邁向水稻半糯基因Du8之定位選殖

Abstract

稻米是人類重要的糧食之一，更是台灣地區之主要糧食。隨著經濟發展，消費型態的改變，台灣稻米的生產出現了生產過剩的情形，且因應加入世界貿易組織WTO，稻米的生產已由量的時代轉變為質的時代，提升稻米品質在近年來成為重要的研究課題。 直鏈澱粉的含量為影響稻米品質的重要因素，低直鏈澱粉的米飯口感濕潤、有彈性，因此較受日本、韓國與台灣等地區消費者青睞。本論文係以來自EMS誘變台稉8號所得之13個突變品系為材料，而這些突變品系擁有不同直鏈性澱粉含量。試驗首先以碘呈色比色法測定13個突變品系之直鏈澱粉含量，範圍從5.11%到15.53%，直鏈澱粉含量存在相當程度之變異。以碘液染色胚乳，呈色顏色隨直鏈增加而加深，直至飽和為止，可成為簡易判斷澱粉含量之方法。為瞭解半糯胚乳顏色和影響直鏈澱粉含量改變的原因，挑選口感食味佳半糯品系CNY921391，其直鏈澱粉含量11.92%，進行觀察胚乳澱粉粒結構與半糯基因定位選殖。CNY921391澱粉結構分別在400 X與3000 X掃描式電子顯微鏡下觀察，於3000 X中，其澱粉粒型態與米粒外觀晶瑩剔透的台稉8號之常見多角型、排列緊實不同，而是圓形、排列鬆散，造成胚乳不透明之乳白色。在CNY921391與台中秈17號雜交建立之F2族群中，胚乳透明型與半糯乳白型的個體比例為3.6:1，顯示半糯為單一隱性基因所控制，並從中挑選30個胚乳外表型呈半糯F2個體，以96個分子標幟進行建構遺傳圖譜，平均間距為16.63 cM, 並進行半糯基因之遺傳連鎖分析，結果將此半糯基因粗略定位於第四條染色體的長臂S13714與C53391之間，是一新發現的半糯性基因，因此命名為Du8，再以26個於標的區域具互換的F2個體進行細微定位，最後將Du8侷限在CH0422與CH0426之間，此一152987 bp的區域中涵蓋12個基因。本論文已完成至Du8的候選基因只剩12個，但尚未達到本研究之最終目標，未來將繼續高解析定位以縮小標的區間與進行候選基因之定序來確認Du8突變點，最後並釐清半糯性狀之基因與直鏈性澱粉合成機制，並應用於提升稻米品質之育種。

Rice is one of the major crops in the world, and it is also a major food in Taiwan. The aim of rice production has been changed from quantity-oriented to quality-oriented because of altered rice consumption along with rapid development of economics and of joining WTO in Taiwan. Researches and breeding for improvement of the rice quality have been the important tasks ever since. The amylose content is one of important factors that affect the quality of rice grain. Low-amylose rice is mostly popular and widely accepted in Japan, Korea and Taiwan, which tends to show fluffy texture and glossy appearance after cooked. In this research, we selected 13 mutant lines of Taikeng 8 (TK8) induced by EMS that were provided by Dr. Yong-Pei Wu. The amylose contents of 13 lines were measured by the absorbance of spectrophotometer at 620 nm after iodine-binding procedures and ranged from 5.11% to 15.53%. The endosperm was stained with iodine which the color of endosperm was proportion to the amylose content. To understand what caused the changes of endosperm color and amylose content, we selected the mutant line CNY921391 for which good taste and low-amylose content of 11.92%. The starch granules of CNY921391, rather than in polyhedron shapes as of TK8, were in sphere shapes by SEM at 3000 X. We established an F2 population by crossing CNY921391 with O. sativa ssp. indica cv. Taichung Sen 17 (TCS17) for applying positional cloning this mutated dull gene. The ratio of transparent endosperm to opaque endosperm was found to be 3.6:1, knowing that this mutant gene is a recessive allele. A total of 30 F2 individuals with opaque endosperm were genotyped with 96 polymorphic markers and subjected for construction of genetic maps with an average interval of 16.63 cM. After linkage analysis, the dull gene was coarsely mapped between S13714 and C53391on the long arm of the chromosome 4 and named the mutated gene as Du8. We proceeded fine mapping by using 26 recombinants and narrowed down the target region between CH0422 and CH0426 encompassing 152987 bp and 12 candidate genes after annotation. We accomplished the fine mapping to narrow down candidate genes to 12, which paved a way to achieve final goal. In the future, high-resolution mapping and DNA sequence candidate genes will be implemented to touch down Du8. Consequently, we can elucidate the gene regulations of dull and metabolism of starch, and further apply in rice breeding to promote rice quality.