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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES ******* PHUNG TON QUYEN STUDY ON RICE BREEDING FOR BROWN PLANTHOPPER RESISTANCE USING MOLECULAR MARKERS Major Field: Genetics and Plant Breeding Code: 62.62.01.11 DOCTORAL THESIS SUMMARY Hanoi - 2014 The Doctoral thesis was completed in: AGRICULTURAL GENETICS INSTITUTE Scientific Supervisors: 1: Assoc. Prof. Vu Duc Quang 2: Dr. Luu Thi Ngoc Huyen Reviewer 1: Reviewer 2: Reviewer 3: The Doctoral thesis was defended at Institute Committee of PhD Dissertation Examination: ………...………………………………………………………………………………… ………………………………………………………………………….....…………….. Date: The Ph.D thesis may be found at: .....................…………………………………….. THE PUBLISHED ARTICLES RELATED TO THE CURRENT THESIS 1 Phung Ton Quyen, Nguyen Thi Lang, Luu Thi Ngoc Huyen, Vu Duc Quang (2010), “Evaluation of some brown planthopper resistance of rice lines/varieties in Red River and Cuulong deltas”, Journal of Vietnam Science and Agricultural Technology, No: 1 (14)/2010, pp. 8-13 (in Vietnamese) 2 Dinh Van Thanh, Lai Tien Dung, Phan Thi Bich Thu, Luu Thi Ngoc Huyen, Phung Ton Quyen (2011), “Results of evaluating research on brown planthopper resistance Nilaparvata lugens (Homoptera: Delphacidea)”, Final Report of Department of Crops Production, Agricultural Publisher, pp. 256-263 (in Vietnamese). 3. Luu Thi Ngoc Huyen, Phung Ton Quyen, Vu Duc Quang (2013), “Application of marker technology to develop inbred rice with brown planthopper resistance”, Journal of Vietnam Science and Agricultural Technology, No:2 (41)/2013, pp. 20-25 (in Vietnamese). INTRODUCTION 1. Imperativeness of the dissertation Rice (Oryza sativa L) is an important plant in Vietnam and is a major food source for half of worldwide population. One of the feasible strategies is to apply biotechnologies to improve rice yield and generate new rice varieties to ensure the food demand of human. Brown Planthopper (Nilaparvata Lugens Stal) is one of serious pests which causes rice yield significant reduction in the rice producing countries, especially in the tropic areas of rice growing in the world. In Vietnam, approximately 10% to 30% out of total rice production are annually loss due to planthopper. The aim of applying marker assisted selection (MAS) is to use the markers which are linked with the desire QTL/gen to enhance selecting efficiency and shorten the selection time. To develop the rice varieties which are resistant with brown planthopper with MAS, mainly depends on the markers have linkage with the brown planthopper resistant gen is possible work. Hence, we have concentrated on the topic entitled “Study on rice breeding for brown planthopper resistance using molecular markers ” Objectives of the dissertation Applying marker assisted selection method to develop inbred-elite rice varieties which shown a stability of brown planthopper resistance in the Red River Delta areas. 2. Time and areas implementation - From 2007 to 2013 - Agricultural Genetics Institute, Institution of Plant Protection, Center of Technologies Exchange and Extension, Thanh Tri- Ha Noi. - Field works was mainly conducted in Nam Dinh, Thai Binh, Ha Nam, Hanoi, provinces. 3. Inovative and its contributions 1 - This is one of initial researches on improving rice planthopper resistance by applying molecular breeding in Vietnam, it is also one of part involving to Agricultural Biotechnology Project of MARD - Within of the current dissertation, it has been successfully pyramided two genes (planthopper resistant genes) into one rice variety by use of SSR markers which linked to planthopper resistant genes. 4. Significant contributions -Scientific significance: Application molecular markes to generate two rice lines carrying two planthopper resistance genes - Practical significance: The improved line KR8 is a promising line which is possibly released for producing in a large scale. 6. Structure of Dissertation The dissertation was presented in 155 pages which included: Introduction 3 pages, Chapter 1: Background and Scientific Basic (51 pages); Chapter 2: Materials and Methods (23 pages); Chapter 3: Results and Discussion (57 pages), included 01 page presented for conclusions and suggestion, and 01 page listed the published article involving the current dissertation result, and reference list (19 pages). Also, the dissertation included 37 Figures, 37 Tables and 167 liturature cited, in which there are 46 Vietnamese research lituratures and 121 English research articles were used, 03 published articles from the current dissertation and 2 appendixes. Chapter 1 OVERVIEWS 1.1. Brown planthopper and its resistant characterization in rice 1.1.1. Bio-charateristics of BPH The Brown planthopper (BPH) is among the most important pests of rice which has a scientific name as Nilaparvata lugens Stal. 1.1.2. The status of rice losses have caused by BPH in recent years in Vietnam 2 BPH infestation has occurred in recent years, especially in 2000, over 540 thousand hectares of rice were infected by BPH, of which 256 thousand hectares were seriously losses. 1.1.3. Characteristics of BPH resistance in rice: 1.1.3.1. Mechanism of pest resistance The pests and insects were become harmful pests when agricultureintensive farming was initially conducted by human. 1.1.3.2. Genetics of BPH rice resistance In the great efforts of research on BPH resistance in rice, some major BPH resistant genes in rice have been identified and reported as the listed in the Table 1.1 Table 1.1. BPH resistant genes and the rice varieties (indicator) carrying the resistant genes Indicator lines TN1 Mudgo ASD7 Rathu Heenati Babawee ARC10550 Swarnalata T12 Chinsaba Pokkali O. australiensis O. officinalis O. officinalis O. officinalis O. officinalis, O. australiensis,. Rathu Heenati O. australiensis O. rufipogon Griff AS20-1 O. minuta O. minuta Resistant gene None Bph1 bph2 Bph3 bph4 bph5 Bph6 bph7 bph8 Bph9 Bph10 bph11(t) bph12(t) Bph6, Bph13 Bph14 và Bph15 Bph16 Bph17 Bph18 bph18(t) bph19 Bph20, Bph21 Bph20(t) và Bph21(t) 3 D/R D R D R R D R R D D R R D - O. rufipogon Griff O. eichingeri, O. latifolia O. officinalis Oryza rufipogon Griff Oryza sativa L Tổ hợp lai TN1xGC9, IS (IR72 x GC9) bph22(t) và bph23(t) Bph25 Bph25(t) Bph 26 Bph26(t) Bph27 Bph27 (t) BphZ Bph3+BphZ. D - Note: D: Dominance; R: Recessive; (-) Unknown 1.2. Some biotechnology applications in genetic research and rice breeding - Marker assisted selection (MAS) MAS for single trait: The QTL/gen is transferred into elite variety by applying Marker assisted backcrossing (MABC) - Pyramiding 1-2 BPH resistant genes into some elite rice varieties F1 F2 BC1F1 BC2F1 F3 BC3F1 Fn BC4F2BC4F4 BC4F1 Figure 1. Diagram of applying molecular marker to select the rice lines with BHP resistance (Huyen et al., 2011) 4 1.3. Genetic researches and selecting BPH resistant rice varieties in Vietnam 1.3.1. Identifying the molecular markers linked with the genes and mapping the resistant genes in Vietnam Some molecular markers have linkage with the BPH resistant genes have been identified. Constructing a linkage map for BPH resistant gene (BphZ(t)) on the chromosome 4, pyramiding the resistant genes Bph3+BphZ(t) and have developed the potential BHP resistant lines namely IS1.2, IS2.3, RS. BphZ(t) gene is being further fine mapped on the chromosome 4. 1.3.2. Improving the BHP resistant rice variety in Vietnam In recent years, the CR203 and CR84-1 were known as medium BPH resistant varieties, however, they were also affected when BPH infestation severely occurred. Chapter 2 MATERIALS AND METHODS 2.1. Materials - BPH resistant rice varieties/lines were used as the donor plants: Two BPH resistant genes Bph3 and BphZ(t) have been pyramided in these lines. - The recipient plant: IR64 (Tam Dien Bien) - SSR markers linked with Bph3 and BphZ(t) . - The BPH resistant rice variety: Ptb33; - The sensitive BPH: TN1 - The controlled variety: KD18 The origin of plant materials: Rice variety/line carrying BPH resistant gene (donor plant) The lines IS1.2, IS2.3 and RS were already pyramided 2 BPH resistant genes Bph3 and BphZ(t), respectively. Based on the Bph3 mapping on the chromosome 6 and BphZ(t) on the chromosome 4, the molecular markers (SSR) which have linked with the genes were selected rầy nâu. 5 NST số 4 ë lóa Bản đồ LK gen BphZ(t) Figure 2.1. Bph3 mapping in detail Figure 2.2. BphZ(t) mapping in detail (Huyen et al, 2003; 2010) The recipient plant: IR64 variety - BPH pests were collected in the Red River Delta and Thap Muoi areas were used in this study. - Chemical agents and other materials were purchased from GenSet, Pharmacia, Sigma, Boehringer, Promega, BioRad, ... 2.2. Methods 2.2.1. Evaluation on BPH resistance and BPH sensitive of the rice varieties/lines Based on the rank standard of IRRI (5 ranks) and Institute of Plant Protection (10 rank) 2.2.2. Backcrossing, pyramiding BPH resistant genes from the donors to the elite rice variety The parental plants included the donor (carrying BPH resistant genes) and the donor without BPH resistant gene, but has some economic traits. 2.2.3. Information collection of molecular markers. 6 Most of molecular markers used in this study were collected from the web http://www.gramen.org/cho and some other publications 2.2.4. Method to improve rice BPH resistance by molecular markers techniques - Evaluating and selecting the F2 to F6 generations on the field test - Using SSR markers to identify BPH resistant gene - Testing and confirming the BPH resistant lines which carried the BPH resistant genes in the net house and the field test. 2.2.5. Other laboratory techniques - DNA extraction, and purification following the CTAB method - PCR techniques: 2.2.6. Field test The experiments on the field were conducted by Thanh (1986). 2.3. Statistical and data analysis IRRISTART 5.0, Excel 2007 were used in this study. Graphical Genotyper 2 (GGT2.0) and methods of statistical analysis another. Chapter 3 RESULTS AND DISCUSSION 3.1. Evaluating the rice germplasm and the BPH resistant lines/varieties .1.1. Results of some rice lines/varieties in 2008 Table 3.1. The effects of some rice varieties on the BPH in the Red River Delta and Cuu Long Delta 1 CR203 Resistant gene 1 2 CR84-1 1 NN NN 3 Khang dan 18 None N NN 4 Q5 None N NN 5 SL12 None NN NN No Name 7 BPH test on RRD BPH test on CLD NV NN 6 SL15 None N NN 7 IR64 None NV N The rice lines carrying BPH resistant genes 8 IS1.2 2 KC KC 9 IS2.3 2 KC KC 10 RS 2 KC KC 11 IS4.8 2 KC KC 12 RS3 2 KC KC 13 RS4 2 KC KC Controlled varieties 14 TN1 Sensitive NN NN 15 Ptb33 Resistant KC KC Note: High resistance (KC), lightly infected (NV); infected (N), severe infection (NN). All lines IS and RS carrying BPH resistant genes shown high resistant rank on the BPH pests in RRD and CLD 3.1.2. Evaluating the effect of BPH pests on the rice lines/varieties in the net houses at Long An and Hanoi in 2011 - Severe infestation of BPH in the northern province showed higher than some previous years occurred - Among total 144 rice lines/varieties, 45 varieties showed BPH resistance in the North, equivalent with 29.58%. There were 9 rice varieties revealed BHP resistance at Moc Hoa-Long An province (4.2%), respectively. 3.1.3. Developing the crossed combinations to select the BPH resistant lines Scheme to develop KR8 by applying molecular markers 8 F1 F2 BC1F2 BC3F5 BC3F6 BC3F7 3.2. Screening the BPH resistant genes by use of molecular markers 3.2.1. DNA extraction and purification Figure 3.1. DNA conconcentration and DNA quality of KR8 variety, eletrophothesis on agarose gel 0,8% The results showed that DNAs were clean and good quality, the concentration reached about 200 – 300ng/l which were enough requirements for further research. 3.2.2. Checking the PCR products on the agarose gel 1% 9 Figure 3.2. Electrophoresis PCR products on the agarose gel 1% with the 1kb ladder. PCR products showed one lane only, they were high resolution. It implied that PCR performance were well done. 3.2.3. Screening molecular markers linked with BPH resistant genes 3.2.3.1. Parental polymorphism with the Bph3 gene Figure 3.3. Screening polymophic markers between the parental plants (the likage of RM588 with Bph3 gene) Figure 3.4. Screening polymorphic markers between the parental plants (Linkage of RM190 with Bph3 gene) 3.2.3.2. Screening the molecular markers linked with the BphZ(t) gen Figure 3.5. Screening polymorphic markers between parental plants 10 (RM5757 marker linked with BphZ gene) Figure 3.6. Screeing the polymophic markers between the parental plants (RM 3367 has linkage with BphZ(t) gene) Figure 3.7. Screeing the polymorphic markers between the parental plants (RM 3288 marker showed linkage with BphZ(t)) Figure 3.8. Screeing the polymorphic markers between the parental plants (RM6997 marker showed linkage with BphZ(t)) Figure 3.9. Screening polymorphic markers between the parental plants (RM 3735 linked with BphZ(t)) Figure 3.10. Screeing polymorphic markers between the parental plants 11 (RM5714 has a linkage with BphZ(t)) Figure 3.11. Screening polymorphic markers between the parental plants (RM5757 linked with BphZ(t)) RM586; RM588; RM589; and RM190 markers showed linkage with Bph3 gene. RM5714; RM3288; RM3367; RM3735; RM5757; and RM6997 exhibited a linkage with BphZ(t) gene, respectively. 3.2.4. Screening the individual plants of BC generations carrying BPH resistant gene: 3.2.4.1. Screening BC lines carrying Bph3-BPH resistant gene Figure 3.12. RM588 marker showed a linkage with Bph3, and selected the individual plants of BC1F1 carrying BPH resistant gene Figure 3.13. RM588 markes linked with Bph3 to screen individual plants carrying the BPH resistant gene in the F2 generation 12 Figure 3.14. RM190 marker linked with Bph3 to screen the individual plant carrying BPH resistant gene from the BC lines Figure 3.15. RM588 markes linked with Bph3 to screen individual plants carrying the BPH resistant gene in the BC3F3 generation from the IR64/IS1.2 3.2.4.2. Analysis and identification of the present of BPH resistant BphZ(t) gene in the BC lines Figure 3.16. Use of SSR marker (RM5757) linked with BphZ(t) gene to identify the individual plants carrying BPH resistant gene in BC1F1 Figure 3.17. Use of RM3367 linked with BphZ(t) gene to identify the individual plants carrying BPH resistant gene in the BC line 13 Figure 3.18. Use of RM3735 linked with BphZ(t) gene to identify the individual plants carrying BPH resistant gene in the BC line 3.2.5. Analysis and identifying the individual plants carrying BPH resistant gene in the specific populations 3.2.5.1. Identifying the individual plant carrying BPH resistant Bph3 gene Figure 3.19. Use of RM190 marker linked with Bph3 to select the individual plants carrying BPH resistant gen of KR8 from the IR64/IS1.2 Figure 3.20. Use of RM588 marker linked with Bph3 from combination of IR64/IS to select the individual plants of 64R8-2 (KR8-2) which carrying BPH resistant gene 14 Figure 3.21. Use of RM190 marker linked with Bph3 gene to select the individual plant of DTR64 line carrying BPH resistant gene 3.2.5.2. Analysis and identifying the individual plants carrying BPH resistant BphZ gene Figure 3.22. Use of RM3735 linked with BphZ(t) to select the individual plants of KR8 carrying BPH resistant gene Figure 3.23. Use of RM3367 linked with BphZ(t) gene to select the individual plants of KR8 carrying BPH resistant gene 3.3. Screening the potential BPH resistant lines in the field 3.3.1. The potential rice lines Total 27 potential rice lines (BPH resistant lines) were selected for the Red River Delta in 2010 as shown in the Table 3.2. Table 3.2. The component yield traits of the potential rice lines in the Summer crops of 2010 (Density 50 plants /m2) Spikelet No Variety/line /plant 1 2 3 4 KD18 đ/c DTR64 64R2-1-1 64R2-1 7,5 6,7 6,2 5,3 Grain /spikelet 119,0 139,6 167,2 188,0 15 Real Filled P1000 yield grain (g) (1000kg/ (%) ha) 86,8 92,9 84,1 84,7 19,2 22,8 24,8 23,8 6,09 6,00 6,18 6,17 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 64R2-2 64R4-3 64R1 64R8-1 64R8-2 64R1-3-5 64R1-3-6 64R1-4-2 64R1-4 -3 64R1-4-4 64R1-4-5 64R1-7- 1 64R1-7-2 64R1-7-3 64R1-7-4 64R1-4-1 64R1-4-3 64R4-4-1 64R4-4-3 64R4- 4-5 64R4-1-1 64R4-2-1 64R4-16-2 64R3-4-16 IR64 IS1.2 IS2.3 CV% 7,3 5,3 5,3 6,2 6,2 7,3 5,4 6,0 5,5 7,3 6,7 6,0 5,6 6,7 5,3 5,3 101,3 188,0 188,0 158,2 158,2 101,3 145,5 165,0 150,2 101,3 190,6 165,8 182,4 175,2 188,0 164,6 93,4 84,7 83,9 82,8 81,9 93,4 85,6 91,0 81,9 93,4 82,8 91,0 84,6 70,4 84,7 92,1 24,3 23,8 23,8 23,2 23,2 24,3 27,2 24,2 23,6 24,3 24,1 24,2 24,9 24,1 23,8 24,1 6,10 6,24 6,11 6,10 6,12 6,12 6,10 6,13 6,05 6,05 6,06 6,11 6,10 6,11 6,09 6,10 5,6 194,2 90,5 23,6 6,08 5,6 174,3 82,8 23,8 6,20 5,6 182,4 84,6 24,9 6,20 4,5 185,3 85,1 22,6 6,20 5,2 4,0 6,1 5,5 5,7 6,6 6,8 184,2 165,8 1756 187,3 102,5 121,3 120,3 83,6 84,1 75,4 87,1 96,4 73,2 72,2 22,6 24,2 24,1 22,6 22,3 19,5 18,5 6,14 6,10 6,17 6,06 6,10 5,61 5,60 5,5 2,1 1,5 0,9 8.90 LSD0,05 0,53 5.42 2.06 0.04 0.03 As the field performent of the potential rice lines were conducted in several the Northern province, there were 3 elite lines were selected as the: DTR64, KR81(KR8) and KR8-2. 3.3.2. Screening the agronomic traits of the 3 elite rice lines 3.3.2.1. Screening the agronomic traits of the 3 elite rice lines in the net house 16 Table 3.3. Evaluation of the BPH resistant/sensitive rice lines in 2011 Rank of BPH resistant pests No Variety/line Hanoi Nam Định Nghe An Đong thap Can tho 1 KR8-1 3 1 3 3 3 1 KR8-2 3 3 3 3 3 2 DTR64 3 3 3 3 3 3 IS1.2 3 1 1 3 3 4 IS2.3 3 1 1 3 3 5 IR64 7 7 7 9 7 6 Ptb 33 1 1 3 3 3 7 TN1 9 9 9 9 9 Table 3.4. Evaluation of the BPH resistant/sensitive rice lines in 2012 Rank of BPH resistant pests No Variety/line Ha noi Nam Nghe Dong Dinh An Thap Can Tho 1 KR8-1 3 1 3 3 3 2 KR8-2 3 3 3 3 3 3 IS1.2 3 1 1 3 3 4 IS2.3 3 1 1 3 3 5 IR 64 7 7 7 7 7 6 Ptb 33 1 1 3 3 3 7 TN1 9 9 9 9 9 During the field trials in the years of 2011 and 2012: 3 rice lines namely KR8, KR8-2 and DTR64 showed the high BPH resistance from rank 1 to 3, while IR64 showed severly BPH influenced as the rank from 7 to 9. 17
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