Investigation of resistance to tobacco mosaic virus desease on tomato by dna marker

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.D Faculty of Biotechnology - Vietnam National University of Agriculture All figures in this thesis are true and 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 i ACKNOWLEDGMENTS ii LIST OF TABLES v LIST OF FIGURES vi ABBREVIATIONS vii ABSTRACT viii CHAPTER I: INTRODUCTION 1.1 Rationale Lỗi! Thẻ đánh dấu không được xác định 1.2 Aims and requirements of study 1.2.1 Aims 1.2.2 Requirements CHAPTER II: LITERATURE REVIEW 2.1 Introduction 2.1.1 The origin 2.1.2 Nutritional values 2.1.3 Characteristics of tomato plants 2.2 The situation of tomato production in the world and in the country 2.2.1 The situation of tomato production in the world 2.2.2 The situation of tomato production in Vietnam 2.3 Tobacco mosaic virus disease on tomato 10 2.3.1 Symptoms and signs of disease 11 2.3.2 The cause to the tobacco mosaic virus disease 11 2.3.3 Disease Cycle and Epidemiology 13 2.3.4 Epidemiology 14 2.4 Status of research on resistance to tomato mosaic virus disease in tomatoes 15 CHAPTER III: RESEARCH MATERIALS AND METHODS 18 3.1 Research Materials 18 3.1.1 Research subjects 18 iii 3.2.1 Place and time of study 19 3.2 Research content 20 3.3.1 Experimental layout 20 3.3.2 Methods for assessing important agronomic traits 21 3.3.3 Evaluation of resistance to tomato mosaic virus disease of selected tomato varieties by using molecular markers 22 CHAPTER IV: RESULTS AND DISCUSSION 25 4.1 Survey on genetic diversity of researched tomato varieties 25 4.1.1 Growth stages of tomato plants 25 4.1.2 Results on some structural features 26 4.1.3 Investigate the characteristics of inflorescence structure, bloom characteristics, yield, and yield factors 29 4.2 Results of the PCR method to detect the Tm-2 gene in the studied tomato group 4.2.1 Test of DNA total 32 4.2.2 Determination of DNA content by OD spectroscopy of total DNA 34 4.2.3 Results of PCR products detecting Tm-2 gene 35 CHAPTER V: CONCLUSIONS AND RECOMMENDATIONS 39 5.1 Conclusions 39 5.2 Recommendations 39 REFERENCES 40 iv LIST OF TABLES Table 2.1 Composition of Dry Matter Content of Tomato5 Table 2.2 Area harvested, yield, and production over the world of tomatoes Table 3.1 Name of tomato varieties stored at the Center for Conservation and Development of Plant Genetic Resources 18 Table 3.2 PCR reaction components 24 Table 3.3 PCR conditions for different primer pairs 24 Table 3.4 List primer 24 Table 4.1 The stages of growth and development of tomatoes 25 Table 4.2 Some growth characteristics, morphological characteristics, and tree structure of the studied varieties 27 Table 4.3 Some characteristics of inflorescence structure, bloom characteristics, yield and yield factor 31 Table 4.4 Determination of DNA content by OD spectroscopy of total DNA 34 Table 4.5 Selection of promising varieties 38 v LIST OF FIGURES Figure 2.1 Anatomical morphology of tomato stem, flower, fruit Figure 2.2 Tomato production of countries in the world in 2020 Figure 2.3 Tobacco mosaic virus disease on tomato leaf 10 Figure 2.4 Signs of tobacco mosaic virus disease on tomato leaf and fruit 11 Figure 2.5 The rod-shaped virus particles of TMV 10 Figure 2.6 A single TMV particle 10 Figure 2.7 Transcriptional process of TMV 13 Figure 4.1 Test of DNA total 32 Figure 4.2 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 36 Figure 4.5 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- 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 The results of the study on the morphological and structural characteristics of the samples are presented in Table 4.2 Table 4.2 Some growth characteristics, morphological characteristics, and tree structure of the studied varieties No Name 3-2 11-3 5-8 10-10 (2) 5-10 x 2-4 2-5 3-1 13-7 6-6(2) 11-9 9-4 14-3 (1) 7-9 10-4 (2) 6-9 (1) 14-5 (1) 6-6 5-9 12-2 14-12 10 11 12 13 14 15 16 17 18 19 20 Leaf type Pinna Pinna Pinna Pinna The Number of height Growth Brush nodes under Greenness of phenotype form* the 1st tree inflorescence (cm) S-f DG 12.6 50.00 S-f G 11.8 56.12 S-f G 10.4 60.80 S-f G 11.0 53.11 Pinna S-f G 9.6 67.05 Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna Pinna S-f S-f F S-f S-f S-f S-f F S-f S-f S-f S-f S-f S-f S-f DG G G G G G G G G DG G G G G G 1 1 2 2 2 1 10.0 11.8 12.4 12.0 11.6 11.2 11.8 12.6 11.8 9.4 10.2 10.4 9.2 10.2 10.4 50.00 56.50 42.67 55.00 58.53 62.14 56.72 48.20 59.27 58.60 59.00 66.85 53.10 51.27 64.12 Note: S-f: Semi-finite; F: finite; G: Green; DG: Dark green;(*): 1-tall, wide canopy: 2-high, narrow canopy; 3-low, wide canopy; 4-low, narrow canopy 27 Determine the growth shape of tomatoes by evaluating plant structural characteristics, particularly using the criterion "plant height." There are three different development patterns in tomatoes: finite, semi-finite, and limitless Plants with an infinite growth phenotype are considered to have plant growth, which is described as plant growth that ceases when the plant is in flower, has a terminal inflorescence on top of the head, and has a restricted height continues while the plant is in bloom, when the plant's height is unrestricted, and when there is no inflorescence on the top of the head Semi-finite growth is a hybrid of the finite and infinite types of growth Based on the results of the evaluation of the growth phenotype, there is an infinite growing variety, which is number and 13, the rest are semifinite phenotypes Evaluation of leaf morphological traits can assist identify cultivar exploitation or similarity since leaf morphological traits are generally cultivardependent The study used the color and form of the leaves as two criteria to assess leaf morphology One of the key morphological traits used to identify different varieties is the color of the leaves However, under the influence of the external environment and fertilizer application rates, particularly nitrogen, the color of the leaves can change significantly without affecting the chlorophyll content The color of the young leaf is significantly influenced by luck Dark green leaves typically contain more chlorophyll and have a greater capability for photosynthetic activity The 20 cultivars examined were classified into two groups based on the degree of greenness in their leaves: green and dark green Leaves of all samples are compound leaves, so there is no difference in leaf shape in the studied varieties Bush form is also an important feature that can be used to group the studied varieties, including types: 1-tall, wide canopy: 2-high, narrow canopy; 3-low, wide canopy; 4-low, narrow canopy Type are the varieties with infinite, semi-finite growth, tall trees for higher biomass yield, thereby affecting economic productivity Types and are tall cultivars corresponding to a finite 28 growth phenotype, plants of low height are preferred due to less labor required to tie the truss and reduce breakage The studied varieties are in forms and The number of nodes from the base to the first inflorescence is correlated with the time of planting to flowering and the time of planting to maturity, that is, the fewer nodes under the first inflorescence, the lower the number of nodes shorten flowering and ripening time, and increase early ripening, and early harvest In addition, the number of nodes under the inflorescence is one of the factors that directly determine the height of the inflorescence, it depends on the genetic characteristics of the variety and the combination of external conditions, and usually ranges from 7-15 nodes in the studied samples It can be relatively divided into groups: group has a number of segments less than 10, including samples, denominators 5, 15, and 18 Group number with a number of nodes greater than 10 includes the remaining groups 4.1.3 Investigate the characteristics of inflorescence structure, bloom characteristics, yield, and yield factors The genetic makeup of the variety determines inflorescence morphology, which is an attribute that is unaffected by environmental factors Inflorescences come in three varieties: simple, intermediate, and complicated Inflorescences of a simple form typically yield larger fruits than those of an intermediate or complicated type The analysis revealed that all 20 experimental kinds exhibited concentrated bloom patterns and simple inflorescences on their leaves Every variety has a straightforward inflorescence In addition to their genetic makeup, plants' flowering features are also influenced by environmental factors The fruit will ripen quickly due to the concentrated bloom, and when the harvest is also concentrated all at once, unfavorable conditions will be avoided Flowers bloom sporadically so that more batches of fruit can be harvested over a longer period of time and with superior nutrition, limiting post-harvest loss To get the best production and quality, the producer chooses a certain crop and the right time to plant it based 29 on this type of bloom Concentrated fruit types are appropriate for manufacturing locations that produce processing materials Regarding the types of scattered fruit, they are appropriate for scattering crops to prevent product stagnation A key factor affecting the variety's output is the pace of the fruit set This is another indicator of how well the variety adapts to the local ecological circumstances in the production area External factors, particularly temperature and rainfall, have a significant impact on the rate of fruit set, with high temperatures and heavy rain significantly slowing down the process The analysis of 20 research samples revealed a range in fruit set rates of 56.60% to 94.28% The variety samples' fruit production per tree ranged from 10 to 30 fruits (due to the late planting time, so the data are not available) Statistics are not complete; usually, the varieties with many fruits have low average weights and vice versa Based on the statistics dated 21/11/2022, the studied varieties can be divided into groups A group with few fruits (