豆類作物種間及種內基因體對應區域之DNA序列變異

Abstract

本研究係針對包括毛豆、紅豆、落花生、綠豆、豇豆、菜豆、蠶豆、豌豆、刀豆及樹豆等重要豆類作物進行基因體對應區域分析。主要研究目的在探討這十種豆類作物(共46品種)基因體演化之微共線性，評估各豆科作物種內及種間變異程度，利用DNA序列變異進行演化遺傳學之分群分析及估算各豆類作物之演化時間，並且討論DNA序列應用在物種及品種鑑別之可行性。分析區域包含細胞核ITS及Adh1基因體片段、葉綠體RbcL及matK基因、粒線體CoxⅠ及19S rDNA基因。本研究共使用13組引子進行PCR擴增，PCR擴增結果顯示取樣之各基因體區域在豆類作物間具有共線性，擴增產物在物種間具有長度及重複數(copy numbers)之變異，顯示各擴增區域在豆類演化過程中所發生之變異。其中豇豆、落花生、菜豆及毛豆分別具有較低的物種內變異，綠豆在葉綠體基因序列具有較高物種內變異。本研究所偵測得到具高度變異之核內DNA片段適用於物種間的群聚分析，而相對保守的葉綠體基因則較適用於作物間演化關係之追尋，序列保守但常發生大片段變異的粒線體基因之DNA序列較不適合於物種變異分析。試驗結果亦證實核外DNA序列變異可應用於追蹤一個體之親本譜系。系統演化分群分析結果顯示，參試十種豆類作物可區分為三個分化枝(clade)，分別為岩黃耆族（Hedysareae）、蠶豆族（Viceae）及菜豆族（Phaseoleae）。其中落花生與其他豆科作物具有最遠的遺傳距離，蠶豆族的碗豆及蠶豆之基因體序列具有高度保守性。以4.2千萬年前菜豆族與蠶豆族的分歧時間作為參考點，利用核內及葉綠體基因所共同建構的演化樹進行蝶型花亞科作物分化時間之估計，結果顯示落花生為最早分化出的物種，約在4.4千萬年前分出。在菜豆族的演化中刀豆屬分化最早，約3.2千萬年前發生，大豆及樹豆約在2.6千萬年前同時分化；菜豆屬與豇豆屬的分歧發生在2.2千萬年前，蠶豆與豌豆的分化約發生在1千萬年前，各作物之屬內分化均在近1千萬年內發生。本研究所探討豆類作物基因體之分子演化關係，及物種間和物種內DNA序列變異之結果，將有助於豆類作物間遺傳資訊之相互參考及利用。

To study the DNA sequence variation between and within legumes, and genomic evolutionary history of legume crops, orthologous genomic regions were sequenced for ten important legume crops (46 varieties), including soybean (Glycine max), peanut (Arachis hypogaea), azuki bean (Vigna angularis), mungbean (V. radiata), cowpea (V. unguiculata), pea (Pisum sativum), broad bean (Vicia faba), jack bean (Canavalia ensiformis/ C. gladiata), common bean (Phaseolus vulgaris/ P. coccineus) and pigeon pea (Cajanus cajan). The analyzed sequences included nuclear ITS region and Adh1 gene, chloroplast genes RbcL and matK, and mitochondria CoxⅠ and 19S rDNA. All surveyed regions were successfully amplified indicating the colinearity of genome. Different sizes and copy numbers of amplified orthologous regions were observed in the legume crops. The cowpea, peanut, Phaseolus and soybean had smaller intra-specific diversity than the other legumes. However, highly diverse DNA sequences were observed in mungbean chloroplast genome. The highly diverse nuclear DNA could supply sufficient variations for classifying closely relative species. Results from cytoplasmic DNA inheritance indicate the DNA sequence variation is applicable for identifying genetically related individuals. Phylogeny constructed with conserved chloroplast genes displayed good resolution for the divergence of species. Due to lack of sufficient nucleotide substitutions, mitochondria DNA sequences are not suitable for phylogenetic analysis. Based on phylogenetic analysis, the surveyed legume crops were grouped into three clades including the tribes Hedysareae, Viceae, and Phaseoleae. The peanut belonging to the tribe Hedysareae was the most diverse to other legumes. The pea and broad bean were grouped into an individual clade. The divergence date of each legume was estimated based on a reference point of the Phaseoleae-Viceae. The divergence of Arachis hypogaea occurred most anciently at 44 Myr ago. In the evolution of the tribe Phaseoleae, the genus Canavalia was first diverged at 32 Myr ago. The divergence of Glycine max occurred at 26 Myr ago, and it was almost at the same time with Cajanus cajan. The date of Vigna-Phaseolus divergence was estimated to be 22 Myr ago. The intra-genus divergence of legumes occurred more recently within the last 10 Myr. Results from DNA sequence variation and genomic evolution would be contributed in sharing and applying the genetic information of legume crops.