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 cultivars8
3.2 PCR products 8
3.2.1. Study of the effect ofsome parameters into the
formation of PCR products of primer RM190 8
3.2.2. Study ofthe effect ofsomeparametersinto formation PCR products ofprimerRM13 and RM27011
3.2.3. Study of the effect of some parametersinto 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
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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
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
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