以基因序列多型性作為臺灣檜木之分子標誌

Abstract

臺灣檜木包含臺灣扁柏 (Chamaecyparis obtusa Sieb. Zucc. var. formosana (Hayata) Rehder) 與紅檜 (C. formosensis Matsum.) 兩種。本論文為了輔助鑑別臺灣檜木物種，透過基因序列分析，找出序列之多型性，單一核苷酸多型性 (single nucleotide polymorphism; SNP)、片段插入及缺失 (insertion-deletion; indel)，開發為分子標誌。選殖臺灣檜木 caffeoyl CoA O-methyltransferase (CCoAOMT) 基因、alpha-pinene synthase (APS) 基因、cadinene synthase (CAS) 基因並進行序列分析，三個基因分別具有5、10、10個顯子。臺灣扁柏及紅檜之 CCoAOMT 基因於Intron 1、2及3具有Indel；CAS基因於Intron 1及5 存在 Indel；而在 APS 基因中僅有 SNP 差異。依據 CCoAOMT 基因序列設計引子進行聚合酶連鎖反應 (polymerase chain reaction; PCR)，除了共同條帶，可合成紅檜特有片段約1.4 kb。針對 CAS 基因 Intron 1之Indel設計的引子，可得兩條紅檜特有PCR產物分別為750及800 bp；另得到紅檜及臺灣扁柏共同條帶為650 bp。若經毛細管電泳分析，臺灣扁柏樣品尚出現另一相近條帶，但紅檜則沒有。將650 bp序列分析後，臺灣扁柏樣品合成之產物比紅檜多 2 bp 片段插入序列，可用以鑑別臺灣扁柏。另根據CAS基因 Intron 5之 Indel 設計的引子，於紅檜及臺灣扁柏均可合成約700 bp片段，序列分析結果指出，11 bp 片段缺失之序列為紅檜特有序列。以上三組引子可應用於臺灣扁柏及紅檜分子標誌鑑定用。

Chamaecyparis obtusa Sieb. Zucc. var. formosana (Hayata) Rehder and C. formosensis Matsum. are known as Taiwan yellow and red cypress, respectively. To develop molecular markers to identify cypress, polymorphic gene sequences such as single nucleotide polymorphism (SNP) and insertion-deletion (Indel) were used as molecular markers to identify the species of timber and wood products. Caffeoyl CoA O-methyltransferase (CCoAOMT), alpha-pinene synthase (APS) and cadinene synthase (CAS) genes were cloned from both Taiwan yellow and red cypress and sequenced. Gene structure analysis revealed that there are 5, 10, 10 exons in CCoAOMT, APS, CAS, respectively. Indels were found in intron 1, 2 and 3 of CCoAOMT and intron 1 and 5 of CAS but only SNPs were found in APS. Specific primers have been designed based on the polymorphic DNA sequences in CCoAOMT, 1.4 kb fragment was synthesized only in red cypress with other common fragments. Two molecular markers based on the CAS indels sequence in intron 1 and 5 have been developed. For the marker based on intron 1 can identify cypress species through electrophoresis, in red cypress two specific bands about 750 and 800 bp were found together with the common 650 bp fragment in both cypresses. One extra fragment was separated away from the 650 bp common fragment by capillary electrophoresis, using DNA extracted from yellow cypress. After sequencing, the yellow cypress could be distinguished with red cypress by 650 bp PCR product by 2 bp insertion. Using primers designed based on intron 5 of CAS, 700 bp fragmemt was synthesized in both red cypress and yellow cypress but with 11bp deletion in red cypress.