利用 Lea 第四群基因族探討大豆屬物種之親緣系統及分子演化關係

Abstract

台灣是一年生野生大豆分佈的南界，同時也是多年生野生大豆分佈的北界，因此我們使用了大豆第四群 LEA 蛋白的 GmPM16 和 GmPM28 基因族 (gene families) 為標誌，嘗試去探討大豆屬物種之分子演化關係。 本試驗使用栽培種大豆、野生大豆及 13 個大豆遠緣種共 15 個品系，根據已知之栽培種大豆 cDNA 序列，設計引子，將參試品系分別選殖並加以定序，定序結果以鄰近連接法 (neighbor-joining tree) 與最小間距網狀圖 (minimum spanning network) 比較分析其系統關係。 試驗結果顯示，大豆屬物種的種子全蛋白以 SDS-PAGE 進行分析，每一個物種均有其獨特的電泳圖譜；但若以 GmPM16 及 GmPM28的抗體進行西方墨點轉漬法進行分析，卻無法明確的區分。Genomic DNA 序列分析結果，GmPM16 和 GmPM28 兩個基因族的基因片段在多年生大豆遠緣種皆比一年生栽培種有較高的歧異度，由其 ks/ka 值推論這兩個基因族在演化動力 (evolutionary forces) 上並未符合中性假說 (neutrality hypothesis)。除此之外，本試驗顯示GmPM16 和 GmPM28 基因族的基因片段具有低的 π 値 (核苷酸歧異度)及高的 Hd 値 (基因型歧異度)，此表示此族群早期曾長時間維持低度的有效族群，之後族群快速成長，經歷的時間足以累積突變，但卻不足以累積大量的序列變異。另外以 genomic DNA、intron DNA 和 ORF 序列，利用不同分析方法所建構的系統關係樹和最小間距網狀圖結果並不一致，由此推測此兩個基因族可能是彼此具有不同的溯祖時間；依據分群結果推論，採集自台灣台東的 tom039 和起源自澳洲的基因組 DDD3D3、AAB’B’、AA的親緣關係為較接近。

Taiwan is the southernmost region of the distribution for annual wild soybean species and the northernmost region of the distribution of the perennial ones. In this study, we used GmPM16 and GmPM28 gene families, which belong to the fourth group of late embryogenesis abundant (LEA) proteins of soybean, as the genetic markers to assess the molecular relationships of the genus Glycine. We surveyed sequence variations in the cultivated soybean, wild soybean, and thirteen accessions of the perennial soybean relatives. The PCR primers were designed based on the soybean cDNA sequences. We examined the phylogenetic relationships by total sequences, introns and open reading frame (ORF) using the neighbor-joining and minimum spanning network methods. In the present study, SDS-PAGE analysis of the seed proteins from many Glycine species suggested that each species had unique protein profiles that varied in the total number of bands and their relative motilities. However, the results of western blot with GmPM16 and GmPM28 antibodies indicated no significant difference among them. According to total sequences of the 2 soybean seed gene families, the genetic diversities were higher in the perennial soybean relative accessions than those of the annual soybeans. The value of Ks/Ka also suggested the evolutionary forces of these two gene families were deviated from the neutrality hypothesis. Furthermore, the species, based on the sequences of those two gene families, were with lowerπ(nucleotide diversity) values and higher Hd (haplotype diversity) values. The results suggested that these populations were expanded after a period of low effective population size; rapid population growth enhanved the retention of new mutations. The evolutionary relationship among the entries was inconsistent as indicated in phylogenetic trees and minimum spanning network, which were constructed based on total sequences, introns, or ORFs. This indicated that the two gene families had their unique coalescence time scale in the phylogenetic trees. On the basis of clustering results, we proposed that the genome of tom039 collected in eastern Taiwan has a close relationship to the Australian-originated genomes DDD3D3, AAB’B’, and AA.