RESEARCH ON THE GENETIC DIVERSITY OF SOME SOYBEAN VARIETIES THAT ARE RESISTANT TO RUST DISEASE IN DIFFERENT WAYS

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THAI NGUYEN UNIVERSITY
COLLEGE OF EDUCATION

Vu Thanh Tra

RESEARCH ON THE GENETIC DIVERSITY OF SOME
SOYBEAN VARIETIES THAT ARE RESISTANT TO
RUST DISEASE IN DIFFERENT WAYS
Speciality: Genetics
Code: 62 42 70 01

ABBREVIATE BIOSCIENCE DOCTORAL THESIS

Thai Nguyen - 2012


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The study was fulfilled at Thai Nguyen University,
College of Education

Supervisor:

A. Prof. Dr. Chu Hoang Mau
Dr. Tran Thi Phuong Lien

Judge 1:..............................................................
..............................................................
Judge 2:..............................................................
..............................................................

The thesis will be defended before Thesis Evaluation Committee at
Thai Nguyen University level at
:.........................................................................
At:......., ……………… 20.....


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PUBLICATIONS
1. T.A. Pham, C.B. Hill, M.R. Miles, B.T. Nguyen, T.T. Vu, T.D.
Vuong, T.T. VanToai, H.T. Nguyen, G.L. Hartman. “Evaluation
of soybean for resistance to soybean rust in Vietnam”. Elsevier –
Field Crops Research 117, pp. 131–138.
2. Vu Thanh Tra, Tran Thi Phuong Lien, Chu Hoang Mau.
“Evaluation of genetic diversity of 50 Vietnam soybean varieties
resists differently to soybean rust using SSR marker” Biological
Journal. 34(2):235-240.
3. Vu Thanh Tra, Tran Thi Phuong Lien, Chu Hoang Mau. “Study
the genetic relationship of some Vietnamese soybean cultivars
having different responses to rust”. Technology and Science
Journal – Thai Nguyen University. 85(9)/2: 11-16.
4. Vu Thanh Tra, Ha Hong Hanh, Tran Thi Phuong Lien, Chu
Hoang Mau. “Isolation and analysis of proteins from soybean
leaves”. Technology and Science Journal – Thai Nguyen
University 85(01)/1: 303-310.
5. Vu Thanh Tra, Ha Hong Hanh, Tran Thi Phuong Lien, Chu Hoang
Mau. “Study on leaf proteins infected soybean rust using two
dimensional gel electrophoresis” (Submitted to Biotechnology journal)


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INTRODUCTION
1. Preface
Soybean (Glycine max (L.) Merrill) is a short-time industrial plant
with high economic value and helpful to soil improvement. It is easy
to grow and particularly adaptable to various ecological areas.
Soybean seed contains 30-55% protein, and many types of nonreplaceable amino acids, 12-25% lipid and necessary vitamins for
human body. Soybean products are popularly used for different
purposes such as food, cooking oil, functional food and materials for
medicine and industries. Besides high nutrition proportion, soybean is
capable of fixing soybean nitrogen by the symbiosis of bacteria R.
Japonicum on root constituting nodules, improving land efficiently.
Therefore, soybean has been interested in and thrived in many
countries around the world. In Vietnam, soybean is the main plant
encouraged to develop priorities, and to produce after the rice, maize
and peanuts.
Vietnam used to be an exporter of soybean in the 1980s, but so
far our country import millions of tons of soybean annually.
Although the cultivated area increases every year, it has low
productivity and unstable yield, bad resistance to disease and stress.
Insects and in particular, the rust disease directly causes the effect of
the planted area and reduction of productivity, quality of soybean
seed, causing huge economic losses.
The rust disease of soybean are caused by fungus Phakopsora
pachyrhizi and are regarded as one of the main threats on soybean
plants, causing significant damage and reducing from 10-80% the
yield and quality of soybean in many countries in the world,
including Viet Nam.
In recent years, studies on the soybean rust have been carried
out and obtained some significant results, but most researches have
only focused on monitoring the process of disease development,

epidemiological studies, assessing the loss of productivity or


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analyzing disease response. They do not pay attention to learning
about resistance against rust disease. The study of genetic diversity
of soybean groups with different responses to the rust is not only
significant in the conservation of the varieties that are resistant to
disease but also have important implications in the work of selecting
breeder with high quality.
Derived from the above reasons, we made the selection of topic:
"Research on the genetic diversity of some soybean varieties that
are resistant to rust disease in different ways".
2. Objectives
Evaluating the potential response of soybean varieties with rust
disease in order to find out new resistant kinds, identifying the
genetic diversity on the basis of DNA polymorphism analysis,
comparing protein polymorphisms and mapping soybean leaf protein
electrophoresis.
3. Activities
- Assessing soybean’s resistance against rust disease.
- Identifying protein, lipid and amino acid composition in seeds of soybean.
- Analyzing genetic diversity of soybean varieties by using
molecular indicators RAPD and SSR.
- Mapping soybean leaf protein electrophoresis and protein identifier
system by means of one-dimensional electrophoresis, two-way and
identifying the protein on chromatography-mass spectrometry system.
Simultaneously analyzing and comparing the protein diverse
components of soybean with rust infection and resistance.
4. Significant results

- The study has classified the level of rust resistance of 50
varieties of soybean collected in Vietnam into 3 groups: group which
is susceptible to rust, the intermediate group and resistance group.
Especially, the study has discovered six new resistant varieties such
as PMTQ, HSP1, HSP2, CNB, ZG and MTD65. At the same time,


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the biochemical compositions of 50 varieties of soybean have also
been analyzed.
- Using molecular indicator RADP and SSR, the study analyzed
genetic relationship of 50 kinds of soybean, the results showed that
the polymorphism of SSR is at higher level.
- With the method of 2-dimensional electrophoresis, the study has
established 2DE electrophoresis maps of soybean protein in resistant
cultivars DT2000 with 119 points of protein on gene, including 35
proteins identified and classified. Especially, three proteins related to
disease resistance / defense, drought and stress tolerance, have been
found. These are the first data statistics of studying leaf protein
system, mapping protein, identifying the protein in resistant
cultivars. This result plays an important role in understanding disease
mechanisms and suggesting approaches the level of protein.
- With the method of comparing the change in protein expressed
on the 2DE gene, the study also found that there are 8 points of
protein increasing and 1 point decreasing compared to samples in
two rust infected soybean to detect and identify 6/9 of protein. Two
protein samples in two susceptible cultivars (DT12, VMK) have 6
points of protein higher than two rust-resistant cultivars (DT2000,
CBU8325), have recognized and identified 4/6 of the protein. These
are the first figures published in Vietnam when comparing different

expression levels of these proteins in infected and samples, among
the infected and resistant cultivars by electrolysis 2DE combined
identifications of mass spectrometry.
5. Scientific and practical meanings
- The thesis has studied soybean, a meaningful plant to our
agriculture. Modern and highly reliable scientific methods have been
used to study characteristics, biochemical components, DNA
diversity and protein analysis of some kinds of soybean which have
different reaction to rust. The thesis has been seriously and carefully
invested in, which has brought ample results. The thesis will be


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reference to following studies on soybean. Such scientific methods as
DNA diversity analysis with RADP, SSR techniques or mass
spectrum and electrophoresis analytical method are new approaches
which are scientifically meaningful and bring various interesting
information on disease infection and resistance as well
- In fact, the thesis has classified different kinds of soybean
which have different reactions with rust into 3 groups. Among them,
new rust-resistance kinds are PMTQ, HSP1, HSP2, CNB, ZG and
MTD65. The above information is very useful and can be applied in
breed selection and creation.
6. The structure of the thesis
The thesis comprises of 115 pages, which are divided into the
following parts: Introduction including 3 pages, Chapter 1:
Theoretical overview, 29 pages; Chapter 2: Materials and methods,
20 pages, Chapter 3: Results and discussion, 46 pages; Conclusion
and recommendations, 2 pages; Announced works of the author: 1
page. Reference: 14 pages; The thesis uses 15 tables, 19 figures and

121 reference materials in Vietnamese and English.
Chapter 1. OVERVIEW
1.1. Soybean and biochemistry of soybean
1.1.1. Soybean
Soybean (Glycine max (L.) Merrill) is a plant of the legume
(Fabaceae). The branch of Glycine has two sub-branches such as
Glycine and Soja. Originally, soybean comes from East Asia. On
morphological characteristics, soybeans are herbaceous, leaves of
three types: cotyledon, leaf and double leaves. The flowers of
soybean are small without flavor, butterfly shaped. Fruits are the
adjacent fruit, difficult separation, slightly curved, at the young fruit
is green, hairy when ripe with brown. Seeds coming in many shapes:
round, oval, flattened circles... have high nutritional value. On
genetic characteristics, soybean with diploid chromosomes 2n = 40 is
a self-pollinated plant, is less cross pollination. Haploid genome of


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soybean is from 1.29 -1.8 x 109bp. Time of soybean growth is
divided into three categories: early ripening, moderate and late.
1.1.2. Biochemical characteristics of soybean
1.1.2.1. Proteins in soybean seed
In soybean seeds, protein accounts for 12-55%. Protein is stored
in organelles with the function of providing acid amino and nitrogen
sources or the enzymes involved in the process of seed germination.
Protein mainly stored in soybean seed is globulin.
1.1.2.2. Lipids, vitamins and other substances in the soybean seed
In soybean, lipid content accounted for 12-25% of dry weight.
The quality of lipids in soybean seed is very good, so it is widely
used in food processing industry. Soybean seeds have lipid-soluble

vitamins, especially vitamin E. In addition, the soybean seed also
contains other substances such as carbohydrate, minerals, nucleic
acids, growth stimulants.
1.1.2.3. Leaf protein and soybean protein system
The leaf soybean protein is the protein involved in metabolism
and energy metabolism. It is the protein involved in electron
transport process, enzymes, the stored proteins, or proteins involved
in the metabolism and metabolism of amino acids or proteins
involved in resistance and stress.
1.2. Rust disease and rust resistance in soybean
1.2.1. Rust disease in soybeans
Rust disease caused by fungus Phakopsora pachyrhizi is one of
the main diseases in soybean (Glycine max) in Asia and causing
significant damage in the yield of soybeans in many countries.
Signs and symptoms of disease: Signs of the disease are the dots
in the leaf blade about 1 mm, growing gradually to turn yellow and
then brown, the size reaching from 2-5mm with diverse, angular. On
the lower side of blade with the lesions, there appeared brown
powder which is the lower of fungal spores.
Lifespan and infected process: The infected process begins
when spores germinate to form a germ tube on the surface of rim
(diameter of 5-400 micrometers) until the formation of fiber. Lower
spores usually develop for 5-8 days, maximum up to 4 weeks after
infection, the spore process can last 3 weeks.
1.2.2. Disease resistance of soybean rust


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The interaction between host and parasite is under the model of
gene for gene. Up to now, scientists have found that there are five

major genes related to rust resistance in soybean, including gene
Rpp1, gene Rpp2; gene Rpp3; gene Rpp4 and gene Rpp5.
1.3. Methods of analysis of genetic diversity on soybean
1.3.1. The method of using form directives
Form directive is a co-dominant directive, usually expressed as
a characteristic controlled by a single locus such as the gene for
flower color and shape of the seeds, skin.
1.3.2. Method of using biological indicators
The biochemical indicators appear in most polymorphic protein
such as isozyme and stored proteins. These isozymes can be
separated by weight, molecular size and the carrier.
1.3.3. The methods use DNA indicators
1.3.3.1. RAPD indicators
In principle, the RAPD technique bases on PCR reaction by
using short sequenced samples, pairs and randomly cloned DNA
fragments with complementary sequences with the sequence of the
samples. The RAPD markers used in the study of diversity and
genetic mapping of the typical locus include the following: (i)
designing sample, (ii) Preparing DNA samples and run PCR, (iii)
checking cloning products on agarose gene, (iv) Analyzing of results
by specialized software (v) Determining genetic coefficients and
setting the map of the genetic relationship of objects.
1.3.3.2 SSR indicators
Technical SSR (Simple Sequence Repeat) was first detected on
the object by Litt and Luty (1989). The use of indicators SSR in
genetic mapping of the typical locus include the following: (i)
Isolating SSR from the genomic library, (ii) Determining the
sequence of the SSR regions; (iii) Identifying typical sample pairs of
DNA sequences limited by the ends of the SSR; (iv) Multiplying
genomic regions corresponding to PCR using specific samples, (v)

Analyzing the size of PCR products in order to determine the
presence of SSR alleles.


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Chapter 2. MATERIALS AND METHOD OF THE STUDY
2.1. Materials
2.1.1. Materials and methods for collecting infected leaves
Using 50 soybean varieties derived from local Vietnam by the
Center for Development and Research of beans and Agricultural
Genetics Institute and Can Tho University is studying materials.
Including 4 resistant cultivars against rust disease provided by the
Institute of Plant Protection. Some varieties were collected from
local Cao Bang, Ha Giang and Thai Nguyen.
2.1.2. Chemicals
Chemicals were purchased from vendors such as Merck,
Invitrogen, Amersham Pharmacia Biotech, New England Biolabs...
2.2. Research methodology
2.2.1. Method of artificial infection
2.2.2. Methods of biochemical analysis
2.2.2.1. Determination of total lipid
2.2.2.2. Determination of total soluble protein
2.2.2.3 Determination of amino acid in soybean seed
2.2.3. The methods of analyzing genetic diversity in DNA
2.2.3.1. Method for extraction and purification of total DNA
2.2.3.2. Method for determination of DNA by spectral
2.2.3.4 Electrophoresis of DNA on polyacrylamide gene and silver staining
2.2.3.5. Polymorphic analysis by RAPD technique
2.2.3.6. Polymorphism analysis by SSR technique
2.2.3.7. Method of processing data

2.2.4. The formulated analytical methods of protein
2.2.4.1 Separation and extraction of protein from soybean leaves
2.2.4.2. Electrophoresis of SDS-PAGE
2.2.4.3. 2 dimensional Electrophoresis - 2DE
2.2.4.4. Protein staining and image analysis of gene
2.2.4.5. Identify proteins on a 2DE electrophoresis with mass spectrometry


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Chapter 3. RESULTS AND DISCUSSION
3.1. Evaluation of soybean’s resistance to rust disease
Evaluating rust disease resistance of 50 varieties of soybeans,
we have divided into three different groups to react with rust:
Rust resistance group including 13 varieties: PI200492,
PI230970, PI462312, PI459025, DT2000, CBU8325, DT95,
MTD65, CNB, PMTQ, HSP2, HSP1, ZG: the behavior is clearly
resistant to the dark brown lesions, less the number of spores, no
yellow halo, slow speed development of disease. Especially, our
research results show that there are six new resistant varieties found
such as PMTQ, HSP1, HSP2, CNB, ZG and MTD65.
The intermediate group consists of 12 varieties: M103, DT96,
PHCB, PS, PT, NS, MT1, HG2, VK2, CT2, VK3, CT1, the
characteristic of two types of wound infection and resistance. In
particular, there are two popular varieties such as M103 and DT96.
Susceptible group includes 25 varieties: DT12, VX92, VX93, DT84,
V79, SL, CV, VMK, HG, DK, CBD, CB7, QHCB, LVG, TTHT,
DBBT, DTBT, MT2, HG1, CSF, MD , DL, MH, ND, CSGL, HN..
3.2. Analyzing characteristics of biochemical of soybean seeds
3.2.1. Analyzing protein and lipid content in seeds of soybean varieties
The results of analyzing total lipid and soluble protein content

of 50 varieties of soybean were presented in Table 3.1 (not showed).
The results show that between varieties, protein content ranged from
30.5% (cultivar PMTQ) to 43.5% (cultivar DT2000, DT96 and
TTHT). Meanwhile, the lipid content ranged from 11.35% (cultivar
PMTQ) to 17.4% (cultivars DT2000, DT96 and TTHT). The result
of analyzing protein and lipid in 50 soybean’s seeds shows the result
is consistent with the author's research Chu Hoang Mau (2002).
In terms of correlation, between 3 soybeans groups with
different response to rust disease, we didn’t find significant
differences in the levels of lipid and protein between the 3 groups.
However, protein and lipid of the hybrid’ seeds within each group
are higher in the local varieties.


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3.2.2. Analyzing amino acid composition in seeds of some soybean
varieties
We chose 20/50 participated in the study analyzed amino acid
composition in seeds. Of these, there are seven resistant varieties, 4
intermediate varieties and 9 infected varieties including hybrids,
local and imported varieties.
The result showed that the presence of 17 types of amino acid
and amino acid content of each type in each soybean sample was
studied; at the same time it showed that the three groups (resistant,
intermediate, and rust infected) didn’t have significant differences in
total content of 17 amino acids. They ranged from 31.81% - 39.54%,
in which SL (31.81%) with the total of amino acid is lowest and
CNB (39.54%), VX92 (39.51%) with the total amino acid is highest.
3.3. Assessment of genetic diversity of soybean varieties response
to the rust

3.3.1. Analyzing DNA polymorphism of soybean varieties using
RAPD molecular indicator
The results of multiplying the DNA fragments by RAPD reaction
from the genome of 50 soybean varieties showed there were 15
samples within 20 samples expressing polymorphisms. For each
random sample, the number of cloned DNA fragments ranged from 210 fragments with the size from 250bp-2000bp. Total DNA fragments
cloned from the genome of 50 soybean varieties is 3380 fragments, in
which sample M14 has the most DNA replication(335 fragments
DNA) (Figure 3.1) and the least is sample M8 (9 fragments).
The result in table 3.2 indicates that the total number of
polymorphic DNA fragments of 20 random samples in analysis of 50
soybean varieties is 113 fragments. It included 73 polymorphic
fragments (count for 64.6%) and 40 fragments without
polymorphism (35.4%). Seven samples (M2, M3, M4, M9, M10,
M13 and M18) are completely polymorphic (100%) and five samples
(M5, M8, M12, M16 and M17) are not polymorphic (0%). In which,


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there are 14/20 samples with polymorphic fragments of 50%, sample
M14 has polymorphic fragments with 50% (Fig. 3.1), sample M18
has polymorphic fragments with 100% (Fig. 3.2) (not showed).
This result is consistent when analyzing polymorphic
information content in the PIC value (table 3.2). Specifically, the
value of sample M14 PIC was 0.27 (polymorphic rate of 50%) and
PIC values of the sample M4 was 0.86 (high polymorphism), in
which 13/20 samples for value PIC ≥ 0.5.
Table 3.2. The ratio of the polymorphic fragments and value PIC
of the samples
Total

Total
%
%
No Sample PIC polymorphic polymorphic No Sample PIC polymorphic polymorphic
fragments fragments
fragments fragments
1
M1 0.74
6
83.3
11
M11 0.80
6
83.3
2
M2 0.76
5
100.0
12
M12 0.00
3
0.0
3
M3 0.67
6
100.0
13
M13 0.80
5
100.0

4
M4 0.86
4
100.0
14
M14 0.27
8
50.0
5
M5 0.00
2
0.0
15
M15 0.35
6
83.3
6
M6 0.85
8
37.5
16
M16 0.00
4
0.0
7
M7 0.79
7
85.7
17
M17 0.00

3
0.0
8
M8 0.00
4
0.0
18
M18 0.50
4
100.0
9
M9 0.84
10
100
19
M19 0.77
8
87.5
10 M10 0.75
4
100
20 TRA4 0.82
10
80.0
Total
113
64.6

Genetic relationship of 50 soybean varieties
Between 50 soybean varieties at the molecular level on the basis

of analyzing RAPD with 20 random samples, we have established a
tree diagram of the 50 soybean varieties (Figure 3.3). The result
showed that 50 soybean varieties distributed into 2 major branches
(Branch I and Branch II). Branch I has only VK2 and DT12 with
genetic distance compared to 48 remaining varieties of 21% (1 0.79). Branch II includes the remaining 48 soybean varieties with
genetic distances ranging from 0% to 15%.


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Compared with the study of Nguyen Duc Thuan et al. (2006)
which was conducted on 30 varieties of soybean (from Mekong
Delta Rice Institute) with 13 pairs of samples RAPD, the result
showed that there are 9 polymorphic samples, and four groups
(groups A, B, C, D) are divided into similar coefficient from 0.8 to
0.98.

Branch II

Branch I

Figure 3.3. Tree diagram of 50 soybean varieties bases on genetic
similar coefficient and UPGMA clustering method
3.3.2. Analyzing DNA polymorphism of soybean varieties uses
SSR molecular indicator
In this study, we used 15 pairs of sample SSR in SSR PCR
reaction to clone DNA fragments from the genomes of 50 soybean
varieties, electrophoresis image of product SSR of 15 pairs (Figure
3.4A, 3.4B, 3.4C and 3.4D). Of the 15 indicators SSR used for
analysis of genetic diversity in 50 soybean varieties there are 14
indicators for genetic polymorphisms (unless Satt460 without

polymorphism result).
Statistics of cloned DNA fragments, we detected 81 alleles at 14
locus, the number of polymorphic alleles at each locus varied from 4


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to 8 and six alleles on average (table 3.3). Indicator Satt489 has the
most number of alleles with 8. Value PIC (polymorphism
information content) of the indicators SSR varied from 0.473 to
0.798, reaching 0.729 on average, and amplitude of the coefficient of
diversity between the indicators is relatively narrow (0.473 to 0.798)
(Table 3.3). This result for polymorphic is higher when comparing to
a study by Tran Thi Phuong Lien et al. on the same object (0.6326).
Comparing with some other authors in the world, the result in genetic
analysis of DNA polymorphism in our soybean is similar to the study
of Abe et al. (2003) on 131 varieties from 14 Asian countries
(average coefficient diversity of 0.782), higher than the research of
Narvel et al. (2000) of 74 soybean varieties in North America
(average coefficient diversity of 0.56).
Table 3.3. The result of analyzing genetic diversity by indicator SSR
Number
PIC
of Alleles
1
Satt005
(ATT)19
D1b
7
0.770
2

Satt009
(ATT)14
N
5
0.690
3
Satt042
(ATT)27
A1
4
0.473
4
Sat_064
(AT)34
G
5
0.767
5
Satt146
(ATT)17
F
7
0.798
6
Sct_187
(CT)10
G
6
0.757
7

Satt150
(ATT)20
M
5
0.686
8
Satt173
(ATT)18
O
5
0.767
9
Satt175
(ATT)16
M
7
0.798
10
Satt373
(ATT)21
L
6
0.753
11
Satt431
(ATT)21
J
5
0.692
12

Satt489
(ATT)23(GTT)
C2
8
0.784
13
Satt557
(ATT)17GAT
C2
5
0.707
14
Satt567
(ATT)14
M
6
0.757
81
0.729
In the 15 pairs of sample SSR, 7 pairs are often used to study
genetic diversity are Satt_042, Satt_005, Satt_146, Satt_173,
No

SSR

SSR Form

Bond

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Satt_175, Satt_009 and Satt_431, with many alleles: from 7-11
alleles. The result of our study shows that these indicators for alleles
range from 4-7 alleles. The level of polymorphism corresponding to
the genetic variation among soybean samples showed genetic
diversity exists in the soybean, but not too large. Highest similar
coefficient was found between the SL and HG1 soybean of 0.97.
This relationship is supported by the tree diagram through group
analysis result (Figure 3.5).
Analyzing tree diagram of Figure 3.5 shows that 50 soybean
varieties were divided into two branches, the genetic distance
between them was 29% (1-0.71):
(1) The first branch, including varieties of rust infection and an
intermediate cultivar VK2, can be divided further into two subbranches. The first sub-branch (group I) includes 9 varieties DBBT,
CV, CSGL, DL, HG, QHCB, HG1, SL and VX93 with genetic
distance close together, with similar coefficients ranging from 0.802
to 0.975. The second sub-branch divided into two small branches,
including: (i) Small sub-branch I has varieties (group II) such as
CSF, MH, DTBT, VMK, LVG, HN, CBD, ND, V79, DT84, DT12,
and (ii) Small sub-branch II has varieties (group III): MD, MT2,
CB7, VX92, TTHT and VK2, with similar coefficient of 0.65 to
0.95.
(2) The second sub-branch includes resistant cultivars and
intermediate varieties. This branch also has two sub-branches: the
first sub-branch consists of the varieties (group IV) such as DT96,
CT2, HG2, VK3, MT1, CT1, PS, NS, M103, PT and PHCB with
similar coefficient of 0 , 75 to 0.95, meanwhile, the second subbranch is split into two small sub-branches. Small sub-branch I has
varieties (group V) such as CBU8325, ZG, CNB, HSP1, PI462312
with similar coefficient from 0.82-0.95. Small sub-branch II has
varieties (group VI) such as MTD65, DT2000, HSP2, PI230970,

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PI459025, PMTQ, DT95, PI200492 with genetic distances in the
range from 0.68 to 0.95.

Brand II

Sub brand II
Sub brand I

Sub brand II

Brand I

Sub brand I

Figure 3.5. Tree diagram of the relationship of 50 soybean
varieties have different reactions to rust disease based on SSR
molecular indicator
The correlation between RAPD and SSR is in the analysis of soybean
To compare the correlation of the two molecular indicators
RAPD and SSR with the polymorphic analysis of 50 soybean, we
noticed that using indicator RADP with 15/20 (75%) samples of
polymorphism, meanwhile, indicator SSR of polymorphism is higher
than 14/15 samples (count for 93.3%). This result can be explained
by the short size and randomness of 20 samples RAPD, so they just
start a random pair with a DNA genome. Meanwhile, the sequence of
SSR is the repeat sequence, studied and known in advance, they
repeat many times in the body; therefore the level of polymorphism

of SSR is higher.
In terms of value PIC, indicator RAPD has PIC ranging from
0.27 to 0.86 while the index of this indicator SSR varied from 0.473
to 0.798. Thus, using SSR makes the amplitude of coefficient
relatively narrow diversity compared to RAPD. On the other hand,


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when using the tree diagram to analyze the genetic diversity, we
noticed that in both RAPD and SSR of 50 soybean varieties are
classified into two large branches with genetic distance, respectively
21 % and 29%, coefficient of genetic variation of SSR is more
demonstrative than RAPD. This result also reflected in large
branches and sub-branch of the 50 soybean using two different
indicators. The result in RAPD is classified into two main groups,
while SST of the 50 varieties are classified into five small groups (I,
II, III, IV, V) with many similar coefficient (Figure 3.5). This result
confirms that the method using SSR is higher and more reliable; this
is the first base to search Vietnam soybean varieties carrying each
specialized resistance gene.
3.4. Analyzing polymorphism of leaf protein of several soybean
varieties with different resistance to the rust
To reach the soybean leaf proteome, we have chosen two pairs
of soybean expressing for the antagonistic rust: imported soybean
pairs are widely produced (DT12 and DT2000) and local soybean
pairs (VMK and CBU8325). Soybean leaves infected with spores
after 1, 3, 6, 9 days are closely monitored. On the leaves of 1, 3 days,
there are no lesions. On the sixth day leaves, they begin to appear,
but 9 days have sporadic lesions blurred. This is the time to find a
powerful expression of proteins such as protein-related PR10 in

soybean leaves by 2-DE electrophoresis and mass spectrometry.
Thus, we use leaves 6, 9 days after infection to study.
3.4.1. Mapping the proteome of soybean leaves by 2-dimensional
electrophoresis technique
3.4.1.1. Separating soybean protein
Soybean leaf protein of 6 and 9 days of VMK, DT12,
CBU8325 and DT2000 (they are not infected sample) were separated
by 2-DE and stained by coomassie G250. Figure 3.6 illustrates the 2DE electrophoresis images of leaves protein DT2000 at the time of 6
days (Figure 3.6A) and 9 days (Figure 3.6B).


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Figure 3.6. 2-DE Electrophoresis images compares to protein
expression levels in leaves of soybean DT2000 at the time of 6
days (A) and 9 days (B). The magnified area expresses the
comparison of different protein over the time
The result shows that soybean DT2000 with 119 points of
protein is detected on 2DE electrophoresis images in 9 day sample
while 6 day sample only has about 103 proteins. This result proved
that leaf protein was expressed more in time of 9 days. Proteins
separate similar positions very well, many proteins form streaks, or
the corresponding position might be due to the same protein but have
different isoform. Soybean leaf proteins are separated on gradient
strip bar with pH 3-10 and the length of 7cm, but electrophoresis
images of proteins concentrated at about pH 5 to 8. This is quite
reasonable, because most of the protein located in the range pH with
slightly alkaline acidic or neutral. On molecular weight, the protein is
widely distributed, there are many proteins with molecular weights>
120 kDa, while some proteins are distributed in areas with low

molecular weight (<14 kDa), protein with density in the region 25
kDa and 80 kDa. In some areas, protein expression levels deep, even
into streak, protein plays important role in the function of leaves,
they have high levels and are created frequently in leaves as protein
RuBisCO or membrane proteins in the photochemical system.


20
3.4.1.2. Mapping electrophoresis of soybean leaf protein
In this experiment, we applied the method of 2DE electrophoresis
for mapping protein electrophoresis of soybean leaf in rust resistant
varieties DT2000 and compared with other varieties such as DT12,
CBU8325 and VMK.
DT2000

VMK

CBU8325

DT2000

Figure 3.7. Compare the diversity of soybean leaf protein of
varieties DT12, DT2000, CBU8325 and VMK
The result of Figure 3.7 shows 2DE electrophoresis of soybean
leaf protein in varieties DT2000. On the electrophoresis of soybean
leaf protein, we noticed that there are 109 points of protein found,
their molecular weight distributed from 6 kDa to 80 kDa, pH ranges
from 4 to 9, in which focused primarily protein from pH 5 to 8.
Points distributed protein stability through the running times of
different electrophoresis. Each protein has molecular and charge

weight characterized by measuring the overall electrophoresis with a
certain position on the gene to form a map of protein electrophoresis.
3.4.1.3. Identifying soybean leaf protein


21
To identify the proteins on the electrophoresis, DT2000 has
119 points of proteins, some of them are isoform so we only select
80 points separately to analyze protein identification. As a result, the
80 points selected proteins to analyze by mass spectrometry, 35
proteins were identified (Table 3.4, not showed). 35 soybean leaf
proteins were identified in the sample DT2000, points of proteins are
identified to fluctuate from 65 to 438 points, while the overall
volume of distribution ranges from 6.5 kDa to 57 kDa.
In the world, the research and map of protein electrophoresis as
well as identifying protein of soybean leaves has been initially
carried out in recent years. But no other publication carried out
analysis of soybean leaf protein with rust disease resistance as well
as electrophoresis basing on map system and identified proteins are
common as a basis for comparison, the study of disease related
symptoms. Our result is one of the first statistics, in which protein on
the gene with 119 points, we identified the 35 protein in soybean rust
disease resistance DT2000, specially related protein 3 to their
defenses / plant disease resistance have been found and needs to be
studied deeply.
3.4.1.4. Grouping of soybean leaf proteome
Using bioinformatic tools GO to predict protein function on the
database EBI and Swiss-Prot, of the 35 protein in soybean leaves is
consistent with the most basic components in the leaves, in which
protein have photosynthetic function with the highest proportion of

29%, then the energy metabolism proteins of photosynthesis cycle
(17%), and the proteins involved in glycolysis system (11%). This
result is consistent, so the leaf protein groups involved in complex
optical systems such as optical systems of PSI, PSII, protein and
other chloroplast proteins involved in glycolysis process at high
percentage in the leaves. These proteins closely related to the
function of the leaf that is receiving sunlight and converted into
sugar during photosynthesis. In addition to proteins involved in


22
photosynthesis, in this result, we also recognize that the group of
storage proteins (9%), proteins involved in muscle metabolism (8%),
electrical transport proteins death (8%), translation (8%), disease
resistance (8%), and unknown (6%). Specifically, in nine groups of
proteins have been classified (see Figure 3.8), disease-related
proteins and resistance, accounting for 8% (Disease / defense).
Ascorbate peroxidase2 is an enzyme detoxifying the peroxide
using ascorbate as the substance. Some researches noted that the
mutations appear to affect expression levels of ascorbate peroxidase 2,
this protein react with the salt tolerant and resistant stress process.
However, their roles are for rust resistance not recorded in any report.
This protein has an important role in rust-resistance of varieties DT2000
or not needed but separate surveys to elucidate further.
Translation protein
Storage protein
Proteins involved in
muscle metabolism

Protein involved in

photosynthesis

Unknown Protein involved in disease resistance
Protein involved in electron
transport
Protein involved in
glycolysis process

Energy

Figure 3.8 Grouping function of soybean leaf protein DT2000
Catalase is an important enzyme found in most other creatures.
This enzyme catalyzes for the decomposition of hydrogen peroxide by
measuring the body detoxify. The research of Balestrasse et al. has
shown that when cadmium stress, the roots and nodules of soybean
expression of this enzyme. Due to their important role in addressing the
unique, and so enhanced the expression of this enzyme against cadmium
stress was confirmed. In other hand, the study by Vasconcelos et al.
(2009) pointed out that when experiencing drought stress, the enzyme
catalase and ascorbate peroxide of expression has changed to help plants
adapt to dry environment. Meanwhile, the stress-induced protein of
group-related protein PR10 pathogens, they are believed to play a role in


23
cystic disease to bear against said medium in soybean (soybean
nematode syst-SCN), diseases popular in America. The authors believe
that this protein expression levels have increased significantly and in a
long stint at the roots of soybean plants infected. As a result, very
important role of this protein in the British defenses and help the body

cope with plant diseases have been described.
3.4.2. Studying polymorphism of the soybean protein by twodimensional electrophoresis technique
3.4.2.1 Comparing protein expression levels in rust infected soybean
In this study, soybean leaf protein in DT12 and VMK at the time of
9 days after exposure of the control and experimental samples, separated
by 2-DE, genes were stained with coomassie G250 (Figure 3.9). The
result showed protein points distributed mainly in the pH from 5 to 9,
many proteins form streaks have the same mass but different charge
(isoform). This is because they have the same protein but different
charge states with the different isomers.
In both varieties DT12 and VMK, test samples with 9-points of
protein indicates the level of protein changes (increase/decrease 1.5
times), including 8 points (points 1, 2, 3, 4, 5, 6, 7 , 9) with
increasing concentration and a point (point 8) with reduced
expression levels when compared to sample (Figure 3.9). Notably, in
addition to four points (2, 6, 8 and 9) are large protein remaining 5
points (1, 3, 4, 5, 7) are relatively low in protein.

Figure 3.9. Comparing the diversity of soybean leaf protein and
the experiment in the infected varieties of 9-day DT12 and VMK


24
In soybean leaves, the concentration of large proteins, mostly
proteins involved in photosynthesis (RuBisCO and the
photochemical system protein) or protein reserves in the leaves. The
increase (or decrease or even disappear) the concentration of protein
in some samples compared to controls was infected by rust (DT12,
VMK) proved in the same transformation has had on metabolic level,
the change of gene activity leads to protein content of some process

of change as infection occurs. The change of the content of these
proteins is related to the disease process or other physiological
responses of plants by the consequences of infection.
Chromatography with mass spectrometry ESI-Q-TRAP, differential
protein spots were identified as Table 3.5
The recognition result showed that between the control samples
and infected samples of two varieties DT12 and VNK have proteins
related to photosynthesis cycle (covered subunit precursor PSI, PSI
reaction center subunit IV A, RuBisCO large subunit), the storage
proteins (28 kDa protein stem, vegetative storage proteins) or proteins
involved in metabolism (ribose 5-phosphate isomerases). Especially at
the point 3,4,5 is not recognized on the basis of protein data.
Table 3.5. Identification of proteins with different
expression levels between experimental and control samples in
two infected samples DT12 and VMK
TNo

Name of protein

1

PSI PsaN subunit
precursor
PSI reaction centre
subunit IV A
Unknown
Stem 28 kDa protein
Vegetative storage
protein
Ribose 5-phosphate

isomerase
Rubisco large subunit

2
3
4
5
6

7

Spot

Registration
Q’ty of
Mark
no
kDa

Function

Increasing/
Decreasing

Increasing

1

gi|5902586

212

15.4

Photosynthesis

2

gi|5606709

179

16.3

Photosynthesis

3, 4, 5
6

gi|169898

128

29.0

Storage

7

gi|170088

122

32.

Storage

8

gi|15285625

89

20.1

Metabolism

9

gi|3114769

235

54.2

Increasing
Increasing
Increasing
Increasing
Increasing

Creating energy
in process of
Decreasing
fixing carbon


25
Currently, there have been some studies of the protein in soybean
leaves, but the results comparing the protein between infected and
non-infected leaves are almost limited. Therefore, according to our
knowledge, the fact that photosynthesis protein and the protein was
increased in reserve, proved of activity at the time of 9 days of
infection, while the related protein metabolism by reduced . As we
know, when competition becomes infected Mature trees will soon pass
and the resulting reduction in photosynthesis, as were spots yellow,
cream shed and grain yield. However, in the period of 9 days of
infection, according to our very understanding of this phase of the
plant may have on physiological changes, such as biochemical process
of photosynthesis takes place strength, protein reserves are synthesized
enhanced in a specific time for trees to mature early. Evidence is the
concentration of these proteins are involved in photosynthesis as
covered subunit precursor PSI, PSI reaction center subunit IV A,
RuBisCO large subunit and the storage proteins such as stem 28 kDa
protein, increased vegetative storage protein. Currently, no studies that
have found no alterations noted concentrations of protein are in an
early stage of infection 9 days. Therefore, our research has supplied
the initial population data simultaneously hint system protein research
interests is to be able to approach closer to the disease mechanism, in
addition to studies of the genetic instructions.

3.4.2.2. Comparing protein expression levels between infected and
soybean rust resistance
To find out the changes of the protein between some soybean
varieties infected and resistant, we have selected and analyzed 02
varieties infected (DT12 and VMK) and 02 resistant (DT2000 and
CBU8325). The results show, there are 06 points in the same infected
leaf protein concentrations increased over the resistance.

Figure 3.10. Comparing protein diversity nine-day-old soybean leaves
between varieties infected with rust DT12, VMK and rust-resistant
varieties DT2000 and CBU8325