微衛星序列(microsatellite)，又稱簡單重複序列(simple sequence repeat)，是一段以2 ~ 6個鹼基對為重複單位的DNA片段。微衛星序列因其共顯性遺傳、多對偶基因、高度多型性、再現性高與易以聚合酵素連鎖反應偵測等優點，成為廣泛應用於分子育種及遺傳多樣性評估的分子標誌。蝴蝶蘭是一種高經濟價值花卉作物且是台灣重要的花卉產業。建立與發展高效率的微衛星標誌系統可有效應用在蝴蝶蘭的品種鑑定與品種專利權的保護。因此，本實驗的目的在於進行蝴蝶蘭微衛星標誌的選殖及特性分析。首先，構築台灣阿媽蝴蝶蘭的小片段插入基因組基因庫及豐富性微衛星序列基因組基因庫，並篩選雙鹼基重複的微衛星序列，總計得到約170個含有微衛星序列的選殖株，目前已完成30個微衛星序列的核酸定序。比較兩種基因庫的微衛星序列選殖效率，發現後者的效率高出10倍左右。分析所選殖出的微衛星序列中，發現連續型微衛星序列佔61.3%；而複合型微衛星序列具有較高的重複次數。根據微衛星序列兩側的游離序列(flanking region)長度，估計有62.5%的微衛星序列可設計引子對。利用6個微衛星基因座引子對檢測不同品系的台灣阿媽蝴蝶蘭，發現這些微衛星基因座之對偶基因數分佈在2 ~ 13之間，平均每一個微衛星基因座會出現7個對偶基因。評估這些微衛星基因座的遺傳變異度後，顯示其範圍分佈在0.47 ~ 0.88之間；而多型性程度則以PIC值(polymorphism information content)表示，分佈在0.19 ~ 0.7之間，而平均值則為0.48。分別將台灣阿媽蝴蝶蘭PaAGS002與PaAGS003基因座之不同對偶基因選殖出並定序，經序列比對分析，發現造成對偶基因大小差異的原因包括重複次數的不同與發生在游離序列的缺失。接著，以聚合酵素連鎖反應偵測6個微衛星基因座在12種原生種蝴蝶蘭與2種同科之蘭花物種的跨物種擴增性，結果顯示微衛星基因座在相近物種間具有高保守性。進一步，由14種原生種蝴蝶蘭與1文心蘭原生種之聚合酵素連鎖反應產物，選殖出PaAGS002相對應的微衛星基因座，並進行核酸定序與親緣演化分析，發現微衛星標誌與傳統型態分類的演化模式大致相同。此外，利用4種蝴蝶蘭商業栽培品種測試5個微衛星基因座的可利用性，初步結果顯示其具有蝴蝶蘭品種鑑定的應用潛力。

Microsatellites, also known as simple sequence repeats (SSRs), are short (2-6 bp) tandemly repeated DNA sequences. Microsatellites are widely used as genetic markers in molecular breeding and genetic diversity assessment because they are codominant, multiallelic, highly polymorphic, high reproducibility and easily scored by PCR. Phalaenopsis orchid is among the most valued ornamentals and considered as an important floriculture industry in Taiwan. The establishment and development of very efficient SSR markers would be very useful for orchid cultivar identification and proprietary variety protection. Therefore, the objectives of this study were to isolate, identify and characterize SSR markers for Phalaenopsis orchids. Firstly, the small-insert genomic library and microsatellite-enriched genomic library of Phalaenopsis amabilis were contructed and screened for dinucleotide, AG/CT or AC/TG- microsatellite sequences. A total of approximate 170 microsatellite-containing clones were obtained. Comparing the efficiency of the two microsatellite isolation strategies, the enrichment approach showed much greater efficiency up to 10-fold. Presently, 30 clones have been sequenced, 61.3% of which are perfect repeats and compound repeats have the higher values of repeat number. According to the length of flanking sequences of microsatellite clones, 62.5% primer pairs could be designed for amplification. Six SSR primer pairs were evaluated for amplification and genetic polymorphism in several Phalaenopsis amabilis cultivars. The number of alleles found per locus varied from 2 to 13 with a mean number of 7. Heterozygosity was ranged from 0.47 to 0.88. The level of polymorphism (polymorphism information content, PIC) ranged from 0.19 to 0.7 with an average of 0.48. Different alleles PaAGS002 and PaAGS003 were cloned from Phalaenopsis amabilis, respectively. These clones were sequenced and aligned, the results revealed that the size differences among the microsatellite alleles are not only due to varization in the numbers of repeats but also due to the occurrence of insertion/ deletion events in the flanking region. Cross-species amplification of six microsatellite loci were tested in 14 orchid species, the results suggested that microsatellite loci were highly conserved in Phalaenopsis species. Furthermore, sequences of PCR products at the homologous microsatellite locus PaAGS002 from 15 orchid species were subjected to phylogenetic analysis, the evolutionary pattern of SSR is highly consistent with that based on morphological characters. In addition, five microsatellite loci were also tested for utility in four commercial orchid varieties. The preliminary results indicated that microsatellites will have potential applications in orchid cultivar identification.

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