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. 2018 Aug 2;19(1):578.
doi: 10.1186/s12864-018-4956-7.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization

Yung-I Lee  1   2 , Jing Wei Yap  3   4   5 , Shairul Izan  6   7 , Ilia J Leitch  8 , Michael F Fay  4   9 , Yi-Ching Lee  10 , Oriane Hidalgo  4 , Steven Dodsworth  4 , Marinus J M Smulders  6 , Barbara Gravendeel  11   12   13 , Andrew R Leitch  3
Affiliations

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization

Yung-I Lee et al. BMC Genomics. .

Abstract

Background: Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA.

Results: SatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26-28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific.

Conclusions: We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.

Keywords: FISH; Fluorescent in situ hybridization; Karyotype; Paphiopedilum; Satellite DNA.

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Conflict of interest statement

1Biology Department, National Museum of Natural Science, No 1, Kuan-Chien Rd., 40,453 Taichung, Taiwan, ROC, 2Department of Life Sciences, National Chung Hsing University, Taichung 40,227, Taiwan, ROC, 3School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, UK, 4Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK, 5Forest Research Institute Malaysia (FRIM), 52,109 Kepong, Selangor Darul Ehsan, Malaysia, 6Plant Breeding, Wageningen University & Research, P.O. Box 386, NL-6700 AJ Wageningen, The Netherlands, 7Department of Crop Science, Faculty of Agriculture, University Putra Malaysia (UPM) Serdang, Selangor, Malaysia, 8Schoolof Plant Biology, University of Western Australia, Crawley, WA 6009, Australia, 9Endless Forms group, Naturalis Biodiversity Center, Vondellaan 55, 2332 AALeiden, The Netherlands, 10Faculty of Science and Technology, University of Applied Sciences Leiden, Zernikedreef 11, 2333 CK Leiden, The Netherlands, 11Institute Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

All the Paphiopedilum species seedlings used in this study are artificially propagated that approved by the Council of Agriculture of Executive Yuan in Taiwan with the regulations of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

Not applicable.

The authors declare that they have no competing interests.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Fluorescent in situ hybridization (FISH) of root tip metaphase chromosomes of species belonging to subgenus Parvisepalum with the SatA (red), 45S (green) and 5S (white) rDNA probes, counterstained with DAPI (blue): (a) P. armeniacum, (b) P. malipoense, (c) P. emersonii, (d) P. hangianum, (e) P. micranthum, (f) P. delenatii, (g) P. vietnamense. Bar = 10 μm
Fig. 2
Fig. 2
Fluorescent in situ hybridization (FISH) of root tip metaphase chromosomes with the SatA, 45S (green) and 5S (white) rDNA probes, counterstained with DAPI (blue): (a) P. concolor (subgenus Brachypetalum), (b) P. villosum (section Paphiopedilum), (c) P. rothschildianum (section Coryopedilum), (d) P. lowii (section Pardalopetalum), (e) P. appletonianum (section Barbata), and (f) P. primulinum (section Cochlopetalum). The absence of FISH signals confirms that SatA is indeed specific to subgenus Parvisepalum. Bar = 10 μm
Fig. 3
Fig. 3
Fluorescent in situ hybridization of karyotypes of Paphiopedilum subgenus Parvisepalum with the SatA (red), 45S (green) and 5S (white) rDNA probes, counterstained with DAPI (blue). Phylogenetic relationships between these species shown on the right-hand side of the figure (see also Additional file 1: Fig. S1)
Fig. 4
Fig. 4
Ideograms of somatic metaphase chromosomes of species belonging to subgenus Parvisepalum.SatA (red), 45S (green) and 5S (white) rDNA signals
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