Adh基因應用於豆科及禾本科物種之鑑別

Abstract

近年來DNA條碼被廣泛使用於親緣分析及物種鑑別上，不過目前使用之葉綠體及核醣體DNA條碼常無法區分植物親緣相近之物種，而為了補充現有植物DNA條碼解析度不足之問題，本研究先以紅豆品種間Adh基因序列變異為基礎，延伸至其他豆科物種，再試圖擴展至禾本科等其他作物，希望能推薦廣適用性高且高鑑別效能之DNA條碼。本研究首先成功地以FPNI-PCR方法擴增出紅豆Adh2基因全長(3826 bp)，其DNA序列結構與大部分物種之Adh序列結構相符，皆含有10個exon、9個intron，exon在長度上十分保守，與其他物種Adh之相似度大多高於70%。而比對9個臺灣紅豆品種之Adh1及Adh2基因發現，Adh1基因在品種間不具多型性，Adh2基因則在品種間具有15個多型性位點，且這些多型性可將此9個臺灣品種區分為三群，由此推測Adh2基因應比Adh1基因更具種內區分之潛力，而後續豆科Adh DNA條碼擴增之結果也應證此推論。本研究共開發出四組Adh DNA條碼，分別針對豆科物種之Adh1、Adh2基因以及禾本科之Adh1基因進行增幅，在豆科方面以EX5F + EX7R引子對為佳，禾本科則以Mex2F + Mex4R引子對為主，此兩組分子標誌皆可以長度多型性區別不同物種，種內也具有定序多型性。整合此四組Adh DNA條碼之擴增結果，其均可有效地應用在物種層級之鑑別及親緣分析，而在種內之鑑別則依物種而異，此顯示本研究所開發之Adh DNA條碼確實可補充現今DNA條碼解析度不足之問題。此外，本研究亦發現相對同源(paralogs)及相鄰同源(orthologs) Adh基因難以判斷之問題，在文中也分析可能之原因及提供改善之方法，期望能對後續Adh基因之應用有所助益。

Recently DNA barcodes have been applied worldwide in phylogeny analysis and species identification. However, chloroplast and nuclear ribosomal sequences, which are commonly used as plant DNA barcodes, usually can not distinguish close-related species. To complement the insufficient resolution of current plant DNA barcodes, we analyzed sequence variations of Adh genes among adzuki bean vatieties, and then extended to investigate leguminous and other gramineous species, hoping to develop high-resolution Adh DNA barcode for Leguminosae and Gramineae speciess. The Adh2 gene of adzuki bean was successfuly sequenced by the FPNI-PCR method. Its gene structure is similar to those of published Adh genes, including the number and the length of exons, and the similarity of coding region (above 70%). Adh1 and Adh2 genes of nine adzuki bean varieties developed in Taiwan were also sequenced and aligned. It suggested that the Adh2 gene had higher potential to distinguish plants within species. Based on the variations of Adh DNA sequences, we developed two Adh DNA barcodes to amplify Adh1 and Adh2 genes of leguminous species and additional two Adh DNA barcodes to amplify Adh1 genes of gramineous species. These Adh DNA barcodes were successfully used to reconstruct phylogenetic tree and identify plants at inter-species and intra-species level. These demonstrated that Adh DNA barcodes complete the DNA barcode system at low taxonomic level. In addition, we discovered that paralogous and orthologous Adh genes were constantly hard to be distinguished and these cause difficulties in phylogeny analysis and species identification. Therefore, the possible causes and improving methods were also discussed in this study.