水稻rolts基因座之精確定位與性狀分析

TNG67 (T) and SA1613.1 (S) both are normal rice lines with high yield. However, their F2 progenies exhibit wide segregations in panicle number and root biomass. A continuous distribution in phenotypes suggests quantitative trait loci (QTL) controls underneath. Primary mapping on a F2 population revealed two major QTLs, qPN1 and qPN11, with epistatic interactions on each other. When both qPN1 alleles are from TNG67, allele of qPN11 from SA1613.1 acts dominantly and generates rootless plant with tiny single tiller, abbreviated as “rolts”. Since qPN1 was found to be very close to the centromere of chromosome 1, qPN11 was chosen as the major target for fine-mapping in this study. To do fine mapping on qPN11 that confer rolts phenotype, a BC2F6 line was established as a backcross recombinant inbred line (BRIL). In the selected BC2F6 line, only the qPN1 and qPN11 loci remained heterozygous while homozygous as TNG67 throughout the genome. Therefore any phenotypic changes in its selfed progenies must be caused by segregation of qPN1 and/or qPN11 but not interfered by other loci. Using 811 BC2F7 progenies as the mapping population, candidate region for qPN11 was narrowed down to be within InDel markers D32693 and I33724A. The theoretical interval between these two markers is 1661 Kb in the Japonia genome with 232 predicted ORFs. RNA-Seq analysis on roots of 5 DAG seedlings were employed to identify genes that located within the candidate region and showed differential expressions between normal [T,T] and rolts[T,S] plants (the symbols before and after comma represent qPN1 and qPN11 allele, respectively). Hydroponic culture of rolts seedlings revealed an early symptom five days after germination. Compared with normal plants, rolts seedlings exhibited a short seminal root, very few lateral root, and absence of adventitious root (crown root). Confocal microscopic studies on the seminal root of rolts seedlings revealed absence of long cells in its elongation region and irregular cell walls all over the root cells. This result suggests that rolts mutation may disturb cell elongation and cell wall biogenesis. As symptom of root defect in rolts can be lessened by providing 1% sucrose, indicates that function of rolts can be modulated by nutrition. Moreover, rolts plant is less responsive to exogenous auxin, implicates a defect in auxin signaling pathway.TNG67與SA1613.1均屬外觀正常且豐產的水稻品系，但其雜交產生的F2子代在分蘗數及根部性狀上卻出現明顯的分離現象，且呈現連續分佈，暗示其受到數量遺傳基因座(QTL)調控。本實驗室利用F2族群進行初步基因座定位分析，得知第1號與第11號染色體上各有一個QTL候選基因座，分別命名為qPN1與qPN11。以逗號前後分別代表qPN1與PN11之基因型(T：TNG67同型接合，S：SA1316.1同型接合，H：heterozygous異型接合)，當qPN1與qPN11的基因型組合為[T,S]時，植株外表型為弱小單分蘗，且幾乎無根，稱此性狀為rolts (rootless plant with tiny single tiller)。 BC2F7為目前精確基因座定位分析的族群，是經由兩次與TNG67回交以及多次的自交所得到的，其染色體組的大部分區塊均已固定為TNG67同型接合狀態，不再分離干擾性狀判定，唯獨qPN1與qPN11之基因型仍維持異型接合狀態，故可分離產生rolts植株，作為精確定位rolts基因之用。共分析811株BC2F7植株的外表型與[qPN1，qPN11]基因型，發現可將qPN11基因座候選區間縮小至InDel分子標誌D32693與I33724A之間 (各有兩個植株提供證據)，該區間在TN67基因組上為1661 Kb，但在SA1613.1基因組上則為1189 Kb。嘗試使用即時定量PCR檢測區間內有興趣基因之表現量。此外，利用次世代定序之技術，分析rolts植株與正常植株5 DAG之種子根全基因表現模式，試圖找出差異所在。 使用水耕栽培[T,S] rolts植株，發現在種子萌芽五天後即出現明顯性狀，與[T,T]植株相比較，其種子根短且不定根少甚至缺乏。以共軛焦顯微鏡觀察rolt植株的種子根，發現在根部延長區(elongation zone)缺乏長細胞(正常株細胞會有長短之分)，且細胞壁形狀摺皺不規則，推測rolts性狀主要為影響根部細胞延長與細胞壁生成。上述rolts性狀在改變營養環境(MS含糖培養基)或是添加外源生長素(NAA-增加不定根的形成)之後，可緩解其異常性狀，分別可以觀察到根部長度增長與不定根根數增加。此試驗指出rolts基因的屬性可能與營養攝取及生長素相關。 中文摘要…………………………………………………………………I Abstract…………………………………………………………………II 目錄………………………………….…………………………………III 圖表目錄…………………………………………………………………V 前言………………………………………………………………………1 前人研究…………………………………………………………………2 一、水稻簡介與利用………………………………………………2 二、水稻根部形態與生長發育……………………………………2 三、與水稻根部生長發育有關之基因……………………………3 四、植物激素對根部的生長調控…………………………………5 五、分子標誌輔助縮限基因候選區間……………………………6 六、利用微陣列分析資料庫定位水稻基因………………………7 七、次世代定序法…………………………………………………8 材料與方法………………………………………………………………9 一、 試驗材料……………………………………………………9 (一) 台農67號 (Oryza sativa L. ssp. japonica, cv. Tainung 67；TNG67)…......................................9 (二) SA1613.1……………………………………………………9 (三) SA1613.1×TNG67的BC1F1至BC2F7世代…………………….9 二、 試驗法法…………………………………………....……10 (一) 水稻基因組DNA之小量萃取………………………………10 (二) 水稻幼苗種子根總量RNA萃取……………………………10 (三) InDel分子標誌的設計……………………………………11 (四) SSR、InDel分子標誌的聚合酶鏈反應（Polymerase chain reaction； PCR）條件及步驟…………………………......11 (五) 多型性分子標誌的分析………………………………….11 (六) 利用MS medium以及 MS salt栽培水稻法…………..…11 (七) 水稻水耕栽培法………………………...............12 (八) 石臘包埋切片法………………………………….………12 (九) mPS-PI (modified Pseudo-Schiff Propidium Iodide)染色………………..........................................13 (十) 檢測基因表現量的Real-time PCR…………………….…13 結 果……………………………………………………………...……14 一、精確定位水稻rolts 基因及構築其分析族群……………………14 (一) 構築定位rolts基因回交重組自交(BRIL)之目標親本株…....14 (二) 利用於候選區間設計InDel分子標誌縮小rolts基因候選區間.15 (三) 利用精確定位分析族群定位水稻十一號染色體rolts基因座..16 (四) 探討qPN1與qPN11 兩個基因候選區間之間的關聯…………….18 二、觀察分析rolts對水稻發育之影響……...........……………19 (一) rolts和正常植株之性狀分析………………………………….19 (二) 解剖觀察分析rolts和正常植株葉片……………………....…20 (三) 解剖觀察分析rolts和正常植株根部……………………………21 三、觀察分析添加外源荷爾蒙或改變生長環境對rolts和正常株之影響........................................................22 (一) 觀察分析醣類對於rolts和正常株生長之影響…………………22 (二) 觀察分析NAA對於rolts和TNG67植株生長之影響………………22 四、觀察分析rolts和正常株基因表現量.......................23 (一) 利用Real-time PCR來分析rolts和TNG67基因表現量的差異…23 (二) 利用次世代定序(next generation sequencing)之技術來分析rolts和TNG67基因表現量的差異..............................24 討 論 ……………………………………………………………………26 參 考 文 獻………………………………………….......…………30 附 錄………………………………………………….…………………5