臺灣地區大豆屬物種之親緣關係研究 I. 六個大豆屬物種之種實、蛋白譜型與基因組特性的調查 II. G. tomentella物種複合群的基因組組成及分類地位

Abstract

本研究主要目的在於探討臺灣地區大豆屬物種的親緣關係，先從種實、蛋白譜型與基因組特性進行六個大豆屬物種的一般性的調查，包括利用掃描式電子顯微鏡觀察大豆屬物種種皮表面的細微構造，找出不同物種特有的構造以助大豆屬物種的分類研究。在細微構造上，栽培種的種皮表面有小孔洞和附著物，而其他物種則有由附著物所形成的網狀構造- 粉霜，不同物種粉霜的孔穴大小存在顯著差異。 本研究利用掃描式電子顯微鏡觀察大豆屬物種子葉表面的特殊構造- 紋孔。栽培種大豆紋孔出現的時間點約在開花後10-15天。紋孔區塊內細胞外觀呈現多裂狀，經共軛焦顯微鏡檢視，發現此處細胞的細胞壁分化出二級結構，為典型傳輸細胞的特徵，可能有助於種子發育時的養分傳送。大豆屬物種種子大小不一，特別是栽培種種子與其他野生種相差極大，但每一物種紋孔區塊面積相差不大。前人研究認為紋孔是大豆屬物種特有的構造，但本研究的結果發現至少在蝶形花亞科的12個族內21個屬的物種演化出紋孔這個構造。 本研究以酵素火燄法來製備大豆物種根尖染色體，配合Giemsa染色進行顯微鏡觀察，顯示在臺灣所採集的大豆屬物種有兩種染色體數目，一年生的Glycine soja體染色體2n = 40，而多年生的G. tabacina、G. tomentella (長莢型與短莢型) 體染色體均為2n = 80。本研究也用細胞流測儀檢視臺灣搜集系的倍數性，所得的結果和染色體計數的結果相符。而四倍體物種的DNA含量與可能的兩個二倍體親本DNA含量加總相當。 多種大豆屬物種的種子全蛋白以SDS-PAGE與西方式墨點轉漬法進行分析，結果指出每一個物種均有獨特的電泳圖譜，可以做為物種鑑識之用。所使用的免疫分析的九個種子蛋白中，GmPM1、GmPM2、GmPM8三個種子成熟蛋白譜型在不同物種間差異明顯，G. tomentella物種複合群搜集系依據這三個種子蛋白譜型進行的分群與前人利用同功酶群的方法所推論出的結果一致。因此，長莢型G. tomentella屬於G. tomentella的T2 race，而短莢型的G. tomentella屬於T4 race。 本研究進行不同組合的人工雜交試驗來找出T2 race的基因組組成。雜交試驗 (正反交) 指出G. dolichocarpa與G. tomentella T2 race之間沒有生殖隔離現象存在，這項證據也說明G. tomentella T2 race就是G. dolichocarpa。在可能的二倍體親本雜交的組合中，本研究只在DD x D3D3的組合得到一個發育不良的豆莢，說明D與D3 基因組有較相近的親緣關係。同時也說明G. dolichocarpa可能具有DDD3D3基因組組成。本研究也利用膠內雜交蛋白電泳方法進一步加以證實。

The main purpose of this research is to study the phylogenetic relationship of Glycine species collected in Taiwan. The morphology and fine structures of seed coat of Glycine species accessions were analyzed by scanning electron microscopy (SEM). The seed coat surface of the cultivar (Shishi) contained pores and deposits. The bloom resulting from the attachment of endocarp was present in all of the species examined except G. max. In addition, the cavity sizes of the bloom were significantly different among these accessions. These characters, including seed weight, seed length, seed coat proportion and cavity density, might be helpful in Glycine species taxonomy. A special structure (pit) on cotyledon surface of Glycine species was also analysed by SEM. Pit appeared on developing G. max seed about 15 DAP. These cells are multilobed, i.e. the signature of transfer cells, that might transfer nutrition from mother tissue to embryo. The size of pit area does not correlate with seed size since the pit sizes of each accession are almost the same while the seed sizes among them are totally different. The pit structure is not unique to Glycine species, it is also present on 21 genera among 12 tribes of Papilionoideae. The flame dry method was used to prepare the somatic chromosome for checking the chromosome numbers of all Taiwanese Glycine accessions, the annual accessions were diploid and the perennial ones were all tetrapolid. The ploidy levels and the DNA contents of Glycine species were also determined by flow cytometry. The polyploid species contained amounts approximately the sums of the respective proposed parental diploid species. The seed protein from many Glycine species were analyzed by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) and Western blot against soybean seed maturation protein and seed storage protein antibodies. Each species yields a unique electrophoretic pattern that varied in the total number of bands and their relative mobilities. Three genes, GmPM1, GmPM2 and Gm PM8 are more varied during the evolution of genus Glycine species, so their protein profiles can be used as a tool to identity Glycine species. In this study, using seed protein to group of G. tomentella species complex provided the same arrangement when compared to using isozyme described before. Hence, the long-pod G. tomentella, that is G. dolichocarpa, belongs to G. tomentella T2 race, and the short-pod one belongs to T4 race. There was no reproductive isolation between G. dolichocarpa and T2 race, and for taxonomy status, T2 race is G. dolichocarpa. Crossing between the respective parental diploid species was also applied to comfirm the genome composition of G. tomentella T2 race. Furthermore, in gel hybridization of seed protein was used to examine the events.