Sử dụng các dấu phân tử thanh lọc các giống lúa mang gen kháng rầy nâu vùng Đồng bằng sông Cửu Long

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 1 1. 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 ( 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. 2 Objectives: * 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. 3 2. 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) 4 16 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. 5 Table 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 ∞ 7 * Digesting PCR products of primer 7312.T4A by restriction enzyme HinfI to find latent polymorphism levels between rice cultivars. The reaction was incubated at 37 0C in 18 hours. Finally, the DNA fragments produced by restriction digestion were resolved electrophoretically in a 3% TE agarose gel. Table 3.7. Chemicals of digestion reaction Chemicals Formula Bi H2O 8 µl RC buffer 2 µl HinfI 2 µl DNA STS 8 µl 8 3. RESULTS AND DISCUSSION 3.1 Determination of DNA concentration and DNA purification of rice cultivars After determining the DNA concentration and purification by measuring the absorption spectra, the results indicated that all DNA samples obtained in the range from 1.8- 2.0.Therefore, these samples were suitable for performing PCR amplification with the primers used in experiment. 3.2 PCR products 3.2.1. Study of the effect of some parameters into formation PCR products of primer RM190 Figure 4.1. Results of PCR amplification with marker RM190 on 3% agarose gel M: ladder 100bp, lane 1: OM4488, 2: TN1, 3: HD1, 4: OM5740 and 5: Ptb33. Performing PCR amplification as formula of Table 3.3 and Table 3.4 above, the amplified fragments visualized on agarose gel electrophoresis were pale. PCR reaction was very 130bp 120bp 600bp 1 2 3 M 4 5 9 sensitive and easily to be affected on many reasons such as: concentration of MgCl2, Taq polymerase, Tm, amount of cycles….We must adjust parameters of PCR to get a completely intact and bright band on gel. * Increasing the amount of Taq polymerase up to 0.5 µl and decreasing the amount of MgCl2 to 2.5 µl. When enzyme concentration was too low, it was not suitable enough to elongate reaction as well as created non-specific products or pale band. Moreover, if MgCl2 concentration was too high, the activity of enzyme could be limited and led to the formation of undesirable products. For this reason, we proceeded to adjust these parameters. Figure 4.2. PCR products of RM190 marker with adjusted formula M: ladder 100bp, lane 1: OM4488, 2: HD1, 3: TN1, 4: OM5740, 5: Ptb33. After changing the amount of Taq and MgCl2 , the bands were brighter and clearer. The result indicated that this formula was appropriated for amplification of primer RM190. Therefore, 120bp 130bp 1 2 3 4 5 M 10 this adjusted formula was chosen for PCR amplification of the rest samples. Figure 4.3. PCR products of primer RM190 with adjusted formula on 3% agarose gel M: Ladder 100bp, lane 1: MNR1, 2: MNR2, 3: MNR3, 4: MNR4, 5: MNR5, 6: 0M4488, 7: OM5740, 8: OM5756, 9 and 19: HD1, 10 and 20 : TN1, 11and 22 : negative control (H2O), 12: MTL601, 13: MTL603, 14: MTL617, 15: MTL638, 16: MTL640, 17: MTL643, 18: MTL663, 19: HD1 and 21: Ptb33. According to the results of Figure 4.3, primer RM190 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 120bp 130bp 600bp M 1 2 3 4 5 6 7 8 9 10 11 M 12 13 14 15 16 17 18 19 20 21 22 600bp 120 bp 130 bp 11 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, 12, 13, 14 and 16. Kawaguchi et al. (2001) defined that RM190 linked tightly with bph4 resistance gene on chromosome 6. Besides, according to Jaripong Jairin (2006), two genes Bph3 and bph4 also linked tightly together on chromosome 6. 3.2.2. Study of the effect of some parameters into formation PCR products of primer RM13 and RM270 When performing PCR amplification with formula of Table 3.3 and Table 3.4 above, we realized that rice cultivars were amplified bands in the size of 140bp and 120bp by RM13 and RM270, respectively. However, the bands were rather pale. Consequently, we carried out the adjustment the amount of Taq and MgCl2 as the same as control of RM190 above (Taq polymerase 0.5 µl and MgCl2 2.5 µl) in order to get desirable products. (A) (B) Figure 4.4. PCR products of primer RM13 (A) and RM270 (B) 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 140bp 120bp 12 Gel A : M: Ladder 100bp, lane 1: MNR2, 2: MNR3, 3: MNR4, 4: MNR5, 5: OM4488, 6: negative control (H2O), 7: OM5740. Gel B: M: Ladder 100bp, lane 1: MNR2, 2: MNR3, 3: MNR4, 4: MNR5, 5: negative control (H2O), 6: OM4488 and 7: OM5740. After altering the amount of Taq and MgCl2, PCR products of RM13 and RM270 were not pale anymore (Figure 4.5). Nevertheless, PCR products amplified by primer RM270 still appeared some sub bands. Therefore, we proceeded to survey annealing temperature so as to decrease these sub bands. (A) (B) Figure 4.5. PCR products of primer RM13 (A) and RM270 (B) with test formula Gel A : M: Ladder 100bp, lane 1: MNR2, 2: MNR3, 3: MNR4, 4: MNR5, 5: OM4488, 6: OM5740, 7: negative control (H2O). Gel B: M: Ladder 100bp, lane 1: MNR2, 2: MNR3, 3: MNR4, 4: MNR5, 5: OM4488, 6: OM5740 and 7: OMCS2000. * Temperature gradient chosen for primer RM270: 56 0C, 570C, 580C, 590C, 600C. 1 2 3 4 5 6 M 7 M 1 2 3 4 5 6 140bp 120bp 13 Figure 4.6. Result of Temperature gradient of RM270 Lane 1 and 6: MNR2 (560C), 2: MNR2 (570C), 3: MNR2 (580C), 4: MNR2 (590C) and 5: MNR2 (600C). Among temperature investigated above, the treatment with 600C for annealing period had the brightest band as well as created product with less primer dimmer. Therefore, this temperature was chosen for PCR amplification of rest samples. Figure 4.7. PCR products of primer RM13 and RM270 with adjusted formula M: ladder 100bp, lane 1: MTL586, 2: MTL601, 3: MTL603, 4: MTL617, 5: HD1, 6: TN1, 7: Ptb33, 8: negative control (H2O), 9: MTL586, 10: MTL601, 11: MTL603, 12: HD1, 13: TN1 and 14: Ptb33. 1 2 3 4 5 6 120bp 140bp RM13 RM270 500bp M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 14 According to Trinh Thi Luy (2008), two primers RM13 and RM270 showed the highly polymorphism between wild rice cultivars. However, the results above (Figure 4.7) indicated that RM13 and RM270 showed unique amplified products of rice cultivars tested and not link tightly to BPH resistance gene. 3.2.3. Study the effect of parameters into formation PCR products of primer 7312.T4A DNA of rice cultivars was amplified by primer 7312.T4A as formulas of Table 3.3 and 3.5 (Figure 4.8). Although all rice cultivars appeared main band in position approximately 100bp on agarose gel, the band was still pale and appeared some sub bands. In order to get accurate analysis for next steps of experiment, the optimization of PCR conditions was carried out. M: ladder 100bp, lane 1: MNR1, 2: MNR2, 3: MNR3, 4: HD1, 5: TN1 and 6: Ptb33 * Increasing the amount of Taq up to 0.4µl and decreasing the amount of MgCl2 to 2.5 µl. Using the higher amount of Taq was the aim for increasing more effectively prolongation and 1000bp M 1 2 3 4 5 6 600bp Figure 4.8. PCR products of primer 7312.T4A 15 amplification. Furthermore, if MgCl2 concentration presented too high, it also created more sub bands in final product. Figure 4.9. PCR products with adjusted the amount of Taq polymerase and MgCl2 Lane 1: MNR1, 2: MNR2, 3: MNR3 and 4: MNR4. * Although target band became darker and brighter after changing the amount of Taq and MgCl2, the sub band also appeared darker. Once Taq concentration was increased, the sub bands were also amplified more beside the increase of target band amplification. Consequently, we kept on adjusting parameters to get desirable products. * Keeping the amount of Taq at 0.4µl and decreasing the amount of MgCl2 to 2.5 µl. Furthermore, we also increased the annealing temperature from 580C into 600C in order to limit the formation of sub bands. 1 2 3 4 M 600bp 1000bp 16 Figure 4.10. PCR products with the amount of MgCl2 and annealing temperature adjusted Lane 1: MNR1, 2: MNR2, 3: MNR3, and 4: MNR4. By altering annealing temperature to 600C and MgCl2 to 2µl, the sub bands were decreased significantly. Because melting temperature (Tm) of primer 7312.T4A 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 to 1µl so as to limit the inhibition substances in the samples for PCR reaction. 1 2 3 4 17 Figure 4.11. PCR products with adjusted cycle and DNA concentration Lane 1: MNR1, 2: MNR2, 3: MNR3 and 4: MNR4. * The band on gel was bright and intact without sub band anymore (Figure 4.11). The result indicated that this formula was appropriate for the amplification of primer 7213.T4. Therefore, this adjustment was amplified for other samples. 1 2 3 4 1000bp M 1 2 3 4 5 6 7 8 9 10 11 M 12 13 14 15 16 17 18 19 20 21 22 1000bp Figure 4.12. PCR products of primer 7312.T4A with adjusted formula 18 M: ladder 1kb, lane 1: MNR1, 2: MTL652, 3: MTL660, 4: MTL662, 5: MTL663, 6: OM5756, 7: OM6018, 8 and 19: HD1, 9 and 20: TN1, 10 and 21: Ptb33, 11 and 22: negative control (H2O), 12: MNR2, 13: MNR3, 14: MNR4, 15: MNR5, 16: OM4488, 17: OM5740 and 18: OM5756. The target gene was identified successfully in rice population based on linked molecular marker in the size of 1000 bp. Then, PCR products were digested by enzyme HinfI in order to estimate the polymorphism between rice cultivars. 3.3. Study of the polymorphism levels between rice cultivars by digestion of DNA STS products with restriction enzyme (HinfI) Figure 4.13. Digestion products of DNA STS with enzyme HinfI M: ladder 100bp, lane 1: MTL586, 2: MTL601, 3: MTL603, 4: MTL617, 5: MTL620, 6: MNR2, 7: MNR3, 8: MNR4, 9: MNR5, 10: OM4488, 11: OM5740, 12: HD1, 13: TN1 and 14: Ptb33. M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ~566bp ~398 bp 500bp 19 Digestion of PCR products with restriction enzyme HinfI found the level of polymorphism in rice. This result was the same as result of Jena K.K. (2005). In his research, PCR products rice cultivars carrying BPH resistance gene (Bph18) also presented two main bands about 566 bp and 398 bp on gel after digested with restriction enzyme HinfI. However, in this study, there were some rice cultivars displaying three main bands on gel (lane 5 and 7) including 900bp, 566 bp and 398 bp, respectively. TN1 variety also displayed a band in the size of 900bp on gel. According to many researches such as Haiyuan yang (2002) or Sogawa and Pathak (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 566bp and 398bp. 20 4. CONCLUSION * Both RM13 and RM270 marker showed unique bands on gel as well as did not express the linkage of BPH resistance gene on rice varieties of Mekong delta. * The most suitable condition for analyzing PCR product of RM13 and primer RM270 was the formula of Table 3.3 adjusted (Taq polymerase 0.5µl, MgCl2 2.5 µl), thermal cycle of Table 3.4 and 3% agarose gel Besides, the optimum anealling temperature of RM270 primer in PCR reaction was 600C. * By formula of Table 3.3 and 3.5 adjusted, RM190 marker 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 (0.4µl for Taq polymerase, 2 µl for MgCl2 and 1µl for DNA). After digesting PCR products of 30 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 21 800bp, respectively. This indicated that these varieties could carry BPH resistance gene which was heterozygous at this locus. 5. SUGGESTIONS From these results, the following recommendations 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 so as to investigate the effect of BPH resistance before applying for rice production in a popular way.jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj REFERENCES In Vietnamese Bùi Chí Bửu và Nguyễn 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, Xiang ren, Qingmei weng, Lili zhu and Guangcun He. 2002. 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