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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY SCREENING OF BPH RESISTANCE GENE ON SOME RICE VARIETIES IN MEKONG DELTA BASED ON MOLECULAR MARKER SUPERVISOR STUDENT Dr.TRAN NHAN DUNG NGUYEN NGOC QUYNH ANH Student code: 3064434 Session: 32 (2006-2010) Can Tho, 2010 APPROVAL SUPERVISOR STUDENT Dr. TRAN NHAN DUNG NGUYEN NG.QUYNH ANH Can Tho, November 25, 2010 PRESIDENT OF EXAMINATION COMMITTEE i ABSTRACT Among the damage insects of rice, the brown planthopper (Nilaparvata lugens Stal.) is a major threat to rice production and causes significant yield loss annually, especially in Asian countries. Host-plant resistance is an important breeding strategy to reduce the damage caused by brown planthopper (BPH) and increase rice productivity. In this study, thirty rice cultivars obtained from Mekong Delta Development Research Institute were detected BPH resistance gene by SSR (Simple sequence repeats) marker RM13, RM279, RM190 and STS (Sequence-tagged site) marker 7312.T4A. Based on the analysis of PCR products on agarose gel, two SSR markers RM13 and RM270 showed unique bands on agarose gel for all rice cultivars tested as well as didn’t link tightly to BPH resistance gene. In contrast, the SSR marker RM190 and STS marker 7312.T4A revealed the tightly linkage to BPH resistance gene Bph3 and Bph18, respectively. Among thirty rice cultivars tested with RM190, there were sixteen rice cultivars showed the BPH resistant ability. By marker 7312.T4A, the analysis of HinfI -digested PCR products indicated that twelve rice cultivars possessed Bph18 gene. Key words: Brown planthopper (BPH), biotype, BPH resistance gene, SSR marker, STS marker. ii CONTENTS Abstract i Content ii 1. Introduction 1 2. Materials and methods 3 2.1 Materials 3 2.1.1 Genetic materials 3 2.1.2. Equipments 4 2.1.3. Chemicals 4 2.2 Methods 4 3. Results and discussion 8 3.1. Result of determining the DNA concentration and DNA purification of rice cultivars 8 3.2 PCR products 8 3.2.1. Study of the effect of some parameters into the formation of PCR products of primer RM190 8 3.2.2. Study of the effect of some parameters into formation PCR products of primer RM13 and RM270 11 3.2.3. Study of the effect of some parameters into formation PCR products of primer 7312.T4A 14 3.3. Study of the polymorphism levels between rice cultivars by digestion of DNA STS products with restriction enzyme (HinfI) 18 4. Conclusions 20 5. Suggestions 21 11. INTRODUCTION Brown plant hopper (Nilaparvata lugens Stal.) (BPH) is the most dangerous insect pest on rice production because it not only sucks the sap and burns the plant but also acts as a vector to transfer dangerous virus diseases such as ragged stunt and grassy stunt virus. In most of Asian countries, BPH is one of the main factors that causes serious yield reduction. Recently, in the Mekong delta of Viet Nam, BPH caused dwarf-yellowing and ragged stunt disease on 70,000 ha of rice in Thu Dong and Monsoon seasons (http://www.voh.com.vn). BPH has ability that can adapt with many kinds of rice cultivars by changing its biotype. Therefore, the management of BPH on rice field is very difficult. Scientists have tried to find effective method for controlling BPH. Recently, the application of BPH resistance rice cultivars into production has been considered as one of the best solutions. Up to now, there are 21 genes for BPH resistance and 4 BPH biotypes that have been investigated from cultivated and wild rice cultivars (Alam and Cohen (1998), Su et al. (2002), Soundararajan et al. (2004), Zhang (2007), Rahman et al. (2009). Therefore, it is very necessary to find specific markers that link tightly to BPH resistance gene as well as supplement new BPH resistance rice varieties for rice production more effectively. In this research, thirty rice cultivars originated from Mekong Delta Development Research Institute will be investigated BPH resistance gene by SSR and STS marker. 2Objectives: * To detect brown plant hopper resistance genes on some rice cultivars of Mekong Delta by STS marker (7312.T4A) and SSR markers (RM13, RM270 and RM190). * To find suitable PCR amplification formulas for detecting BPH resistance genes of primers used in experiment. 32. MATERIALS AND METHODS 2.1 Materials 2.1.1 Genetic materials: 30 rice cultivars were collected from Mekong Delta Development Research Institute. HD1 and TN1 variety were used as resistant control and susceptible control, respectively. Table 3.1.List of rice cultivars No Rice Cultivars No Rice cultivars 1 MTL586 18 MNR2 2 MTL601 19 MNR3 3 MTL603 20 MNR4 4 MTL617 21 MNR5 5 MTL620 22 OM4488 6 MTL621 23 OM5740 7 MTL638 24 OM5756 8 MTL640 25 OM6018 9 MTL641 26 OM6379 10 MTL642 27 OM6599 11 MTL643 28 NANG HOA 9 12 MTL650 29 ĐMT126 13 MTL652 30 ĐMT129 14 MTL660 31 PTB33 (A0 control) 15 MTL662 32 HD1( resistant control) 416 MTL663 33 TN1 (susceptible control) 17 MNR1 34 OMCS2000 (A1 control) 2.1.2. Equipment: microwave, PCR BIORAD C2000 device, centrifuge, OD Beckman Coulter, vortex machine, electric balance, grinding machine, micropipette (USA), tubes (Germany), . 2.1.3. Chemicals * DNA extraction: Nitrogen liquid, extraction buffer, SDS10%, Isopropanol, TE, CTAB, Chloroform, Isoamylalcohol, Ethanol 70% and 96% (Merck)… * Electrophoresis: TE 1X, Ethidium Bromide (Bio-Rad), loading buffer, agarose (Fermentas). * PCR amplification and enzyme digestion: Taq polymerase (BiRDI), BiH2O, primer RM13, RM270, RM190 and 7312.T4A (Invitrogen), MgCl2 (Merck), dNTPs (Promega), Buffer (Fermentas), BSA 1% (Fermentas), RC buffer, HinfI (Invitrogen) 2.2 Methods * Collecting young leaves of rice cultivars and extracting DNA as CTAB method of Rogers and Bendich (1988). DNA extraction of each of rice cultivar was repeated 2 times. * Measuring DNA concentration and DNA purification by OD Beckman Coulter device. * Amplifying DNA of rice cultivars by primers including: RM13, RM270, RM190 and 7312.T4A, respectively. 5Table 3.2. List of primers used Primer Primer’s sequences Chromosome Repeat motif Author RM190 For. 5’ CTT TGT CTA TCT CAA GAC AC 3’ Rev. 5’ TTG CAG ATG TTC TTC CTG ATG 3’ 6 (bph4) (TC)36 (Kawaguchi et al., 2001) RM13 For. 5’ TCC AAC ATG GCA AGAGAG AG 3’ Rev. 5’ GGT GGC ATT CGA TTC CAG 3’ 5 (Bui Chi Buu et al., 2005) RM270 For. 5’ GGC CGT TGG TTC TAA AAT C 3’ Rev. 5’ TGC GCA GTA TCA TCG GCG AG 3’ 12 (Bui Chi Buu et al., 2005) 7312.T4A For. 5’ ACG GCG GTG AGC ATT GG 3’ Rev. 5’ TAC AGC GAA AAG CAT AAA GAG TC 3’ 12 (Bph18(t)) (Jena K. K. et al ., 2005) * Preparing PCR amplification primer RM190, RM13, RM270 by the formula (Table 3.3 and Table 3.4) and 7312.T4A formula (Table 3.3 and Table 3.5) below. Then, PCR reaction parameters were adjusted in order to obtain desirable product on agarose gel. * PCR products were tested on agarose gel with different concentrations, namely 3% agarose gel (PCR products of primer RM13, RM270 and RM190) and 2% agarose gel (PCR products of primer 7312.T4). 6 Table 3.3. PCR components Chemicals Stock (µl) Volume (µl) BiH20 11 Taq Buffer with KCl 1X 2.5 MgCl2 25mM 3 dNTP 200µM 2 Forward 100ρmol/µl 1 Primer Reverse 100ρmol/µl 1 BSA 0.1X 0.25 Taq polymerase 5U/µl 0.25 DNA 50-200ng/µl 2 Total 25 Table 3.4. PCR thermal test cycle of primer RM13, RM270 and RM190 Temperature Time Cycle 95oC 2 min 95oC 45 min 54oC 45 min 72oC 1 min 35 72oC 10 min 10oC ∞ Table 3.5. PCR thermal test cycle of primer 7312.T4A Temperature Time Cycle 95oC 2 min 95oC 30 s 58oC 30 s 72oC 1 min 30 s 35 72oC 10 min 10oC ∞ [...]... Thị Lang 2005 Nghiên cứu và ứng dụng marker phân tử để phát hiện gen kháng rầy nâu trên cây lúa (Oryza sativa L.) Hội nghị khoa học toàn quốc về Công nghệ sinh học năm 2005, trang: 165-169 In English Alam S.N and Cohen M.B 1998 Detection and analysis of QTLs for resistance to the brown planthopper, Nilaparvata lugens, in a doubled haploid rice population Theor Appl Genet 97: 1370-1379 Haiyuan yang,... mapping and genetic analysis of a rice brown planthopper (Nilaparata lugens Stal) resistance gene Hereditas 136: 39–43 Jena K K et al 2005 High-resolution of a new brown planthopper (BPH) resistance gene, Bph18(t), and marker-assisted selection for BPH resistance in rice (Oryza sativa L) Theor Appl Genet 112: 288–297 Jirapong Jairin et al 2006 Mapping of a broad-spectrum brown planthopper resistance gene,... was about 600C, we did not keep on controlling Tm anymore The estimation of the cycle and elongation time of PCR was proceeded If the cycle of PCR was repeated too much, it also created sub bands Besides, too long elongation time also gave the same result * Adjusting repeated cycle from 35 to 30 and altering elongation time into 1minute and 25 seconds Besides, the amount of DNA template was also decreased... are presented: * Keeping on the use of 7312.T4A marker for researching BPH resistance gene on other rice cultivars of Mekong Delta Analyzing PCR products through digestion with enzyme HinfI in order to investigate the linkage between marker and BPH resistance gene * Using marker RM190 for detecting BPH resistance gene on other rice cultivars * Transferring BPH resistance rice varieties into field trail... showed the polymorphism between rice cultivars very clearly HD1 cultivar carried bph4 resistance gene and was amplified a band in the size of 130bp on gel In contrast, TN1and Ptp33 cultivar didn’t carry this gene and were amplified bands in the 10 size of 120bp on gel Other rice cultivars carried bph4 gene including: lane 6, 7, 15, 17 and 18 BPH susceptible rice varities included lane 1, 2, 3, 4, 5,... (1976) were concluded that TN1 did not carry any BPH resistance gene as well as infected with all BPH biotypes Therefore, we could judge that these varieties carried Bph18 gene which was heterozygous at this locus Besides, in research of Jena K.K (2005), the digestion product of purebred variety IR65482-7-216-1-2 (the donor of BPH resistance gene, Bph18) also displayed two main bands in the size about... 2214-2220 Su C.C et al 2002 Detection and analysis QTL for resistance to brown planthopper, Nilaparvata lugens (Stal), in rice (Oryza sativa L.) using backcross inbred lines Acta Genet Sin 29: 332-338 Trinh Thi Luy, Pham Thi Thu Ha, Nguyen Thi Lang, Bui Chi Buu 2008 Introgression of a resistance gene to brown plant hopper from Oryza rufipopon to cultilars OmonRice 6 Zhang Q 2007 Strategies for developing... showed the polymorphism result very clearly on 3% agarose gel Among thirty rice cultivars tested, there were sixteen varieties carried Bph3 gene * In this research, we also found suitable thermal cycle for marker 7312.T4 ( 600C for annealing time, 1min 25seconds for elongation time and 30 cycles for PCR reaction) as well as adjusted PCR parameters successfully in order to obtain optimum amplification result... rice cultivars by enzyme HinfI, we gained twelve varieties carried Bph18 gene Furthermore, the digestion PCR product of MTL620, MNR3, OM6599, Nang Hoa 9, OMCS2000 and MTL640 variety presented three main band with the size of 398bp, 566bp and 20 800bp, respectively This indicated that these varieties could carry BPH resistance gene which was heterozygous at this locus 5 SUGGESTIONS From these results,... Qiao Y., Ham T.H., Woo M.K., Lee J., Khanam M.S., Chin J.H., Jeung J.U., Brar D.S., Jena K.K., Koh H.J 2009 High-resolution mapping of two brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta.Theor Appl Genet 119: 1237-1246 Rogers S.O., Bendich A.J (1988) Extraction of DNA from plant tissues Plant Molecular Biology 6: l-10 Sogawa, K., and Pathak M.D 1976 Mechanism . cycle and elongation time of PCR was proceeded. If the cycle of PCR was repeated too much, it also created sub bands. Besides, too long elongation time. that twelve rice cultivars possessed Bph18 gene. Key words: Brown planthopper (BPH), biotype, BPH resistance gene, SSR marker, STS marker. ii CONTENTS