臺灣博碩士論文加值系統

稻稈是水稻收穫後的副產物、大部分是木質素、纖維素、在土壤中難以降解。脆性突變體在第二層的細胞壁有厚度降低現象、脆性水稻可能較容易降解。利用疊氮化鈉誘變本研究室建立了 IR64 和 TNG67 二個突變庫純系。經外外表型篩選、本研究分別獲得 21 個和 23 個 IR64 和 TNG67 脆性突變體。脆性可以分為 0、1、3和 5 四個等級、IRB3 是一個 IR64 的脆性突變品系、為 3 等級的脆性、手指可以輕易折斷。遺傳分析顯示、IRB3 的脆性是一個隱性基因 (不脆: 脆； 3: 1; χ2 = 0.223) 所調控的性狀。基因定位分析將脆性基因標定在第 10 號染色體、 RM271 和 RM5392 之間 666 kb 區間內。使用生物資訊學、在此一區域有七個候選基因被選出來、包括一個可能控制纖維素生合成的 OsCesA7 基因、可能是脆性的基因。將進一步的選殖定序以探討確定脆性基因、並開發供分子輔助育種使用的的功能標誌提高選拔效率。白葉枯病是水稻的重要病害、本研究將 IRB3 與帶有 4 個抗白葉枯病基因 (xa5/ Xa7/ xa13/ Xa21) 的 TN11/IRBB66 BC2F4 高級世代純系雜交、導入抗性基因、擬結合白葉枯病之抗性與脆性育成新的品種。在育種程序中利用分子標誌輔助選拔、應用五組與五個Xa基因連鎖的簡單序列重複 (SSR) 標誌進行抗病性之基因型選拔。配合剪葉接種法進行外表型選拔中、在最高分蘗期接種 Xoo-F2 和 XF89-b 菌株後、於發病後21天測定病斑危害長度、判斷抗病程度、目前已進行至第F5世代、獲得脆性與強抗病性之品系數個。

Straw is a by-product of rice after harvest, it is majorly consisted of lignocelluloses and is difficult to be degraded in the soil. The brittle culm mutants have mutation related with decreased secondary cell wall thickness and maybe easier for degradation. IR64 and TNG67 mutation pools were developed by sodium azide in our previous breeding programs. There were 21 and 23 brittle mutant lines selected from IR64 and TNG67 mutant pools. The brittle lines have differences in brittleness score from 0 to 5. IRB3 is a brittle culm mutant of IR64, it showed a Score 3 brittleness and easily to fracture by finger-bending. Bacterial blight disease is an important disease in rice, IRB3 was crossed with TN11/IRBB66 BC2F4 harboring with four bacterial blight resistant genes (xa5/Xa7/xa13/Xa21) to breed a variety with both blight resistance and brittle culm. In marker-assisted selection, the simple sequence repeat (SSR) markers linked to four Xa genes were applied in the breeding program. In phenotyping selection, plants were inoculated with Xoo-F2 and XF89-b strains by leaf-clipping method and investigated the disease lesion on leaves. The brittleness of IRB3 was controlled by a single recessive gene (non-brittle: brittle; 3: 1; χ2 = 0.223). The brittle gene of IRB3 was mapped on the chromosome 10 in a region of 666 kb between RM271 and RM5392 markers using a F2 population of IRB3 cross TN11/IRBB66 BC2F4. Using bioinformatics, seven candidate genes including the OsCesA7, which relates to the cellulose synthesis may control the brittle culm trait. Further study will be conducted to identify the gene responsible for the brittleness and to develop the functional marker for MAS to introduce the brittleness trait through MAS.

Table of contents

中文摘要 i
English abstract ii
Table of contents iii
List of tables v
List of figures vi
List of appendixes vii
Chapter 1: Introduction 1
Chapter 2: Literature reviews 3
1. Brittle culm trait and related genes identified in rice 3
2. Cell wall composition of rice brittle culm lines 14
3. Bacterial blight disease and resistant genes in rice 15
4. Marker assisted selection 17
Chapter 3: Materials and Methods 19
1. Plant cultivation and management 19
a) Seedling preparation 19
b) Transplanting 19
c) Field management 19
2. Characterization of brittle lines 20
a) Brittleness score investigation and screening for brittle lines from TNG67 mutant pool 20
b) Investigation of agronomic traits 21
c) Yield potential 26
d) Biomass investigation of brittle lines in different growth stage 26
e) Level of damage after typhoon investigation 27
f) Cluster analysis 27
g) Cell wall composition analysis 27
3. Genomic DNA extraction 29
4. Polymerase chain reaction (PCR) 30
5. Polyacrylamide gel electrophoresis (PAGE) 30
6. Genetic analysis 31
a) Rice pollination for crossing 31
b) Allelism test 31
c) Mapping and identification of the brittle gene of IRB3 31
7. Breeding to improve bacterial blight disease resistance of the brittle lines 33
a) Xa gene selection 33
b) Bacterial blight disease inoculation 33
Chapter 4: Results 34
1. Characterization of brittle lines 34
a) Brittleness investigation and screening for brittle lines from IR64 and TNG67 pools 34
b) Agronomic traits investigation 34
c) Biomass of brittle lines at different growth stage 35
d) Level of leaf brittleness influenced by typhoon 36
e) Cluster analysis 36
f) Cell wall composition analysis 38
2. Genetic analysis 39
a) Allelism test 39
b) Mapping and identification of the brittle gene 39
3. Breeding to improve bacterial blight disease resistance for the brittle lines 41
a) Xa gene selection 41
b) Bacteria blight disease inoculation 41
Chapter 5: Discussion 43
Chapter 6: Conclusion and Future Prospects 47
References 48
Tables and Figures 57
Appendixes 89

Reference

Antony, G., J. H. Zhou, S. Huang, T. Li, B. Liu, F. White and B. Yang. 2010. Rice xa13 recessive resistance to bacterial blight is defeated by induction of the disease susceptibility gene Os-11N3. The Plant Cell. 22: 3864-3876.
Aohara, T., T. Kotake, Y. Kaneko, H. Takatsuji, Y. Tsumuraya and S. Kawasaki. 2009. Rice BRITTLE CULM 5 (BRITTLE NODE) is involved in secondary cell wall formation in the sclerenchyma tissue of nodes. Plant Cell Physiol. 50: 1886-1897.
Arshad, H. M. I., S. T. Sahi, M. Atiq and W. Wakil. 2016. Appraisal of resistant genes and gene pyramid lines of rice against indigenous pathotypes of Xanthomonas oryzae pv. oryzae in Punjab, Pakistan. Pak. J. Agri. Sci. 53: 365-370.
Basavaraj, S. H., K. S. Vikas, S. Atul, S. Ashutosh, S. Anita, A. Deepti, Y. Sheel, K. E. Ranjith, S. Devinder, S. G. Krishnan, M. Nagarajan, T. Mohapatra, K. V. Prabhu, K. S. Ashok. 2010. Marker-assisted improvement of bacterial blight resistance in parental lines of Pusa RH10, a superfine grain aromatic rice hybrid. Mol Breeding. 26: 293-305.
Blair, M. W., A. J. Garris, A. S. Iyer, B. Chapman, S. Kresovich and S. R. McCouch. 2003. High resolution genetic mapping and candidate gene identification at the xa5 locus for bacterial blight resistance in rice (Oryza sativa L.). Theor Appl Genet. 107: 62-73.
Central Weather Bureau. 2016. (Online). (2016, September). Retrieved from http://www.cwb.gov.tw/V7/climate/dailyPrecipitation/dP.htm
Chang, C. F. 2014. Development of new varieties with bacterial blight resistance genes by using marker-assisted selection. National Chung Hsing University master thesis. Taiwan, Taichung city.
Chen, S., Z. H. Huang, L. X. Zeng, J. Y. Yang, Q. G. Liu and X. Y. Zhu. 2008. High-resolution mapping and gene prediction of Xanthomonas Oryzae pv. Oryzae resistance gene Xa7. Mol Breeding. 22: 433-441.
Chu, Z., Y. Ouyang, J. Zhang, H. Yang and S. Wang. 2004. Genome-wide analysis of defense-responsive genes in bacterial blight resistance of rice mediated by the recessive R gene xa13. Mol Gen Genomics. 271: 111-120.
Collard, B. C. Y. and D. J. Mackill. 2008. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Phil. Trans. R. Soc. B. 363: 557-572.
Duan, Z. Q., J. M. Wang, L. Bai, Z. G. Zhao and K. M. Chen. 2008. Anatomical and chemical alterations but not photosynthetic dynamics and apoplastic transport changes are involved in the brittleness culm mutation of rice. J Integr Plant Biol. 50: 1508-1517.
Fukushimaa, R. S., M. S. Kerleya, M. H. Ramosa, J. H. Portera and R. L. Kallenbach. 2015. Comparison of acetyl bromide lignin with acid detergent lignin and Klason lignin and correlation with in vitro forage degradability. Anim. Feed Sci. Technol. 201: 25-37.
Gramene. 2017. (Online). Retrieved from http://www.gramene.org/about-gramene
Hansey, C. N., A. J. Lorenz and N. de Leon. 2010. Cell wall composition and ruminant digestibility of various maize tissues across development. Bioenerg. Res. 3:28-37.
Hirano, K., T. Kotake, K. Kamihara, K. Tsuna, T. Aohara, Y. Kaneko, H. Takatsuji, Y. Tsumuraya and S. Kawasaki. 2010. Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis. Planta. 232: 95-108.
Huang, N., E. R. Angeles, J. Domingo, G. Magpantay, S. Singh, G. Zhang, N. Kumaravadivel, J. Bennett and G.S. Khush. 1997. Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR. Theor Appl Genet. 95: 313-320.
IRRI (International Rice Research Institute). 2014. Standard evaluation system for rice (SES). 5th edition. Los Baños, Philippines.
Ishimaru, K., E. Togawa, T. Ookawa, T. Kashiwagi, Y. Madoka and N. Hirotsu. 2008. New target for rice lodging resistance and its effect in a typhoon. Planta. 227: 601-609.
Iyer-Pascuzzi, A. S., H. Jiang, L. Huang, and S. R. McCouch. 2008. Genetic and functional characterization of the rice bacterial blight disease resistance gene xa5. Phytopathology. 98: 289-295.
Jia, X. L., G. L. Wang, F. Xiong, X. R. Yu, Z. S. Xu, F. Wang and A. S. Xionga. 2015. De novo assembly, transcriptome characterization, lignin accumulation, and anatomic characteristics: novel insights into lignin biosynthesis during celery leaf development. Sci Rep. 5: 14 p.
Jung, H. G., D. R. Mertens and A. J. Payne. 1997. Correlation of acid detergent lignin and klason lignin with digestibility of forage dry matter and neutral detergent fiber. J Dairy Sci. 80: 1622-1628.
Kaur, S., P. S. Panesar, M. B. Bera and V. Kaur. 2015. Simple sequence repeat markers in genetic divergence and marker-assisted selection of rice cultivars: a review. Crit. Rev. Food Sci. Nutr. 55: 41-49.
Khan, M. W., F.M. Abbasi, M.S. Masood, A. Rabbani, M.F. Abbasi, M. Sajid, U. Khan and H. Ahmad. 2015. Identification of bacterial blight resistance gene Xa7 in rice (Oryzae sativa L.) through STS marker. Int. J. Biosci. 6: 318-324.
Kotake, T., T. Aohara, K. Hirano, A. Sato, Y. Kaneko, Y. Tsumuraya, H. Takatsuji and S. Kawasaki. 2011. Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. J. Exp. Bot. 62: 2053-2062.
Kovach Computing Services (KCS). 2017. (Online). Wales, U.K. Retrieved from https://www.kovcomp.co.uk/contact.html
Laughlin, H. H., 1918. Modifications of the 9:3:3:1 ratio. Am. Nat. 52: 353-364.
Li, Y. H., Q. Qian, Y. H. Zhou, M. X. Yan, L. Sun, M. Zhang, Z. M. Fu, Y. H. Wang, B. Han, X. M. Pang, M. S. Chen and J. Y. Li. 2003. BRITTLE CULM1, which encodes a COBRA-Like Protein, affects the mechanical properties of rice plants. The Plant Cell. 15: 2020-2031.
Mertens, D. R. 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study. J. AOAC Int. 85: 1217-1240.
Mosleh, M. K., Q. K. Hassan and E. H. Chowdhury. 2015. Application of remote sensors in mapping rice area and forecasting its production: a review. Sensors. 15: 769-791.
Muhammad, M. H., Y. R. Mohd, R. I. Mohd, M. Maziah, A. R. Harun, Md. A. Alam, S. Ashkani, Md. A. Malek and A. L. Mohammad. 2015. Marker-assisted backcrossing: a useful method for rice improvement. Biotechnol. Biotechnol. Equip. 29: 237-254.
National Agriculture and Food Research Organization. 2017. (Online). Retrieved from http://rapdb.dna.affrc.go.jp/
National Institute of Agrobiological Sciences. 2017. (Online). Ibaraki, Japan. Retrieved from http://qtaro.abr.affrc.go.jp/
Oba, M. and M. S. Allen. 1999. Evaluation of the importance of the digestibility of neutral detergent fiber from forage: effects on dry matter intake and milk yield of dairy cows. J Dairy Sci. 82: 589-596.
Ookawa, T., R. Aoba, T. Yamamoto, T. Ueda, T. Takai, S. Fukuoka, T. Ando, S. Adachi, M. Matsuoka, T. Ebitani, Y. Kato, I. W. Mulsanti, M. Kishii, M. Reynolds, F. Piñera, T. Kotake, S. Kawasaki, T. Motobayashi and T. Hirasawa. 2016. Precise estimation of genomic regions controlling lodging resistance using a set of reciprocal chromosome segment substitution lines in rice. Sci. Rep. 6: 12 p.
Porter, B.W., J.M. Chittoor, M. Yano, T. Sasaki, and F.F. White. 2003. Development and mapping of markers linked to the rice bacterial blight resistance gene Xa7. Crop Sci. 43: 1484-1492.
Pradhan, S. K., D. K. Nayak, S. Mohanty, L. Behera, S. R. Barik, E. Pandit, S. Lenka and A. Anandan. 2015. Pyramiding of three bacterial blight resistance genes for broad-spectrum resistance in deepwater rice variety, Jalmagna. Rice. 8: 19 p.
Raffrenato, E. and M. E. Van Amburgh. 2011. Technical note: Improved methodology for analyses of acid detergent fiber and acid detergent lignin. J. Dairy Sci. 94: 3613-3617.
Rajpurohit, D., R. Kumar, M. Kumar, P. Paul, A. Awasthi, P.O. Basha, A. Puri, T. Jhang, K. Singh and H.S. Dhaliwal. 2011. Pyramiding of two bacterial blight resistance and a semidwarfing gene in Type 3 Basmati using marker-assisted selection. Euphytica. 178: 111-126.
Rao, Y. C., Y. L. Yang, D. D. Xin, X. J. Li, K. E. Zhai,B. J. Ma, J. W. Pan, Q. Qian and D. L. Zeng. 2013. Characterization and cloning of a brittle culm mutant (bc88) in rice (Oryza sativa L.). Chin Sci Bull. 58: 3000-3006.
Rasool, F. U., R. Habib and M. I. Bhat. 2012. Evaluation of plant spacing and seedlings per hill on rice (Oryza sativa L.) productivity under temperate conditions. Pak. J. Agri. Sci. 49: 169-172.
Sarker, D., M. R. A. Redoy, N. C. Sarker, M. T. Kamal and M. Al-Mamun. 2016. Effect of used rice straw of mushroom cultivation on growth performance and plasma metabolites in beef cattle. Bang. J. Anim. Sci. 45: 40-45.
Schulze, A. K. S., M. R. Weisbjerg, A. C. Storm and P. N?rgaard. 2014. Forage fiber effects on particle size reduction, ruminal stratification, and selective retention in heifers fed highly digestible grass/clover silages. J. Anim. Sci. 92: 2511-2521.
Sher, A., F. U. Hassan, H. Ali, M. Hussain and A. Sattar. 2016. Enhancing forage quality through appropriate nitrogen dose, seed rate and harvest stage, in sorghum cultivars grown in Pakistan. Grassl Sci. 63: 15-22.
Song, W. Y., G.L. Wang, L.L. Chen, H.S. Kim, L.Y. Pi, T. Holsten, J. Gardner, B. Wang, W.X. Zhai, L.H. Zhu, C. Fauquet and P. Ronald. 1995. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science. 270: 1804-1806.
Sorkheh, K., B. Shiran, T. M. Gradziel, B. K. Epperson, P. Martínez-Gómez and E. Asadi. 2007. Amplified fragment length polymorphism as a tool for molecular characterization of almond germplasm: genetic diversity among cultivated genotypes and related wild species of almond, and its relationships with agronomic traits. Euphytica. 156: 327-344.
Su, Y. J., G. Q. Zhao, Z. W. Wei, C. J. Yan and S. J. Liu. 2012. Mutation of cellulose synthase gene improves the nutritive value of rice straw. Asian-Aust. J. Anim. Sci. 25: 800-805.
Suh, J. P., J. U. Jeung, T. H. Noh, Y. C. Cho, S. H. Park, H. S. Park, M. S. Shin, C. K. Kim and K. K. Jena. 2013. Development of breeding lines with three pyramided resistance genes that confer broad-spectrum bacterial blight resistance and their molecular analysis in rice. Rice. 6: 5-15.
Sundaram, R. M., M. R. Vishnupriya, S. K. Biradar, G. S. Laha, G. A. Reddy, N. S. Rani, N. P. Sarma and R. V. Sonti. 2008. Marker assisted introgression of bacterial blight resistance in Samba Mahsuri, an elite indica rice variety. Euphytica. 160: 411-422.
Tsai, T. H. 2011. Phenotype characterization and genetic analysis of brittle culm mutants of IR64 and TNG67 varieties. National Chung Hsing University master thesis. Taiwan, Taichung city.
Tseng, Y. C. 2012. Pyramiding bacterial blight resistance genes into TK9 rice variety using marker-assisted selection. National Chung Hsing University master thesis. Taiwan, Taichung city.
Van Soest, P. J., J. B. Robertson and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-3597.
Wang, D. F., Y. L. Qin, J. J. Fang, S. J. Yuan, L. X. Peng, J. F. Zhao, X. Y. Li. 2016a. A missense mutation in the zinc finger domain of OsCESA7 deleteriously affects cellulose biosynthesis and plant growth in rice. PLOS ONE. 11: 1-16.
Wang, G. L., C. Wu, L. Zeng, C. He, M. Baraoidan, F. de Assis Goes da Silva, C. E. Williams, P. C. Ronald and H. Leung. 2004. Isolation and characterization of rice mutants compromised in Xa21-mediated resistance to X. oryzae pv. oryzae. Theor Appl Genet. 108: 379-384.
Wang, H., Y. Wu, J. Liu, and Q. Qian. 2006. Morphological fractions, chemical compositions and in vitro gas production of rice straw from wild and brittle culm1 variety harvested at different growth ages. Anim. Feed Sci. Technol. 129: 159-171.
Wang, X. L., Z. J. Cheng, Z. C. Zhao, L. Gan, R. Z. Qin, K. N. Zhou, W. W. Ma, B. C. Zhang, J. L. Wang, H. Q. Zhai and J. M. Wan. 2016b. BRITTLE SHEATH1 encoding OsCYP96B4 is involved in secondary cell wall formation in rice. Plant Cell Rep. 35: 745-755.
Wenzel, C. L., P. M. Chandler, R. B. Cunn Ingham and J. B. Pass Ioura. 1997. Comparative leaf epidermal anatomy of mutants of barley (Hordeum vulgare L. ‘Himalaya’) which differ in leaf length. Annals of Botany 79: 47-52.
Wu, B., B. C. Zhang, Y. Dai, L. Zhang, K. K. Shang-Guan, Y. G. Peng, Y. H. Zhou and Z. Zhu. 2012. Brittle Culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice. Plant Physiol. 159: 1440-1452.
Xu, J. D., Q. F. Zhang, T. Zhang, H. Y. Zhang, P. Z. Xu, X. D. Wang and X. J. Wu. 2008. Phenotypic characterization, genetic analysis and gene-mapping for a brittle mutant in rice. J Integr Plant Biol. 50: 319-328.
Xu, J. Y., J.F. Jiang, X. F. Dong, J. H. Ali and T. M. Mou. 2012. Introgression of bacterial blight (BB) resistance genes Xa7 and Xa21 into popular restorer line and their hybrids by molecular marker-assisted backcross (MABC) selection scheme. Afr. J. Biotechnol. 11: 8225-8233.
Yan, C. J., S. Yan, X. H. Zeng, Z. Q. Zhang, M. H. Gu. 2007. Fine mapping and isolation of Bc7(t), allelic to OsCesA4. J Genet Genomics. 34: 1019-1027.
Ye, Y. F., B. M. Liu, M. Zhao, K. Wu, W. M. Cheng, X. B. Chen, Q. Liu, Z. Liu, X. D. Fu and Y. J. Wu. 2015. CEF1/OsMYB103L is involved in GA‑mediated regulation of secondary wall biosynthesis in rice. Plant Mol Biol. 89: 385-401.
Ying, J. F., S. B Peng, Q. R. He, H. Yang, C. D. Yang, R. M. Visperas and K. G. Cassman. 1998. Comparison of high-yield rice in tropical and subtropical environments i. determinants of grain and dry matter yields. Field Crops Research. 57: 71-84.
Zhan, X. D., H. P. Zhou, R. Y. Chai, J. Y. Zhuang, S. H. Cheng and L. Y. Cao. 2012. Breeding of R8012, a rice restorer line resistant to blast and bacterial blight through marker-assisted selection. Rice Sci. 19: 29-35.
Zhang, B. C., L. W. Deng, Q. Qian, G. Y. Xiong, D. L. Zeng, R. Li, L. B. Guo, J. Y. Li and Y. H. Zhou. 2009. A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice. Plant Mol Biol. 71: 509-524.
Zhang, B. C., X. L. Liu, Q. Qian, L. F. Liu, G. J. Dong, G. Y. Xiong, D. L. Zeng and Y. H. Zhou. 2011. Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice. PNAS. 108: 5110-5115.
Zhang, M., B. C. Zhang, Q. Qian, Y. C. Yu, R. Li, J. W. Zhang, X. L. Liu, D. L. Zeng, J. Y. Li and Y. H. Zhou. 2010. Brittle Culm 12, a dual-targeting kinesin-4 protein, controls cell-cycle progression and wall properties in rice. Plant J. 312-328.
Zhou, Y. H., S. B. Li, Q. Qian, D. L. Zeng, M. Zhang, L. B. Guo, X. L. Liu, B. C. Zhang, L. W. Deng, X. F. Liu, G. Z. Luo, X. J. Wang and J. Y. Li. 2009. BC10, a DUF266-containing and Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.). Plant J. 57: 446-462.
Zhu, H. L., Z. Q. Fang, C. Preston, Y. Y. Li and L. B. Hu. 2014. Transparent paper: fabrications, properties, and device applications. Energy Environ. Sci. 7: 269-287.