在蘭科(Orchidaceae)植物中，扇形文心蘭(Erycina pusilla, 2n = 10)因具有多個優良的特性，例如基因組與染色體數目少、生殖世代短、全年皆可開花，且其成株個體小可在生長箱內培養而不受氣候影響，因此被認為非常具有潛力作為蘭科的模式物種。而在細胞遺傳研究中，已經透過分析染色體長度、長短臂比例的方式建立了扇形文心蘭的簡易核型，然而目前尚缺乏專一性的標誌能更精準的辨識扇形文心蘭的每一對染色體。本實驗中，我們以蘭嶼姬蝴蝶蘭的全基因序列作為參考，設計出數個扇形文心蘭的基因片段，包含EpPEPC、EpEFS、EpCMT、EpERS、EpMDAR與EpSOC1，將這些基因片段作為酪胺訊號放大螢光原位雜合(Tyramide-FISH)的探針，試圖在染色體上標記具專一性的訊號。酪胺訊號放大螢光原位雜合(Tyramide-FISH)比ㄧ般的螢光原位雜合技術所需的探針長度更小(<1 kb)，因此只需要設計部分基因片段序列，便能作為探針。實驗結果顯示，EpPEPC的訊號位於每對染色體的末端。EpCMT與EpERS的訊號分別位於第五對染色體長臂末端以及第一對染色體短臂接近末端位置。而EpMDAR與EpEFS的訊號則分別位於第二對及第三對染色體短臂末端。因此綜合以上的結果，我們成功的建立了用來辨識扇形文心蘭染色體的專一性標誌。

Erycina pusilla is a good plant model in orchid species since it has several advantages, such as a tiny adult plant, shorter generation time than other wild and commercialized orchids, all year blooming, a relatively small genome size and low chromosome number. Although the conventional karyotype analysis in E. pusilla has been reported, neither the chromosome pattern nor the arm ratio can be used for precise chromosome identification. Therefore, it needs specific markers to help distinguishing chromosomes. In this study, we selected gene fragments of EpPEPC, EpEFS, EpCMT, EpERS, EpMDAR and EpSOC1 as tyramide-FISH probes using the full-length corresponding gene sequences of Phalaenopsis equestris as references. Tyramide-FISH can be used to detect smaller probes (less than 1 kb) than conventional FISH. In tyramide-FISH analysis, signals of EpPEPC were located on the terminal sites of every pair of chromosomes. The signals of EpERS and EpCMT, which were located on all same chromosomes with the signals of 5S rDNA, were respectively located on the short arm of the first pair of chromosomes and the long arm of the fifth pair of chromosomes. The signals of EpEFS were located on the short arm of the third pair of chromosomes, and the signals of EpMDAR were located on the short arm of the second pair of chromosomes. With these discriminative signals on chromosomes, we have established the specific markers for chromosome identification in E. pusilla.

Ahmad, Q.N., Britten, E.J. and Byth, D.E. (1983) A Quantitative Method of Karyotypic Analysis Applied to the Soybean, Glycine max. Cytologia, 48: 879-892.
Anderson, L.K., Stack, S.M., Fox, M.H. and Chuanshan, Z. (1985) The relationship between genome size and synaptonemal complex length in higher plants. Exp. Cell Res., 156, 367-378.
Bennett, M.D. and Leitch, I.J. (1995) Nuclear DNA Amounts in Angiosperms. Annals of Botany, 76, 113-176.
Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. and Litt, G.J. (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods, 125, 279-285.
Cabral, J.S., Felix, L.P. and Guerra, M. (2006) Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae). Genet. Mol. Biol., 29, 659-664.
Cai, J., Liu, X., Vanneste, K., Proost, S., Tsai, W.C., Liu, K.W., et al. (2015) The genome sequence of the orchid Phalaenopsis equestris. Nat. Genet., 47, 65-72.
Chandler, S.F. and Sanchez, C. (2012) Genetic modification; the development of transgenic ornamental plant varieties. Plant Biotechnol J, 10, 891-903.
Chao, Y.T., Chen, W.C., Chen, C.Y., Ho, H.Y., Yeh, C.H., Kuo, Y.T., et al. (2018) Chromosome-level assembly, genetic and physical mapping of Phalaenopsis aphrodite genome provides new insights into species adaptation and resources for orchid breeding. Plant Biotechnol J.
Chase, M.W., Hanson, L., Albert, V.A., Whitten, W.M. and Williams, N.H. (2005) Life History Evolution and Genome Size in Subtribe Oncidiinae (Orchidaceae). Annals of Botany, 95, 191-199.
Chen, C., Chen, K., Liu, C. and Kao, Y. (2002) Mapping of DNA markers to arms and sub-arm regions of Nicotiana sylvestris chromosomes using aberrant alien addition lines. Theor Appl Genet, 105, 8-15.
Chiu, Y.T., Lin, C.-S. and Chang, C. (2011) In vitro fruiting and seed production in Erycina pusilla (L.) N. H. Wiliams & M. W. Chase.
Chou, M.L., Shih, M.C., Chan, M.T., Liao, S.Y., Hsu, C.T., Haung, Y.T., et al. (2013) Global transcriptome analysis and identification of a CONSTANS-like gene family in the orchid Erycina pusilla. Planta, 237, 1425-1441.
Corput, M.P., Dirks, R.W., van Gijlswijk, R.P., van Binnendijk, E., Hattinger, C.M., de Paus, R.A., et al. (1998) Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and 20
tyramide signal amplification. J. Histochem. Cytochem., 46, 1249-1259.
Dressler, R. (1993) Phylogeny and Classification of Orchid Family.
Endo, R.T. and Gill, B.S. (1984) Somatic karyotype, heterochromatin distribution, and nature of chromosome differentiation in common wheat, Triticum aestivum L. em Thell. Chromosoma, 89, 361-369.
Felix, L.P. and Guerra, M. (1999) Chromosome analysis in Psygmorchis pusilla (L.) Dodson & Dressier: the smallest chromosome number known in Orchidaceae. Caryologia, 52, 165-168.
Felix, L.P. and Guerra, M. (2010) Variation in chromosome number and the basic number of subfamily Epidendroideae (Orchidaceae). Botanical Journal of the Linnean Society, 163, 234-278.
Gerlach, W.L., Miller, T.E. and Flavell, R.B. (1980) The nucleolus organizers of diploid wheats revealed by in situ hybridization. Theor Appl Genet, 58, 97-100.
Gijlswijk, R.P., Zijlmans, H.J., Wiegant, J., Bobrow, M.N., Erickson, T.J., Adler, K.E., et al. (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J. Histochem. Cytochem., 45, 375-382.
Hizume, M., Shibata, F., Matsusaki, Y. and Garajova, Z. (2002) Chromosome identification and comparative karyotypic analyses of four Pinus species. Theor Appl Genet, 105, 491-497.
Hoshi, Y., Pląder, W. and Malepszy, S. (1998) New C‐banding pattern for chromosome identification in cucumber (Cucumis sativus L.). Plant Breeding, 117, 77-82.
Iovene, M., Wielgus, S.M., Simon, P.W., Buell, C.R. and Jiang, J. (2008) Chromatin Structure and Physical Mapping of Chromosome 6 of Potato and Comparative Analyses With Tomato. Genetics, 180, 1307.
Kato, A., Albert, P.S., Vega, J.M. and Birchler, J.A. (2006) Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation. Biotech. Histochem., 81, 71-78.
Kato, A., Lamb, J.C. and Birchler, J.A. (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.
Kerbauy, G.B. (1984) In vitro flowering of Oncidium varicosum mericlones (orchidaceae). Plant Science Letters, 35, 73-75.
Khrustaleva, L., Jiang, J. and Havey, M.J. (2016) High-resolution tyramide-FISH mapping of markers tightly linked to the male-fertility restoration (Ms) locus of onion. Theor Appl Genet, 129, 535-545.
Khrustaleva, L., Kirov, I., Romanov, D., Budylin, M., Lapitskaya, I., Kiseleva, A., et al. (2012) The Chromosome Organization of Genes and Some Types of Extragenic DNA 21
in Allium.
Khrustaleva, L. and Kik, C. (2002) Localization of single‐copy T‐DNA insertion in transgenic shallots (Allium cepa) by using ultra‐sensitive FISH with tyramide signal amplification. The Plant Journal, 25, 699-707.
Kirov, I., Divashuk, M., Van Laere, K., Soloviev, A. and Khrustaleva, L. (2014a) An easy "SteamDrop" method for high quality plant chromosome preparation. Mol. Cytogenet., 7, 21.
Kirov, I., Van Laere, K., De Riek, J., De Keyser, E., Van Roy, N. and Khrustaleva, L. (2014b) Anchoring linkage groups of the Rosa genetic map to physical chromosomes with tyramide-FISH and EST-SNP markers. PLoS One, 9, e95793.
Kirov, I., Van Laere, K. and Khrustaleva, L. (2015) High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa. BMC Genet., 16, 74.
Koumbaris, G.L. and Bass, H.W. (2003) A new single-locus cytogenetic mapping system for maize (Zea mays L.): overcoming FISH detection limits with marker-selected sorghum (S. propinquum L.) BAC clones. The Plant Journal, 35, 647-659.
Krylov, V., Tlapakova, T. and Macha, J. (2007) Localization of the single copy gene Mdh2 on Xenopus tropicalis chromosomes by FISH-TSA. Cytogenet. Genome Res., 116, 110-112.
Krylov, V., Tlapakova, T., Macha, J., Curlej, J., Ryban, L. and Chrenek, P. (2008) Localization of human coagulation factor VIII (hFVIII) in transgenic rabbit by FISH-TSA: identification of transgene copy number and transmission to the next generation. Folia Biol. (Praha), 54, 121-124.
Kuo, Y.T., Hsu, H.L., Yeh, C.H. and Chang, S.B. (2016) Application of a modified drop method for high-resolution pachytene chromosome spreads in two Phalaenopsis species. Mol. Cytogenet., 9, 44.
Lamb, J.C., Danilova, T., Bauer, M.J., Meyer, J.M., Holland, J.J., Jensen, M.D., et al. (2007) Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes. Genetics, 175, 1047-1058.
Lee, S.H., Li, C.W., Liau, C.H., Chang, P.Y., Liao, L.J., Lin, C.S., et al. (2014) Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla. Plant Cell, Tissue and Organ Culture (PCTOC), 120, 211-220.
Li, D.J. (2016) The distribution of repetitive sequences on metaphase chromosomes of Erycina pusilla. In Institute of Life Sciences: National Cheng Kung University, pp. 1-51.
Lin, C.S., Chen, J.J., Huang, Y.T., Hsu, C.T., Lu, H.C., Chou, M.L., et al. (2013) 22
Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families. Plant Mol. Biol., 82, 193-204.
Lin, C.S., Hsu, C.T., Liao, D.C., Chang, W.J., Chou, M.L., Huang, Y.T., et al. (2016) Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla. Plant Biotechnol J, 14, 284-298.
Moraes, A.P., Leitch, I.J. and Leitch, A.R. (2012) Chromosome studies in Orchidaceae: karyotype divergence in Neotropical genera in subtribe Maxillariinae. Botanical Journal of the Linnean Society, 170, 29-39.
Moscone, E.A. (1990) Chromosome Studies on Capsicum (Solanaceae) I. Karyotype Analysis in C.chacoense. Brittonia, 42, 147-154.
Pan, I.C., Liao, D.-C., Wu, F.-H., Daniell, H., Singh, N.D., Chang, C., et al. (2012) Complete Chloroplast Genome Sequence of an Orchid Model Plant Candidate: Erycina pusilla Apply in Tropical Oncidium Breeding. PLoS One, 7, e34738.
Perez, R., de Bustos, A., Jouve, N. and Cuadrado, A. (2009) Localization of Rad50, a single-copy gene, on group 5 chromosomes of wheat, using a FISH protocol employing tyramide for signal amplification (Tyr-FISH). Cytogenet. Genome Res., 125, 321-328.
Raap, A.K., van de Corput, M.P., Vervenne, R.A., van Gijlswijk, R.P., Tanke, H.J. and Wiegant, J. (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum. Mol. Genet., 4, 529-534.
Rahman, A., Khan, M., Jobair Hossain, S.M., S. Hossain, M., M.A, H. and S.S, H. (2002) Quantitative Karyotype Analysis of Lycopersicon esculentum cv. Oxheart. PJBC. 5, 581-584.
Salina, E.A., Sergeeva, E.M., Adonina, I.G., Shcherban, A.B., Afonnikov, D.A., Belcram, H., et al. (2009) Isolation and sequence analysis of the wheat B genome subtelomeric DNA. BMC Genomics, 10, 414.
Sanz, M.J., Loarce, Y., Ferrer, E. and Fominaya, A. (2012) Use of tyramide-fluorescence in situ hybridization and chromosome microdissection for ascertaining homology relationships and chromosome linkage group associations in oats. Cytogenet. Genome Res., 136, 145-156.
Schriml, L.M., Padilla-Nash, H.M., Coleman, A., Moen, P., Nash, W.G., Menninger, J., et al. (1999) Tyramide signal amplification (TSA)-FISH applied to mapping PCR-labeled probes less than 1 kb in size. Biotechniques, 27, 608-613.
Schwarzacher, T., Ambros, P. and Schweizer, D. (1980) Application of Giemsa banding to orchid karyotype analysis. Plant Systematics and Evolution, 134, 293-297.
Shibata, F., Matsusaki, Y. and Hizume, M. (2005) AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus
23
densiflora. Theor Appl Genet, 110, 1253-1258.
Shindo, K. and Kamemoto, H. (1963) Karyotype Analysis of Some Sarcanthine Orchids. Am. J. Bot., 50, 73-79.
Siroky, J. (2008) Chromosome landmarks as tools to study the genome of Arabidopsis thaliana. Cytogenet. Genome Res., 120, 202-209.
Stephens, J.L., Brown, S.E., Lapitan, N.L. and Knudson, D.L. (2004) Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique. Genome, 47, 179-189.
Tek, A.L. and Jiang, J. (2004) The centromeric regions of potato chromosomes contain megabase-sized tandem arrays of telomere-similar sequence. Chromosoma, 113, 77-83.
Wang, C.-J.R., Harper, L. and Cande, W.Z. (2006) High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9. The Plant Cell, 18, 529.
Yeh, H.-Y., Lin, C.-S. and Chang, S.-B. (2015) Cytogenetic and cytometric analyses in artificial intercytotypic hybrids of the emergent orchid model species Erycina pusilla. Euphytica, 206, 533-539.
Yeh, H.-Y., Lin, C.-S., de Jong, H. and Chang, S.-B. (2017) Two reported cytotypes of the emergent orchid model species Erycina pusilla are two different species. Euphytica, 213.
Zhang, P., Li, W., Fellers, J., Friebe, B. and Gill, B.S. (2004) BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements. Chromosoma, 112, 288-299.
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