蘭嶼姬蝴蝶蘭粗絲期染色體螢光原位雜交與核型分析

Abstract

蝴蝶蘭為國內重要的花卉產業，但蝴蝶蘭生長世代較長，遺傳圖譜尚未建立，細胞遺傳研究也相當缺乏。蘭嶼姬蝴蝶蘭為台灣原生種且具代表性的蘭花，基因組較小適合作為未來蝴蝶蘭基因組定序計畫的材料。蘭嶼姬蝴蝶蘭體細胞中期染色體大小型態相似，以傳統染色法難以鑑別。本實驗分析減數分裂十九對粗絲期染色體，由於收縮程度較低，解析力提升，有助於核型的建構。分析染色體平均長度大小、染色質的分佈、中心節位置以及有無次級收縮區，可描述單一染色體的型態。根據核型分析的結果顯示，姬蝴蝶蘭粗絲期染色體平均長度為21.95 µm，最大與最小相差2.35倍。就染色體型態而論，異染色質皆集中在中心節兩側，但異染色質的大小與分佈位置在不同的染色體之間有很大的差異，有些異染色質節清晰，可辨識不同的染色體。姬蝴蝶蘭多屬於中位中節或次中位中節染色體，只有一對染色體為近端中節，該染色體的短臂末端可觀察到次級收縮區。另外與有絲分裂中期染色體相較，粗絲期染色體提高12倍以上的解析力，可提升染色體圖譜的應用性。進一步以螢光原位雜交技術將5S rDNA、45S rDNA、阿拉伯芥型端粒序列與SOC1基因定位到染色體上，發現5S rDNA 與45S rDNA分別位於第17對及第13對染色體的短臂末端，SOC1位於第1對染色體的短臂上，端粒序列則分佈在每對染色體的端點。依據這些序列在染色體上的分佈位置與訊號的差異，可結合分子標誌建立出更完整的核型，辨別型態相近的染色體。 有別於蝴蝶蘭早期多以有絲分裂中期染色體為主的染色體研究，本實驗首先建立了姬蝴蝶蘭粗絲期高解析核型，可成為模式植物供其他蘭花作比較分析，以了解染色體在蝴蝶蘭屬植物演化過程中核型的演變與排列重組的可能演進。

Phalaenopsis orchid is the important floral industry in Taiwan. It however has a long period of life cycle and deficiency of genetic map as well as cytogenetic research. P. equestris is a native and representive orchid in Taiwan, and the kind of its relatively small genome is suitable for analysis in Phalaenopsis genome sequencing project in the future. The size and morphology among somatic metaphase chromosomes were close, and it is difficult to identify each with conventional staining. Ninteen pachytene chromosomes of meiotic stage were studied in this thesis showing their lower contration and high resolution that could contribute to construct a karyotype. Single chromosome morphology can be described by analyzing average length, distribution of chromatin, centromere position and presence of secondary constriction of chromosomes. Based on the results of karyotype, the average length of each pachytene chromosome was 21.95 µm and a 2.35-fold difference between the largest and smallest ones was found. Regarding each chromosome morphology, all heterochromatin were restricted around centromere, but the amount and distribution had significant differences that could distinguish these chromosomes clearly with some heterochromatin knobs. Almost all P. equestris chromosomes are metacentric or submetacentric while only one chromosome which includes a secondary constriction on the end of short arm belonging to subtelocentric. In comparison with mitotic metaphase, pachytene chromosomes possessed more than 12-fold higher resolution and thus they can facilitate the construction of chromosome maps. Furthermore, 5S rDNA,45S rDNA, Arabidopsis-type telomeric sequences and SOC1 gene were mapped to chromosomes using fluorescence in situ hybridization. The results showed that chromosome 17 had a 5S rDNA locus and chromosome 13 had a 45S rDNA locus on the ends of their short arms. Furthermore, SOC1 was located on the short arm of chromosome 1, and telomeric sequences located on both ends of every chromosomes. Based on the variation in the spatial and quantitative distribution of DNA sequences, a complete karyotype could be constructed combined with molecular markers to distinguish similar chromosomes.

In this study we first developed P. equestris high-resolution karyotype better than previous studies which focus on mitotic metaphase chromosomes, and we hope it will become a model plant used for other orchids. In addition, in the future with the research of chromosome rearrangements of Phalaenopsis species, we may understand how Phalaenopsis chromosomes evolved.