針對臺灣小米種原探討影響澱粉生合成之主效基因

Abstract

小米 (Setaria italica (L.) P. Beauv.) 為多種粟類穀物中全世界栽培第二大宗者，且因其耐旱、耐貧瘠、對於環境之適應力強而廣泛遍布於世界各地，尤其為乾旱與半乾旱地區的重要作物。小米在臺灣的栽種歷史可以追溯至大約五千年前，對原住民而言是最古老的栽培作物，歷史悠久的主食，營養成分高，澱粉含量大約75%，其中，糯小米具有較高的黏滯性和良好的適口性，製成品有甜香味，應用於釀酒、炊飯、煮粥、製餅等。穀粒胚乳中澱粉粒的合成是由一系列酵素參與形成之複雜網絡，但是相較於主要作物，例如水稻、玉米、小麥等，對於小米的澱粉特性以及其生合成的相關研究並不甚多。本研究利用123個臺灣小米收集系，分析其糯性基因型，澱粉合成相關基因 (SSRGs) 之表現量，以及Waxy基因所編碼的澱粉顆粒結合性澱粉合成酶 (GBSSI) 之功能性研究。以不同跳躍子 (transposable element) 插入Waxy為依據將Waxy歸類為四個對偶基因型，type I 為高直鏈澱粉含量的野生型，type III與IX屬於低直鏈澱粉含量，而大多數收集系屬於糯性外表型的type IV。Type III，IX與IV的 Wx 之mRNA含量顯著低於type I，但這些對偶基因型的轉錄片段並未出現異常剪切，長度皆與野生型相同。Type I的GBSSI蛋白質含量顯著高於其他對偶基因型，這樣的差異源自於type III，IX與IV因為跳躍子插入基因中而導致mRNA含量下降，進而造成直鏈澱粉含量降低。Wx對偶基因型在臺灣的地理分布似乎與原住民對於不同小米特性的偏愛所造成的選拔息息相關，尤其在蘭嶼地區展現了地區獨特性。另外，依據水稻中澱粉生合成的路徑，針對小米之澱粉生合成相關基因(SSRGs)包括AGPase large subunit1 (AGPL1)、starch synthase I (SSI)、SSIIa、SSIIIa、branching enzyme2 (BE2)、與pullulanase1 (PUL1)，在穀粒充實期間中挑選開花後 (DAF) 5、10、15與22天進行表現量分析。澱粉合成相關基因的表現量在挑選的收集系中並沒有明顯的差異，不過SSI、SSIIa與SSIIIa 的表現量在後兩階段超越了GBSSI，其中SSIIa為澱粉合成酶中表現量最高者，不過其表現模式在type III有些不同，且PUL1在type IX也有些微不同，表示這些收集系在特定的SSRGs可能攜帶著不同的對偶基因。此研究針對小米中調控澱粉生合成之主要基因進行了初步的探討，未來將結果與澱粉理化特性相互對應後能進一步探討收集系中其他澱粉合成相關基因之對偶基因組成，對偶基因之多型性可應用於分子標誌輔助選拔，以選育優良食味或加工品質且農藝性狀佳的臺灣地方種小米，使小米不僅具有地方特色亦能多元應用發展。

Foxtail millet (Setaria italica (L.) P. Beauv.), the second largest crop among millets, is extensively adapted to diverse environments and remains an important crop in arid and semiarid regions worldwide serving as both cereal foods and forage. In Taiwan, foxtail millet is a traditional crop for indigenous people and its cultivation can date back around 5,000 years ago. However, less information is available about the characteristics and biosynthesis of starch in foxtail millet in comparison with other major cereals such as rice, corn and wheat. In this study, a total of 123 accessions collected within Taiwan were assessed by Wx genotypes, functional studies of GBSSI, and gene expressions of selected SSRGs. Four Wx genotypes were identified: I (non-waxy wild type), III (low AC), IV (waxy), and IX (low AC), while the majority of accessions belonged to the waxy type. The expression level of Wx was significantly lower in type III, IX and IV, whereas the length of their transcripts were the same as wildtype without inaccurate splicing. The GBSSI content of type I was significantly higher than other three genotypes, suggesting the variance was caused by low mRNA level in other three types due to reduced splicing efficiency and then resulted in lower AC. The geographic distribution of Wx genotypes seemed to be associated with the preferences for specific waxy phenotypes under human selection, especially in the Orchid Island (Lanyu). According to the starch biosynthesis pathway in rice, expressions of selected orthologous of foxtail millet SSRGs were estimated during grain filling stages at 5, 10, 15, and 22 DAF (days after flowering), including AGPL1, SSI, SSIIa, SSIIIa, BE2, and PUL1. There was no significant variation in SSRGs among these accessions. Expression levels of SSI, SSIIa, and SSIIIa mainly surpassed GBSSI in the last two stages; besides, the level of SSIIIa was the highest among SSs. However, the expression pattern of SSIIIa was slightly different in type III and so was PUL1 in type IX, which indicated the possibility that these types might carry different alleles of specific SSRGs. The relationship among SSRGs, structure of grain starch, and its physicochemical properties will be further studied. The study provided preliminary information on major genes controlling starch biosynthesis in foxtail millet, which can be applied to breeding programs for elite eating and cooking quality and various utilizations of foxtail millet in the food industry.