石斛蘭屬植物新鮮或乾燥的莖具有清熱生津及滋陰補胃等功效，作為傳統中藥已有很長的歷史。目前市售之石斛市場品來源複雜，品質良莠不齊，因此藥用石斛藥材基原鑑定是石斛藥材品質控制的首要問題。微衛星序列(microsatellites)又稱為簡單重複序列(simple sequence repeats, SSRs)，以2-6個鹼基為重複單位的DNA片段，普遍存在於植物基因組中，相較於其他分子標誌，微衛星序列標誌具有高度變異性、高度多型性、易利用聚合酵素連鎖反應偵測等優點，是目前應用最為廣泛的分子標誌。本實驗之研究目的在於發展藥用石斛蘭微衛星DNA分子標誌，作為鑑定藥用石斛基原的依據。首先，先構築銅皮石斛的小片段插入基因組基因庫及豐富性微衛星基因組基因庫，分別以AG/TC及AC/TG微衛星序列為探針進行篩選，共獲得33個微衛星序列，並進行特性分析，結果顯示豐富性微衛星基因組基因庫具有較高之選殖效率。由其中篩選出8個適合設計引子對的微衛星基因座，分別針對不同及相同採集地的銅皮石斛樣本進行特性分析，並計算其遺傳變異度之觀察值及期望值和多型性程度，結果顯示不同採集地樣本的多型性程度高於同採集地的樣本。偵測微衛星基因座在其他藥用石斛屬植物之移轉性程度高達91%，在其他石斛蘭屬植物中亦有84%的可擴增性，更進一步進行序列分析，顯示微衛星基因座在相近物種間具有高度保守性。以DmTCS001微衛星基因座進行親緣演化分析，發現微衛星標誌與傳統形態分類的模式大致相同。檢測微衛星分子標誌在石斛蘭藥材的可應用性，以DmTCS001、DmACE001及DmACE002微衛星基因座針對石斛蘭藥材進行分析。此外，篩選出DmTCS001及DmACE005兩個基因座利用自動核酸遺傳分析系統(ABI PRISMTM 310 genetic analyzer)進行自動化核酸片段分析。綜合本研究結果顯示，石斛蘭微衛星分子標誌具有鑑定藥用石斛蘭物種及可進一步確認其種原和基原的應用潛力。

　Stems of several Dendrobium species have long been used in traditional Chinese medicine (TCM) as a tonic to nourish the stomach, to promote the production of body fluid, and to reduce fever. However, the supply sources of Dendrobium species on the markets are various and their quality levels are dissimilar as well. Therefore, there is a need to properly authenticate Dendrobium species used in Chinese medicine in order to provide measures for quality control and therapeutic application. Microsatellites or simple sequence repeats (SSRs) are short DNA tandem repeats (2-6 bp) interspersed throughout the plant genomes. Microsatellite fingerprinting is now considered as the most suitable technique in application of molecular markers because of its high resolution and efficiency. Compared to other molecular markers, microsatellites are highly polymorphic and easily detectable by polymerase chain reaction (PCR). The aim of this study was to develop SSR markers from Dendrobium species for authentication applications. Firstly, the small-insert genomic library and microsatellite-enriched genomic library of Ddendrobium. moniliforme (L.) Sweet were constructed and screened for dinucleotide, AG/CT or AC/TG- microsatellite sequences. Thirty-three microsatellite markers were identified and characterized. Comparing the efficiency of the two microsatellite isolation strategies, the enrichment approach showed much greater efficiency. Among these, eight microsatellites were suitable for designing PCR primer sequence to analyze D. moniliforme samples which had been obtained from either the same or different supply sources. To each microsatellite marker, observed and expected heterozygosities, and polymorphism information content (PIC) were calculated. The level of PIC of D. moniliforme samples sampled from different localities was higher than that within-localities. In addition, the transferability of these microsatellites to other medicinal Dendrobium species was high, being 91% and to other Dendrobium species was 84%. Furthermore, the PCR products that contained microsatellite sequence were sequenced. The microsatellite locus DmTCS001 was used for the phylogenetic analysis. SSR phylogeny is consistent with the morphological taxonomy. In order to test the applicability of these SSR markers in authentication of herba Dendrobii, three loci DmTCS001, DmACE001 and DmACE002 were used for the analysis of market samples. Furthermore, the loci DmTCS001 and DmACE005 were selected and subjected to semi-automated fluorescent microsatellite analysis using ABI PRISMTM 310 genetic analyzer. Results from these investigations suggested that Dendrobium SSR markers have potential applications in medicinal Dendrobium species identification and in further confirmation of its botanical identity and origin.

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