New Karyotype Information of Hymenocallis Littoralis, Amaryllidaceae

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New Karyotype Information of Hymenocallis littoralis, Amaryllidaceae

Tawatchai Tanee1,2, Runglawan Sudmoon1,3, Pornarong Siripiyasing1,4, Kittiya Silawong Suwannakud1,5, Pansa Monkheang1 and Arunrat Chaveerach1,6*

1 Genetics and Environmental Toxicology Research Group, Khon Kaen University, Muangkhonkaen, Khon Kaen 40002, Thailand 2 Faculty of Environment and Resource Studies, Mahasarakham University, Kantarawichai, Mahasarakham 44000, Thailand 3 Faculty of Law, Khon Kaen University, Muangkhonkaen, Khon Kaen 40002, Thailand 4 Faculty of Science and Technology, Mahasarakham Rajabhat University, Muangmahasarakham, Mahasarakham 44000, Thailand 5 Graduate School, Khon Kaen University, Muangkhonkaen, Khon Kaen 40002, Thailand 6 Department of Biology, Faculty of Science, Khon Kaen University, Muangkhonkaen, Khon Kaen 40002, Thailand

Received July 18, 2018; accepted August 31, 2018

Summary Hymenocallis littoralis was previously reported various chromosome numbers of 2n=44, 46, 48, 49 and 68 by a conventional staining. In order to analyze these chromosomes in detail, karyotype analysis by the conventional staining and a ﬂuorescence in situ hybridization (FISH) using probes of 45S and 5S rDNA,

and telomere repeats of human-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n was performed on chromosomes of H. littoralis from Thailand. The plants have 26 chromosome pairs (2n=52), and this chromosome number was not consistent with any of the previous reports. The 45S rDNA signals were localized at the terminal region of short arms of two chromosome pairs. Heteromorphic signals of 5S rDNA were detected at the proximal region of the short arms of one long chromosome, and at the interstitial region of the long arm of one short chromosome. The signals of human-type telomere repeat were detected at all chromosome ends, but FISH with Arabidopsis-type telomere repeat showed no signal.

Key words Hymenocallis littoralis, Aceto-orcein staining, FISH, 45S rDNA, 5S rDNA, Telomere, Chromo- some.

Hymenocallis composing 70 species is one of genera tion of H. littoralis using conventional staining and FISH in the subfamily Amaryllidoideae (Chase et al. 2009, methods. Tapia-Campos et al. 2012). H. littoralis (Jacq.) Salisb. is native to warm coastal areas of Southern Mexico and Materials and methods Central America, but widely cultivated and naturalized in many tropical regions as a horticultural and a medici- Plant materials and chromosome preparation nal plant (Ogden 2007). Chromosome numbers of the H. Bulbs from six plants of H. littoralis cultivated in littoralis were reported as 2n=44, 46, 48, 49 and 68, and Khon Kaen province, northeastern Thailand were placed karyotype based on conventional staining methods has in distilled water at room temperature for rooting. The been reported (Sato 1938, Flory 1976, Lakshmi 1978, root tips were kept in cold water for 1 h at 4°C, trans- Nwankiti 1985, Vijayavalli and Mathew 1990). Compar- ferred to 0.05% colchicine for 4 h at room temperature, ative karyotype analysis of this species having various and fixed in ethanol : acetic acid (3 : 1, v/v) for at least chromosome number is desired to reveal relationships 24 h at 4°C. Chromosome preparations were performed among karyotypes with different chromosome number. following the Steam-Drop method (Kirov et al. 2014) In order to establish the tool of karyotype analysis in H. with a few modifications. Briefly, fixed root tips were littoralis, the FISH technique is applied to chromosomes washed twice with an enzyme buffer (0.01 M citric acid, using universal FISH probes of rDNA and telomeres. 0.01 M sodium citrate, pH 4.7). Then they were digested This report was investigated that FISH procedure is in an enzyme solution [0.7% cellulase R10 (Duchefa), adaptable or not to chromosomes of this species. The 0.7% cellulase (Merck), 1% pectolyase (Sigma) and 1% aim of this study is to obtain basic karyotype informa- cytohelicase (Sigma) in the enzyme buffer] at 37°C for 4 h. Then, the meristems were washed twice with * Corresponding author, e-mail: [email protected] distilled water and 96% ethanol and the ethanol was DOI: 10.1508/cytologia.83.437 removed by centrifugation. The meristems were broken 438 T. Tanee et al. Cytologia 83(4) with a dissecting needle within a tube in the fixative. and sealed by rubber gum. The co-denaturation of probe The cell suspension was dropped on a glass slide and air and chromosome DNAs was done at 80°C for 2 min, and dried. Preparations with well-spread metaphases were then hybridization was performed in a moist chamber at selected for further analyses. 37°C for 18 h. After hybridization, the slides were washed in 2×SSC Aceto–orcein staining and karyotype analysis and 50% formamide in 2×SSC for 5 min each at 42°C, Conventional staining was carried out using 2% dehydrated in an ethanol series, air dried, and counter- aceto–orcein for 5 min at room temperature; afterwards, stained with an antifade mounting medium (Vectashield, slides were covered with a coverslip. Ten well-spread Vector Laboratories) with DAPI (Vector Laboratories). metaphases were selected for photomicrography. The FISH signals were detected using an epifluorescence mi- length of short and long chromosome arms were mea- croscope (Axioplan 2, Zeiss) with appropriate filters. Af- sured separately and added to calculate the total length. ter the signals of slides were microscopically recorded, The relative length (RL=100× chromosome length/total the hybridized probes were removed under a high-strin- haploid length), centromeric index (CI=length of short gent condition before reprobing following the method arm/total chromosome length) and standard deviation of reported by Tran et al. (2017). Then another round of RL and CI were calculated (Martínez et al. 2002). The counterstaining and signal detection was performed. CI between 0.50–0.59, 0.60–0.69, 0.70–0.89 and 0.90– 0.99 were considered as metacentric, submetacentric, Results and discussion acrocentric and telocentric chromosomes, respectively (Turpin and Lejeune 1965). All six plants of H. littoralis examined in this study had a chromosome number 2n=52 (Fig. 1A) which DNA probes and labeling constituted 15 small and 11 medium chromosomes. Plasmid DNA of 45S rDNA (BAC T15P10 accession The range of total arm length, centromeric index, and AF167571, Mathieu et al. 2003, Lysak et al. 2006) and relative chromosome length were 1.55–5.58 µm, 0.669– 5S rDNA amplified by PCR (Mathieu et al. 2003) from 0.553 and 1.9–6.9%, respectively. The chromosome Arabidopsis thaliana were provided by Quantitative number in this study did not consequence to any previ- Genetics and Chromosome Structure and Function Re- ous reports with 2n=44, 46, 48, 49 and 68 (Sato 1938, search Groups of Leibniz Institute of Plant Genetics and Flory 1976, Lakshmi 1978, Nwankiti 1985, Vijayavalli Crop Plant Research (IPK), Germany. The 45S rDNA and Mathew 1990). The diversity of the chromosome was labeled with Alexa 488-dUTP (Invitrogen) and the number within the species not dependent on polyploidi- 5S rDNA was labeled with Cy3-dUTP (Invitrogen) by a zation was very interesting in considering the evolution nick translation mix (Roche). The telomeric repeats were of chromosomes. amplified by polymerase chain reaction in absence of a For exact comparison among karyotypes of plants DNA template using a set of primers for Arabidopsis- with different chromosome numbers, FISH karyotyping type telomere repeat (TTT AGG G)5 and for human-type was necessary. Well-spread metaphase chromosomes telomere repeat (CCC TAA A)5 (Ijdo et al. 1991, Cox were hybridized with 45S rDNA and 5S rDNA (Fig. 1B). et al. 1993, Shibata and Hizume 2011) and labelled with The signals of the 45S rDNA probe were observed at ter- Cy3-dUTP (Invitrogen) by the nick translation mix. minal of region of the short arms of chromosomes pairs 5 and pair 7 in the two plant individuals and the weaker FISH procedure signals were observed on chromosome pairs 6, 10, 13, FISH was performed following Lysak et al. (2006) 14, 17, 18, 23, 24, 25, 26, these weak signals showed and Monkheang et al. (2016) with minor modifications. heteromorphy. Either this heteromorphic signal repre- Slides were washed in 2×SSC (300 mM Na–citrate, sents the presence or absence of 45S rDNA or difference 30 mM NaCl, pH 7.0) and fixed with 45% acetic acid for of detection sensitivity, was not revealed. Heteromorphic 3 min, then washed twice in 2×SSC for 5 min at room signals of 5S rDNA probe were detected at a proximal temperature and treated in a pepsin solution (10 mg mL-1 region of the short arms of chromosome pairs 2 and 26 in 0.01 M HCl) for 1 min at 37°C. After rinsing twice in in the two plant individuals and all plants show the same 2×SSC, the slides were post-fixed in 4% formaldehyde heteromorphic signals (Fig. 1B). in 2×SSC for 5 min at room temperature and washed When the chromosomes were hybridized with two twice in 2×SSC. The slides were dehydrated in ethanol types of telomere repeat respectively, only chromo- series (70, 90, 100%) for 2 min each, at room tempera- somes that hybridized with human-type telomere repeat ture and air dried. Simultaneously, the labeled probe showed signals at all chromosome ends (Fig. 1C), but (3 ng µL-1) in a hybridization mixture (20% dextran sul- the Arabidopsis-type telomere repeat did not show any fate, 50% formamide in 2×SSC, pH 7.0) was pre-dena- signal. In most of plant species chromosome ends con- tured at 95°C for 5 min, then 20 µL of the hybridization sist of short tandem repeats, such as Arabidopsis-type mixture was added to the slide, covered with a coverslip telomere repeat (TTT AGG G)n or human-type telomere 2018 New Karyotype Information of Hymenocallis littoralis 439

Fig. 1. Mitotic metaphase plates and karyotypes of H. littoralis (2n=52). A: Aceto–orcein stained chromosomes, B: FISH image probed with 45S (green) and 5S (red) rDNA probes, showing heteromorphy at 5S rDNA signal sites, C: FISH signals (red)

of human-type telomere (TTAGGG)n were located on chromosome ends. Scale bars=10 µm.

repeat (TTA GGG)n, or none of any type telomere repeat Khon Kaen University. Thanks to Prof. Ingo Schubert, (Richards and Ausubel 1988, Moyzis et al. 1988, Tran the Leibniz Institute of Plant Genetics and Crop Plant et al. 2015, Fajkus et al. 2016). Some species of Amaryl- Research (IPK), Germany for providing laboratory fa- lidaceae have human-type telomere repeats (Sykorova cilities (including probes) and assistances and Prof. Dr. et al. 2003, Shibata and Hizume 2011). The result that Thomas Liehr, Institute of Human Genetics, Univer- only human-type telomere repeat was detected might sitätsklinikum Jena, Germany for critical comments and be an important clue of the evolution of telomere repeat helpful suggestions. in the family Amaryllidoideae. Conventional karyotype analysis and FISH analysis using 45S and 5S rDNA References probes are expected to be applied to chromosomes with different chromosome numbers to reveal chromosome Chase, M. W., Reveal, J. L. and Fay, M. F. 2009. A subfamilial classi- evolution in H. littoralis. ﬁcation for the expanded asparagalean families Amaryllidaceae, Asparagaceae and Xanthorrhoeaceae. Bot. J. Linn. Soc. 161: 132–136. Acknowledgements Cox, A. V., Bennett, S. T., Parokonny, A. S., Kenton, A. Y., Cal- limassia, M. A. and Bennett, M. D. 1993. Comparison of plant The authors are thankful for the research fund from telomere locations using a PCR-generated synthetic probe. Ann. Student Exchange Program (2015), Faculty of Science, Bot. 72: 239–247. 440 T. Tanee et al. Cytologia 83(4)

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