Chromosome Karyotyping of Senna Covesii and S. Floribunda Based on Triple‑Color FISH Mapping of Rdnas and Telomeric Repeats
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Plant Breed. Biotech. 2018 (March) 6(1):51~56 Online ISSN: 2287-9366 https://doi.org/10.9787/PBB.2018.6.1.51 Print ISSN: 2287-9358 RESEARCH ARTICLE Chromosome Karyotyping of Senna covesii and S. floribunda based on Triple‑color FISH Mapping of rDNAs and Telomeric Repeats Sung Min Youn, Hyun Hee Kim* Department of Life Sciences, Chromosome Research Institute, Sahmyook University, Seoul 01795, Korea 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. Keywords 5S and 45S rDNAs, Telomeric repeat, Senna covesii, Senna floribunda, FISH INTRODUCTION species (Kim et al. 2005). The information can be useful in identifying species, tracing phylogenetic relationship The genus Senna in the family Fabaceae (Monkheang et among the related species, and breeding and development al. 2011) has various uses such as food, flower decoration of plant species (Koo et al. 2003b; Kim et al. 2005). and improvement of barren land (Elaine et al. 2005; Despite the economic and health benefits of Senna, Resende et al. 2014). It is especially well known as a cytogenetic studies are still limited in this genus (Kim et al. medicinal plant not only as ophthalmic medication but also 2005). in relieving skin diseases, alleviating poor digestion, Fluorescence in situ hybridization (FISH) technique is healing of colds and even heart disease. In addition, it has an efficient and powerful tool in molecular cytogenetics to expanded its usefulness as a living food such as coffee and visualize specific DNA sequences on chromosomes and gum (Singh et al. 2013). Because of these advantages, allows to construct chromosome map of the sequences. Senna is widely cultivated around the world including Highly repetitive DNA sequences such as rDNA, Brazil, America, Asia and Africa (Marazzi et al. 2006; centromeric-, and telomeric-DNA have been used as FISH Singh et al. 2013). probes owing to their different number and distinct Cytogenetic studies provide information on chromo- distribution on chromosomes according to the species somal number, length, type, ploidy, and chromosomal (Hwang et al. 2009). Especially 5S rDNA, 45S rDNA, and distribution of specific sequences that are essential to telomeric repeat DNA are highly repetitive DNAs and elucidate genome structure and constitution in plant usually used as primary probes for FISH experiment in Received February 12, 2018; Revised February 14, 2018; Accepted February 14, 2018; Published March 1, 2018 *Corresponding author Hyun Hee Kim, [email protected], Tel: +82-2-3399-1715, Fax: +82-2-3399-1729 Copyright ⓒ 2018 by the Korean Society of Breeding Science This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 52 ∙ Plant Breed. Biotech. 2018 (March) 6(1):51~56 plants (Koo et al. 2003a; Han et al. 2013). et al. (2018). Mitotic metaphase chromosome spreads were Here, triple-color FISH karyotype was established using obtained following the procedure of Waminal et al. (2012). 5S rDNA, 45S rDNA, and Arabidopsis-type telomeric Thirty-two μL of a FISH hybridization master mix (50% repeat DNA probes to elucidate the chromosomal formamide, 10% dextran sulfate, and 2× SSC) were mixed constitutions of the genome of S. covesii and S. floribunda. with 25 ng of each of PLOP probes. Distilled water was This is the first report on FISH karyotype of the two species added to a total volume of 40 µL. Chromosomes with and this basic information will be useful to construct added PLOP probes on a glass slide were denatured at phylogenetic relationships among Senna species and 80°C for 5 minutes. Slides were incubated at RT, 30 breeding program of the medicinal species. 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. MATERIALS AND METHODS Slides were dehydrated in ethanol series of 70%, 90%, and 100%, air-dried and counterstained with a premixed Plant samples 4',6-diamidino-2-phenylindole (DAPI) solution (1 μg/mL; Seeds of S. covesii and S. floribunda were obtained from DAPI in Vectashield, Vector Laboratories, Burlingame, Agricultural Research Service, USDA (PI 675048 and PI CA, USA). Images were captured with a model BX53 601948). Seeds were germinated in the greenhouse. About fluorescence microscope (Olympus, Tokyo, Japan) equipped 2 cm roots were harvested, pretreated with 2 mM with a DFC365 FS CCD camera (Leica Microsystems, 8-hydroxyquinoline for five hours at room temperature, Wetzlar, Germany) and adjusted by using Cytovision ver. fixed in aceto-ethanol (acetic acid : ethanol = 1 : 3, v/v) for 7.2 (Leica Microsystems). Final images were edited using more than two hours and transferred to 70% ethanol. The Adobe Photoshop CC (Adobe Systems, San Jose, CA, stock materials were kept in the refrigerator until use. USA). Chromosome types were determined according to the description of Levan et al. (1964). Length of Chromosome spread preparation chromosomes were measured using ImageJ (NIH, USA). 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 RESULTS 1% pectolyase (Sigma, Japan) and 2% cellulase (MB Cell, Korea) solution at 37°C for 75 minutes. Meristematic Conventional karyotype tissue (∼2 mm) of the roots were dissected using a needle, Both S. covesii and S. floribunda had chromosome transferred into a 1.5 mL tube with 40 μL chilled number of 2n = 28. The chromosome length of S. covesii aceto-ethanol fixative solution (acetic acid : ethanol = 1:3, ranged from 3.58 to 5.67 μm (Table 1) and those of S. v/v) and vortexed for 30 seconds at room temperature. floribunda ranged from 2.37 to 3.52 μm (Table 2). Both After centrifugation, pellets were resuspended with species had 10 metacentrics and four submetacentric aceto-ethanol (acetic acid : ethanol = 9 : 1, v/v) solution and chromosomes, but S. floribunda chromosomes were drop on a slide glass in the humid chamber and then shorter than those of S. covesii. One pair of satellite air-dried. chromosomes were observed in chromosome 13 of both species (Figs. 1 and 2). Fluorescence in situ hybridization The 5S and 45S rDNAs and Arabidopsis-type telomeric Chromosomal distribution of rDNA and telomeric repeat sequences repeat sequences (TTTAGGG)n were used as probes for FISH karyotype analysis. The pre-labelled oligoprobes As a result of triple-color FISH karyotype analysis using (PLOP) were designed and prepared according to Waminal 5S rDNA, 45S rDNA, and telomeric repeat probes, three Triple-color FISH in Senna Species ∙ 53 Table 1. Karyotypic analysis of mitotic metaphase chromo- Table 2. Karyotypic analysis of mitotic metaphase chromo- some of S. covesii. some of S. floribunda. Arm length (μm) Arm length (μm) Chromosome Chromosome Arm Arm number Short Long Total Type number Short Long Total Type ratio ratio (2n = 28) (S) (L) (S ＋ L) (2n = 28) (S) (L) (S ＋ L) (L/S) (L/S) 1 2.60 3.07 5.67 1.18 mz) 1 1.31 2.21 3.52 1.68 mz) 2 2.09 2.54 4.63 1.21 m 2 1.19 2.11 3.30 1.77 smy) 3 2.11 2.42 4.53 1.14 m 3 1.07 2.07 3.14 1.93 sm 4 1.97 2.43 4.40 1.23 m 4 1.05 2.01 3.06 1.91 sm 5 1.96 2.44 4.40 1.24 m 5 1.27 1.62 2.89 1.27 m 6 2.10 2.12 4.22 1.01 m 6 1.22 1.64 2.86 1.34 m 7 1.14 3.01 4.15 2.64 smy) 7 1.58 1.27 2.85 0.80 m 8 1.84 2.28 4.12 1.23 m 8 1.21 1.61 2.82 1.33 m 9 1.70 2.30 4.00 1.35 m 9 1.41 1.31 2.72 0.92 m 10 1.79 2.17 3.96 1.21 m 10 1.11 1.61 2.72 1.45 m 11 2.02 1.90 3.92 0.94 m 11 1.07 1.64 2.71 1.53 m 12 1.35 2.49 3.84 1.84 sm 12 1.09 1.45 2.54 1.33 m 13x) 1.10 2.53 3.63 2.30 sm 13x) 0.85 1.67 2.52 1.96 sm 14 1.12 2.46 3.58 2.19 sm 14 0.99 1.38 2.37 1.39 m Total 24.89 34.16 59.07 10 m ＋ Total 16.42 23.6 40.02 10 m ＋ 4 sm 4 sm z)metacentric, y)submetacentric, x)satellite chromosome with z)metacentric, y)submetacentric, x)satellite chromosome with secondary constriction.