ISSR分子標誌與DNA條碼應用於茶葉之品種鑑定

Abstract

茶為我國重要之經濟作物，為確保國內比賽茶品種純正及落實生產履歷政策，開發快速且穩定之品種鑑定方法實為必要。本研究篩選出7條ISSR引子及1條功能性基因引子可產生12個核心標誌及6個輔助標誌做為國內重要茶栽培種之DNA指紋，此工具可應用於檢測國內比賽茶及市售茶葉。利用ISSR DNA分子標誌，本研究所檢測比賽茶之烏龍茶及東方美人茶均發現部分之未知品種名稱樣品。此外為提升檢測效率，將不同引子混合進行PCR分析，結果均不能獲得穩定條帶；但將不同引子產生之PCR產物混合進行電泳分析，則可偵測預期之分子標誌，便可提昇檢測效率。 本研究由4個細胞核及8個細胞質（含4個葉綠體及4個粒線體）序列變異較大之片段篩選出具較大序列變異性之ITS2及M8片段，目前開發出ITS2序列與粒線體部分序列，其中品種間ITS2具有33個單一核苷酸多型性，而品種間之M8具有5個核苷酸缺失/插入及2個單一核苷酸多型性。此外分析不同品種之定序訊號結果，發現M8的定序訊號可偵測到不同比例的品種混合（50%、40%、30%、20%及10%）。為了將DNA條碼應用於成茶的品種鑑定，故將引子設計成可擴增小片段DNA的引子，如此定序一次即可得到所要分析的片段。本研究推薦以ITS2及M8作為茶葉商品之DNA條碼，以DNA條碼驗證比賽茶中有疑問之樣本，發現東方美人茶確實有品種混合的樣本。 本研究顯示ISSR分子標誌可應用於成茶的品種鑑定，但它缺乏可偵測品種混合的敏感度，而另一方面，DNA條碼可偵測品種混合，故我們建議DNA條碼可應用在商業茶葉的產銷履歷上，而ISSR分子標誌則可輔助DNA條碼重複確認樣本之品種鑑定。

Tea is one of the most important economic crops in Taiwan. To ensure the authenticity of competition teas and to achieve traceability policy, an efficient method of variety identification for processed teas is urgently expected. In this study, twelve core markers and six auxiliary markers generated from seven ISSR primers and one functional gene were recommended to fingerprint prevailing tea varieties grown in Taiwan. The core markers are also suggested to examine the varieties of competition teas and commercial teas. According to ISSR DNA markers, some unknown varieties were found in the competition tea samples of Oolong tea and oriental Beauty tea. To enhance the efficiency of variety identification, two ISSR primers were randomly selected and mixed for PCR amplification. However, the unstable band patterns were found in many attempts. But stable markers could be obtained from electrophoresis with PCR products. ITS2 (nuclear) and M8 (cytoplasmic) DNA sequences with large variations were screened out of twelve DNA fragments (four nuclear, four chloroplastic and four mitochondrial) to identify tea varieties in Taiwan. Among varieties of tea, thirty-three SNPs were found in the ITS2 fragments, and five InDels and two SNPs were found in the M8 fragments. DNA mixtures of different varieties with various ratios (50%, 40%, 30%, 20%, and 10%) were identified based on sequencing signals of SNPs or InDels of M8 fragments. In order to apply DNA barcodes in variety identification of made teas, the primers were designed to amplify short fragments within the length limitation of one confident sequencing. The ITS2 and M8 sequences were recommended as DNA barcodes for commercial teas. Mixture of different varieties that was unable detected by ISSR DNA markers was verified by ITS2 and M8 DNA barcodes. Our studies have demonstrated that ISSR DNA markers are useful in variety identification of teas. However, they lack in sensitivity to detect variety mixtures. On the other hand, DNA sequence barcodes could detect variety mixtures. In this study, DNA sequence barcodes are suggested to be applied in labeling, tracing or primarily detecting commercial tea products, and ISSR DNA markers are prepared for reconfirmation of variety identification.