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

“Evaluation of genetic diversity of 50 Vietnam soybean varieties resists differently to soybean rust using SSR marker” Biological Journal.. The study of genetic diversity of soybean gro

THAI NGUYEN UNIVERSITY

ABBREVIATE BIOSCIENCE DOCTORAL THESIS

Thai Nguyen - 2012

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

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)

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 non-replaceable 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

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,

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

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

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

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

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

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

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

10 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,

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

No Sample PIC

Total polymorphic

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%