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標題: 開發文心蘭EST-SSR分子標誌與其特性分析
Development and Characterization of EST-SSR Markers in Oncidium
作者: 梁哲豪
Liang, Jhe-Hao
Contributors: 呂維茗
生物科技學研究所
關鍵字: 文心蘭;EST;SSR;分子標誌
Oncidium;EST;SSR;marker
日期: 2013
Issue Date: 2013-11-18 10:43:59 (UTC+8)
Publisher: 生物科技學研究所
摘要: 文心蘭外銷以切花為主,具有高經濟價值,是台灣重要花卉產業之一。透過人工雜交,迄今已育成許多雜交品種。目前文心蘭屬已有310個種(species)被確認,然而尚有一千種以上未能被確實的分類。基於上述兩點,在分類上,文心蘭被認為是一個複雜難辨的屬,因此希望藉由發展DNA分子標誌以利於進行有系統的分類。透過合作計畫與454定序技術,實驗室已經獲得文心蘭的轉錄體資訊,以及由其合併成之50,908條單一合併序列 (contig sequences)。本研究利用上述資料庫,透過SSRIT (Simple Sequence Repeat Identification Tool) 篩選出3,207條含有簡單重複序列 (Simple Sequence Repeats, SSR) 之單一合併序列,其中AG/CT類型的重複序列所占比率最高,達到30.2%。接著,我們以這3,207條單一合併序列設計出55組DNA分子標誌,應用於25種不同的文心蘭,其中含有10個原生種與15個雜交種。在55組分子標誌中,有12組對於25種文心蘭之間具有多型性,19組對於部分文心蘭有多型性,24組分子標誌不適合用於此分析。另一方面,我們將聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 的產物定序以及分析其序列。結果顯示,造成PCR產物片段長度不同的因素除了重複次數差異之外,亦有未知片段插入,此外也有觀察到單核苷酸多型性 (Single-Nucleotide Polymorphism, SNP) 之現象。為了檢驗本研究所設計之分子標誌的準確性,我們利用軟體NTSYSpc (Numerical Taxonomy System)以UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) 演算法建立親緣關係樹,再進一步與傳統分類系統做比較。
Oncidium is economically important in floriculture industry and also a famous cut flower in Taiwan. To date, lots of hybrid orchids have been bred through artificial hybridization. Containing 310 species and more than 1000 unconfirmed records, Oncidium Sw. is considered as a complex and difficult genus in taxonomy. To develop DNA molecular markers for breeding and classification purposes, we make use of transcriptome information of Oncidium Gower Ramsey generated by 454 sequencing technology. Using Simple Sequence Repeat Identification Tool (SSRIT) to search the assembled 50,908 unique contig sequences, 3,207 contigs revealed simple sequence repeats (SSR). Among them, the most common repeat motif was AG/CT (30.2%). Using several criteria including high repeat numbers (>5), enough distance from both ends (>30 nucleotides), etc., fifty-five primer pairs were designed for the di- or trinucleotide SSRs. After screening against 25 Oncidium including 10 native and 15 hybrid species, 12 primer pairs generated banding successfully for most samples, 19 markers were polymorphic only between some samples, and 24 markers were either unstable or totally failed in polymerase chain reaction (PCR). Sequencing analysis employed on several selected PCR products revealed that the size differences were caused by both simple sequence repeats and unknown insertions. Moreover, single-nucleotide polymorphisms (SNP) were also detected frequently. A phylogenetic tree was constructed based on the genetic similarity coefficient using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) method with the software NTSYSpc (Numerical Taxonomy System).
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