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

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

APPROVAL

SUPERVISOR STUDENT

Dr TRAN NHAN DUNG NGUYEN NG.QUYNH ANH

Can Tho, November 25, 2010

PRESIDENT OF EXAMINATION COMMITTEE

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

3.1 Result of determining the DNA concentration and

DNA purification of rice cultivars

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

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 (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

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

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

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

Table 3.2 List of primers used

Primer Primer’s sequences Chromosome Repeat

Rev 5’ GGT GGC ATT CGA TTC CAG 3’

et al., 2005)

RM270

For 5’ GGC CGT TGG TTC TAA AAT C 3’

Rev 5’ TGC GCA GTA TCA TCG GCG AG

3’

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’

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)

Table 3.5 PCR thermal test cycle of primer 7312.T4A

* 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

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

130bp

120bp 600bp

1 2 3 M 4 5

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

120bp

130bp

1 2 3 4 5 M

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

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

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

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

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

500bp

M 1 2 3 4 5 6 7 8 9 10 11 12 13 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

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 MgCl 2

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

Figure 4.10 PCR products with the amount of MgCl 2 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

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