臺灣博碩士論文加值系統

TNG67與SA1613.1這兩個純品系栽培種具有正常的外表型，但雜交產生的F2子代在分蘗數及根部性狀上出現連續分佈的分離現象，暗示有一數量遺傳基因座(QTL)控制此性狀變化。本實驗室利用F2族群進行初步基因座定位分析，得知第1號及11號染色體上各有一個主效基因座，分別命名為qPN1、qPN11(各自對性狀的影響約為22%)。當qPN1與qPN11的基因型組合為[T,S] (逗號前後分別代表qPN1與 qPN11; T:TNG67, S: SA1613.1, H: heterozygous)時，外表型為單分蘗且少根，稱這種性狀為rolts (rootless plant with tiny single tiller)
為了避免其他QTL干擾，並對qPN11進行精確基因座定位分析，所以想利用回交的方式建立近同源品系(NIL)，但是發現回交產生的近同源品系為單分蘗，無法獲的足夠的子代。因此，改變計畫以連續自交方式，獲得在qPN1及qPN11皆為異型接合狀態，其餘部分皆為TNG67基因型的回交重組自交品系(BRIL)，育種過程則以分子標誌篩選(MAS)輔助進行。目前利用140株BC2F5植株，可將qPN11基因座候選區間縮小至2 Mb；且判斷候選基因座最有可能落在InDel分子標誌I32635-1與D32674之間，約300 kb的區間內。
使用水耕栽培針對rolts性狀進行觀察，結果顯示基因型是[T,S]的植株在種子萌芽5天後就出現種子根較短、側根數降低且無不定根的情況。持續以水耕方式種植，發現分蘗數目可以回復，但不定根在長度及數量上遠低於親本，據以判斷qPN11主要影響根部發育，而非分蘗數。由於水稻根的形成起始於節點(node)，，於是計數rolts植株的節點數目，結果顯示與正常植株並無差異，表示根部性狀應是本身結構異常所致。進一步的，我們利用冷凍掃描式電子顯微鏡觀察14天的秧苗的種子根，發現其橫向結構(radial patterning)並無異常；以共軛焦顯微鏡觀察，發現種子根縱向表層細胞的大小與組織構造基本上均正常，惟細胞壁較厚且多摺皺，細胞形狀較不規則，最明顯的是，在根部延長區(elongation zone)沒有發現長細胞，因此推測rolts性狀主要是由細胞在延長上的缺陷所導致。

Two rice lines, TNG67 and SA1613.1, usually produce 6~7 panicles each plant but their F2 progenies have wide segregations in panicle number and root biomass, indicate quantitative trait loci (QTL) involved in this trait. Primary mapping on a F2 population revealed two major QTL, qPN1 and qPN11, each with ~22% phenotypic variation explained (PVE). When both qPN1 alleles are from TNG67, allele of qPN11 from SA1613.1 acts dominantly to produce rootless plant with tiny single tiller, abbreviated as “rolts”.
To characterize effect of the qPN11 allele independently, and to construct materials for its fine-mapping, a Near-Isogenic Line (NIL) was supposed to be established by backcrossing. However, the tiny single tiller phenotype for such a NIL hindered this process. A Backcrossed Recombinant Inbreed Line (BRIL) which heterozygous on both qPN1 and qPN11 but mostly homozygous as TNG67 throughout the genome was therefore established instead. Marker-assisted selections (MAS) were employed during the breeding process. Finally, using 146 BC2F5 segregating progenies from the selected BC2F4 BRIL line, candidate genes for qPN11 was narrowed down to a 2 Mb region, most likely reside between two InDel markers, I32635-4 and D32674, which is 300 kb apart.
To characterize phenotypic defects of rolts, BC2F5 segregating seedlings were screened by markers that associated with qPN1 and qPN11. Hydroponic culture of rolts seedlings revealed an early symptom on 5 DAG, showing short seminal root, very few lateral root and absence of adventitious root. Noticeably, formation of tiller but not adventitious root resumed if rolts seedlings were remained within hydroponic culture, indicates that no major defect for its tiller development. Moreover, node seems to be formed normally in rolts plant as its node number reached ~15 in mature plants, although very tiny. Cryo scanning electron microscopic examination of 14 DAG seedlings revealed a similar radial structure between normal and rolts plants, suggesting that pattern formation and differentiation are not affected. Further confocal microscopic studies suggest that rolts plant is basically normal in cell size and organization, with thicker cell wall and more varied cell shape. However, no long cell can be found in the root elongation zone and almost all cell form root hair. Therefore, the major defect for rolts seems to be on cell elongation.

前言………………………………………………………………………1
前人研究…………………………………………………………………2
一、水稻外觀與生活史……………………………………………2
二、水稻根部型態與生長發育……………………………………2
三、影響水稻根部生長知相關基因………………………………3
四、植物荷爾蒙對根的生長調控…………………………………4
五、基因據圖選殖法………………………………………………5
六、分子標誌輔助選拔……………………………………………5
七、水耕栽培………………………………………………………6
材料與方法………………………………………………………………7
一、試驗材料………………………………………………………7
(一) 台農67號 (Oryza sativa L. ssp. japonica, cv.
Tainung 67；TNG67……………………………………7
(二) SA1613和SA1613.1………………………………………7
(三) SA1613.1×TNG67的BC1F1至BC2F5世代…………………7
二、試驗方法………………………………………………………7
(一) 水稻基因組DNA之小量萃取……………………………7
(二) InDel分子標誌的設計.…………………………………8
(三) InDe、SSRl分子標誌的聚合酶鏈反應（Polymerase
chain reaction；PCR）條件及步驟…………………8
(四) 分子標誌的分析…………………………………………8
(五) 水稻水耕栽培法…………………………………………8
(六) 冷凍斷裂與蝕刻法………………………………………9
(七) 石蠟包埋切片法…………………………………………9
(八) mPS-PI染色法……………………………………………9
結果………………………………………………………………………11
一、建立TNG67回交重組自交系(Bacross recombinant inbreed
line；BRIL)…………………………………………………11
二、利用InDel分子標誌縮小qPN11候選區間……………………12
三、建立水耕栽培方法……………………………………………13
四、觀察少分蘗植株之根部生長發育……………………………13
五、解剖並分析少分蘗植株根部發育之缺陷……………………14
討論………………………………………………………………………16
一、找尋I32635-4至I33724A (2.04 Mb)的重組植株…………16
二、qPN11候選區間內分子標誌之設計…………………………16
三、使用水耕或土耕栽培qPN11精確定位族群…………………16
四、判斷qPN11基因座所影響之性狀……………………………16
五、根系顯現差異之時間點與作用之部位………………………17
六、針對幼苗期種子根進行解剖分析……………………………17
七、導致幼苗期種子根缺陷之原因………………………………18
參考文獻…………………………………………………………………19
附錄………………………………………………………………………38

林雍凱。2007。調控水稻SA1613.1與台農67號之F2子代穗數性狀主要基因座的定位。碩士論文。國立中興大學生物科技學研究所
朱俊穎。2008。以表現輔助蛋白增進植物農桿菌轉殖法的效率與建立帶有qPN11(S)基因座之TNG67近同源品系與其性狀分析。碩士論文。國立中興大學生物科技學研究所
Bai JY, Zhang Q, Jia XP (2006) Comparison of Different Foreground and Background Selection Methods in Marker-Assisted Introgtression. Acta Genetica Sinica 33: 1073-1080
Bleecker AB, Rose-John S, Kende H (1987) An evaluation of 2,5-norbornadiene as a reversible inhibitor of ethylene action in deepwater rice. Plant Physiol 84: 395-398
Burg SP (1973) Ethylene in plant growth. Proc Natl Acad Sci U S A 70: 591-597
Busch M, Mayer U, Jurgens G (1996) Molecular analysis of the Arabidopsis pattern formation of gene GNOM: gene structure and intragenic complementation. Mol Gen Genet 250: 681-691
Celenza JL, Jr., Grisafi PL, Fink GR (1995) A pathway for lateral root formation in Arabidopsis thaliana. Genes Dev 9: 2131-2142
Chakravarthi BK, Naravaneni R (2006) SSR marker based DNA fingerprinting and diversity study in rice (Oryza sativa. L). African Journal of Biotechnology 5: 684-688
Collard BCY, DJ Mackill (2008) Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Phil. Trans. R. Soc. B 363: 557-572
Ding X, (2005) Advances in map-based Cloning for Corp Quantitative Trait Loci. JOURNAL OF PLANT GENETIC RESOURCES 6: 464-468
Dharmasiri S, Swarup R, Mockaitis K, Dharmasiri N, Singh SK, Kowalchyk M, Marchant A, Mills S, Sandberg G, Bennett MJ, Estelle M (2006) AXR4 is required for localization of the auxin influx facilitator AUX1. Science 312: 1218-1220
Duan YH, Zhang YL, Ye LT, Fan XR, Xu GH, Shen QR (2007) Responses of rice cultivars with different nitrogen use efficiency to partial nitrate nutrition. Ann Bot 99: 1153-1160
Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jurgens G (2003) Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426: 147-153
Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jurgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112: 219-230
Haecker A, Gross-Hardt R, Geiges B, Sarkar A, Breuninger H, Herrmann M, Laux T (2004) Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 131: 657-668
Horii H, Nemoto K, Mijyamoto N, Harada J (2006) Shoot communication quantitative trait loci for adventitious and lateral root in rice. PlantBreeding 125: 198-200
Hoshikawa k (1989) THE GROWING RICE PLANT. Nobunkyo, Tokyo,Japan
Inukai Y, Miwa M, Nagato Y, Kitano H, Yamauchi A (2001) Characterization of rice mutants deficient in the formation of crown roots. Breeding Science 51: 123-129
Inukai Y, Sakamoto T, Ueguchi-Tanaka M, Shibata Y, Gomi K, Umemura I, Hasegawa Y, Ashikari M, Kitano H, Matsuoka M (2005) Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling. Plant Cell 17: 1387-1396
Jia L, Zhang B, Mao C, Li J, Wu Y, Wu P, Wu Z (2008) OsCYT-INV1 for alkaline/neutral invertase is involved in root cell development and reproductivity in rice (Oryza sativa L.). Planta 228: 51-59
Kobayashi T, Suzuki M, Inoue H, Itai RN, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2005) Expression of iron-acquisition-related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements. J Exp Bot 56: 1305-1316
Leyser O (2005) The fall and rise of apical dominance. Curr Opin Genet Dev 15: 468-471
Li J, Zhu S, Song X, Shen Y, Chen H, Yu J, Yi K, Liu Y, Karplus VJ, Wu P, Deng XW (2006) A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem. Plant Cell 18: 340-349
Liu H, Wang S, Yu X, Yu J, He X, Zhang S, Shou H, Wu P (2005) ARL1, a LOB-domain protein required for adventitious root formation in rice. Plant J 43: 47-56
Liu SP, Wang JR, Wang L, Wang XF, Xue YH, Wu P, Shou HX (2009) Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family. Cell Research 19: 1110-1119
Lorbiecke R, Sauter M (1999) Adventitious root growth and cell-cycle induction in deepwater rice. Plant Physiol 119: 21-30
Marchant A, Kargul J, May ST, Muller P, Delbarre A, Perrot-Rechenmann C, Bennett MJ (1999) AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J 18: 2066-2073
Ortega-Martinez O, Pernas M, Carol RJ, Dolan L (2007) Ethylene modulates stem cell division in the Arabidopsis thaliana root. Science 317: 507-510
Pelosi A, Lee MCS, Chandler SF, Hamill JD (1995) Hormonal contral of root primordia differentiation and root formation in cultured explants of Eucalyptus globules seedlings. Aust. J. Plant Physiol. 22: 409-415
Shevell DE, Leu WM, Gillmor CS, Xia G, Feldmann KA, Chua NH (1994) EMB30 is essential for normal cell division, cell expansion, and cell adhesion in Arabidopsis and encodes a protein that has similarity to Sec7. Cell 77: 1051-1062
Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, Galweiler L, Palme K, Jurgens G (1999) Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286: 316-318
Sturm A (1999) Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning. Plant Physiol 121: 1-8
Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett M (2001) Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev 15: 2648-2653
Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthelemy J, Palauqui JC (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of Phloem development and structure in Arabidopsis. Plant Cell 20: 1494-1503
Wang C, Tseng T, Lin C (2002) Rice biotech research at the Taiwan Agricultural Research Institute. Asia Pacific Botech News 6: 950-956
Xu M, Zhu L, Shou H, Wu P (2005) A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol 46: 1674-1681
Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD, Weigel D, Chory J (2001) A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291: 306-309
Zhao Y, Hu Y, Dai M, Huang L, Zhou DX (2009) The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice. Plant Cell 21: 736-748