MINISTRY OF EDUCATION
AND TRAINING
MINISTRY OF AGRICULTURE
AND RURAL DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
DONG THI KIM CUC “Research and application of molecule
markers in breeding salt-tolerant Bacthom 7
rice variety”
Major: Genetics and Breeding
Code: 62.62.01.11 DOCTORAL THESIS SUMMARY OF
AGRICULTURE
1

INTRODUCTION
1. Imperativeness of the thesis
Rice production and yield are significantly losses due to diseases and pests infestation and the
environmental impacts. Of these, the noticeable factor is salt-influenced to rice. The cultivated lands
have been affected from the salinity by 380 million ha, accounted for 1/3 total worldwide cultivated
areas.
Salt – affected lands is the main factor which has curbed to develop productivity of rice, and also
caused influence to food security in general. Hence, to reduce the salinity affect to the rice plant has
paid much attention to research [124].
To meet this demand, to generate the salinity tolerance rice variety is necessary work. It is
needed to explore the natural plant resource against salinity tolerance by either directly selecting or
by genetic selection, or marker assisted selection. Application of molecular markers may help to
identify the present of salinity tolerance gene(s), which is very useful for the breeder to select the
effective crossed combination. Hence, It would be accelerated to breed rice salinity tolerance,
shortened the selection time, and expenses and labour. As aforementioned, the topic entitled
“Application the molecular markers to improve salinity tolerance of Bac Thom 7”
2. Objectives
2.1. General objectives
Study on evaluating and developing some salinity tolerance of rice which derived from the the
IRRI and India, and some grown rice varieties in Vietnam were used in this thesis.
Applying marker assisted backcrossing to improve salinity tolerance of rice which are adapted
for the Red River Delta.
2.2. Specific Objectives
Identifying the salinity tolerance and phenotype of the rice lines which carrying the Saltol
locus (donor plant), imported from International Rice Research Institute, as well as selecting the
polularly grown rice cultivar were used as the receipient plant
Applying marker assisted backcrossing (MABC) to pyramid salinity tolerance Saltol locus
into Bac Thom 7, in order to generate the high quality rice variety with salinity tolerance for growing

Institute, (Tu Liem, Hanoi); and the Center of Technological Exchange and Extension (Thanh Tri,
Hanoi), and Giao Thuy, Nam Dinh Province.
Time period: From 2010 to 2013
5. Significant Contribution
Application of molecular assisted backcrossing (MABC) is one of the initial research to
improve Bac Thom 7 with salinity tolerance for growing in the coastal areas of Red River Delta.
Applying MABC method which can be transferred the target gen/QTL in the other variety via
2-3 generations, while, traditional backcrossing has needed about 8 breeding generations

3

Application of molecular assisted backcrossing could pyramid the saltol into Bac thom 7
which also has carried the enough desire traits of Bac Thom 7, but can be grown in the salinity
affected areas upto 6 ‰
6. Structure of the Thesis
The current thesis was presented by 159 pages, of which included 25 Tables and 31 Fingures,
and separated into 4 chapters: Chapter I: An overview (50 pages), Chapter II: Materials and Methods
(15 pages); Chapter III: Results and Discussion (88 pages); Chapter IV: Conclusion and Suggestions
(2 pages). One hundred and ten literature references were used to cite for this thesis, in which there
are 23 Vietnamese references and 89 English reference and 16 link webpage were also used.


areas will be Red River delta and Cuulong delta.
1.2. Salt affected land and salt affected areas in Vietnam
1.2.1. Salt affected land
The land consists of 50-60% ratio of argillaceous. The land shows high tight level and poor
absorbent level, tought patter, and chapped and difficult to do tillage.
Because the salt land is composing of much Na+ under the NaCl dissovel, thus, the pressure of
Na
2
SO4 endosmosis is so high that can be influenced to water and nutrition absobtion of the plant.
Also, neutral and alkali in salt land are causing low activation of the microoganism
1.2.2. General introduction of the rice field affected salinity in Vietnam
Accoring to Hoang Kim and Bien and Howeler (2003), in Vietnam, there are two larger rice
field affected salt is: Red River Delta included some sub-area such as Thai Binh, Hai Phong, Nam
Dinh, Ninh Binh, while approximately 1.8 to 2.1 millio ha of land have been affected by salinity
where are located in Ca Mau, Bac Lieu, Ben Tre, Kien Gian, Tien Giang, Tra Vinh and Soc Trang
Provinces. Most of cultivated land areas have been affected salinity and alum and flooding [4].
1.3. Genetic reseaches on salinity tolerance of rice
1.3.1. Mechanism of salinity tolerance of rice
• Phenomenom of salt prevention • Phenomenom of leaf to leaf patition
• Phenomenom of re-absorbance • Tolerance ability by the tissues

5

• Moving from root to bud •Dillution influence
1.3.2. Genetics of Salinity tolerance
1.3.2.1. Reseach on genetics of quantitative traits of salinity tolerance
According to Mishra et al (1998), the trait of salinity tolerance in plant is polygenic trait,
negligible causing effect from the parental plant (recipient plant) because these genes are not located
in the cytoplasm [72]. During development stage of rice, the plant height, and rice production under
salt affection are controlled by the additative genes (Mishra et al 1990)[73].

1.4.3.3. Studying on plant breeding
Along with the advanced development of molecular breeding technologies, the breeders have paid more
attention to the issue as Marker assisted selection (MAS), has implied to use the markers that have linkage
with the interested QTL/genes in plant breeding programe
1.4.4. Application of Marker assisted backcrossing (MABC)
MABCis an a practical and efficient technique in transferring the interested QTL/gene into the elite rice
variety to generate the improved rice variety in a short time carrying the desire QTL/gene and attain
approximately 100% genetic background of the elite rice variety: The breeding programe may only
implement at the BC3 or even thought in BC2 generation, respectively.
1.5. Some archievements in improving rice salinity tolerance
1.5.1. Some results and archivements in research on rice salinity tolerance in the world
During the year 1977 to 1980, International Rice Research Institute (IRRI), was successfully
selected the good rice salinity tolerance such as IR42, IR4432-28-5, IR4595-4-1, IR463-22-2, and
IR9884-54-3 with the yield at 3,6 tones/ha. Gregorio et al (2002)[45] developed TCCP226-2-49-B-B-
3 rice cultivar with high salinity tolerance ability.
Some local rice varieties which were derived in the East Asia have often been high salinity tolerance
such as Nona Bokra (India), Pokkali (Sri Lanka), Getu (India), SR26B, Damodar, Cheriviruppu, Pat
and Solla (India), Ketumbar (Indonesia), Khao Seetha (Thailand). Some rice varieties were in the
template (subtropical countries) such as Harra (Spanish), Agami (Egypt), and Daeyabyeo (Korea).
Several Japonica rice varieties such as Moroberekan have high salinity tolerance, which were
origined in the affected salt areas. This variety has been researched and used as the donor plant
(salinity tolerance) and population mapping (Kim et al, 2009)[55]. The rice varieties were Oryza
glaberrima, which are mostly grown in the West African show lower salinity tolerance ability to
compare with the rice varieties (Oryza sativa) (Awala và cs, 2010)[2].
Recently, in 2013, researchers in IRRI have successfully developed the high-super salt
tolerance that could be very useful for the farmers to grow this rice cultivar in the affected salt areas
such as the coastal areas.
1.5.2. Application of molecular markers in improving salinity tolerance of rice
The fine mapping of Saltol QTL was made on the chromosome 1 by the researcher groups
(Gregorio 1997; Bonille et al 2002; and Niones 2004) which explained about 40-65% salinity
8

CHAPTER III
MATERIALS, CONTENTS AND METHODS
2.1. Materials
The rice materials included:
 Total 14 rice lines/varieties carrying Saltol QTL salinity tolerance were imported from
IRRI and some popularly grown rice varieties in Red River Delta
 Chemical argents and research facilities:
SSR markers used: 447 markers
Research instruments: Experimental tools of Agricultural Genetics Institute
2.2. Areas to conduct experiments
The Laboratory of Molecular Biology Division-Agricultural Genetics Insititute. Tuliem-Hanoi
The Net house and paddy fields conducted for experiment at the Center of Technology
Exchange and Extension, Vinh Quynh, Thanh Tri, Hanoi
Experiments for evaluation of growth and development of the imported rice lines/varieties
were conducted in two provinces: Nam Dinh and Hanoi
Perious to implement: From 2010 to 2013
2.3. Contents
2.3.1. Content 1: Research, evaluation of the salinity tolerance and agronomical traits, rice yield and
some sub-traits involving in rice yield of the Salton-lines/varieties imported from IRRI and some
popularly grown in the Red River Delta. It has been an important for further research on rice salinity
tolerance for the coastal areas in North Vietnam
2.3.2. Content 2: Application of marker assisted backcrossing to transfer the Saltol QTL into
Bacthom 7, an elite rice cultivar

No
Line/variety
Effect after 2 weeks treated NaCl 6‰
Effect after 3 weeks treated NaCl 6‰
Rep 1
Rep 2
Rep 3
Aver
Rep 1
Rep 2
Rep 3
Aver
1
IR72046-B-R-8-3-1-3
3
5
3
3.7
7
5
5
5.7
2
IR52713-2B-8-2B-1-2
3
3
3
3.0
7
5

5
6.3
6
IR55179-3B-11-3
3
3
3
3.0
7
5
7
6.3
7
IR65196-3B-5-2-2
5
5
3
4.3
7
7
7
7.0
8
IR74099-3R-3-3
3
3
3
3.0
7
5

9
8.3
12
Khang dan 18
7
7
7
7.0
9
9
9
9.0
13
Pokkali (salinity tolerance )
1
1
3
1.7
3
1
3
2.3

10

14
IR29 (Sensitive)
7
9
7


96.3
h
24.2
ab

23.0
cd

2
IR52713-2B-8-2B-1-2
127
115
109.0
c

110.0
c

24.5
a

25.0
a

3
IR77674-3B-8-2-2-AJY5
155
130
109.0

120
92.5
g

94.0
i

23.5
bc

22.3
e

6
IR55179-3B-11-3
145
120
113.0
b

115.3
b

23.7
c

24.0
b

7


9
IR 4630-22-2-5-1-3
142
115
113.0
b

106.3
e

21.3
d

20.3
f

10
FL478
135
120
103.3
d

102.3
f

20.3
e


CV (%) 0.47
0.46
2.04
1.63
LSD
0.05
0.84
0.87
0.8
0.63
3.1.2.2. Results of evaluation of the growth and development of some imported rice varieties grown
in Gia Thuy, Nam Dinh in 2010
Table 3.5. Several agronomical traits and morphology of the rice varities grown at Giao Thuy,
Nam Dinh Province in 2010
No
Line/variety
Days to heading (day)
Plant height (cm)
Panicle length (cm)
Spring
Summer
Spring
Summer
Spring

25.7
a

3
IR77674-3B-8-2-2-AJY5
160
134
110.7
cd

105.0
cde

23.3
abc

23.0
bcd

4
NSIC Rc 106
140
110
95.0
f

87.7
g

22.3


113.7
bc

24.0
abc

24.3
ab

7
IR65196-3B-5-2-2
142
125
119.0
b

115.0
b

23.3
abc

23.0
bcd

8
IR74099-3R-3-3
140
115

132
115
105.0
de

99.0
def

20.7
d

20.7
ef

11
Pokkali
-
140
-

188.7
a
-
25.3
a
12
Bac thom 7(control)
135
120
111.0

3.2. Application of Marker assisted backcrossing to improve salinity tolerance of Bac Thom 7
3.2.1. Results of identification of parental plants to improve QTL Saltol rice line
In order to improve salinity tolerance of rice varieties grown in the Red River delta, we have
used Marker assisted backcrossing method to transfer QTL Saltol into the receipient plant, but
attaining its agronomical traits such as quality of rice. Based on the obtained results, the Bac Thom 7
is the variety that need to be improve salinity trait and used as the receipient Saltol material.
3.2.2. Resukts of applying Marker assisted backcrossing to pyramid QTL saltol into BT7
3.2.2.1. Identification of the markers linked with Saltol and polymorphic markers between BT7 and
FL478
In this study, total 30 markers at the target gene of Saltol were used to identify the linkage markers
between the donor and receipient plants. Fifteen polymorphic markers between the parental plants at the target
gen were AP3206, RM3412b, RM10748, RM493, RM140, RM10825. G1a, G6a, G11a, Salt 4a, SCK1b,
SCK1d, SCK2, SCK10, and SCK10a. The information of the polymorphic markers have shown in the Figure
3.2 and Figure 3.5

Figure 3.2. Polymophic markers between BT7 and FL478 with 3 markers as
RM493, RM3421b and RM140
Note:: P1: Bacthom 7; P2: FL478

12 Figure 3.4. Position of QTL/gen Saltol located on the chromosome 1
3.2.2.2. The results of identification of polymorphic markers that were out of QTL Saltol region
between BT7 and FL478 on the 12 chromosomes
To identify the polymorphic markers which were located out of region of Saltol on the 12
chromosomes for determining the genetic background of the selected individual plants from the
crossed population. Total 447 SSR markers were used to screen to find out the polymorphic markers,
102 polymophic markers were identified (accounted for 21,38%) between Bacthom 7 and FL478


H: Heterozygote

14 Figure 3.11. Results of electrophothesis on 94 individual plants of BC1F1 (RM3412b
markers).
From 1-94 the individual plants BC1F1, BT7: Bac thom 7,FL: FL478 A: BT7, B:FL478, H:
Heterozygote
Combination of 2 markers namely RM493 and RM3412b, 14 individual plants have been
screened to carry Saltol as the plant number: 5, 10, 11, 14, 19, 28, 29, 32, 36, 42, 45, 50, 71, 83.
3.2.3.3. Results of selecting the individual plants in the BC2F1 population by applying molecular
markers
-Results of selection of the individual plants carrying locus Saltol in the BC2F1 population
In this experiment, the successfully crossed 141 individual plants in BC2F1. To identify the individual
plants carrying Saltol in the population of BC2F1, two closely linked markers with Saltol as RM493
and RM3412b were consecutively used to select the individual plants that carrying the target gen. The
results of selection of the individual plants carrying Saltol were shown in the Figure 3.13 and 3.14.

Figure 3.13. Results of electrophoresis of 141 individual plants from BC2F1 (RM3412b)
From 1-141, the individual plants BC2 F1- BT7:BT 7; FL: FL478; A: BT 7; B:FL478; H: Heterogyzote

Figure 3.14. Results of electrophoresis of 141 individual plants from BC2F1 (RM493)
From 1-141, the individual plants BC2 F1, BT7: BT 7,FL: FL478 A: BT 7, B:FL478, H:Heterogyzote
The results of selection of the individual plants which have been carrying the target gen by
applying markers RM3412 and RM493, we have selected 34 individual plants as the plant number: 1,
2, 7, 9, 11, 13, 15, 22, 23, 24, 30, 34, 36, 42, 45, 47, 51, 53, 57, 59, 60, 65, 74, 77, 81, 92, 93, 94, 96,
112, 114, 117, 136, 141.
- Evaluation of the background of the individual plants carrying Saltol in the BC2F1 population
Results of identifying the individual plants carrying Saltol and attained the maximum genetic

To identify the individual plants carrying Saltol in the population of BC3F1 by using 2
markers RM493 and RM3412, total 115 individual plants have obtained: 6, 7, 8, 10, 14, 16,
18, 22, 28, 29, 30, 32, 35, 36, 38, 41, 42, 45, 50, 54, 63, 64, 65, 70, 72, 73, 74, 75, 80, 82,
83, 84, 94, 101, 102, 109, 111, 112, 116, 122, 123, 135, 148, 157, 158, 166, 169, 174, 176,
178, 184, 188, 190, 192, 194, 197, 198, 200, 211, 215, 217, 218, 221, 234, 233, 237, 238,
246, 248, 254, 257, 259, 260, 263, 270, 273, 274, 275, 276, 277, 284, 289, 290, 293, 300,
302, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 317, 320, 324, 331, 332, 333,
335, 336, 344, 345, 351, 353, 357, 358, 359, 361, 366, 367.
Among 115 individual plants carrying Saltol to identify by use of 2 markers RM493 and
RM3412, 88 individual plants were to backcrossed as accounted from the individual plants
number 8 (in the population of BC2F1).
* Evaluation of the genetic background of the individual plants carrying Satol in the population of
BC3F1

17

To identify the genetic background of the individual plants carrying Saltol in the
population BC3F1, only 88 individual plants which were developed from the plant number 8
(genetic background 80,7% of BC2F1).
Figure 3.29. Satistical analysis of genetic background of 88 individual plants from BC3F1 on 12
chromosomes by use of GGT1
As the result shown in Figures 3.30; 3.31, it was identified two individual plants number 30
and 32 which have had the highest genetic background 99,3% and 100% as the BT7.

Figure 3.30. Genetic map of individual plant number 30

Figure 3.31. Genetic map of the individual plant number 32
Note that figures 3.30 and 3.31: The numberal chromosomes were expressed the below
number, and the list of markers used to screen the genetic background was on the left side, equivalent
with position of marker that was on the right of chromosome. Red region was indicated genetic

(ear)
Color awn
1
IL32-1
135
112.9
jk
20.6
bcde
2.9
ef
10.7
hi
Light yellow
2
IL32-2
135
111.2
mn
20.7
de
2.9
bcdef
10.4
i
Light yellow
3
IL32-3
135
113.2

12.1
a
Light yellow
6
IL32-6
135
109.5
o
20.5
de
3.5
a
10.4
hi
Light yellow
7
IL32-7
135
117.9
a
21.5
bcd
2.8
f
10.4
hi
Light yellow
8
IL32-8
135

11.7
efghi
Brown
11
IL32-11
135
113.2
ijk
21.2
ab
3.3
ab
12.1
ab
Dark yellow
12
IL32-12
135
114.5
ef
20.4
de
3.3
ab
11.1
defghi
Light yellow
13
IL32-13
135

12.1
abc
Dark yellow
16
IL32-16
135
111.9
lm
21.1
bcde
2.8
cde
11.4
abcdef
Light yellow
17
IL32-17
135
108.9
o
20.5
de
2.6
ef
12.1
abc
Light yellow

19


bc
3.1
abcd
10.4
hi
Light yellow
21
IL32-21
135
113.2
hijk
20.9
cde
3.1
abc
10.1
hi
Dark yellow
22
IL32-22
135
111.2
n
21.9
bcd
3.1
ab
12.1
ab
Dark yellow

bcd
3.4
a
11.7
defghi
Brown
26
IL32-26
135
114.2
efg
21.0
bcd
3.2
a
12.7
abcd
Brown
27
IL32-27
135
113.2
hijk
20.2
de
2.8
def
11.7
bcdefg
Light yellow

bcde
2.8
ef
10.4
hi
Brown
BT7
(control)

135
116.2
bc
21.2
bcde
3.1
abcde
11.6
abcdef
Light yellow

CV (%)
0.48
2.3
9.82
4.13 LSD5%
0.89
0.79

IL32-3
15.3
76.5
3-5
Tolerance
4
IL32-5
15.7
78.5
3-5
Tolerance
5
IL32-7
17.3
86.5
1-3
High tolerance
6
IL32-8
17.3
86.5
1-3
Tolerance
7
IL32-9
16.0
80.0
3-5
Tolerance
8

IL32-17
17.3
86.5
1-3
High tolerance

20

14
IL32-18
17.7
88.5
1-3
High tolerance
15
IL32-19
16.7
83.5
3
Tolerance
16
IL32-20
15.7
78.5
3-5
Tolerance
17
IL32-23
15.3
71.5

1-3
High tolerance
Cv%

0.37
0.45
Thus, evaluation of the growth and development and salinity tolerance in the artificial condition of
the individual plants of BT7 - Saltol showed that most of the carried QTL/gen Saltol that shown in
the laboratory exhibited good growth and development to compare with the controlled variety.
3.3.3. Results of evaluation of some agronomical traits of the BT7- Saltol (BC3F3) under the
paddy field. 21

Tabe 3.20. Indicators of some growth and development of some BT7–Saltol lines (BC3F3) in the summer crop in 2012 tại
at Giao Thuy, Nam Dinh province
No
Name
DTH
(days)
Leaf green
level
(point)
Blade
pubescence
(điểm)

111.8
3
21.0
7.7
29.1
Light yellow
3
IL32-3
112
3
3
107.9
3
20.8
5.3
27.8
Light yellow
4
IL32-5
112
5
3
106.7
5
22.1
5.3
32.0
Light yellow
5
IL32-7

Light yellow
8
IL32-12
112
7
3
104.3
3
21.0
6.0
28.0
Light yellow
9
IL32-13
112
5
3
110.3
5
22.0
6.3
28.0
Light yellow
10
IL32-14
112
5
5
104.4
1

3
105.6
1
22.1
8.0
33.8
Light yellow
14
IL32-18
112
3
3
101.0
1
21.0
4.8
25.8
Light yellow
15
IL32-19
112
7
1
101.9
3
23.0
5.4
31.4
Light yellow
16

23.5
Light yellow
20
IL32-29
112
5
5
99.9
3
20.6
5.0
25.9
Light yellow
BT7

112
5
3
104.2
3
21.5
6.7
25.3
Light yellow
Cv%

4.22

length (cm)
Panicle
exsertion (cm)
Flag leaf length
(cm)
BT7(control)

132
113.2

24.0
a

3.9
b

24.8
d

Line 1
IL32-1
132
112.8

22.3
bc

6.0
a



Line 5
IL32-5
132
112.9
21.6
bc

4.6
b

25.4
c

Line 9
IL32-9
132
112.7

22.8
b

5.9
a

23.8
e

Line 10
IL32-10


22.5
b

5.7
a

25.3
c

CV%
2.74
9.48
0.58
LSD
0.05
1.06
0.43
0.25

As the results shown in Table 3.22, the potential BT7- Saltol lines showed days
to heading that were similar with BT7 (132 days). The plant height of the lines were
ranged about 112,7 cm (line number 9) to 114,5 cm (line number 12). Hence, there is
no significant difference to compare with the BT7 as 113.2 cm.

Medium
5,57
2,04
2,73
5 line
69,00
89,22
55,85
17,52
Medium
5,70
1,98
2,88
12 line
68,74
87,28
57,03
17,00
Medium
5,52
1,94
2,84
BT 7 -
(control)
68,96
87,54
56,22
16,02
Medium
5,61