1 Introduction

Senna Mill., a representative genus from the family Fabaceae Lindl. (Resende et al. 2014), comprises approximately 350 morphologically diverse species of herbs, shrubs, and trees (Cordeiro and Felix 2018). Senna species are distributed throughout circumtropical regions with an extremely wide range of habitats (Marazzi et al. 2006; Pellerin et al. 2019). They are morphologically distinguished based on their androceu, corolla, floral architecture, bracteole, and fruit characteristics (Marazzi et al. 2006). Many Senna species have been recognized for their medicinal and industrial uses such as for treating diverse diseases (e.g., digestive ailments, skin disorders, respiratory illnesses, visual problems, and even heart disease) and producing compounds used in commercial goods, flavoring, perfume, pet food, and coffee (Rahman et al. 2013; Pellerin et al. 2019). Despite their economic and health benefits, a paucity of molecular cytogenetic data has impeded comparative analyses for the evaluation of Senna genome evolution. Although a few comparative cytogenetic studies have been reported, only a few species have data based on fluorescence in situ hybridization (FISH) of 5S and 45S rDNA and telomeric repeats (Rosato et al. 2018; Youn and Kim 2018; Pellerin et al. 2019).

Karyotype data can be used to identify species, reveal past genome rearrangements, and infer taxonomic relationships among related species (Guerra 2008; Jo et al. 2019; Chen et al. 2020). A karyotype, which is a genetically stable characteristic unique to a given species, provides the number, shape, size, and morphology of an organism’s chromosome complement (Pellerin et al. 2019; Zhou et al. 2019a). Chromosomal rearrangements can alter karyotype features resulting in changes in chromosome number (dysploidy) or organization, which often reflect evolutionary events such as speciation (Wölk et al. 2015). Descending dysploidy occurs when chromosomal fusion leads to species with fewer chromosome numbers, whereas ascending dysploidy results from retention of centromere function after a chromosome fission (Winterfeld et al. 2020; Ta et al. 2021; Waminal et al. 2021).

FISH is an invaluable technique in karyotyping (Waminal et al. 2018; Youn and Kim 2018). The 5S rDNA and 45S rDNA sequences are commonly used as FISH probes because they are highly repetitive and widely conserved across taxonomic groups (Pellerin et al. 2018a, b; Waminal et al. 2018; Zhou et al. 2019b). The Arabidopsis thaliana-type telomeric repeat (TTTAGGG)n, the canonical plant telomeric repeat most commonly found at chromosome termini, is also widely conserved across taxonomic groups (Watson and Riha 2010; Peska and Garcia 2020). Interspecies divergence in the chromosomal distribution of rDNA and the telomeric repeat sequences provide phylogenetically useful information for analyzing genome dynamics.

While the predominant diploid chromosome number in Senna is 2n = 28 (Rice et al. 2015), descending dysploid karyotypes of 2n = 22–26 are also commonly observed (Cordeiro and Felix 2018; Pellerin et al. 2019). Published data on chromosome number is lacking in a number of Senna species. To broaden the karyotype information in Senna, we performed triple-color FISH using rDNA and telomeric repeat sequence probes in 11 Senna species. To our knowledge, there are currently no reports of FISH karyotyping using rDNA and telomeric probes in these Senna species. This analysis revealed interspecific karyotype variations that provide insight into karyotype dynamics in Senna. These preliminary data will also facilitate cytogenetic mapping of major species-specific repeats, improve our understanding of taxonomic relationships and evolutionary history, and provide useful information for future Senna genomic research and breeding projects.

2 Materials and methods

2.1 Plant materials and chromosome preparation

Seeds of the 11 Senna species were purchased from the National Plant Germplasm System (NPGS, USA) and Rare Palm Seeds (RPS, Germany) (Table 1). Concentrated sulfuric acid (Sigma-Aldrich Co., St. Louis, MO, USA) was used to treat the seeds before germination to break seed dormancy and expedite germination (Baskin et al. 1998). Root tips were collected and pre-treated in 2 mM 8-hydroxyquinoline for 5 h at 18 °C then stored in 70% ethanol at 4 °C until use.

Table 1 List of Senna species used in this study with their published chromosome information
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Chromosome preparation was performed according to our published protocol (Waminal and Kim 2012; Peniton et al. 2019) with minor alterations. Briefly, fixed root tips were washed in distilled water and digested in an enzyme solution containing 1% pectolyase Y-23 (Duchefa, Haarlem, The Netherlands) and 2% cellulase R-10 (Phytotechnology Laboratories, USA) for 60–90 min at 37 °C. Chromosomes were then fixed in chilled Carnoy’s solution and centrifuged. Supernatants were aspirated, and the precipitates were resuspended in aceto-ethanol (9:1 v/v) and mounted onto slides in a humid chamber. After air drying, slides were soaked in 2% (v/v) formaldehyde fixative (Merck Schuchardt OHG, Hohenbrunn, Germany) for 5 min to preserve the chromosomes, quickly dipped into distilled water, and finally dehydrated in a series of ethanol concentrations (70, 90, and 100%) (Vrána et al. 2012).

2.2 Fluorescence in situ hybridization (FISH)

FISH was performed according to Waminal and Kim (2012) with some modifications. Pre-labeled oligoprobes (PLOPs) for 5S rDNA, 45S rDNA, and Arabidopsis-type telomeric sequences are described in Waminal et al. (2018). Hybridization solutions consisted of 50% formamide, 10% dextran sulfate, 2 × saline sodium citrate buffer (SSC), 50 ng/µL of each PLOP, and nuclease-free water to a total volume of 40 µL. Slides were denatured at 80 °C for 5 min, then placed in a humid chamber at 37 °C for at least 45 min. Slides were then washed carefully in 2 × SSC and dehydrated in a series of ethanol concentrations (70, 90, and 100%) for 3 min each at room temperature. Finally, chromosomes were counterstained with DAPI premixed in Vectashield antifade solution. Chromosome images were captured using a BX53 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a DFC365 FS CCD camera (Leica Microsystems, Wetzlar, Germany), and analyzed with Cytovision ver. 7.2 software (Leica Microsystems). Images were finalized using Photoshop CS6 (Adobe Inc., San Jose, California, USA).

2.3 Karyotyping

We used at least three metaphase spreads with the best morphology for total chromosome length (TCL, 2n) measurements using Image J software ver. 1.51 k (Schneider et al. 2012). Homologous chromosomes were paired and arranged based on rDNA and telomeric repeat FISH signals, chromosome length, and centromere position. Chromosome type was classified according to the criteria of Levan et al. (2009). Karyograms and idiograms were generated using Adobe Photoshop CS6.

3 Results

3.1 Chromosome counts

The 11 species could be grouped into three groups according to their diploid chromosome number, 2n = 22, 24, and 28 (Table 2). Because 2n = 28 is considered the predominant chromosome number in Senna (Cordeiro and Felix 2018), and was most frequently represented in our data, all species with different 2n numbers were regarded as descending dysploidy karyotypes (Winterfeld et al. 2020; Ta et al. 2021; Waminal et al. 2021).

Table 2 Triple-target FISH karyotype analysis of 11 Senna species
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Chromosome morphology, differential intensities, and chromosomal distribution of rDNA and telomeric repeats enabled the identification of homologous chromosomes in the 11 Senna species. The total chromosome length (TCL) among these 11 species ranged from 54.16 to 133.2 μm (Table 2). S. uniflora had the longest chromosomes, whereas S. notabilis had the shortest chromosomes. The average chromosome length (TCL/2n) also differed among the species, indicating that cyclic changes in genome size may have occurred during genus diversification (Seijo and Fernández 2003).

Homologous chromosome complements of the 11 species included metacentric, submetacentric, and sub-telocentric chromosomes. Only S. alata had all metacentric chromosome pairs. Other species included metacentric and submetacentric chromosomes. In addition, S. siamea, S. corymbosa, and S. lindheimeriana had one pair each of sub-telocentric chromosomes (Table 2, Fig. 3).

3.2 Chromosomal distribution of rDNA and telomeric probes

The rDNA probes displayed varied chromosomal distributions and signal intensities across the 11 Senna species (Fig. 1). All species presented a single 5S rDNA locus, except for S. uniflora, which possessed two loci (Fig. 2). The 5S rDNA signals were frequently detected in the penultimate chromosome number and were generally localized to proximal chromosome regions (Figs. 2 and 3). The number and intensity of 45S rDNA signals varied considerably among the species. Three pairs were detected in S. alata, S. alexandrina, S. auriculata, S. notabilis, S. polyphylla, and S. siamea. Two pairs were found in S. uniflora, and one pair each was found in S. corymbosa, S. hirsuta var. leptocarpa, S. lindheimeriana, and S. marilandica. Most of these signals were found in the terminal regions of the short arms of the respective chromosomes. A hemizygous 45S rDNA locus was observed in chromosome 6 of S. auriculata (Fig. 2), and we did not observe any juxtaposition between 5S rDNA and 45S rDNA signals in any species.

Fig. 1
figure 1

Triple-color FISH images of 11 Senna species. The predominant chromosome number was 2n = 28. The exceptions were S. marilandica and S. uniflora which have 2n = 22 and 2n = 24, respectively. One pair of 5S rDNA (green) and one to three pairs of 45S rDNA (red) were detected. Scale bar = 10 µm

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Fig. 2
figure 2

FISH karyograms of the 11 Senna species. Green, red, and blue signals indicate 5S rDNA, 45S rDNA, and telomeric repeats, respectively. Yellow arrowheads point to ITR signals. The white arrowhead points to the hemizygous 45S rDNA locus in S. auriculata. Scale bar = 10 µm

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Fig. 3
figure 3

Idiogram of the 11 Senna species. Red, green, and blue bars represent 45S rDNA, 5S rDNA, and telomere repeat, respectively

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The Arabidopsis-type telomeric repeat hybridized to the terminal regions of all chromosomes in all species (Table 2, Figs. 1, 2, and 3). In addition, some chromosomes also displayed weak interstitial telomeric repeat (ITR) signals in peri-centromeric regions in S. alexandrina, S. corymbosa, and S. hirsuta (Fig. 2). A pair of ITR signals was detected on chromosome 1 in all three species. The remaining pairs were localized to chromosome 12 in S. alexandrina, 2 and 6 in S. corymbosa, and 9 and 12 in S. hirsuta (Fig. 2).

4 Discussion

Data on chromosome number and FISH-based karyotype in Senna are relatively scarce (Cordeiro and Felix 2018). In our previous work, we presented FISH karyotypes using 5S and 45S rDNA and telomeric repeats in 12 Senna species, including S. tora whose genome has been recently sequenced (Youn and Kim 2018; Pellerin et al. 2019; Kang et al. 2020; Ta et al. 2021; Waminal et al. 2021). To complement previous data and improve our understanding of karyotype diversity in Senna, we further analyzed the karyotypes of 11 additional Senna species. To our knowledge, this is the first report on the chromosome numbers of S. lindheimeriana, S. marilandica, S. polyphylla, and S. uniflora.

Most Senna species investigated in this study were diploid, with a predominant chromosome number of 2n = 28, corresponding with previous reports (Souza and Iseppon 2004; Cordeiro and Felix 2018). However, S. marilandica (2n = 22) and S. uniflora (2n = 24) showed descending dysploid karyotypes, which may have resulted from post-polyploidy dysploidization after a polyploidization of ancient karyotypes with 2n = 14 (Biondo et al. 2012; Shchapova 2013; Winterfeld et al. 2020). Similar processes have also occurred in several other plants such as Brassica, Cucumis, Nothoscordum, Brachyscome, and Senna tora (Maluszynska and Heslop-Harrison 1993; Watanabe et al. 1995; Koo et al. 2010; Pellerin et al. 2019; Waminal et al. 2021). Indeed, changes in chromosome count may have played a role in the occurrence of reproductive isolation and speciation in Senna (Freyman and Höhna 2018).

Using FISH, we observed interspecific differences in the signal patterns of our markers, indicating species specificity and the usefulness of our probes in distinguishing each species. A hemizygous 45S rDNA pattern similar to that observed in the short arm of chromosome 6 in S. corymbosa has been observed in other Senna and non-Senna species (Lan and Albert 2011; Mancia et al. 2015; Waminal et al. 2016; Pellerin et al. 2019). This hemizygous locus may be explained by homology-mediated unequal crossing over between non-allelic homologous repeat units, which significantly shortened one site, making it undetectable by FISH (Pellerin et al. 2019).

Most species displayed a single locus proximal distribution of 5S rDNA, except for S. uniflora, which showed two loci. These results corroborate the reduced copy number and interstitial distribution of 5S rDNA often observed in flowering plants (Roa and Guerra 2012, 2015). Our results also revealed that 5S and 45S rDNA are not linked in the same chromosomal region; thus, genomic rearrangements by conversion and crossing-over should occur with greater frequency (Waminal and Kim 2012). Independent localization suggests that 5S and 45S rDNA experienced distinct evolutionary processes (Mantovani et al. 2005). Variations in the distribution pattern of rDNA repeats in groups of related species have been explained via structural rearrangement events such as translocations, inversions, duplications, and deletions. All of these events commonly result in structural changes in the karyotype (Silvestri et al. 2020).

Although telomeric sequences are normally located at chromosomal termini (Fuchs et al. 1995), some ITRs were detected in either three or two chromosome pairs in S. hirsuta, S. corymbosa, and S. alexandrina (Fig. 2). ITRs have been observed in a few chromosomes in several Senna species, especially in S. tora, where they are extensively amplified in all chromosomes (Pellerin et al. 2019). ITR signals have also been discovered in animals and some other plant species (Uchida et al. 2002; He et al. 2013; Souza et al. 2016). ITR size, number, and distribution could vary inter- or intra-specifically. Although the origin and evolution of ITRs remain largely unexplored in plants, some proposed mechanisms to explain ITR formation include unequal gene conversion, chromosomal fusion, crossing-over, DNA replication, transposition of telomeric repeats by mobile elements, or the translocation of an ITR during genetic recombination (He et al. 2013; Aksenova and Mirkin 2019). The ITRs observed in Senna species suggest that telomere-mediated inter-chromosomal rearrangements are a major pathway in the evolutionary dynamics of most Senna species (Sousa et al. 2014). This observation is supported by the high frequency of descending dysploids in Senna, as dysploidy often arises from inter-chromosomal rearrangements including end-to-end translocations and nested chromosome insertions (Winterfeld et al. 2020; Ta et al. 2021; Waminal et al. 2021). Recent studies have shown that ITRs are dynamic elements that play essential roles in telomere maintenance and the regulation of gene expression through interactions with telomeres (Ruiz-Herrera et al. 2008; Aksenova and Mirkin 2019).

If these ITRs are formed by chromosomal fusion with reciprocal translocation, the product of this translocation would be a submetacentric chromosome with a weakly detectible ITR, plus a single chromosome and a small fragment (Schubert and Lysak 2011). However, we did not find such small chromosomes in Senna. Another mechanism, called a fusion–fission cycle or a Robertsonian rearrangement, has been used to explain ITRs in other plants (Schubert et al. 1995). With these mechanisms, both centromeric and telomeric sequences are retained, although one of the centromeres and the interstitial telomeric sequences must be inactivated for proper mitosis (Sousa et al. 2014).

We observed that S. corymbosa and S. hirsuta had similar numbers and distributions of 5S rDNA, 45S rDNA, telomeric repeats, and even ITR signals (Figs. 2 and 3). This similarity was also observed in S. occidentalis (Pellerin et al. 2019; Ta et al. 2021), suggesting a closer relationship between S. hirsuta, S. corymbosa, and S. occidentalis. Based on FISH signal similarity, we speculate that S. alata, S. alexandrina, S. auriculata, S. notabilis, S. polyphylla, and S. siamea are closely related, whereas S. corymbosa is closely related to S. hirsuta and S. lindheimeriana in another clade. Molecular phylogenomic data will further clarify these relationships.

5 Conclusion

FISH karyotypes of 11 Senna species were established using three-color probes targeting 5S rDNA, 45S rDNA, and telomeric repeat sequences. The interspecific karyotypic variation in the species studied constitutes useful data for identifying each species and elucidating interspecific relationships in Senna. FISH karyotype analysis using major species-specific repeats as probes, and phylogenomic analyses using chloroplast genomes may provide a clearer picture of the genome dynamics in Senna. The determination of highly abundant repeats using next-generation sequencing data and application of such markers in more Senna species are essential for further studies.