Abstract
The genus Senna in the family Fabaceae is distributed and cultivated around the world and has been used as sources of medicine and commercial goods. Molecular cytogenetic study based on FISH technique is useful for breeding and genomic research of the species. Triple-color FISH karyotype analyses were carried out to understand the genome structure of two Senna species, S. covesii (A. Gray) H. S. Irwin & Barneby and S. floribunda (Cav.) H. S. Irwin & Barneby using 5S rDNA, 45S rDNA, and telomeric repeat probes. Three and one pairs of 45S and 5S rDNA signals were detected, respectively, in S. covesii, whereas one pair each of 45S and 5S rDNA signals, were detected in S. floribunda. Telomeric signals appeared at all chromosome termini in both species. This result provides basic cytogenetic information which will be useful for future breeding and genomic research of Senna species.
-
Key words: 5S and 45S rDNAs, Telomeric repeat, Senna covesii, Senna floribunda, FISH
INTRODUCTION
The genus
Senna in the family Fabaceae (
Monkheang et al. 2011) has various uses such as food, flower decoration and improvement of barren land (
Elaine et al. 2005;
Resende et al. 2014). It is especially well known as a medicinal plant not only as ophthalmic medication but also in relieving skin diseases, alleviating poor digestion, healing of colds and even heart disease. In addition, it has expanded its usefulness as a living food such as coffee and gum (
Singh et al. 2013). Because of these advantages,
Senna is widely cultivated around the world including Brazil, America, Asia and Africa (
Marazzi et al. 2006;
Singh et al. 2013).
Cytogenetic studies provide information on chromosomal number, length, type, ploidy, and chromosomal distribution of specific sequences that are essential to elucidate genome structure and constitution in plant species (
Kim et al. 2005). The information can be useful in identifying species, tracing phylogenetic relationship among the related species, and breeding and development of plant species (
Koo et al. 2003b;
Kim et al. 2005). Despite the economic and health benefits of
Senna, cytogenetic studies are still limited in this genus (
Kim et al. 2005).
Fluorescence
in situ hybridization (FISH) technique is an efficient and powerful tool in molecular cytogenetics to visualize specific DNA sequences on chromosomes and allows to construct chromosome map of the sequences. Highly repetitive DNA sequences such as rDNA, centromeric-, and telomeric-DNA have been used as FISH probes owing to their different number and distinct distribution on chromosomes according to the species (
Hwang et al. 2009). Especially 5S rDNA, 45S rDNA, and telomeric repeat DNA are highly repetitive DNAs and usually used as primary probes for FISH experiment in plants (
Koo et al. 2003a;
Han et al. 2013).
Here, triple-color FISH karyotype was established using 5S rDNA, 45S rDNA, and Arabidopsis-type telomeric repeat DNA probes to elucidate the chromosomal constitutions of the genome of S. covesii and S. floribunda. This is the first report on FISH karyotype of the two species and this basic information will be useful to construct phylogenetic relationships among Senna species and breeding program of the medicinal species.
MATERIALS AND METHODS
Plant samples
Seeds of S. covesii and S. floribunda were obtained from Agricultural Research Service, USDA (PI 675048 and PI 601948). Seeds were germinated in the greenhouse. About 2 cm roots were harvested, pretreated with 2 mM 8-hydroxyquinoline for five hours at room temperature, fixed in aceto-ethanol (acetic acid: ethanol = 1: 3, v/v) for more than two hours and transferred to 70% ethanol. The stock materials were kept in the refrigerator until use.
Chromosome spread preparation
Mitotic metaphase chromosome spreads were prepared using the technique described by
Kato et al. (2004), with some modifications. Root tips (~2 cm) were digested in 1% pectolyase (Sigma, Japan) and 2% cellulase (MB Cell, Korea) solution at 37°C for 75 minutes. Meristematic tissue (~2 mm) of the roots were dissected using a needle, transferred into a 1.5 mL tube with 40 μL chilled aceto-ethanol fixative solution (acetic acid: ethanol = 1:3, v/v) and vortexed for 30 seconds at room temperature. After centrifugation, pellets were resuspended with aceto-ethanol (acetic acid: ethanol = 9: 1, v/v) solution and drop on a slide glass in the humid chamber and then air-dried.
The 5S and 45S rDNAs and
Arabidopsis-type telomeric repeat sequences (TTTAGGG)
n were used as probes for FISH karyotype analysis. The pre-labelled oligoprobes (PLOP) were designed and prepared according to
Waminal et al. (2018). Mitotic metaphase chromosome spreads were obtained following the procedure of
Waminal et al. (2012). Thirty-two μL of a FISH hybridization master mix (50% formamide, 10% dextran sulfate, and 2× SSC) were mixed with 25 ng of each of PLOP probes. Distilled water was added to a total volume of 40 μL. Chromosomes with added PLOP probes on a glass slide were denatured at 80°C for 5 minutes. Slides were incubated at RT, 30 minutes. The slides were washed with 2× SSC at room temperature for 5 minutes, 0.1× SSC at 42°C for 20 minutes and 2× SSC for 5 minutes at room temperature. Slides were dehydrated in ethanol series of 70%, 90%, and 100%, air-dried and counterstained with a premixed 4′,6-diamidino-2-phenylindole (DAPI) solution (1 μg/mL; DAPI in Vectashield, Vector Laboratories, Burlingame, CA, USA). Images were captured with a model BX53 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a DFC365 FS CCD camera (Leica Microsystems, Wetzlar, Germany) and adjusted by using Cytovision ver. 7.2 (Leica Microsystems). Final images were edited using Adobe Photoshop CC (Adobe Systems, San Jose, CA, USA). Chromosome types were determined according to the description of
Levan et al. (1964). Length of chromosomes were measured using ImageJ (NIH, USA).
RESULTS
Conventional karyotype
Both
S. covesii and
S. floribunda had chromosome number of 2
n = 28. The chromosome length of
S. covesii ranged from 3.58 to 5.67 μm (
Table 1) and those of
S. floribunda ranged from 2.37 to 3.52 μm (
Table 2). Both
species had 10 metacentrics and four submetacentric chromosomes, but
S. floribunda chromosomes were shorter than those of
S. covesii. One pair of satellite chromosomes were observed in chromosome 13 of both species (
Figs. 1 and
2).
As a result of triple-color FISH karyotype analysis using 5S rDNA, 45S rDNA, and telomeric repeat probes, three pairs of 45S rDNA signals were observed on chromosomes 7, 11, and 13, and one pair of 5S rDNA signals, on chromosome 1 in
S. covesii (
Figs. 1 and
2). In case of
S. floribunda, only one pair of 45S and 5S rDNA signals were detected on chromosome 13 and 14, respectively. The satellite chromosome 13 showed major 45S rDNA signals in both species. The
Arabidopsis-type telomeric sequence distributed to the terminal regions of all chromosomes in both species.
DISCUSSION
The chromosome number of
S. floribunda (2
n = 28) in this study corroborates the finding in previous reports (
Rice et al. 2015;
Cordeiro and Felix 2018), whereas that of
S. covesii has not been reported yet and was revealed as 2n = 28 in this study.
Souza and Benko-Iseppon (2004) and
Biondo et al. (2005) reported the sizes and types of chromosomes on eleven
Senna species. The average length was in a range of 0.62–2.50 μm. The chromosome sizes of
S. covesii and
S. floribunda were larger than those of the previously reported
Senna species.
Souza and Benko-Iseppon (2004) and
Biondo et al. (2005) analyzed the chromosome types of
Senna species according to the ratio of long arm per short arm. Generally, prevalent chromosome types of the
Sennas were metacentrics while submetacentrics were only few. In this research,
S. covesii and
S. floribunda, consisted of 10 pairs of metacentrics and 4 pairs were submetacentrics.
Senna species have been regarded as effective medicinal plants. Breeding and development of the species has been expected, and the molecular cytogenetic research based on chromosomal constitution should precede genomic research to get information of the chromosomal organization of a genome. FISH technique has been useful molecular cytogenetic tool which can localize specific DNA sequences on chromosomes making it possible to visualize chromosomal landscape of a genome and construct cytogenetic physical maps (
Park et al. 2010). Tandem repeat sequences or multigene families can be used as FISH probes for chromosome characterization (
Waminal et al. 2011). 5S and 45S rDNA genes form the multi-gene clusters which commonly exist in eukaryotes as several hundred to thousand copies of highly conserved tandem repeats (
Gerlach and Bedbrook 1979).
Arabidopsis-type telomeric tandem repeats (TTTAGGG)
n distribute majorly on the eukaryotic chromosome ends (
Fuchs et al. 1995). Owing to their sequence repetitiveness, specific distribution on the chromosomes, and varying distribution among species and populations, 5S and 45S rDNA genes and
Arabidopsis-type telomeric repeats were used as primary FISH probes to elucidate the chromosome level genomic constitution of a species and to trace the phylogenetic and comparative genomic relationship among plant species (
Koo et al. 2003a;
Han et al. 2013). Nevertheless, FISH karyotype on the
Senna chromosomes using these probes has not been reported until now. In this paper, 5S rDNA, 45S rDNA and telomeric repeat sequences were used as probes for triple-color FISH karyotype analysis in
S. covesii and
S. floribunda.
The nucleolar organizing region which appeared as secondary constriction on the satellite chromosome was detected by 45S rDNA probe on the chromosome 13 in both species. Distribution of 5S rDNA was different between two species and additional two pairs of 45S rDNA repeats were detected in
S. covesii. Due to highly copied heterochromatin regions comprising satellite DNA, varying distribution numbers of 5S and 45S rDNAs among species in the same genus may occur through unequal crossing-over (
Dubcovsky and Dvorák 1995;
Murata et al. 1997;
Roy et al. 2005;
Roa and Guerra 2012;
Han et al. 2013). It can be deduced to undergo different chromosomal rearrangement in two species on the varying appearance of 5S and 45S rDNAs. Additional comparative repeatomics and cytogenomics studies will be helpful to explain the relationship of these two species and other species in the genus in more detail.
This result is the first FISH karyotype report applying 5S rDNA, 45S rDNA and telomeric repeat sequences as probes to S. covesii and S. floribunda. This result will be useful for Senna plant breeding and provide basic chromosomal backbones for Senna genome sequencing analysis.
ACKNOWLEDGEMENTS
This study was carried out by a grant from National Research Foundation of Korea (NRF 2017R1A2B2004778).
Fig. 1Triple-color FISH metaphases of two Senna species. (A) S. covesii, (B) S. floribunda using 5S rDNA, 45S rDNA and telomeric repeat probes. Green, red, and yellow signals indicate 5S rDNA, 45S rDNA, and telomeric repeats, respectively. The scale bar is 10 μm.
Fig. 2FISH karyotypic idiogram of S. covesii (A) and S. floribunda (B). The number stands for chromosome number. Green, red, and yellow signals indicate 5S rDNA, 45S rDNA, and telomeric repeats, respectively. The scale bar is 10 μm.
Table 1Karyotypic analysis of mitotic metaphase chromosome of S. covesii.
Table 1
|
Chromosome number (2n = 28) |
Arm length (μm) |
Type |
|
|
Short (S) |
Long (L) |
Total (S + L) |
Arm ratio (L/S) |
|
1 |
2.60 |
3.07 |
5.67 |
1.18 |
mz)
|
|
2 |
2.09 |
2.54 |
4.63 |
1.21 |
m |
|
3 |
2.11 |
2.42 |
4.53 |
1.14 |
m |
|
4 |
1.97 |
2.43 |
4.40 |
1.23 |
m |
|
5 |
1.96 |
2.44 |
4.40 |
1.24 |
m |
|
6 |
2.10 |
2.12 |
4.22 |
1.01 |
m |
|
7 |
1.14 |
3.01 |
4.15 |
2.64 |
smy)
|
|
8 |
1.84 |
2.28 |
4.12 |
1.23 |
m |
|
9 |
1.70 |
2.30 |
4.00 |
1.35 |
m |
|
10 |
1.79 |
2.17 |
3.96 |
1.21 |
m |
|
11 |
2.02 |
1.90 |
3.92 |
0.94 |
m |
|
12 |
1.35 |
2.49 |
3.84 |
1.84 |
sm |
|
13x)
|
1.10 |
2.53 |
3.63 |
2.30 |
sm |
|
14 |
1.12 |
2.46 |
3.58 |
2.19 |
sm |
|
Total |
24.89 |
34.16 |
59.07 |
|
10 m + 4 sm |
Table 2Karyotypic analysis of mitotic metaphase chromosome of S. floribunda.
Table 2
|
Chromosome number (2n = 28) |
Arm length (μm) |
Type |
|
|
Short (S) |
Long (L) |
Total (S + L) |
Arm ratio (L/S) |
|
1 |
1.31 |
2.21 |
3.52 |
1.68 |
mz)
|
|
2 |
1.19 |
2.11 |
3.30 |
1.77 |
smy)
|
|
3 |
1.07 |
2.07 |
3.14 |
1.93 |
sm |
|
4 |
1.05 |
2.01 |
3.06 |
1.91 |
sm |
|
5 |
1.27 |
1.62 |
2.89 |
1.27 |
m |
|
6 |
1.22 |
1.64 |
2.86 |
1.34 |
m |
|
7 |
1.58 |
1.27 |
2.85 |
0.80 |
m |
|
8 |
1.21 |
1.61 |
2.82 |
1.33 |
m |
|
9 |
1.41 |
1.31 |
2.72 |
0.92 |
m |
|
10 |
1.11 |
1.61 |
2.72 |
1.45 |
m |
|
11 |
1.07 |
1.64 |
2.71 |
1.53 |
m |
|
12 |
1.09 |
1.45 |
2.54 |
1.33 |
m |
|
13x)
|
0.85 |
1.67 |
2.52 |
1.96 |
sm |
|
14 |
0.99 |
1.38 |
2.37 |
1.39 |
m |
|
Total |
16.42 |
23.6 |
40.02 |
|
10 m + 4 sm |
References
- Biondo E, Miotto STS, Schifino-Wittmann MT. 2005. Cytogenetics of species of Chamaecrista (Leguminosae- Caesalpinioideae) native to southern Brazil. Bot J Linn Soc. 150: 429-439.
- Cordeiro JMP, Felix LP. 2018. Intra-and interspecific karyotypic variations of the genus Senna Mill. (Fabaceae, Caesalpinioideae). Acta Bot Bras. 32: 128-134.
- Dubcovsky J, Dvorák J. 1995. Ribosomal RNA multigene loci: nomads of the Triticeae genomes. Genetics. 140: 1367-1377.
- Elaine B, Miotto STS, Schifino-Wittmann MT, De Castro B. 2005. Cytogenetics and cytotaxonomy of Brazilian species of Senna Mill. (Cassieae - Caesalpinioideae - Leguminosae). Caryologia. 58: 152-163.
- Fuchs J, Brandes A, Schubert I. 1995. Telomere sequence localization and karyotype evolution in higher plants. Plant Syst Evol. 196: 227-241.
- Gerlach W, Bedbrook J. 1979. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7: 1869-1885.
- Han SE, Kim HH, Heo K. 2013. A cytogenetic study of Astragalus koraiensis YN Lee. Korean J Pl Taxon. 43: 139-145.
- Hwang YJ, Kim HH, Kwon SJ, Yang TJ, Ko HC, Park BS, et al. 2009. Karyotype analysis of three Brassica species using five different repetitive DNA markers by fluorescence in situ hybridization. Kor J Hort Sci Technol. 27: 456-463.
- Kato A, Lamb JC, Birchler JA. 2004. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proc Natl Acad Sci U S A. 101: 13554-13559.
- Kim S-Y, Choi H-W, Koo D-H, Kim C-S, Bang JW. 2005. Karyotype analysis and physical mapping of rDNAs using McFISH in Jeffersonia dubia Benth. Korean J Med Crop Sci. 13: 48-51.
- Koo D-H, Kim S-Y, Bang K-H, Seong N-S, Bang JW. 2003a. Cytogenetic analyses of Angelica plants using feulgen staining and multicolor fluorescence in situ hybridization. J Plant Biotechnol. 30: 123-127.
- Koo D-H, Seong N-S, Seong J-S, Bang K-H, Bang JW. 2003b. Karyotype analysis and physical mapping of rDNAs in Bupleurum longeradiatum. Korean J Med Crop Sci. 11: 402-407.
- Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas. 52: 201-220.
- Marazzi B, Endress PK, de Queiroz LP, Conti E. 2006. Phylogenetic relationships within Senna (Leguminosae, Cassiinae) based on three chloroplast DNA regions: patterns in the evolution of floral symmetry and extrafloral nectaries. Am J Bot. 93: 288-303.
- Monkheang P, Sudmoon R, Tanee T, Noikotr K, Bletter N, Chaveerach A. 2011. Species diversity, usages, molecular markers and barcode of medicinal Senna species (Fabaceae, Caesalpinioideae) in Thailand. J Med Plants Res. 5: 6173-6181.
- Murata M, Heslop-Harrison JS, Motoyoshi F. 1997. Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones. Plant J. 12: 31-37.
- Park HM, Jeon EJ, Waminal NE, Shin KS, Kweon SJ, Park BS, et al. 2010. Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization. Genes & Genomics. 32: 527-531.
- Resende K, Prado C, Davide L, Torres G. 2014. Polyploidy and apomixis in accessions of Senna rugosa (GDon) H.S.Irwin & Barneby. Turkish J Biol. 38: 510-515.
- Rice A, Glick L, Abadi S, Einhorn M, Kopelman NM, Salman-Minkov A, et al. 2015. The Chromosome Counts Database (CCDB) – a community resource of plant chromosome numbers. New Phytol. 206: 19-26.
- Roa F, Guerra M. 2012. Distribution of 45S rDNA sites in chromosomes of plants: structural and evolutionary implications. BMC Evol Biol. 12: 225
- Roy V, Monti-Dedieu L, Chaminade N, Siljak-Yakovlev S, Aulard S, Lemeunier F, et al. 2005. Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster. Heredity (Edinb). 94: 388-395.
- Singh S, Singh SK, Yadav A. 2013. A review on Cassia species: Pharmacological, traditional and medicinal aspects in various countries. American Jouranl of Phytomedicine and Clinical Therapeutics. 1: 291-312.
- Souza MGC, Benko-Iseppon AM. 2004. Cytogenetics and chromosome banding patterns in Caesalpinioideae and Papilionioideae species of Pará, Amazonas, Brazil. Bot J Linn Soc. 144: 181-191.
- Waminal NE, Kim N-S, Kim HH. 2011. Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species. Genes & Genomics. 33: 521-528.
- Waminal NE, Park HM, Ryu KB, Kim JH, Yang TJ, Kim HH. 2012. Karyotype analysis of Panax ginseng C.A.Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution. Comp Cytogenet. 6: 425-441.
- Waminal NE, Pellerin RJ, Jang W, Kim HH, Yang TJ. 2018. Characterization of chromosome-specific microsatellite repeats and telomere repeats based on low coverage whole genome sequence reads in Panax ginseng. Plant Breed Biotech. 6: 74-81.
