植物DNA分析於鑑識科學應用之研究

Abstract

植物之分子鑑定由於缺乏適當的DNA資料庫以資比對而受到很大的限制，本研究乃以葉綠體基因組（cpDNA）中的trnL intron 及trnL-trnF IGS 等兩個基因位建立植物DNA資料庫以應用於植物之種屬鑑定。本資料庫包含台灣地區常見植物79科206屬264種共365株個體。這些植物之種屬幾乎可經由此二基因位之序列多型及長度多型加以鑑別，經由盲樣測試發現以本研究所建立之資料庫比對後大部分盲樣可搜尋到正確的種別，以鄰近歸群法進行80個樣品之歸群分析後幾乎所有的樣品均歸屬於正確的科內及屬內，顯示此二基因位所建立之DNA資料庫適於應用在刑事植物之種屬鑑別。 鬼針草(Bidens pilosa L.)之種子具有帶刺的鉤毛可經由動物傳播，故此類廣泛分佈的植物亦有機會出現在犯罪現場。本研究採集鬼針草之三個變種（大花咸豐草、小白花及白花鬼針）共11個族群161個樣品，加上7株當控制組的鬼針舅（Bidens biternata）共168個樣品，分別進行trnL intron及trnL-trnF IGS等兩個cpDNA的基因位與ITS1、5.8S及ITS2等三個細胞核核糖體DNA（nrDNA）的基因位之序列分析。其中trnL intron、ITS1、5.8S及ITS2 等四個基因位之序列分析均可有效的鑑別鬼針舅及鬼針草兩種間之差異。將此五個基因位之型別整合為84種單倍型幾乎可鑑別大花咸豐草與其它兩個變種間之差異。根據核苷酸變異位點、序列型、基因型歧異度、核苷酸歧異度、遺傳歧異度等相關指數，於nrDNA表現之遺傳多樣性均遠高於cpDNA。AMOVA分析之結果顯示鬼針草其遺傳變異的主要貢獻為族群內個體間的變異，於nrDNA及cpDNA均佔50 ％以上。大花咸豐草與其他兩變種間的基因交流值均遠低於此二變種間的基因交流值，極可能在較早歸化的白花鬼針與小白花鬼針間已出現明顯的雜交現象，而較晚歸化的大花咸豐草與此二變種間的雜交現象則可能尚不明顯。 於可疑種子之種屬鑑定的案例報告中，本研究以cpDNA之trnL-trnF IGS及nrDNA之ITS1等二基因位之序列分析成功的進行其種屬鑑定，顯示本系統為大麻種子進行種屬鑑定之有效方法。Forensic botanical comparison can be hampered by the lack of appropriate DNA databases. In this study, we report on the use of the trnL intron and the trnL-trnF IGS in the chloroplast genome (cpDNA) and establish a DNA sequence database for plant species identification. The database comprises 365 individual sequences representing 79 families, 206 genera and 264 species. These plant species can be grouped to species level using both sequence and length polymorphisms at these loci. In blind trial testing, most of the blind samples matched their respective species from our local DNA database. Cluster analysis with Neighbor joining method of the two DNA regions from 80 samples of this study were constructed and clustered to both family and genus. The sequence database described in this study can be used to identify plant species using DNA sequences of the two loci and illustrates its value in plant species identification. Bidens pilosa L.with barbed fruits is dispersed via animal species resulting in a widespread distribution and therefore frequently collected when attached to evidential samples during criminal investigation. In this study, 161 specimens were used from these varieties comprising 11 different populations and 7 samples of Bidens biternata were included as an out-group control. DNA fragments of all samples at the trnL intron and trnL-trnF IGS loci of cpDNA, ITS1, 5.8S and ITS2 of nrDNA were amplified and sequenced. From the sequence types of the 5 loci, trnL intron, ITS1, ITS2 and 5.8S were found to be useful markers to differentiate Bidens biternata and Bidens pilosa. There were 84 haplotypes at the 5 loci from 161 specimens. Among 84 haplotypes, B. radiata could be almost differentiated from the varieties of Bidens pilosa using these 5 loci. It showed that the genetic diversity of nrDNA sequence was higher than that of cpDNA from the data of the number of polymorphic sites (S), number of haplotypes (H), gene diversity (Hd), nucleotide diversity (π) and genetic diversity (θ). From the results of the analysis of molecular variance (AMOVA), there are more than 50 % variance contributed by the variance of “within populations” in the loci of nrDNA and cpDNA. The gene flow between var. radiata and var. minor or var. pilosa showed lower than those between var. minor and var. pilosa, it indicated the significant hybridization phenomenon between var. minor and var. pilosa which naturalized earlier than var. radiata. In the case report of suspect seed samples, species analysis was performed by DNA analysis using the trnL-trnF IGS of cpDNA, and the ITS1 of nrDNA. Based on these analyses, it was determined that all seeds were from the species Cannabis sativa. The system established in this study provides a robust tool for cannabis seed identification.