VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------- GRADUATION THESIS TITLE: “INVESTIGATION OF RESISTANCE TO TOBACCO MOSAIC VIRUS DISEASE ON TOMATO BY DNA MARKER” Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------- GRADUATION THESIS TITLE: “INVESTIGATION OF RESISTANCE TO TOBACCO MOSAIC VIRUS DISEASE ON TOMATO BY DNA MARKER” Student : Nguyen Thi Hong Hai Student’s code : 637410 Class : K63CNSHE Major : Biotechnology Supervisor : Pham Thi Dung, Ph.D Hanoi - 2022 GUARANTEE I hereby declare that this thesis is completely done by my scientific research under the guidance of Mrs. Pham Thi Dung Ph. Faculty of Biotechnology - Vietnam National University of Agriculture. All figures in this thesis are true and do not duplicate the results of any previous graduate report.
The graduation thesis has references to documents, citation information is recorded in the references section. I take responsibility for my promises to the faculty and the university. Hanoi, December 2022 Student Nguyen Thi Hong Hai i Acknowledgments First, in completing this research paper, I received plenty of help, guidance, and encouragement from teachers and friends. First of all, I would like to express my deepest thanks to my supervisor, Mrs.
Pham Thi Dung who has given me suggestions on how to shape the study and has always been most willing and ready to give my valuable advice, helpful comments as well as correction of my research paper. Next, I would like to express my gratitude to all teachers in the Biotechnology faculty- at the Vietnam National University of Agriculture for their lectures for 4,5 years that help me 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 thesis.
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 Hanoi, December 2022 Student Nguyen Thi Hong Hai ii TABLE OF CONTENT GUARANTEE. ii LIST OF TABLES. v LIST OF FIGURES. viii CHAPTER I: INTRODUCTION.
Lỗi! Thẻ đánh dấu không được xác định. Aims and requirements of study. 3 CHAPTER II: LITERATURE REVIEW. Characteristics of tomato plants.
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. Tobacco mosaic virus disease on tomato.
Symptoms and signs of disease. The cause to the tobacco mosaic virus disease. Disease Cycle and Epidemiology. Status of research on resistance to tomato mosaic virus disease in tomatoes.
15 CHAPTER III: RESEARCH MATERIALS AND METHODS. Place and time of study. Methods for assessing important agronomic traits. Evaluation of resistance to tomato mosaic virus disease of selected tomato varieties by using molecular markers.
22 CHAPTER IV: RESULTS AND DISCUSSION. Survey on genetic diversity of researched tomato varieties. Growth stages of tomato plants. Results on some structural features.
Investigate the characteristics of inflorescence structure, bloom characteristics, yield, and yield factors. Results of the PCR method to detect the Tm-2 gene in the studied tomato group 4. Test of DNA total. Determination of DNA content by OD spectroscopy of total DNA.
Results of PCR products detecting Tm-2 gene. 35 CHAPTER V: CONCLUSIONS AND RECOMMENDATIONS. 40 iv LIST OF TABLES Table 2.1 Composition of Dry Matter Content of Tomato5 Table 2. Area harvested, yield, and production over the world of tomatoes .1 Name of tomato varieties stored at the Center for Conservation and Development of Plant Genetic Resources.
PCR reaction components. PCR conditions for different primer pairs. The stages of growth and development of tomatoes. Some growth characteristics, morphological characteristics, and tree structure of the studied varieties.
Some characteristics of inflorescence structure, bloom characteristics, yield and yield factor. Determination of DNA content by OD spectroscopy of total DNA. Selection of promising varieties. 38 v LIST OF FIGURES Figure 2.
Anatomical morphology of tomato stem, flower, fruit. Tomato production of countries in the world in 2020. Tobacco mosaic virus disease on tomato leaf. Signs of tobacco mosaic virus disease on tomato leaf and fruit.
The rod-shaped virus particles of TMV. A single TMV particle. Transcriptional process of TMV. Test of DNA total .2 Results of PCR products detecting Tm-2 gene by primer Tm2RS-2 35 Figure 4.3 Results of PCR products detecting Tm-2 gene by primer Tm2RS-3 36 Figure 4.4 Results of PCR products detecting Tm-2 gene by primer Tm2S-1 .5 Results of PCR products detecting Tm-2 gene by primer Tm2S-2.
37 vi ABBREVIATIONS ToMV Tomato mosaic virus TMV Tobacco virus MAS Marker-assisted selection CAPS Cleaved amplified polymorphic sequence SNP Single nucleotide polymorphism RdRP RNA-dependent RNA polymerase MP The movement protein FAO The Food and Agriculture Organization of the United Nations CP Coat protein RAPD Random amplified polymorphic DNA SCAR Sequence characterized amplified region vii ABSTRACT Tomato mosaic virus (ToMV) is one of the most serious virus diseases not only in tomatoes, but also in many plants, such as cucumber. Infecting ToMV disease, plants always reduce growth, development, and yield. Using pesticide, one of the methods, can control the development of this disease. However, this way can affect tohuman, environmental condition, etc, due to the pesticitide may resist in product and soil.
Finding the resistant genes to ToMV disease from wild tomato type using DNA markers are optimal method. By the way, can reduce time and cost for creating new tomato varieties. From the survey of 20 tomato varieties, some tomato varieties have been drawn with many good agro-biological characteristics, high yield is 5-8, 10-10(2), 5-10 x 2-4, 2-5, 3-1, 13-7, 11-9, 10-4(2), 14-5(1), 5-9, and 12-2. By using DNA markers for research and analysis, two varieties with outstanding agro-biological characteristics and containing genes for resistance to tobacco mosaic virus disease were found such as 11- 9 and 10-4(2).
These two tomato varieties will be used as a prerequisite for the selection process of breeding resistant to tobacco mosaic disease on tomato. viii CHAPTER I: INTRODUCTION 1. Rationale On a worldwide scale, the tomato (Lycopersicon esculentum) continues to increase in importance for consumption as a fresh crop, for inclusion as a major constituent in many prepared foods, and also for research into the fundamental principles of growth and development in plants. Members of the Lycopersicon are tolerant of a wide range of environmental and nutritional conditions.
A few of the species have been crossed to provide a large number of varieties directed towards either the production of a single-harvest field crop or, particularly under protection, a succession of fruit for the fresh market over quite a long timespan. Field tomatoes are amenable to mechanical harvesting through the combined efforts of plant breeders and agricultural engineers, thus allowing vast tonnages to be grown and processed economically. The products are incorporated into a wide range of canned, frozen, preserved, or dried foods. Given their economic importance, and because they are amenable to molecular biology and genetic engineering techniques, tomato fruit has been selected for intense study at the molecular level over the last decade.
However, the tomato production process is still facing many difficulties, one of the causes limiting product development and reducing tomato yield is tobacco mosaic virus disease. Tobacco mosaic virus disease has been studied for a long time, but so far, the disease is still a common pest and greatly affects tomato yield and quality. Many vegetable and ornamental crops can be infected by tobamoviruses. Infections usually give rise to characteristic mosaic symptoms and lead to considerable cosmetic damage and yield losses.
Tobamoviruses, including tobacco and tomato mosaic virus (TMV and ToMV, respectively) belong to the α-like supergroup of viruses. They consist of a characteristic proteinaceous rod comprising 2140 coat protein (CP) copies, which envelop the positive-stranded linear RNA genome. After infection of the plant cell the RNA genome is uncoated and the viral gene products, the RNA-dependent RNA polymerase 1 (RdRP), the movement protein (MP), and the coat protein (CP), are produced. Infection of neighboring cells commences with the movement of RNA-MP complexes through plasmodesmata with MP-induced altered size exclusion limits.
Long-distance transport of the virus proceeds through the vascular tissue and depends both on MP and CP. Tobamoviruses, especially TMV, are the classical model system for the study of virus infection in plants (Mamidala & Nanna, 2009). Currently, disease control is still mainly by chemical methods. However, the abuse of chemical drugs has caused consequences such as environmental pollution, creating resistance to pests and diseases, and affecting human and livestock health.
Therefore, finding effective safety measures to prevent tobacco mosaic virus disease is one of the urgent issues today. Breeding new varieties with genetic resistance to tobacco mosaic virus disease is the most effective way to minimize the damage. Therefore, the efficient use of new resistance genes by breeders is essential. Researchers have identified tobacco mosaic virus disease resistance genes for decades, including Tm-1, Tm-2, Tm-22 in wild tomato species (Pelham, 1965).
Today, in plant breeding, the selection method thanks to molecular markers linked to the target genes have become an effective tool that can quickly, early, and accurately select traits regardless of the environment. Although some tomato lines resistant to late blight have been selected, the results are still very limited and need to be further researched. Starting from the above basis, to serve the selection and breeding of tomato varieties carrying tobacco mosaic virus disease resistance genes, we conducted the project: " Investigation of resistance to tobacco mosaic virus disease on tomato by DNA marker. Aims and requirements of the study 1.1 Aims Detecting genetic resources to identify tomato varieties with good yield and quality, containing genes for resistance to tobacco mosaic virus disease for breeding program.
Requirements - Evaluation of some important agrobiological traits in tomato corporations. - Using molecular markers to identify tobacco mosaic virus disease resistance genes in tomato consortium. 3 CHAPTER II: LITERATURE REVIEW 2.1 The origin of tomato Almost all tomato cultivars are members of the Lycopersicon esculentum species. The beginnings and the early events of domestication are mostly unknown, much like with domesticated plants in general.
Three things are certain enough to be fairly assumed. First off, all related wild tomato species are endemic to the Andean area, which today includes parts of Chile, Colombia, Ecuador, Bolivia, and Peru. This means that the cultivated tomato is a New World invention. Second, before being brought to Europe, the tomato was domesticated to a very advanced state.
Early herbals' woodcut illustrations show that the first varieties grown in Europe produced big fruit. The fruit (in all wild species) is tiny. The descriptions indicated that a good number of sizes, forms, and colors were known. Third, the most likely ancestor, the wild cherry tomato (L.
esculentum variety cerasiforme), is spontaneous throughout tropical and subtropical America and has spread throughout the Tropics of the Old World(Pelham, 1965). The time, place, and other aspects of domestication are far less certain. Mexico is likely the region of domestication, even though concrete evidence is absent. This is supported by the weight of data from a variety of fields.
Hereditary enzyme comparisons show much greater similarity between older European cultivars and the indigenous cherry tomatoes of Mexico and Central America than between European cultivars and the indigenous plants of the Andes, which is unquestionably the genus Lycopersicon's distribution center and the other potential domestication region. Portions of the tomato plant have not been discovered in the archaeological remains of the Andean region, but parts of the majority of the locally cultivated plants have. Neither have representations of the tomato been discovered in ancient pottery or other artifacts from the region.