MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE
AND TRAINING
AND RURAL DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
.....................................
NGUYEN THI THUY ANH
RESEARCH ON IMPROVING THE YIELD OF SOME RICE
LINES/VARIETIES BY MOLECULAR MARKERS
Specialization: Biotechnology
Code number: 9420201
SUMMARY OF AGRICULTURAL DOCTORAL THESIS
HA NOI, 2018
The dissertation has been completed at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
Supervisors:
1. Prof. Dr. Tran Trung
2. Assoc.Prof. Dr. Tran Dang Khanh
Reviewer 1:
Reviewer 2:
Reviewer 3:
The dissertation will be defended at the Dissertation committee at
the National level
Vietnam Academy of Agricultural Sciences
Date
month
year
The full text of the dissertation can be found in the following
libraries:
1. The National Library of Vietnam
2. The Library of Vietnam Academy of Agricultural Sciences
RELATED PUBLICATIONS COMPLY WITH THESIS
1. Nguyen Thi Thuy Anh, Tran Trung, Khuat Huu Trung, Le Hung
Linh, Ta Hong Linh, Hoang Kim Thanh, Than Thi Thanh, Nguyen Nhu
Toan, Nguyen Thi Loan, Tran Dang Khanh, (2016), "Marker-assisted
backcrossing to develop elite rice lines from KD18/KC25 concerning
high grains/ panilce ", the Second National Plant Sciences Conference
on Crop Scienes, pp. 331-336.
2. Nguyen Thi Thuy Anh, Tran Trung, Khuat Huu Trung, Tran Dang
Khanh, (2017), "Application of molecular breeding to select individual
plants of BC2F1 population carrying the QTL/gene (increasing the
number of grains per panicle) to improve yield of the Khang Dan 18
variety, Journal of Agricultural Science and Technology, Vol. 6, pp. 16.
3. Nguyen Thi Thuy Anh, Tran Trung, Khuat Huu Trung, Le Hung
Linh, Tran Dang Khanh, (2017), "Application of molecular breeding to
select the individual plants carrying QTL/gene (increasing the number
of grains per panicle) from the BC2F1 population improving the yield
of NPT1 variety”, Journal of Science and Technology, Vol. 12, pp. 3740.
INTRODUCTION
1. The essentials of the thesis
Rice (Oryza sativa L.) is the most important food crop in
Vietnam, and is also the main source of food for more than half of the
world wide population. Nowadays, with the rapidly growing
population, the significant reduction of agricultural land and the
extreme effects of climate change have caused adverse impacts on rice
productivity. In some recent years, with the rapid development of
modern biotechnology, numerous molecular markers linked to the
important traits of rice have been identified. Scientists and breeders
have paid much attention to those molecular markers for rice breeding
programs. Marker assisted backcrossing (MABC) is a power tool and
proposed for rapid and effective in plant breeding. As a fore mentioned
issues, the application of molecular markers and backcrossing to
introgress and pyramid the high yield QTLs/genes into traditional and
elite rice varieties is imperative work. Therefore, the main objective of
this thesis is to improve the yield of some rice lines/varieties by use of
molecular marker.
2. Objectives of the study
The objectives of the current study are to apply the molecular
breeding to introgress the yd7 QTL/gene (increase the number of grains
per panicle), into some rice lines/varieties in order to improve and
develop the new and promising rice lines/varieties for the Red River
Delta.
3. Research content
3.1. Evaluation of rice plant materials for rice breeding.
3.2. Using molecular markers to identify the polymorphism between the
QTL/gene of the donor and recipient lines/cultivars.
1
3.3. Improving the yield of Khang dan rice cultivar and promising NPT
(new plant type) line for the Red River Delta by use of MABC
3.4. Field trials and evaluation of rice yield of some promising
lines/varieties developed from
4. The scientific and practical significance
4.1. Scientific significance
The obtained results will provide useful information and
scientific data for the rice breeding by use of MABC methods.
Successful application of MABC to transfer yd7 QTL/gene
controlling the trait of increasing seed number per panicle in some
lines/varieties which will be helped to widely apply in rice breeding
programe.
4.2. Practical significance
Using marker assisted backcrossing method has facilitated to
rapidly and accurately selected QTL/gene controlling the increase of
number of seeds per panicle as well as pyramiding them on the elite and
promising rice lines/varieties, are being grown in the Red River Delta,
can minimize the time selection and labor force in the field and
partially help overcome the limitation of traditional breeding methods.
The selected rice lines/cultivars will be an initially important
plant materials for breeding to improve rice yield for the Red River
Delta as well as help to ensure national food security and enhance
income for rice growers.
5. New contributions of the thesis
- This thesis is one of the first reports in Vietnam on applying
MABC method to successfully transfer yd7 QTL/gene (increasing the
number of grains per panicle) for rice yield improvement.
2
- The polymorphic markers at yd7 QTL/gene of the donor and
recipient lines were identified to select the individual plants carrying
the target QTL/gene as well as polymorphic markers which spread over
on 12 chromosomes for genetic background selection.
- Successfully improved yield of some rice lines/varieties by
use of MABC. The developed rice lines carrying the target QTL/gens
and simultaneously retained the genetic background of the recipient
plants. Specifically, the yield of improved K2 line was 28,3% higher
than that of control variety Khang Dan 18, the N8 yield was 9,4%
higher than that of control variety NPT1, respectively.
6. Structure of the thesis
The dissertation included 137 pages without annexes, 55
photographs and 33 tables. The dissertation uses 120 references,
including 21 Vietnamese, 98 English and 1 website.
CHAPTER 1. LITERATURE REVIEWS
1.1. Application of genetic marker for rice breeding
1.1.1. Concept and classification of genetic markers
Genetic marker is known as a landmark on the chromosomes
which often use for genome analysis, to distinguish different
individuals of a species. Genetic markers are often associated with
genes and are genetically inherited [17], [54].
Genetic marker can be divided into two categories: traditional
markers and DNA markers. From the end of the 20 th century, genetic
markers have been classified into three groups: morphological marker;
biochemical marker; DNA marker [17], [47].
1.1.2. Application of molecular marker in research and breeding
3
Molecular markers can be used in genetic studies, mutations in
genome, evolution and for selection of the linkage between alen and
alleles with phenotypic traits.
Marker assisted selection or marker assisted backcrossing is
considered as the simplest form of molecular breeding. The aim of
MABC is to one or more QTLs/genes of interest from the specific
materials into elite lines/varieties for the target trait improvement.
1.2. Application of molecular marker to improve rice yield
1.2.1. Overview of productivity and components of productivity
The rice yield is a complex trait determined by its following
component traits including: number of panicle per plant, number of
grains per panicle and grain weight. The number of panicle per plant
depends on the ability to branch, effective branch number. The number
of grains per panicle is also determined by two factors: the number of
offspring, the rate of grain production of the panicle. The grain weight
is determined by three factors: length, breadth and thickness seed [45],
[48], [78], [106].
1.2.2. The QTL/gene linked to the rice yield traits
The composition of rice yield is a multiple agronomic trait that
is controlled by numerous genes, and is a quantitative trait (QTL). So
far, hundreds of QTLs related to rice yield traits have been identified
and cloned which distributed across the whole rice genome.
1.2.3. Application of molecular markers to improve rice yield
Rice is one of the most important food crops for human
consumption. Therefore, rice is the most studied subject, applied
advanced techniques to improve productivity have recently made. With
information on cloning the QTLs, along with a marker system linking
4
closely to QTLs/genes, many molecular breeding programs have been
developed for rice yield improvement.
Since 1995, based on molecular marker analysis and field
experiments, Chinese researchers have identified two QTLs from O.
rufipogon wild rice lines yld1 and yld2. Each of these QTLs contributed
to an 18% increase in rice yields compared to the Weiyou 64 rice lines.
Ashikari et al. (2005) made a cross between Japonica Koshihikari and
Indica Habataki rice and developed two line which were carried Gn1
and Ph1 genes contributed with a 23% increase in yield and a 20%
decrease in plant height to compare with Koshihikari rice variety.
In other studies, Zhang et al. (2012) reported that there was a
major QTL defining the particle length as qGL3. Under field
conditions, NIL carrying the qGL3 allele showed a 16,20% increase in
yield [115]. Studies on the number of branches on panicle, Sasaki et al.
(2017) found a new QTL namely qTSN12 derived from the new
phenotype YTH83. In the NIL populations, the yield of rice increased
from 18% to 36% [79].
1.3. Research on the application of molecular markers in rice
breeding in Vietnam
In Vietnam, the application of molecular markers for rice
breeding has been implemented since the last years of the century.
Traits of interest in rice research in Vietnam include yield, rice quality,
aroma, disease resistance (leaf blight, rice blast disease, plant hopper,
virus) and tolerance (flooding, salinity, drought, heat) as well as studies
on the rice genome have been made for rice breeding programs.
However, currently, unavailable report on QTL/gene controlling rice
yield is reported, while international breeders have so far achieved good
results in molecular marker applications to improve rice yield.
5
Therefore, the objective of the thesis is to apply MABC to transfer
QTL/gene (increase the number of grains per panicle) into some
Vietnamese rice varieties for rice yield improvement.
CHAPTER 2. MATERIALS, CONTENTS AND
METHODOLOGY
2.1. Materials
2.1.1. Rice lines/varieties
- Thirteen donor lines carrying yd7 QTL/gene which
controlling the increase grains per per panicle were used as the donor
plant materials in this study.
- The selected recipient plants were Khang Dan 18 variety. This
variety is being cultivated in the Red River Delta and the promising
NPT1 rice line provided by the Institute of Agricultural Genetics were
used.
2.1.2. Molecular marker used in this study
Six SSR molecular markers linked to the target yd7 QTL/gene
and 156 SSR molecular marker were used to examine the target gene
and their parental polymorphisms and for the genetic background in
different generations.
2.1.3. Chemicals and equipment use
2.2. Location and time of study
*Research location:
- Experiment was conducted at: Department of Molecular
Biology, Department of Genetic Engineering - Institute of Agricultural
Genetics (AGI), Center for Technology Transfer and Agriculture
Extension - Thanh Tri - Hanoi, Experimental field in Lai Yen-Hoai
Duc-Hanoi.
*Research time: From 2013 to 2017
6
2.3. Research methods
Field experiment methods
Conventional breeding, backcross between donor and recipient
plants
Molecular breeding method
Develop F1 plants by made cross between the donor and
recipient plants. BCnFn populations were developed by conventional
breeding method. Using the SSR molecular markers to identify the
individual plants carrying QTL/gene of interest and attaining the
highest genetic background of the recipient plant in the populations of
BC1F1, BC2F1, and BC3F1.
Methods to evaluate stress tolerances, and yield of the studied rice
lines/varieties
The experiments were done following to the method of
National Technical Regulation on the Valuation of Cultivation Value
and Use Value of Rice issued by the Ministry of Agriculture and Rural
Development in 2011.
Some techniques used in the laboratory
DNA extraction and purification technique, PCR method with
SSR primer, Electrophoretic method
Statistical analysis
Data in field were analyzed by use of the IRRISTAT 5.0
program; Excel version 2007.
Data collection and analysis techniques in the laboratory were
analyzed by using Graphical Genotypes 2 (GGT 2.0) software and other
necessarystatistical methods.
7
CHAPTER 3: RESULTS AND DISCUSSION
3.1. Evaluation of plant materials for rice breeding
- Evaluation of some main agronomic traits of 13 donor lines
was made. The results showed that the KC25 line showed the most
yield potential, with good resistance to pests and diseases, especially
KC25 line revealed better field purity than the other lines. Thus, the
KC25 line was chosen as donor QTL/gene that controlling the number
of grains per panicle.
- Based on the growth and development as well as rice yield
components of the recipient rice varieties, two lines/varieties namely
Khang Dan 18 and NPT1 were selected for recipient plants
3.2. Applying themolecular markers to examine the polymorphism
between the donor and recipient rice varieties on the chromosomes
3.2.1. Identifying polymorphism markers at position of QTL/gene
controlling the number of grains per panicle between the donor
and recipient plants
In this study, six molecular markers on chromosome 7 were
used to examine the polymorphism between the Khang Dan 18 and
NPT1, which could then be used to select individual plants carrying yd7
QTL/gene in the backcrossed population. As the results shown, three
markers showing polymorphism at the target QTL/gene position.
Specifically, RM445 marker is located on QTL/gene yd7 at 17,46Mb,
the RM500 and RM21615 are the flanking markers at 15,91Mb,18,25
Mb on the both QTL/gen sides, respectively.
Figure 3.1: Polymorphic DNA markers found between the donor
and recipient plant by use of RM445, RM500 and RM21615
L: Ladder 50bp; 1: Khang dan 18; 2: NPT1; 3: KC25
8
3.2.2. Examination of polymorphism on the 12 chromosomes
between the donor and recipient plants
Identification of polymorphisms between genes donor and
recipient rice varieties by use of 156 SSR markers which evenly
distributed on 12 chromosomes for selection of genetic background of
breeding population.
The results revealed that 62/156 SSR markers showed
polymorphism between KC25 and Khang Dan 18 combination; and
63/156 SSR markers were polymorphism between NPT1 and KC25,
respectively. The polymorphic markers between the QTL/gene of donor
and recipient plants on the 12 chromosomes are used to determine the
genetic background of the individual plants in backcrossed polulations.
3.3. Improving rice yield of Khang dan 18 and promising line the
NPT by use of MABC
3.3.1. Use of polymorphic markers to select the individual plants of
Khang Dan 18/KC25
- Select the individual plants in BC1F1 population
There were 32/84 individuals showing heterozygote genotypes by
use of RM500 marker. This heterozygous plants in BC1F1 was selected
and further examine for genetic background
Figure 3.9: Electrophoresis of 84 individuals of BC1F1by use of
RM500 marker
1-84: BC1F1 individuals, M: KD18; B: KC25; L: Ladder 50bp
9
By use of the software GGT2 to analyze data, the individual
plant number 74 showed the highest genetic background by 83,4%,
followed by the plant number 109 was 80,2% respectively.
- Select the individual plants in BC2F1 population
In this step, we used two markers RM500 and RM21615 to
identify the individual plants carrying QTL/gene plant. The results
showed that 15 BC2F1 individual plant were determined including plant
numbers: 8, 18, 22, 32, 35, 42, 56, 59, 60, 61, 70, 86, 88, 97 and 101.
Figure 3.14: Electrophoresis to examine the individuals carrying
the target gene in BC2F1 population by use RM500 marker
L: 50bp ladder, M: KD18, B: KC25, A: Homozygous; H: zygote;
1-61: Individual in BC2F1 population
Figure 3.15: Electrophoresis to examine the individuals
carrying the target gene in BC2F1 population by use RM21615
marker
L: 50bp ladder; M: KD18; B: KC25; A: homozygous; H: zygote;
1-61: Individual BC2F1
10
Fifteen individual plants in BC2F1 population carrying
QTLs/genes were used for checking their genetic background by use of
62 polymorphic markers distributed on 12 chromosomes. The result
showed that individual plant number 61 and 59 carrying QTL/genes
increased the number of seeds per panicle and retained the highest
genetic background, were 91,8% and 92,3%, respectively
- Select the individual plants in BC3F1 population
Polymorphism markers including RM445, RM500, RM21615
at the target yd7 QTL/gene were used. The results demonstrated that 24
individuals carrying the target QTL/gene were the plant number as
following: 9, 10, 14, 19, 21, 22, 25, 28, 29, 30, 37, 38, 46, 48, 61, 67,
70, 74, 79 and 95 respectively
Figure 3.20: Electrophoresis to examine the individuals
carrying the target gene in BC3F1 population by use RM500
marker
1-95: BC3F1 individuals, M: Khang dan 18; B: KC25; L: Ladder 50bp
Further analyze of the 24 individuals carrying QTL/genes
indicated by use of 62 polymorphic markers evenly distributed on 12
chromosomes was made, the data were analyzed using the Graphical
Genotyper 2 (GGT2) program. Among them, the plant number C1-74-
11
59-14 carrying target gene, simultaneously had the highest background
which are similar with Khang Dan 18 (approximately 100%).
Figure 3.23: Genetic map of individuals number C1-74-59-14
BC3F1 population of breeding combination KD18 / KC25
A: Homozygous with Khang dan 18; B: Homozygous with KC25;
H: Heterozygous; U: Sample does not show
- Select the individual plants in BC3F2 population
Eleven individual plants BC3F2 population carrying the target
gene were identified by use of markers RM445, RM500 and RM21615,
which showed homozygous with KC25 and closely linked to yd7
QTL/gen. Individual plant number included: 12, 21, 23, 24, 26, 34, 53,
56, 73, 85 and 95. These individuals were selfing as well as evaluate
their field purity.
12
3.3.2. Use of polymorphic markers to select the individual plants
carrying the target QTL/gene of the NPT1/KC25 combination
- Select the individual plants in the BC1F1 populations
Figure 3.27: Electrophoresis to examine the individuals in
BC1F1 population by use RM445 marker (NPT1/KC25 crossed
combination)
1-62: BC1F1 individuals; M: NPT1; B: KC25; L: Ladder 50bp
Thus, the use of RM445, RM500 has identified 19
heterozygote with NPT1, they are the individual plant number: 1, 5, 6,
10, 11, 15, 19, 20, 24, 25, 26, 27, 31, 33, 40, 46, 48, 51 and 54. These
individual plants carrying the target gene increasing the number of
grains per panicle, and further examine for their genetic background.
Figure 3.28: Electrophoresis to screen the individual plants in
BC1F1 population by use of RM500 marker
1-62: BC1F1 individuals, M: NPT1, B: KC25, L: Ladder 50bp
Analysis of genetic background showed that individual plant
number 54 had the highest genetic background of the recipient plant of
NPT1 by 81,2%. Therefore, the plant number 54 is selected to continue
developing BC2F1 population.
13
- Select the individual plants in the BC2F1 population
By use of the markers RM445, RM500 and RM21615, we
identified 10 individual plants carrying QTL/gene. They were following
plant number: 9, 13, 14, 30, 98, 99, 122, 124, 142 and 143. These
individuals consecutively examine for their genetic background. The
results showed that the plant number C37-54-122 had the highest
genetic background by 96,2% which are similar with recipient plant
NPT1.
Figure 3.33: Electrophoresis to examine individuals in BC2F1 by use
of RM500 marker
1-36: BC2F1 individuals, M: NPT1, B: KC25, L: Ladder 50bp
Figure 3.34: Electrophoresis to screen individuals plants in BC2F1
by applying RM21615 marker
1-36: BC2F1 individuals, M: NPT1, B: KC25, L: Ladder 50bp
- Select the individual plants in the BC3F1 populations
Figure 3.38: Electrophoresis to examine individuals in BC3F1 by
RM500 marker
1-27: BC3F1, M: NPT1; B: KC25; L: Ladder 50bp
14
Figure 3.39: Electrophoresis to examine the individuals in BC3F1 by
RM21615 marker
1-27: BC3F1, M: NPT1; B: KC25; L: Ladder 50bp
Figure 3.41: Genetic map of C37-122-51 BC3F1 generation
of NPT1/ KC25 crossed combination
A: Homozygous with NPT1; B: Homozygous with KC25;
H: Heterozygote; U: Sample does not show
So, following the next step, we combined three markers
RM445, RM500 and RM21615 to screen 13 individuals BC3F1 carrying
15
yd7 QTL/gene increase the number of grains perpanicle include
individual plant number: 1, 30, 35, 39, 40, 46, 47, 48, 49, 51, 52, 59 and
individual 76.
These individuals were tested for their genetic background. The
results obtained two individual plant numbers: C37-122-51 and C37122-59, which showed the closest genetic background approximately
100% loci to the recipient NPT1.
- Select the individual plants in BC3F2 populations
By applying 3 markers RM445, RM500 and RM21615, we
found 10 individuals showing homozygous genotypes with the donor
plant in all three markers. They are the number: 19, 32, 38, 51, 57, 78,
99, 103, 107 and 121. These plants were next selfing and develop in
field for further purity evaluation.
3.4. Field trials and evaluating agronomic traits of some promising
lines
3.4.1. Field trials to evaluate agronomic traits of some promising
lines developed from the KD18 x KC25 crossed combination
- Evaluation of rice yield of some promising lines obtained
from KD18 x KD18 x KC25 (BC3F3 generation)
Figure 3.44: Reality yield of some promising lines
(KD18/KC25combination) compared to the controlled variety in
the Spring season 2016
16
Table 3.12: Components of rice yield of promising lines
(KD18/KC25 combination) in the Spring season 2016 in Hoai DucHanoi
NPP
TG
FGP
FGP
rate
(%)
P1000
(g)
TY
(ton/ha)
AY
(ton/ha)
K1
5,8
298,8
227,1
76,0
20,0
10,65
7,14
K2
6,0
279,5
229,8
82,2
20,4
11,32
7,23
K3
5,3
250,3
216,8
86,6
20,0
9,26
6,43
K4
4,6
241,2
191,9
79,6
24,8
8,66
6,07
K5
6,8
237,8
188,2
79,1
20,7
10,77
7,39
K6
4,8
250,5
208,6
83,3
22,2
8,91
6,18
K7
5,6
291,1
252,5
86,7
20,1
11,55
7,51
K8
5,4
281,6
224,9
79,9
20,8
10,15
7,04
K9
5,8
282,5
230,9
81,7
20,1
10,72
7,29
K10
6,5
249,4
143,2
57,4
22,3
8,38
5,57
K11
6,3
248,9
216,7
87,1
20,3
11,24
7,03
KD18
(đ/c)
5,5
192,1
168,5
87,7
20,1
7,47
5,48
Line/
Variety
name
CV%
10,7
LSD0,05
13,4
NPP: number of panicle per plant; FGP: filled grains per
panicle; P1000: grain weight; TG: Total grain; TY: theoretical yield;
AY: attained yield.
The table 3.12 showed that most of major agronomic traits have
had significant differences between the improved lines and control
KD18. In particular, the promising lines were found to be relatively
high number of grains/panicle and were higher than the control variety
KD18. This implied that the application of the molecular marker has
successfully introgress yd7 QTL/gene increase of panicle seeds in
popularly rice grown in Red River Delta.
17
- Xem thêm -
.PDF 
27 
105 
57 
