Research Article |
Corresponding author: Arunrat Chaveerach ( raccha@kku.ac.th ) Academic editor: Lorenzo Peruzzi
© 2016 Pansa Monkheang, Arunrat Chaveerach, Runglawan Sudmoon, Tawatchai Tanee.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Monkheang P, Chaveerach A, Sudmoon R, Tanee T (2016) Karyotypic features including organizations of the 5S, 45S rDNA loci and telomeres of Scadoxus multiflorus (Amaryllidaceae). Comparative Cytogenetics 10(4): 637-646. https://doi.org/10.3897/CompCytogen.v10i4.9958
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Scadoxus multiflorus Martyn, 1795 is an ornamental plant with brilliantly colored flowers. Even though its chromosomes are rather large, there is no karyotype description reported so far. Therefore, conventional and molecular cytogenetic studies including fluorescence in situ hybridization (FISH) with 45S and 5S rDNA, and human telomere sequence (TTAGGG)n probes (Arabidopsis-type telomere probes yielded negative results) were carried out. The chromosome number is as reported previously, 2n = 18. The nine chromosome pairs include two large submetacentric, five large acrocentric, one medium acrocentric, two small metacentric and eight small submetacentric chromosomes. Hybridization sites of the 45S rDNA signals were on the short arm ends of chromosomes #1, #3 and #8, while 5S rDNA signals appeared on the long arm of chromosome 3, in one homologue as a double signal. The telomere signals were restricted to all chromosome ends. Three chromosome pairs could be newly identified, chromosome pair 3 by 5S rDNA and chromosomes #1, #3 and #8 by 45S rDNA loci. In addition to new information about rDNA locations we show that the ends of S. multiflorus chromosomes harbor human instead of Arabidopsis-type telomere sequences. Overall, the S. multiflorus karyotype presents chromosomal heteromorphy concerning size, shape and 45S and 5S rDNA positioning. As Scadoxus Rafinesque, 1838 and related species are poorly studied on chromosomal level the here presented data is important for better understanding of evolution in Amaryllidaceae.
Scadoxus multiflorus , aceto-orcein staining, fluorescence in situ hybridization (FISH), rDNA, telomere
Scadoxus multiflorus Martyn, 1795 (also known as Haemanthus multiflorus Martyn, 1795 and H. kalbreyeri Baker, 1878) is a species belonging to the family Amaryllidaceae (
Scadoxus multiflorus is economically important as it is popular as cultivated ornamental plant. Nonetheless, this species was not studied yet for its karyotype in details. There are only two previous reports based on conventional chromosome staining of S. multiflorus. Both studies reported 2n = 18 chromosomes, however,
As for S. multiflorus reported karyotypic details are contradictory and no FISH studies have been performed so far the present study aimed to close this gap of knowledge using conventional staining and FISH.
Scadoxus multiflorus plant was collected in Khon Kaen Province, Northeastern, Thailand (A. Chaveerach et al. 903, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand). The roots were collected from bulbs placed in distilled water at room temperature. The root tips were excised and kept in cold water for 1 h at 4°C, after that transferred to 0.05% colchicine solution for 4 h at room temperature to accumulate metaphase chromosomes before fixation in ethanol:acetic acid (3:1, v/v) for at least 24 h at 4°C. The protocol for the SteamDrop method (
Conventional staining was carried out on slides using 2% aceto-orcein for 5 min at room temperature and then covered with a coverslip. Ten well-spread metaphases were selected for photomicrography with a digital camera under oil immersion by light microscope. The length of short and long chromosome arms (p and q) were measured separately and added to calculate the total length (LT). The relative length of chromosome (RL), the centromeric index (CI) and standard deviation (SD) of RL and CI were calculated according to
45S and 5S rDNAs which were isolated from Arabidopsis thaliana Schur, 1866 and telomere repeat sequences from Arabidopsis (TTTAGGG)n and human (TTAGGG)n were applied in this study. The plasmid of 45S rDNA cloned in the vector T15P10IV1 was labelled with Alexa 488-dUTP, while the 5S rDNA probe was labelled with Cy3-dUTP by Nick translation (Roche Cat No 11745808910). The telomeric probes were generated by polymerase chain reaction (PCR) in absence of a DNA template using primers (TTTAGGG)5 and (CCCTAAA)5, and (TTAGGG)5 and (CCCTAA)5 according to
A FISH protocol according to
A 3 µl (60 ng) of labelled probe was dissolved in 17 µl of hybridization mixture (20% dextran sulfate, 50% formamide in 2 × SSC, pH 7.0), and pre-denatured at 95°C for 5 min. Then the solution was added to the slide, covered with a coverslip and sealed by rubber gum. Now, the slide was placed on a heating plate at 80°C for 2 min for co-denaturation of probe and target DNA and incubated in a moist chamber at 37°C for 18 h for hybridization.
After hybridization, slides were washed in 2 × SSC for 5 min at 42°C and three times with 50% formamide in 2 × SSC for 5 min at 42°C. After that, slides were washed three times with 2 × SSC for 5 min at 42°C for 5 min each. Finally, the slides were dehydrated in an ethanol series, air dried, counterstained with 4, 6-diamino-2-phenylindole (DAPI) plus Vectashield antifade mounting medium (Vector Laboratories, USA) and covered with a coverslip. Signals were detected using an epifluorescence microscope with Triple filter (UV, Texas Red and FITC) and photographed (microscope: Axioplan2, Zeiss; Camera: Hammamatsu-ORCA-ER C4742-80, Japan; Lamp: Flouarc, Zeiss).
The idiogram and karyotype analyses established from the metaphases confirmed the diploid chromosome number of S. multiflorus to be 2n = 18. The karyotype analysis of this species is summarized in Table
Mitotic metaphase chromosomes of Scadoxus multiflorus (2n = 18). A aceto-orcein staining of two cells B karyogram showing four large and five small pairs of chromosomes C idiogram.
Mean length of the short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI, RL from metaphase chromosomes in 10 cells of the blood lily (Scadoxus multiflorus), 2n = 18.
Chr. | Ls | Ll | LT | CI ± SD | RL ± SD | Type | Size |
---|---|---|---|---|---|---|---|
1a* | 4.86 | 11.14 | 16.007 | 0.696 ± 0.068 | 0.148 ± 0.010 | sm | L |
1b* | 4.66 | 9.81 | 14.465 | 0.678 ± 0.100 | 0.134 ± 0.025 | sm | L |
2a* | 2.85 | 9.89 | 12.741 | 0.776 ± 0.085 | 0.118 ± 0.008 | a | L |
2b* | 2.86 | 9.00 | 11.865 | 0.759 ± 0.073 | 0.110 ± 0.015 | a | L |
3 | 1.49 | 9.52 | 11.008 | 0.865 ± 0.131 | 0.102 ± 0.015 | a | L |
4a* | 2.29 | 8.58 | 10.870 | 0.790 ± 0.087 | 0.101 ± 0.013 | a | L |
4b* | 2.15 | 7.33 | 9.480 | 0.773 ± 0.074 | 0.088 ± 0.016 | a | M |
5 | 1.62 | 3.17 | 4.784 | 0.662 ± 0.066 | 0.044 ± 0.006 | sm | S |
6 | 1.95 | 2.53 | 4.485 | 0.565 ± 0.034 | 0.042 ± 0.004 | m | S |
7 | 1.50 | 2.90 | 4.402 | 0.659 ± 0.063 | 0.041 ± 0.004 | sm | S |
8 | 1.50 | 2.65 | 4.153 | 0.639 ± 0.065 | 0.038 ± 0.004 | sm | S |
9 | 1.19 | 2.59 | 3.779 | 0.685 ± 0.069 | 0.035 ± 0.005 | sm | S |
Comparison of different estimators of intrachromosomal asymmetry including mean centromeric asymmetry (MCA) and coefficient of variation of chromosome length (CVCL) from metaphase chromosomes in 10 cells of the blood lily (Scadoxus multiflorus), 2n = 18.
Chr. | Mean from 10 metaphases | SD of chr. length | MCA | CVCL | |||||
---|---|---|---|---|---|---|---|---|---|
Ll-Ls | Ls Ll | Ls (Ll+Ls) | Ll (Ll+Ls) | (Ll-Ls) Ll | (Ll-Ls) (Ll+Ls) | ||||
1a* | 6.28 | 0.44 | 0.30 | 0.70 | 0.56 | 0.39 | 2.21 | 39.23 | 13.82 |
1b* | 5.15 | 0.47 | 0.32 | 0.68 | 0.53 | 0.36 | 3.01 | 35.61 | 20.82 |
2a* | 7.03 | 0.29 | 0.22 | 0.78 | 0.71 | 0.55 | 2.30 | 55.20 | 18.02 |
2b* | 6.14 | 0.32 | 0.24 | 0.76 | 0.68 | 0.52 | 2.02 | 51.72 | 17.05 |
3 | 8.03 | 0.16 | 0.14 | 0.86 | 0.84 | 0.73 | 1.85 | 72.94 | 16.78 |
4a* | 6.29 | 0.27 | 0.21 | 0.79 | 0.73 | 0.58 | 1.71 | 57.90 | 15.71 |
4b* | 5.18 | 0.29 | 0.23 | 0.77 | 0.71 | 0.55 | 1.98 | 54.60 | 20.89 |
5 | 1.55 | 0.51 | 0.34 | 0.66 | 0.49 | 0.32 | 1.02 | 32.34 | 21.35 |
6 | 1.40 | 0.52 | 0.34 | 0.66 | 0.48 | 0.32 | 0.77 | 31.74 | 17.44 |
7 | 0.58 | 0.77 | 0.44 | 0.56 | 0.23 | 0.13 | 0.95 | 12.93 | 21.09 |
8 | 1.15 | 0.57 | 0.36 | 0.64 | 0.43 | 0.28 | 0.73 | 27.79 | 17.60 |
9 | 1.40 | 0.46 | 0.31 | 0.69 | 0.54 | 0.37 | 0.78 | 37.04 | 20.73 |
The metaphase spreads were hybridized with 45S (Alexa, green) and 5S (Cy3, red) rDNA probes as shown in Fig.
Organizations of 45S rDNA and 5S rDNA loci on metaphase chromosomes of Scadoxus multiflorus (2n = 18). AFISH signals of 45S (Alexa, green) and 5S (Cy3, red) rDNA probes on two cells B karyogram C idiogram.
The hybridization of the Arabidopsis-type telomeric probe yielded no obvious FISH signal, while the human-type (Cy3, red) revealed small signals exclusively at the end of all S. multiflorus chromosomes (Fig.
Localization of the human telomere repeat sequence (TTAGGG)n on metaphase chromosomes of Scadoxus multiflorus (2n = 18). AFISH signals of the (TTAGGG)n probe on two cells B karyogram C idiogram.
Overall, the results show chromosomal heteromorphy in sizes and shapes.
Here we provide the first study of S. multiflorus chromosomes by means of molecular cytogenetics. Furthermore, the previously reported chromosome number could be confirmed to be 2n = 18 (
Although the regions of 5S and 45S were not observed with conventional staining, due to several limitations, such as oil-immersion light microscopic methods, genetic processing and analysis standards, the signal intensities of rDNA probes in FISH showed clear variation in copy numbers. The copy number and distributed position on chromosome are very important as species markers (
In a group of families of the monocot order Asparagales, the telomeric sequence (TTAGGG)n of the human-type was found to be maintained (
Overall, our results allow now distinguishing five of the nine S. multiflorus chromosome pairs individually. Development of suitable genomic single-copy FISH probes might allow discrimination of all chromosome pairs and to use them for identification of homologous chromosomes in other species of genus, or even of related genera. As Scadoxus and related species are poorly studied on chromosomal level the here presented data is important for better understanding of evolution in Amaryllidaceae.
The authors thanks to Ingo Schubert, for providing plant materials and labelled probes, and all members of the Quantitative Genetics and the Chromosome Structure and Function Research Group of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany for offering laboratory facilities and assistance. Thanks to the Research and Study in Abroad Fund for Graduated Students from Faculty of Science, Khon Kaen University for providing financial support to research in Germany. We are thankful for critical comments and helpful suggestions by Thomas Liehr, Institute of Human Genetics, Universitätsklinikum Jena, Germany.