VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FALCUTY OF BIOTECHNOLOGY ------- ------- GRADUATION THESIS TOPIC: “INVESTIGATION OF RESISTANCE TO LEAF MOLD DISEASE ON TOMATO BY DNA MARKER” Ha Noi- 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FALCUTY OF BIOTECHNOLOGY ------- ------- GRADUATION THESIS TOPIC: “INVESTIGATION OF RESISTANCE TO LEAF MOLD DISEASE ON TOMATO BY DNA MARKER” Student : Pham Quy Tung Student’s code : 637087 Class : K63CNSHE Major : Biotechnology Supervisor : Pham Thi Dung, Ph.D Ha Noi- 2022 GUARANTEE I hereby declare that this thesis is completely done by my own scientific research under the guidance of Mrs. Pham Thi Dung Ph.D, Faculty of Biotechnology - Vietnam Academy of Agriculture. All figures in this thesis are truthful, and do not duplicate the results of any previous graduate report. Graduation thesis has references to documents, citation information is recorded in the references section.
I take responsibility for my promises to the board and the university. Hanoi, December 2022 Student Pham Quy Tung i Acknowledgements First words, in the process of completing this research paper, I have received great deal of helps, guidance and encouragements from teachers and friends. First of all, I would like to express my deepest thanks to my supervisor, Mrs. Pham Thi Dung who given me suggestions on how to shape the study and always been most willing and ready to give my valuable advice, helpful comments as well as correction of my reseach paper.
Next, I would like to express my gratitude to all teachers in Biotechnology falcuty- Vietnam university of Agriculture for their lectures 5 years that help me much in completing this paper. Besides that, I’m so thankful for all the support and encouragement of Mr. Phan Huu Ton and your employees at the Center for Conservation and Development of Plant Genetic Resources who helped and created the most favorable conditions for me during my internship. Last but not least, I would like to thank my family and my friends who have always encouraged, supported and helped me to complete this paper Ha Noi, December 2022 Student Pham Quy Tung ii TABLE OF CONTENTS GUARANTEE.
ii TABLE OF CONTENTS. iii LIST OF TABLES. v LIST OF FIGURES. viii CHAPTER I: INTRODUCTION.
Aim and requirements of the study. 2 CHAPTER II: LITERATURE REVIEW. Introduce about tomato. Characteristics of tomato plants.
Popular varieties of tomatoes. The situation of tomato production in the world and in the country. The situation of tomato production in the world. The situation of tomato production in Vietnam.
Leaf mold disease on tomato plants. Symptoms and signs of disease. Causes of leaf mold disease. Developmental features of the disease.
Measures to prevent leaf mold disease. Status of research on resistance to leaf mold disease in tomatoes. Studies on resistance to leaf mold in the world. RESEARCH MATERIALS AND METHODS.
Place and time of study. Methods for assessing important agronomic traits. Evaluation of resistance to leaf mold disease of selected tomato varieties by using molecular markers. 25 CHAPTER IV: RESULTS AND DISCUSSION.
Survey on genetic diversity of researched tomato varieties. Morphological survey of tomato samples studied .1 Survey on some growth characteristics, morphological characteristics and structure of tomato .2 Investigate the characteristics of inflorescence structure, bloom characteristics, yield and yield factors.3 Fruit morphology survey. Results of PCR method to detect Cf-19 gene in the studied tomato group. DNA total of leaf tomato.
Determination of DNA content by nano drop. Results of PCR products detecting Cf-19 gene .3 Selection of a number of promising seed varieties with good characteristics. CONCLUSIONS AND RECOMMENDATIONS. 49 iv LIST OF TABLES Table 2.1 Nutrition facts in 100g tomatoes .2 Area harvested, yield and production over the world of tomatoes .1 Some growth characteristics, morphological characteristics and tree structure of the studied varieties .2 Some characteristics of inflorescence structure, bloom characteristics, yield and yield factor .3 Survey about some characteristics of fruit morphology .4 Survey about some characteristics of fruit quality.5 Determination of DNA content by nano drop .6 Results of PCR products detecting Cf-19 gene by primer qrt- 5760A .7 Results of PCR products detecting Cf-19 gene by primer qrt- 5780A .8 Results of PCR products detecting Cf-19 gene by primer qrt- 5760A and primer qrt-5780A.9 Selection of promising varieties.
42 v LIST OF FIGURES Figure 2.1 Structure of tomato .2 Leaf mold caused by Passalora fulva on commercially and hom grown tomatoes. The disease develops rapidly, spreading from lower to upper leaves .3 Leaf mold on a tomato leaflet. Yelloe diffuse spots or patches are present on the upper leaflet surface and olive green to brown fungal spores are found on the corresponding underside of the leaflet .4 The Cf-9/Cf-4/Cf-1 (Milky Way) and Cf-2/Cf-5 Loci in Tomato .5 Recognition Specificity in Cf-2 and Cf-5 .2 DNA total of leaf tomatos.3 Results of PCR products detecting Cf-19 gene by primer qrt- 5760A .4 Results of PCR products detecting Cf-types gene by primer qrt-5780A. 41 vi ABBREVIATIONS Avr: Avirulence gene C.
fulvum: Cladosporium fulvum CTAB: Cetyl Trimethyl Ammonium Bromide Hcr2: homologues of Cladosporium resistance gene Cf-2 Hcr9: homologues of Cladosporium resistance gene Cf-9 LRR: leucine-rich repeat R gene: resistance gene vii ABSTRACT Leaf mold disease, caused by the fungus Passalora fulva, is one of the most devastating diseases in most tomato growing regions worldwide. To date, many disease resistance genes have been published and the use of natural resistance genes has been identified as one of the most effective solutions in breeding tomato varieties resistant to leaf mold disease. This study used DNA markers to detect Cf-19 resistance genes in 20 tomato cultivars stored at the Center for Conservation and Development of Plant Genetic Resources - Vietnam National University of Agriculture. To select promising tomato cultivars, selection was based on important agro-biological characteristics such as fruit set rate, fruit shape index, and presence of resistance genes.
The result show that there were 4 varieties suitable for breeding and producing 2- *, 10- , 2- and KT. viii CHAPTER I: INTRODUCTION 1. Rationale Tomato (Lycopersicon esculentum Mill), belonging to the Solanaceae family, native to Middle and South America, is one of the most widely grown and consumed fruit vegetables today. Tomato is a crop with high economic value, promoting investment and development to improve the yield and quality of tomato products in order to increase income for farmers, contributing to hunger eradication and poverty alleviation is one of the current agricultural development policies of our country.
However, the tomato production process in our country is still facing many difficulties, one of the causes limiting the production development and reducing tomato yield is leaf mold disease. Tomato leaf mold disease has been studied for a long time, but so far the disease is still a common pest and greatly affects the yield and quality of tomatoes. Tomato leaf mold disease caused by the fungus Passalora fulva (previously called Fulvia fulva or Cladosporium fulvum) is one of the most harmful diseases in most tomato growing areas in Vietnam and around the world. The disease can cause damage throughout the growing period of the plant.
The fungus damages many parts of the tomato plant such as stems, leaves, flowers and fruits. Spores of the fungus can survive for 6 months to a year above ground at room temperature. Tomato production is mainly focused on winter and winter-spring crops, these are the most suitable time for leaf mold disease to arise and develop, causing great losses to economy and productivity. Currently, disease control is mainly by chemical methods.
But it also comes simultaneously with the consequences of environmental pollution, creating resistance to diseases and affecting human and livestock health. Therefore, finding safe and effective measures to prevent leaf mold disease on tomatoes is one of the urgent task nowaday. Breeding new varieties with genetic resistance to Passalora fulva is the most effective way to minimize the damage caused by leaf mold disease. 1 Therefore, effective use of new resistance (R) genes by breeders is essential.
Over the decades, researchers have identified resistance genes for leaf mold disease, including Cf-2, Cf-4, Cf-5, Cf-8, Cf-9, Cf-11 and Cf-19. Although there have been some lines of tomato resistant to leaf mold disease have been selected, but the results are very limited and need to be further researched. Starting from the above basis, in order to serve the work of selecting and creating tomato varieties carrying the gene for resistance to leaf mold disease, we carried out the reseach: ―Investigation of resistance to leaf mold disease on tomato by DNA marker” 1. Aim and requirements of the study 1.
Aim Detecting genetic resources to identify tomato varieties with good yield and quality, containing genes for resistance to leaf mold disease for breeding work. Requirements Evaluation of some important agrobiological traits in tomato corporation. Using molecular markers to identify leaf mold resistance genes in tomato consortium 2 CHAPTER II: LITERATURE REVIEW 2. Introduce about tomato 2.
The origin Tomato originated in Peru, a country in South America, is vegetable of the family "Solanaceae", scientifically known as "Lycopersicon sculentum Mill". Tomato was popularized in China and Southeast Asian countries around the 17th century and became one of the important vegetables in many countries. However, Mexico was the first country to cultivate the tomato plant. Nutritional value Tomatoes are succulent, watery (about 90-95%), have a different number of cells (also known as seed chambers) (2-3 sometimes more).
Fruit shape can be flat, round, elliptical, long oval, plum shape, pear shape. (Trinh Thi Le,2016) Tomatoes are sweet, cool, have the effect of creating energy, balancing cells and increasing vitality. In ripe tomatoes contain many nutrients such as sugar, vitamins A, C. phosphorus, calcium, copper, magnesium, β-catoten.
Due to its rich nutritional composition, tomatoes have become a popular dish in many countries and are widely grown food crops across the continent.1 Nutrition facts in 100g tomatoes Element Unit Content Element Unit Content H2O g 94.88 Vitamin PP Mg 0.50 Zinc, copper, Total digestible fiber G 1.10-100 magnesium Calcium Mg 10-12.09 Β-caroten Mg/kg 4.00 Lycopene Mg/kg 20-200 Vitamin C mg 40. Characteristics of tomato plants Tomato is a diploid plant with 2n=24 chromosomes, which is characterized by the following characteristics: Roots: The root system is clustered, deeply rooted and strongly branched, the ability to develop secondary roots is very large. After 2 months of planting, the roots were widely distributed in the topsoil with a thickness of 60 cm, most concentrated in the soil layer 30 cm from the surface (Duong Hong Dat, 2003). Under field conditions, tomato roots can be eaten up to 1.3 m wide and up to 1 m deep (Thompson et al.
With such a large root mass, tomatoes are classified as drought tolerant. The root system is shallow or deep, strong or weak growth is related to the degree of branching and development of the aboveground parts. Therefore, it is necessary to prune the branches, press the appropriate tops for the favorable development of tomato roots. Stem: The stem is round, succulent, covered with many dragons, when the tree is large, the trunk turns to wood.
The characteristic of tomato is that it spreads around or grows into a bush. Based on the growth characteristics, tree height can be classified into 3 types: dwarf type (under 65cm), medium type (65-120cm), tall type (from 120-200cm). In the process of growing, tomato plants will grow a lot of axillary buds making the tree dense, so in production it is necessary to prune branches to concentrate nutrients on fruit.( Dinh Thi Hue, 2013) Leaves: Tomato leaves are the morphological feature that distinguishes one variety from another. Most tomato leaves are compound leaves, each complete leaf consists of 3-4 pairs of leaflets.
In between pairs of leaflets there is also a middle leaf, on the base of the leaflet there are small leaves called lateral leaves.