Karyotype Description and Comparative Chromosomal Mapping of 5S rDNA in 42 Species

This study was conducted to evaluate the 5S rDNA site number, position, and origin of signal pattern diversity in 42 plant species using fluorescence in situ hybridization. The species were selected based on the discovery of karyotype rearrangement, or because 5S rDNA had not yet been explored the species. The chromosome number varied from 14 to 160, and the chromosome length ranged from 0.63 to 6.88 μm, with 21 species having small chromosomes (<3 μm). The chromosome numbers of three species and the 5S rDNA loci of nineteen species are reported for the first time. Six 5S rDNA signal pattern types were identified. The 5S rDNA varied and was abundant in signal site numbers (2–18), positions (distal, proximal, outside of chromosome arms), and even in signal intensity. Variation in the numbers and locations of 5S rDNA was observed in 20 species, whereas an extensive stable number and location of 5S rDNA was found in 22 species. The potential origin of the signal pattern diversity was proposed and discussed. These data characterized the variability of 5S rDNA within the karyotypes of the 42 species that exhibited chromosomal rearrangements and provided anchor points for genetic physical maps.


Introduction
Ribosomal DNA (rDNA) is essential to all cell types that code rRNA.rDNAs are detected by exercising considerable parts of chromosomes, including 45S and 5S rDNA [1].There are numerous copies of 5S rDNA in genomes and this rDNA has been used in hundreds of cytogenetic investigations as an important marker for fluorescence in situ hybridization (FISH) analysis [2,3].The 5S rDNA is not only for essential crops [4], but also for woody plants, such as walnuts [5], the Chinese pepper [6][7][8], and wintersweet [9].
The sequence lengths of 5S rDNA range from 48 bp [10] to 854 bp [11] according to the copies and variations available when searching for "5S rDNA" in the Nucleotide database of the National Center for Biotechnology Information (NCBI).Furthermore, the lengths of 5S rDNA as a FISH probe in the NCBI PubMed database varied considerably, at 41-1193 bp [12,13].
In summary, 5S rDNA is a valuable cytogenetic marker owing to the tandem repeats and multiple copies with unusual chromosomal locations.These discoveries may provide a method for surveying the locations and number of rDNA diversifications among species and their respective relational accessions.Such findings will enhance understanding of the developmental and phylogenetic links of studied species.Patent oligonucleotide FISH (oligo-FISH) technology is claimed to be problematic in related cytogenetic investigations compared to conventional FISH analysis because of its low cost [34].Moreover, oligonucleotide probes (oligo-probes) have been developed and successfully applied to many plants based on available DNA sequencing data [7,33,[35][36][37].This study aimed to analyze and compare the polymorphism of the signal patterns of 5S rDNA among 42 plant species, examining the 5S rDNA signal number, position, and intensity, and the chromosome number of each plant.Furthermore, several contributing factors caused by the diversity of the 5S rDNA signal pattern remain to be addressed.

Materials and Methods
The species used for this experiment were chosen due to the discovery of karyotype reshuffling [5][6][7]9,12,[28][29][30]32,33,[37][38][39].In addition, the 5S rDNA of the species had not been explored previously.Information on the seeds or seedlings of these 64 plants (52 woody plants and 12 herbaceous plants belonging to 42 species, 30 genera, and 20 families) is provided in Table 1.All specimens of the 64 plants were collected from 23 counties or districts in six Chinese provinces.

Chromosome and Oligonucleotide Probe Preparation
The seeds of the analyzed species were germinated in Petri dishes with moistened filter paper.These seeds were maintained at 25 • C during the day and 18 • C at night until their roots reached approximately 2 cm in length.Subsequently, the roots of the germinated seeds were removed and the collected seedlings were grown in soil at room temperature (15-25 • C) until they produced new roots, which were cut again.The cut root tips were incubated in nitrous oxide (N 2 O) gas for 3-5 h, with a processing time according to cell wall lignification and chromosome size.The root tips were then soaked in glacial acetic acid for 5 min and in 75% ethyl alcohol afterward.Chromosome preparation was based on a study by Luo et al. [14].Briefly, an enzymolysis procedure was performed at 1 mm of the meristematic zone of the root tip (cut root cap) at 37 • C by 45 min using pectinase and cellulase.There was a total of 50 mL (buffer was covered with 0.4324 g citric acid and 0.5707 g trisodium citrate, 1 mL buffer, 0.02 g pectinase, and 0.04 g cellulase).The enzymes were purchased from Kyowa Chemical Products Co., Ltd.(Osaka, Japan) and Yakult Pharmaceutical Ind. Co., Ltd.(Tokyo, Japan).The enzymes were then blended into suspension for dropping onto clean slides.These slides were air dried at room temperature and examined using an Olympus CX23 microscope (Olympus, Tokyo, Japan).

FISH
Slides with well-spread chromosomes were used to hybridize the above oligo-probe.Chromosome samples were fixed (4% paraformaldehyde at room temperature, for 10 min), dehydration (75%, 95%, and 100% ethanol at room temperature for 5 min), denatured (deionized formamide at 80 • C for 2 min), and dehydrated again (75%, 95%, and 100% ethanol, at −20 • C for 5 min), Subsequently the chromosome samples were hybridized (0.375 µL of 5S rDNA, 4.675 µL of 2× SSC, and 4.95 µL of ddH 2 O in a total of 10 µL hybridization mixture) for 2 h in an incubator at 37 • C. The hybridized slides were then rinsed with 2× SSC and ddH 2 O twice for 5 min at room temperature and air dried before counterstaining with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, CA, USA) for 5 min according to the step described by Luo et al. [12].Chromosomes were traced using an Olympus BX-63 microscope (Olympus Corporation, Tokyo, Japan), and FISH photographs were obtained using a DP-70 CCD camera allocated to the microscope.

Karyotype Analysis
Karyotype data were executed using Photoshop CC 2015 (Adobe Systems Inc., San Jose, CA, USA) and DP Manager (Olympus Corporation, Tokyo, Japan).More than eight slides of each plant were observed, and at least 15 well-spread cells were selected to determine the chromosome number and length.All examined chromosomes were assembled from longest to shortest.The chromosome ratio was controlled by the length of the longest chromosome to that of the shortest chromosome.Exhaustive and deep karyotype analysis could not be conducted because of the obscure centromere position and small chromosome size of many of the analyzed species.
slides of each plant were observed, and at least 15 well-spread cells were selected to de-termine the chromosome number and length.All examined chromosomes were assembled from longest to shortest.The chromosome ratio was controlled by the length of the longest chromosome to that of the shortest chromosome.Exhaustive and deep karyotype analysis could not be conducted because of the obscure centromere position and small chromosome size of many of the analyzed species.

Diverse Signal Patterns of 5S rDNA Reveal a Complex Genome Architecture
To further investigate the diversity of 5S rDNA, different types of ideograms for the 42 species were drawn (Figures S5-S8) based on the FISH karyograms shown in Figures S1-S4.The first diversity of 5S rDNA was the signal location.Proximal signals were observed at several chromosomes in 31 species (74%), while distal signals were observed at several chromosome terminus in 28 species (67%).Interstitial signals were observed at several chromosomes in 19 species (45%), whereas distal signals deviated from the chromosome in four species (10%, the fourth class): C. chinensis (A1), S. japonicum (A6), K. paniculata (B14), and Z. nitidum (D7).The second diversity was the signal number.The largest number of chromosomes with a 5S rDNA signal was 18 in T. sebifera (D6), while the smallest number was two in 22 species (52%).Seven species (17%) contained 10-16 chromosomes with a 5S rDNA signal and 18 species (43%) had 4-8 chromosomes with a 5S rDNA signal.The ratio of chromosomes with a 5S rDNA signal to the total chromosome was assessed to signal cover.The largest ratio was 0.89 in P. nepalensis (A2), and the smallest ratio was 0.03 in Z. nitidum (D7) and Z. armatum 'Putaoqingjiao' (D16).The ratio for 31 species (74%) ranged from 0.03 to 0.20, while the ratio for eight species (19%) ranged from 0.20 to 0.50.Only two species had ratios ranging from 0.50 to 0.89: R. pseudoacacia (A3) and T. wallichiana var.mairei (B5).
We summarized the results in Figures S5-S8 to produce the 5S rDNA signal pattern in Figure 5.The results for 42 species belonging to 20 families are shown in different colors.There were six 5S rDNA signal pattern types: type I, chromosome includes the proximal signal location; type II, chromosome consists of the distal signal location; type III, chromosome consists of the proximal and distal signal locations; type IV, chromosome only consists of signal outside of the chromosome; type V, chromosome consists of the distal signal location and signal outside of the chromosome; and type VI, satellite chromosome consists of distal signal location.These types of signal patterns indicate that there is various diversity in the 5S rDNA signal arrangement.
We summarized the results in Figures S5-S8 to produce the 5S rDNA signal pattern in Figure 5.The results for 42 species belonging to 20 families are shown in different colors.There were six 5S rDNA signal pattern types: type I, chromosome includes the proximal signal location; type II, chromosome consists of the distal signal location; type III, chromosome consists of the proximal and distal signal locations; type IV, chromosome only consists of signal outside of the chromosome; type V, chromosome consists of the distal signal location and signal outside of the chromosome; and type VI, satellite chromosome consists of distal signal location.These types of signal patterns indicate that there is various diversity in the 5S rDNA signal arrangement.In the 42 species examined in this study, 10 signal pattern types or type combinations were present (Figure 5).Twenty six plants only possessed signal pattern type I; nine plants only possessed signal pattern type II; sixteen plants had a combination of type I + type II; six plants only had type III; R. pseudoacacia f. decaisneana had a combination of type I + type III; T. wallichiana var.mairei had a combination of type I + type II + type III; S. japonicum only had type IV; C. chinensis and K. bipinnata had a combination of type II + type IV; Z. nitidum only had type V; and P. nepalensis had a combination of type I + type II + type III + type VI.
There were diverse signal patterns of 5S rDNA among the 42 species, indicating a complex genome architecture.In the family Rutaceae, four varieties of Zanthoxylum had type I, but five varieties of Zanthoxylum had a combination of type I + type II, and Z. nitidum had type V.In Fabaceae, A. fruticose and E. crista-galli had type I, but R. pseudoacacia and R. pseudoacacia 'Idaho' had type III, R. pseudoacacia f. decaisneana had combination of type I + type III, S. japonicum only had type IV, C. chinensis had a combination of type II + type IV, and P. nepalensis had a combination of type I + type II + type III + type VI.In the family Taxaceae, four species of Taxus had type III, but T. wallichiana var.mairei had a combination of type I + type II + type III.In Oleaceae, F. pennsylvanica had type II, but L. lucidum, L. × vicaryi and S. oblata had a combination of type I + type III.In Berberidaceae, B. diaphana had type II, but B. soulieana had a combination of type I + type II.In the family Malvaceae, F. simplex had type I, but H. mutabilis had a combination of type I + type II.In Lauraceae, L. baviensis had type I, but L. baviensis had a combination of type I + type II.
A summarized 5S rDNA signal diversity is illustrated in Figure 6.There were three major groups contributing to this diversity: (i) signal number: increases or decreases; (ii) signal location: on distal chromosome or proximal chromosome, or deviation from the chromosome; (iii) signal size: increases or decreases, or the normal signal size.

Karyotype Analysis of the 42 Species
Traditional karyotype analysis involves counting chromosome numbers, determining centromere location, and measuring chromosome length and long/short arm ratio.
Intraspecific chromosome number variation, even in the entire population, has also been found in species such as Cuscuta epithymum (L.) L. and Cuscuta planiflora Ten.[57], where most variation was attributable to auto-or allopolyploidy.The additional numbers can be explained by ascending or descending dysploidy.Thus, the accumulation of repetitive DNA can lead to an increase in chromosomes and, consequently, to an increase in genome size, especially in subgenus Monogynella [58].In our study, chromosome numbers varied in the interspecific and intraspecific populations of the genus Zanthoxylum.The cause of the variation was probably similar to that of Cuscuta and Monogynella.Furthermore, the stable differentiation in the 5S rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in splitting the two subgenera.Rather than major structural changes, transpositional events are responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes [25].Zanthoxylum genomes have complex chromosome rearrangements, such as chromosomal fission, reversal, and translocations [8], which also explains the chromosome number variation in this genus.Chromosome polymorphisms within species in natural populations of vertebrates are far less common and are believed to be temporary transitions during chromosomal evolution [59,60].Likewise, the genus Zanthoxylum may be experiencing chromosomal evolution.
The longest chromosome length of the 64 plants evaluated in this study ranged from 1.23 to 6.88 µm.In contrast, the shortest chromosome length of each plant ranged from 0.63 to 3.85 µm, exhibiting striking differences among the examined species.Previous research has shown the accumulated chromosome lengths of hundreds of plant species [6,9,12,28,30,32,33,37,38,[61][62][63].Analyzing these data, it is not difficult to find slight differences in chromosome length, even for the same accession of the same species.For example, R. pseudoacacia has reported chromosome lengths of 1.12-1.74µm [37] and 0.94-1.67µm [28].Nonetheless, these two chromosome lengths were small (<3 µm).Hence, chromosome length was more suitable for qualitative rather than for quantitative analyses.Thirty-seven plant species analyzed (more than half) had chromosome lengths < 3 µm, placing them in the small chromosome rank.Owing to the hazy centromere mark and tiny chromosomes in many of the investigated plants, the chromosome size was determined by metaphase and the measurement method.A more delicate karyotype analysis (e.g., arm length, karyotype, and cytotype) was unavailable and limited.

Occurrence and Diversity of 5S rDNA in Plants
The 5S rDNA, which occurs in all cellular life forms, is a highly stable tandem repeat sequence that ubiquitously exists in plants [64].With the evolution and development of the plant, 5S rDNA also underwent simultaneous changes.The length of 5S rDNA in the NCBI Nucleotide database ranged from 48 to 854 bp [10,11], while its length as a FISH probe in the PubMed database of NCBI ranged from 41 to 1193 bp [12,13,31].This study was the first time that the 41-bp oligoprobe had been used to analyze 5S rDNA for 19 species from 13 families.Overall, 5S rDNA occurred in at least two chromosomes in all 42 species.With advances in science and technology, 5S rDNA has been confirmed in an increasing number of species [3,22,65,66].However, whether the reported length of 5S rDNA is a complete or partial sequence, there are no doubt considerable differences among these 5S rDNAs, including the length and base pair [17,67,68].

Potential Origin of 5S rDNA Diversity in Plants
The diversity of signal patterns means 5S rDNA has been used as an excellent and dynamic marker in the species of F. pennsylvanica, Iris versicolor L., L. vicaryi, L. lucidum, P. nepalensis (P.concolor, former name in Flora of China), R. pseudoacacia, and S. oblata [12,28,29,70].After comparing the 5S rDNA obtained in previous studies with ours, both perfectly reflect the extensive diversity in P. nepalensis, in which all 18 chromosomes can be distinguished according to the signal position, signal intensity, and signal number.Nevertheless, 5S rDNA was quite conserved and dormant in numerous species, such as C. campanulatus, C. sativa, H. rhamnoides, J. regia, M. atropurpureum, P. stratiotes, P. trichocarpa, and Sanguisorba L. [9,16,22,33,36,71,72]; leaving a question about the big difference in 5S rDNA among the above species.There are several plausible hypotheses under investigation, such as (i) chromosome rearrangement (e.g., deletion, duplication, inversion, translocation), (ii) polyploidization, (iii) self-incompatibility, and (iv) chromosome satellites.
The 5S rDNA position is a hot topic for chromosomal reshuffling because of its system into long reaches of the standpat tandem repetition unit and its active transcription.This feature implies that they are impressionable to chromosomal destruction or non-allelic homologous recombination, thus raising the feasibility of chromosomal reorganization, such as fissions, inversions, and fusions [73][74][75][76][77].The 5S rDNA diversification was regarded as variable genomic areas, compliant with double-strand break and chromosomal reorganization, facilitating karyotypic reconstruction [77][78][79].The 5S rDNA position was also diversified by translocation or transposition events of repeats in those chromosomes [80].An interstitial 5S rDNA position with a diverse location is presumed to indicate a thin inversion.A plus 5S rDNA implies the presence of a replication [81].In addition, the 5S rDNA site of one parent was either excluded from the chromosome or shifted into gene silencing and then disappeared, which could decrease the 5S rDNA site number [82,83].These studies demonstrate that chromosome rearrangement causes variations in 5S rDNA diversity.
The 5S rDNA has a polyploidization-relevant preference to the distal from a proximal position but keeps a stable loci number [14].The 5S rDNA sites are proximal, a highly transparent direction in chromosomes with a single site [84].Consequently, the presence of the 5S rDNA site in the distal chromosomes and the abundance of microsatellites in adjacent areas provide conducive conditions for additional reshuffling.These results emphasize the effect of variable chromosomal 5S rDNA loci in generating assignments [85].There are no associations between the number of 5S loci and chromosome number, but there is correspondence with ploidy level and genome size [20,84,86], although anomalies still occur [64,87,88], such as the genus Cuscuta L. [84].These studies demonstrate that polyploidization causes variation in 5S rDNA diversity.
In summary, the variations 5S rDNA in signal number, location, and size were caused by chromosome rearrangement (e.g., deletion, duplication, inversion, translocation), recombination, self-incompatibility, as well as chromosome satellites.

Figure 5 .
Figure 5. Signal pattern of 5S rDNA in 42 species.Signal patterns type I-VI are summarized based on Figures S5-S8.The number between the signal pattern and the species represents the species

Figure 5 .
Figure 5. Signal pattern of 5S rDNA in 42 species.Signal patterns type I-VI are summarized based on Figures S5-S8.The number between the signal pattern and the species represents the species number.The number after the species represents the ratio of longest to shortest chromosome length.Rutaceae includes 10 plants (grey); Orchidaceae includes nine plants (light blue); Fabaceae includes eight plants (cyan); Elaeagnaceae includes six plants (light yellow); Taxaceae includes five plants (light pink); Juglandaceae and Oleaceae both include four plants (green and orange); Asparagaceae, Berberidaceae, Euphorbiaceae, Malvaceae, and Lauraceae all include two plants (yellow, blue, red, magenta, pink); Aquifoliaceae, Calycanthaceae, Cupressaceae, Podocarpaceae, Fagaceae, Poaceae, Salicaceae, and Sapindaceae each include one plant, respectively.

Table 1 .
Details of all 64 plants used in this work.

Table 2 .
Chromosome number and length of the 42 species used in this work.
Note: asterisk (*) in Table2indicates chromosome number of three species are first reported.