. Chromosome Sci. 2002, 6, 13–16. [Google Scholar]
  33. El-Twab, M.H.; Kondo, K. Physical mapping of 45S rDNA loci by fluorescent in situ hybridization and evolution among polyploid Dendranthema species. Chromosome Sci. 2003, 7, 71–76. [Google Scholar]
  34. Huang, X.; Zhu, M.; Zhuang, L.; Zhang, S.; Wang, J.; Chen, X.; Wang, D.; Chen, J.; Bao, Y.; Guo, J.; et al. Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high-resolution multiplex oligonucleotide FISH. Appl. Genet. 2018, 131, 1967–1986. [Google Scholar] [CrossRef] [PubMed]
  35. Zhu, M.; Du, P.; Zhuang, L.; Chu, C.; Zhao, H.; Qi, Z. A simple and efficient non-denaturing FISH method for maize chromosome differentiation using single-strand oligonucleotide probes. Genome 2017, 60, 657–664. [Google Scholar] [CrossRef]
  36. He, J.; Yu, Z.; Jiang, J.; Chen, S.; Fang, W.; Guan, Z.; Liao, Y.; Wang, Z.; Chen, F.; Wang, H. An eruption of LTR retrotransposons in the autopolyploid genomes of Chrysanthemum nankingense (Asteraceae). Plants 2022, 11, 315. [Google Scholar] [CrossRef]
  37. Waminal, N.E.; Pellerin, R.J.; Kim, N.S.; Jayakodi, M.; Park, J.Y.; Yang, T.J.; Kim, H.H. Rapid and efficient FISH using pre-labeled oligomer probes. Sci. Rep. 2018, 8, 8224. [Google Scholar] [CrossRef] [Green Version]
  38. Mahmoudi, S.; Mirzaghaderi, G. Tools for drawing informative idiograms. bioRxiv 2021. [Google Scholar] [CrossRef]
  39. He, Q.; Cai, Z.; Hu, T.; Liu, H.; Bao, C.; Mao, W.; Jin, W. Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.). BMC Plant Biol. 2015, 15, 105. [Google Scholar] [CrossRef] [Green Version]
  40. Shirabe, K.; Ryota, E.; Kaori, S.; Akio, K.; Scoles, G. Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure. Genome 2013, 56, 131–137. [Google Scholar]
  41. Luo, X.; Liu, J.; Wang, J.; Gong, W.; Chen, L.; Wan, W. FISH analysis of Zanthoxylum armatum based on oligonucleotides for 5S rDNA and (GAA)6. Genome 2018, 61, 699–702. [Google Scholar] [CrossRef] [PubMed]
  42. Diao, Y.; Zhou, M.Q.; Hu, Z.L. Chromosomal localization of the 5S and 45S rDNA sites and 5S rDNA sequence analysis in Nelumbo Species. Agric. Sci. China 2011, 10, 679–685. [Google Scholar] [CrossRef]
  43. Liu, D.; Thakur, S.; Kumar, U.; Malik, R.; Bisht, D.; Balyan, P.; Mir, R.R.; Kumar, S. Physical localization of 45S rDNA in Cymbopogon and the analysis of differential distribution of rDNA in homologous chromosomes of Cymbopogon winterianus. PLoS ONE 2021, 16, e0257115. [Google Scholar]
  44. Pontes, O.; Neves, N.; Silva, M.; Lewis, M.S.; Madlung, A.; Comai, L.; Viegas, W.; Pikaard, C.S. Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proc. Natl. Acad. Sci. USA 2004, 101, 18240–18245. [Google Scholar] [CrossRef] [Green Version]
  45. Li, K.P.; Wu, Y.X.; Zhao, H.; Wang, Y.; Lu, X.M.; Wang, J.M.; Xu, Y.; Li, Z.Y.; Han, Y.H. Cytogenetic relationships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbitaceae) as inferred from rDNA distribution patterns. BMC Evol. Biol. 2016, 16, 85. [Google Scholar] [CrossRef] [Green Version]
  46. Martins, C.G.P.M., Jr. Conservative distribution of 5S rDNA loci in Schizodon (Pisces, Anostomidae) chromosomes. Chromosome Res. 2000, 8, 353–355. [Google Scholar] [CrossRef]
  47. Kamisugi, Y.; Nakayama, S.; O’Neil, C.M.; Mathias, R.J.; Trick, M.; Fukui, K. Visualization of the Brassica self-incompatibility S-locus on identified oilseed rape chromosomes. Plant Mol. Biol. 1998, 38, 1081–1087. [Google Scholar] [CrossRef]
  48. Taketa, S.; Harrison, G.E.; Heslop-Harrison, J.S. Comparative physical mapping of the 5S and 18S-25S rDNA in nine wild Hordeum species and cytotypes. Appl. Genet. 1999, 98, 1–9. [Google Scholar] [CrossRef]
  49. Abd El-Twab, M.H.; Kondo, K. Genome Mutation revealed by artificial hybridization between Chrysanthemum yoshinaganthum and Chrysanthemum vestitum assessed by FISH and GISH. J. Bot. 2012, 2012, 480310. [Google Scholar] [CrossRef] [Green Version]
  50. Matoba, H.; Uchiyama, H. Physical mapping of 5S rDNA, 18S rDNA and telomere sequences in three species of the genus Artemisia (Asteraceae) with distinct basic chromosome numbers. Cytologia 2009, 74, 115–123. [Google Scholar] [CrossRef] [Green Version]
  51. Liu, X.; Gituru, W.R.; Wang, Q.F. Distribution of basic diploid and polyploid species of Isoetes in East Asia. J. Biogeogr. 2004, 31, 1239–1250. [Google Scholar] [CrossRef]
  52. Dai, X.K.; Yang, Y.J.; Liu, X. Transplanting experiment and transcriptome sequencing reveal the potential ecological adaptation to plateau environments in the allopolyploid Isoetessinensis. Aquat. Bot. 2021, 172, 103394. [Google Scholar] [CrossRef]
  53. Huang, Y.; An, Y.M.; Meng, S.Y.; Guo, Y.P.; Rao, G.Y. Taxonomic status and phylogenetic position of Phaeostigma in the subtribe Artemisiinae (Asteraceae). J. Syst. Evol. 2017, 55, 426–436. [Google Scholar] [CrossRef]
  54. Chen, X.; Wang, H.; Yang, X.; Jiang, J.; Ren, G.; Wang, Z.; Dong, X.; Chen, F. Small-scale alpine topography at low latitudes and high altitudes: Refuge areas of the genus Chrysanthemum and its allies. Hortic. Res. 2020, 7, 184. [Google Scholar] [CrossRef] [PubMed]
  55. Bernardes, E.C.; Benko-Iseppon, A.M.; Vasconcelos, S.; Carvalho, R.; Brasileiro-Vidal, A.C. Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae). Genet. Mol. Biol. 2013, 36, 357–363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. De Melo, N.F.; Guerra, M. Variability of the 5S and 45S rDNA sites in Passiflora L. species with distinct base chromosome numbers. Ann. Bot. 2003, 92, 309–316. [Google Scholar] [CrossRef] [Green Version]
  57. Chiarini, F.E. Variation in rDNA loci of polyploid Solanum elaeagnifolium (Solanaceae). N. Z. J. Bot. 2014, 52, 277–284. [Google Scholar] [CrossRef]
  58. Roa, F.; Guerra, M. Non-Random Distribution of 5S rDNA sites and its association with 45S rDNA in plant chromosomes. Cytogenet. Genome Res. 2015, 146, 243–249. [Google Scholar] [CrossRef]
  59. Rosato, M.; Alvarez, I.; Nieto Feliner, G.; Rossello, J.A. High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae). PLoS ONE 2017, 12, e0187131. [Google Scholar] [CrossRef] [Green Version]
  60. Mazzella, C.; Rodriguez, M.; Vaio, M.; Gaiero, P.; Lopez-Carro, B.; Santinaque, F.F.; Folle, G.A.; Guerra, M. Karyological Features of Achyrocline (Asteraceae, Gnaphalieae): Stable karyotypes, low DNA content variation, and linkage of rRNA genes. Cytogenet. Genome Res. 2010, 128, 169–176. [Google Scholar] [CrossRef]
  61. Matoba, H.; Mizutani, T.; Nagano, K.; Hoshi, Y.; Uchiyama, H. Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization. Hereditas 2007, 144, 235–243. [Google Scholar] [CrossRef] [PubMed]
  62. Dydak, M.; Kolano, B.; Nowak, T.; Siwinska, D.; Maluszynska, J. Cytogenetic studies of three European species of Centaurea L. (Asteraceae). Hereditas 2009, 146, 152–161. [Google Scholar] [CrossRef] [PubMed]
  63. Kobayashi, T.; Ganley, A.R. Recombination regulation by transcription-induced cohesin dissociation in rDNA repeats. Science 2005, 309, 1581–1584. [Google Scholar] [CrossRef] [PubMed]
  64. Raskina, O.; Barber, J.C.; Nevo, E.; Belyayev, A. Repetitive DNA and chromosomal rearrangements: Speciation-related events in plant genomes. Cytogenet. Genome Res. 2008, 120, 351–357. [Google Scholar] [CrossRef]
  65. Hanson, R.E.; Islam-Faridi, M.N.; Percival, E.A.; Crane, C.F.; Ji, Y.; McKnight, T.D.; Stelly, D.M.; Price, H.J. Distribution of 5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors. Chromosoma 1996, 105, 55–61. [Google Scholar] [CrossRef]
  66. Huang, M.; Li, H.; Zhang, L.; Gao, F.; Wang, P.; Hu, Y.; Yan, S.; Zhao, L.; Zhang, Q.; Tan, J.; et al. Plant 45S rDNA clusters are fragile sites, and their instability is associated with epigenetic alterations. PLoS ONE 2012, 7, e35139. [Google Scholar] [CrossRef] [Green Version]
  67. Rosato, M.; Moreno-Saiz, J.C.; Galian, J.A.; Rossello, J.A. Evolutionary site-number changes of ribosomal DNA loci during speciation: Complex scenarios of ancestral and more recent polyploid events. AoB Plants 2015, 7, plv135. [Google Scholar] [CrossRef] [Green Version]
  68. Schubert, I.; Wobus, U. In situ hybridization confirms jumping nucleolus organizing regions in Allium. Chromosoma 1985, 92, 143–148. [Google Scholar] [CrossRef]
  69. Srisuwan, S.; Sihachakr, D.; Siljak-Yakovlev, S. The origin and evolution of sweet potato (Ipomoea batatas Lam.) and its wild relatives through the cytogenetic approaches. Plant Sci. 2006, 171, 424–433. [Google Scholar] [CrossRef]
  70. Pikaard, C.S. Nucleolar dominance and silencing of transcription. Trends Plant Sci. 1999, 4, 478–483. [Google Scholar] [CrossRef]
  71. Chen, Z.J. Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu. Rev. Plant Biol. 2007, 58, 377–406. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Genes 13 00894 g001 550
Figure 1. Identification of materials using cytogenetics (black bar = 10 μm) and morphological analysis (white bar = 1 cm): (a) Chrysanthemum indicum (Wuhan, Hubei) (NAU031); (b) C. lavandulifolium var. aromaticum (NAU010); (c) C. indicum (Botanical Garden, Beijing) (NAU077); (d) C. mongolicum (NAU011); (e) C. indicum (Lushan, Jiangxi) (NAU033); (f) C. indicum (Tianzhushan, Shaanxi) (NAU047); (g) C. indicum (Nanchang, Jiangxi) (NAU032); (h) C. indicum (Taibaishan, Shaanxi) (NAU079); (i) C. nankingense (NAU172); (j) C. naktongense (NAU015).
Figure 1. Identification of materials using cytogenetics (black bar = 10 μm) and morphological analysis (white bar = 1 cm): (a) Chrysanthemum indicum (Wuhan, Hubei) (NAU031); (b) C. lavandulifolium var. aromaticum (NAU010); (c) C. indicum (Botanical Garden, Beijing) (NAU077); (d) C. mongolicum (NAU011); (e) C. indicum (Lushan, Jiangxi) (NAU033); (f) C. indicum (Tianzhushan, Shaanxi) (NAU047); (g) C. indicum (Nanchang, Jiangxi) (NAU032); (h) C. indicum (Taibaishan, Shaanxi) (NAU079); (i) C. nankingense (NAU172); (j) C. naktongense (NAU015).
Genes 13 00894 g001
Genes 13 00894 g002 550
Figure 2. Geographic distribution of related Chrysanthemum accessions: (a) whole geographic distribution map of Chrysanthemum accessions; (b) a detailed map of the blue dotted area; (c) detailed topography amplitude map of the blue dotted area.
Figure 2. Geographic distribution of related Chrysanthemum accessions: (a) whole geographic distribution map of Chrysanthemum accessions; (b) a detailed map of the blue dotted area; (c) detailed topography amplitude map of the blue dotted area.
Genes 13 00894 g002
Genes 13 00894 g003 550
Figure 3. Distribution of 5S (red) and 45S (green) rDNA sites detected by Oligo-FISH: (a) Chrysanthemum indicum (Wuhan, Hubei) (NAU031); (b) C. lavandulifolium var. aromaticum (NAU010); (c) C. indicum (Botanical Garden, Beijing) (NAU077); (d) C. mongolicum (NAU011); (e) C. indicum (Lushan, Jiangxi) (NAU033); (f) C. indicum (Tianzhushan, Shaanxi) (NAU047); (g) C. indicum (Nanchang, Jiangxi) (NAU032); (h) C. indicum (Taibaishan, Shaanxi) (NAU079); (i) C. nankingense (NAU172); (j) C. naktongense (NAU015). Scale bars, 10 µm.
Figure 3. Distribution of 5S (red) and 45S (green) rDNA sites detected by Oligo-FISH: (a) Chrysanthemum indicum (Wuhan, Hubei) (NAU031); (b) C. lavandulifolium var. aromaticum (NAU010); (c) C. indicum (Botanical Garden, Beijing) (NAU077); (d) C. mongolicum (NAU011); (e) C. indicum (Lushan, Jiangxi) (NAU033); (f) C. indicum (Tianzhushan, Shaanxi) (NAU047); (g) C. indicum (Nanchang, Jiangxi) (NAU032); (h) C. indicum (Taibaishan, Shaanxi) (NAU079); (i) C. nankingense (NAU172); (j) C. naktongense (NAU015). Scale bars, 10 µm.
Genes 13 00894 g003
Genes 13 00894 g004 550
Figure 4. The number of rDNA sites versus the chromosome number (N = 18). The lines show the grouping of the ploidy levels into diploids and tetraploids according to t-test: (a) *** p < 0.001; (b) ns p > 0.05 (the hollow blue scatter point is the outlier and was excluded in t-test).
Figure 4. The number of rDNA sites versus the chromosome number (N = 18). The lines show the grouping of the ploidy levels into diploids and tetraploids according to t-test: (a) *** p < 0.001; (b) ns p > 0.05 (the hollow blue scatter point is the outlier and was excluded in t-test).
Genes 13 00894 g004
Genes 13 00894 g005 550
Figure 5. Idiograms of the rDNA sites of Chrysanthemum mitotic metaphase chromosomes. The 5S rDNA sites (red), 45S rDNA sites (green), and colocalized sites are marked by boxes with white dotted lines.
Figure 5. Idiograms of the rDNA sites of Chrysanthemum mitotic metaphase chromosomes. The 5S rDNA sites (red), 45S rDNA sites (green), and colocalized sites are marked by boxes with white dotted lines.
Genes 13 00894 g005
Table
Table 1. Investigated materials with numbers and locations of 5S and 45S rDNA sites in the Chrysanthemum genus.
Table 1. Investigated materials with numbers and locations of 5S and 45S rDNA sites in the Chrysanthemum genus.
Accession
Number		TaxonSourceNo. of 5S rDNA SitesNo. of 45S rDNA SitesChromosome NumberSource of Data
NAU001Chrysanthemum nankingenseNanjing, Jiangsu272n = 2x = 18He et al. [5]
NAU002C. dichrumXingtai, Hebei282n = 2x = 18He et al. [5]
NAU004C. rhombifoliumWushan, Chongqing452n = 2x = 18He et al. [5]
NAU006C. potentilloidesTianzhushan, Shaanxi292n = 2x = 18He et al. [5]
NAU007C. lavandulifoliumBotanical garden, Beijing272n = 2x = 18He et al. [5]
NAU010C.lavandulifolium
var. aromaticum		Shennongjia, Hubei232n = 2x = 18This study
NAU011C.mongolicumMeiligeng, Neimenggu332n = 2x = 18This study
NAU015C.naktongenseZhangjiakou, Hebei682n = 6x = 54This study
NAU027indicumYuntaishan, Henan282n = 2x = 18He et al. [5]
NAU029C. indicumWuyishan, Fujian262n = 2x = 18He et al. [5]
NAU030C. indicumShennongjia, Hubei2102n = 2x = 18He et al. [5]
NAU031C. indicumWuhan, Hubei362n = 2x = 18This study
NAU032C. indicumNanchang, Jiangxi462n = 4x = 36This study
NAU033C. indicumLushan, Jiangxi442n = 4x = 36This study
NAU047C. indicumTianzhushan, Shaanxi492n = 4x = 36This study
NAU077C. indicumBotanical garden, Beijing282n = 2x = 18This study
NAU079C. indicumTaibaishan, Shaanxi452n = 4x = 36This study
NAU172C. nankingenseNanjing, Jiangsu4172n = 4x = 36This study
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

He, J.; Zhao, Y.; Zhang, S.; He, Y.; Jiang, J.; Chen, S.; Fang, W.; Guan, Z.; Liao, Y.; Wang, Z.; et al. Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions. Genes 2022, 13, 894. https://doi.org/10.3390/genes13050894

AMA Style

He J, Zhao Y, Zhang S, He Y, Jiang J, Chen S, Fang W, Guan Z, Liao Y, Wang Z, et al. Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions. Genes. 2022; 13(5):894. https://doi.org/10.3390/genes13050894

Chicago/Turabian Style

He, Jun, Yong Zhao, Shuangshuang Zhang, Yanze He, Jiafu Jiang, Sumei Chen, Weimin Fang, Zhiyong Guan, Yuan Liao, Zhenxing Wang, and et al. 2022. "Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions" Genes 13, no. 5: 894. https://doi.org/10.3390/genes13050894

APA Style

He, J., Zhao, Y., Zhang, S., He, Y., Jiang, J., Chen, S., Fang, W., Guan, Z., Liao, Y., Wang, Z., Chen, F., & Wang, H. (2022). Uneven Levels of 5S and 45S rDNA Site Number and Loci Variations across Wild Chrysanthemum Accessions. Genes, 13(5), 894. https://doi.org/10.3390/genes13050894

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop