微衛星序列(microsatellite)，又稱簡單重複性序列(simple sequence repeat, SSR)，由於在基因組序列上重複次數的改變，具有高度變異、高多型性及易利用聚合酵素連鎖反應(polymerase chain reaction, PCR)偵測等特性使其成為非常有效的分子標誌(molecular marker)。葉綠體(chloroplast)是植物特有的胞器，高等植物的葉綠體基因組大小介於120 kb至160 kb，其差異主要來自於葉綠體上一對具重複相反的序列以及基因區間和重複性序列的長度變異。葉綠體序列的演化速率較核基因組慢，但其具有單親遺傳、非重組以及多套單元體(haploid)存在等特性，因此可應用在族群親緣地理以及釐清雜交親本的研究。蝴蝶蘭(Phalaenopsis)是台灣高經濟價值的花卉作物，發展高效率的分子標誌鑑定系統是必須的。由於蝴蝶蘭台灣阿嬤(P. aphrodite subsp. Formosana)的葉綠體基因組已被完全解序，本實驗之研究目的為開發蝴蝶蘭葉綠體微衛星序列分子標誌及進一步探討其應用性。分析葉綠體基因組搜尋得到664個微衛星序列，以單鹼基重複的微衛星序列最多。由其中篩選出12組適合設計引子對的微衛星基因座並檢測其是否符合葉綠體母系遺傳(maternal inheritance)的特性。偵測葉綠體微衛星序列於台灣阿嬤與姬蝴蝶蘭(P. equestris)的種內遺傳變異度與多型性，所產生的對偶基因分布在2-6個間，並發現其中5組葉綠體微衛星基因座能明顯區分不同花色的姬蝴蝶蘭變異種。多型性分析顯示其PIC (polymorphism information content)值介於0.37-0.81之間。分析葉綠體微衛星序列在25種蝴蝶蘭原生種間的可擴增率高達99%，顯示葉綠體微衛星基因座具有高度的保守性。以不加權平均重方式(unweighted pair group arithmetic mean method, UPGMA)分析蝴蝶蘭複合群種的結果發現，可對外觀不易辨識的近緣種進行區分。進一步利用微衛星基因座對市售商業品種所產生的不同大小的對偶基因組成葉綠體單核型(haplotype)，結果每一個栽培種皆具有特定的單核型。綜合以上結果，葉綠體微衛星序列在蝴蝶蘭屬植物種內以及種間具有豐富的歧異度，除可應用於品種鑑定、親本分析外，亦可應用於蝴蝶蘭屬植物親緣演化的研究。

Microsatellites, also known as simple sequence repeats (SSRs), are powerful molecular markers due to their different repeat times and their properties of high variation and high level of polymorphism as well as easily detectable by PCR. Chloroplasts are specialized organelles in higher plants, which range in size from 120-160 kb, primarily reflecting differences in the length of the inverted repeat, intergenic region and repeat sequence fragment. Chloroplast DNAs generally exhibit lower mutation rates than nuclear DNAs, but they are typically uniparentally inherited, nonrecombinant and present in multiple copies of haploid. Therefore, chloroplast SSR can be used in the study of phylogeography and parentage discrimination in plants. Phalaenopsis orchids are the most valued plant in floriculture industry of Taiwan and the development of efficient SSR markers would be very useful for orchid cultivar identification and proprietary variety protection. The complete chloroplast genome sequence of P. aphrodite subsp. formosana has been determined. The aim of this study was to develop chloroplast microsatellites and further investigate the applications of these cpSSRs on Phalaenopsis orchids. 664 cpSSRs in the chloroplast genome were identified, while mononucleotide SSRs were the most predominant. 12 chloroplast miscrosatellite primer pairs were designed and tested their maternal inheritance. All of the microsatellites revealed some degree of polymorphism in P. aphrodite and P. equestris accessions, with the number of alleles per locus ranging from two to six. Five of the chloroplast microsatellites revealed specific allele patterns in the different floral types of P. equestris accessions. The polymorphism information content (PIC) values ranged between 0.37 and 0.81. The cross-species amplification of these cpSSRs in 25 Phalaenopsis species was 99%, suggesting that these cpSSR loci were highly conserved in Phalaenopsis. The analysis of combined SSR data was used in UPGMA analysis to group the close species of Phalaenopsis complex based on alleles and generally able to separate chloroplast types into specific groups. Moreover, all cpSSRs were further analyzed on commercial varieties and multi-locus haplotypes were observed. None of these haplotypes was shared by more than one commercial variety. In conclusion, these chloroplast SSRs reveal a wealth of diversity within Phalaenopsis at intra-specific and inter-specific taxonomic levels that could facilitate not only in cultivar identification and parentage detection but also provide a further evolutionary studies of this genus.

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