微衛星序列(microsatellite)，又稱簡單重複序列(simple sequence repeats, SSRs)或稱短縱列重複序列(short tandem repeats, STRs)，是以2-6個鹼基對為重複單位，頭尾相接形成的小片段DNA。微衛星序列因具有共顯性遺傳、再現性高、多對偶基因、高度多型性和易以聚合酵素連鎖反應偵測等優點，成為廣泛應用於栽培種指紋圖譜建立、遺傳多樣性評估和育種的分子標誌。微衛星序列存在於基因轉錄區和非轉錄區，位在基因轉錄區的微衛星序列與表現性狀連鎖的可能性高，且跨物種的可擴增性高。蝴蝶蘭(Phalaenopsis spp.)是一種高經濟價值花卉作物且是台灣重要的花卉產業，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。本實驗之目的在於選殖蝴蝶蘭互補DNA之簡單重複序列與特性分析。首先構築台灣阿媽(P. amabilis var. formosa)栽培種花部及姬蝴蝶蘭(P. equestris)原生種葉片豐富性微衛星序列cDNA基因庫，分別以AG/TC、AC/TG和AGG/TCC微衛星序列為探針進行篩選，共獲得42個微衛星序列並進行特性分析。利用8組cDNA-SSR引子對分別檢測不同編號台灣阿媽或姬蝴蝶蘭原生種，發現其對偶基因數分佈在2-7之間，平均每一個cDNA-SSR會出現4.1個對偶基因；PIC值(polymorphism information content)分佈在0.62-0.99之間，平均值約為0.85。8組cDNA-SSR引子對在18種蝴蝶蘭原生種間的可擴增性為76.4%，顯示cDNA-SSR基因座在蝴蝶蘭屬內的保守性高。以PecAG002基因座在18個蝴蝶蘭原生種的對偶基因進行核酸定序及親源演化分析，發現cDNA-SSRs與傳統形態分類的演化模式大致相同。PecAG001基因座僅在P. equestris、P. × intermedia和P. lindenii可擴增出產物，具有物種特異性；因此利用PecAG001基因座分析30株商業品種，結果顯示可檢測出姬蝴蝶蘭遺傳貢獻度在3.22%以上的商業品種。另外，將PecAG001及PecAG002基因座以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，得到正確的對偶基因大小。綜合上述結果，本實驗所發展的微衛星標誌來自基因表現序列，可用來鑑定蝴蝶蘭品種和分子育種。

Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are short (2-6 bp) tandemly repeated DNA sequences. Microsatellites are widely used as molecular markers in cultivar fingerprinting, genetic diversity assessment and marker-assisted selection because of their properties of genetic co-dominance, high reproducibility, multiallelic variation and high level of polymorphism as well as easily detectable by PCR. Microsatellites are present in both gene transcribed and nontranscribed regions. cDNA-SSRs may be associated with phenotypic traits and have higher transferability across related species. Phalaenopsis orchid is the most valued ornamentals and considered as an important floriculture industry in Taiwan. The development of very efficient SSR markers would be very useful for orchid cultivar identification and proprietary variety protection. The aim of this study was to isolate and characterize cDNA-SSRs for Phalaenopsis orchids. Firstly, the cDNA libraries enriched for microsatelltes of P. amabilis var. formosa and P. equestris were constructed and screened for AG/TC, AC/TG or AGG/TCC- microsatellite sequences. In total, 42 cDNA-SSR makers were obtained and characterized. Eight cDNA-SSR primer pairs were evaluated for amplification and genetic polymorphism in several P. amabilis or P. equestris accessions. The number of alleles per locus varied from 2 to 7 with a mean number of 4.1. The polymorphisms information content (PIC) ranged from 0.62 to 0.99 with an average of 0.85. The cross-species amplification of these cDNA-SSRs in 18 Phalaenopsis species was 76.4%, suggesting that the cDNA-SSR loci were highly conserved in Phalaenopsis. Sequences of the alleles at the locus PecAG002 from 18 Phalaenopsis species were used for phylogenetic analysis. The evolutionary pattern of cDNA-SSRs is consistent with that based on morphological characters. Positive amplifications at locus PecAG001 were obtained only in P. equestris, P. × intermedia and P. lindenii, indicating that this locus is species specific. Therefore, the locus PecAG001 was further analyzed on 30 commercial orchid accessions. This locus produced amplifications limited to accessions whose genetic contribution by P. equestris are above 3.22%. Moreover, the loci PecAG001 and PecAG002 were subjected to semi-automated fluorescent microsatellite analysis using ABI PRISMTM 310 genetic analyzer and accurate sizes of alleles were observed. Collectively, since these microsatellite makers developed in this study are primarily from expressed sequences, they can be used not only for variety identification but also for molecular breeding in orchids.

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