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Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.Nghiên cứu ứng dụng chỉ thị phân tử trong chọn tạo giống cà chua kháng bệnh xoăn vàng lá và mốc sương ở miền Bắc Việt Nam.
MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIET NAM ACADEMY OF AGRICULTURAL SCIENCES TONG VAN HAI RESEARCH AND APPLICATION OF MOLECULAR MARKER IN BREEDING TOMATO VARIETIES RESISTANCE TO YELLOW LEAF CURL AND LATE BLIGHT DISEASE IN THE NORTH, VIETNAM Research major: Biotechnology Code: 94 20 201 SUMMARY OF DOCTORAL THESIS IN AGRICULTURE HA NOI - 2022 The study was completed at: VIET NAM ACADEMY OF AGRICULTURAL SCIENCES Scientific instructors: Prof Dr Phan Huu Ton Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended at the Institute's Thesis Evaluation Council at the Vietnam Academy of Agricultural Sciences Time: ………, date: ……/… /2022 The thesis can be found at the library: National Library of Vietnam Library of Vietnam Academy of Agricultural Sciences INTRODUCTION The c urgency of the thesis Tomato is a vegetable plant with the scientific name Lycopercicum esculentum Mill, grown in most countries worldwide, including Vietnam In Vietnam, tomatoes are grown and widely consumed throughout the country According to the statistics of 2020, the tomato growing area in the whole country in 2019 was 23,719 thousand hectares and the output was 673,194.5 tons 4 Tomato production brings high economic efficiency; for every hectare of tomatoes, the income is from 120-200 million VND/ha/crop In the spring-summer and autumn-winter crops, the economic efficiency is 3-5 times higher than that of the main crop 18, 22 However, the yield and quality of Vietnam's tomatoes are still precarious and not high, which is caused by harmful diseases, of which leaf curl and late blight are the two most serious diseases Tomato Yellow Leaf Curl Virus (TYLCV) is caused by several viruses belonging to the genus Begomovirus, family Geminiviridae, first discovered in Israel in 1939 112 This disease causes severe damage to the yield and quality of tomatoes Yield losses average from 55 - 90%, even 100%, when the plant is severely infected with this disease 14, 38 TYLCV is spread by the whitefly Bemisia tabaci, a fast-reproducing insect that is very difficult to control Currently, there is no effective medicine to prevent this disease if infected plants can only be uprooted Tomato blight, caused by the fungus Phytophthora infestants, is one of the most destructive diseases in most tomato-growing regions worldwide The control of late blight is mainly based on fungicides and farming practices However, the effectiveness of these measures is not high due to the variation of P infestants, the emergence of new strains, and the increased resistance to fungicides of the pathogens To prevent these two diseases, using resistant tomato varieties is the most effective measure, both cost-effective and safe for humans, livestock and the environment 22, 37, 99 Currently, the set of tomato varieties resistant to yellow leaf curl and late blight in Vietnam is still relatively modest; the cultivated varieties are mostly heavily infected with these two diseases Therefore, breeding varieties resistant to yellow leaf curl disease and late blight is urgently needed In order to successfully breed disease-resistant tomato varieties, the first thing to is to determine the number of resistant genes and effective resistance genes in Vietnam Scientists have discovered six genes for resistance to yellow leaf curl disease, named Ty1, Ty2, Ty3, Ty4, ty5 and Ty6 Ty1, Ty2, and Ty3 are the main genes used extensively in tomato breeding programs resistant to yellow leaf curl disease 125 Besides, resistance genes for late blight Ph1, Ph2, Ph3, Ph4 and Ph5 gene were detected 143 DNA molecular markers associated with the above genes have also been developed Therefore, based on PCR technique to detect and select resistance genes has been widely used in tomato breeding programs, making the selection of resistance genes convenient and accurate The Vietnam National University of Agriculture has collected 230 samples of domestic and foreign tomato varieties in the cooperation and exchange program of genetic resources To exploit this genetic resource for research and breeding tomato varieties resistant to yellow leaf curl and late blight, the first thing to is to evaluate the genetic resources and apply molecular markers to identify variety samples containing disease resistance genes, crossing and use of molecular markers for selecting To breed tomato varieties resistant to yellow leaf curl and late blight, we conducted a study on “Research and application of molecular markers in breeding tomato varieties resistant to yellow leaf curl disease and late blight disease in the North, Vietnam” Objective of the study Supplement valuable materials for several traits such as yield, fruit quality, and genes for resistance to yellow leaf curl and late blight disease, serving the breeding programs of tomatoes resistant to the two diseases mentioned above Breed several excellent tomato lines and varieties with high yield (over 50 tons/ha), carrying genes for resistance to yellow leaf curl disease and genes for resistance to late blight disease by DNA molecular markers, meeting the demand for tomato varieties that is resistant to two diseases Scientific and practical significance 3.1 Scientific significance Supplement scientific data in research in breeding high-yielding, good-quality, resistant to yellow leaf curl and late blight tomato varieties to meet production requirements The study's results open the possibility of widely applying molecular markers in the selection and breeding of tomatoes according to the target traits, especially in breeding tomato varieties resistant to yellow leaf curl and late blight The thesis is a closed scientific research work: From research, evaluation of starting materials, detection of yellow leaf curl resistance genes and late blight resistance genes by molecular markers, artificial infection for effective disease resistance gene detection, hybridization and application of DNA molecular markers to select resistance genes, assessment of elite line selection, basic testing, ecological testing to select elite lines/varieties, thereby development in production 3.2 Practical significance The research results create a diverse and rich source of materials to effectively serve in breeding tomato varieties resistant to yellow leaf curl disease and late blight disease Selected to breeding new tomato lines/varieties, of which two lines TP130 and TP135 are resistant to yellow leaf curl disease, line P7 is resistant to late blight, all lines give stable yields and thereby developing production to meet the practice's needs Subjects and scope of the study 4.1 Subjects 230 tomato variety samples collected at home and abroad are currently kept at the Center for Conservation and Developments of Crop Genetic Resource, Vietnam National University of Agriculture The molecular markers associated with resistance genes to yellow leaf curl virus Ty1, Ty2, Ty3, Ty4 and ty5, genes for resistance to late blight Ph2, Ph3 have been published in domestic and foreign journals 4.2 Location and time The experiments were carried out at the experimental area of the Center for Conservation and Developments of Crop Genetic Resource and the laboratory of the Faculty of Biotechnology - Vietnam National University of Agriculture The testing trials testing were conducted in Soc Son - Hanoi, Moc Chau - Son La and Vinh Bao - Hai Phong Project implementation period: From April 2016 to April 2022 New contributions of the thesis Building a database on agro-biological characteristics of the group 230 tomato varieties, serving the conservation and exploitation of tomato genetic resources in Vietnam, especially genetic resources that carrying disease resistance genes to leaf curl and late blight Identify good resistance genes to yellow leaf curl disease in Vietnam are the Ty1 and Ty3 genes, and good resistant to late blight are Ph2 and Ph3 through artificial infection This is the scientific basis for the selection and breeding of tomato varieties resistant to yellow leaf curl and late blight disease in the North of Vietnam Successfully breded inbrid tomato lines, of which two lines, TP130 carrying Ty1 gene and TP135 carying Ty3 gene, good resistance to yellow leaf curl disease The P7 line carrying the Ph3 gene, good resistance to late blight diseseas All lines have stable yields, reaching over 50 tons/ha in spring-summer and over 60 tons/ha in winter crops, with good fruit quality, resistance to yellow leaf curl and late blight, meeting the practical needs of production in the North of Vietnam Thesis layout: The thesis consists of 136 pages (excluding references and appendices): Introduction (5 pages), Chapter 1: Literature review (41 pages); Chapter 2: Materials, contents and methods (14 pages), Chapter 3: Research results and discussion (75 pages), Conclusion and recommendations (2 pages), References (27 Vietnamese references, 117 English references) The thesis has 43 tables, 47 pictures and 02 appendices, 03 Publications related to the thesis CHAPTER OVERVIEW OF THE DOCUMENTATION AND SCIENTIFIC BASIS OF THE TOPIC The thesis has consulted and summarized Vietnamese and English documents, with relevant contents including The situation of tomato production in the world and Vietnam; Research on leaf curl disease and late blight disease; Research on resistance genes to yellow leaf curl disease, late blight disease and associated molecular DNA markers; Breeding applied MAS varieties; Some research and achievements on breeding resistance to yellow leaf curl and late blight in Vietnam 1.1 The situation of tomato production in the world and Vietnam 1.1.1 The situation of tomato production in the world According to the Food and Agriculture Organization of the United Nations, the area of tomatoes in the five years of 2016-2020 fluctuated around 4,900,000 hectares; the yield was about 36.0 tons/ha In production, it gradually increased from 177,382,876 tons (in 2016) to 186,821,216 tons (in 2020) 50 With the above output, the average per capita consumption is about 22 kg of fruit/person/year Also, in the years from 2016 to 2020, Asia had the largest tomato area and production in the world, accounting for about 61.3% of total production, followed by the Americas, accounting for 13.9% of total output; Europe, about 12.9%; Africa about 11.6%, and elsewhere 0.2% 1.1.2 The situation of tomato production in Vietnam In Vietnam, according to statistics from the Department of Crop Production, Ministry of Agriculture and Rural Development, in the years from 2015 to 2019, the area of tomato cultivation fluctuates between 23-25 thousand hectares, and the yield fluctuates around 25-28 tons/ha Tomatoes are primarily produced in the provinces of the Red River Delta and Lam Dong area The area and production of tomatoes in these two regions account for over 62% of the national tomato production 1.2 Research on leaf curl disease and late blight disease 1.2.1 Research on leaf curl disease 1.2.1.1 Cause Tomato Yellow Leaf Curl Disease (TYLCD) caused by begomovirus was first recognized worldwide in 1959 in Israel In Vietnam, yellow leaf curl disease was first detected in 1970 1.2.1.2 Symptom When diseased, the leaves curl downwards to the inside; in the later stages, the leaves are shapeless, narrow, and yellow from the edges and the tip spreads into the middle of the veins; leaves curl upward into a boat shape; Young leaves turn intense yellow, brittle and narrow Symptoms of yellowing are especially evident in young leaves The petiole can be twisted Dwarf plant stunted, growing many small branches, short stem nodes Plants infected early often not bear fruit due to flower drops 13, 45, 102 1.2.1.3 Pathogens The causative agent of tomato yellow leaf curl is considered a complex of many different begomoviruses, collectively known as Tomato yellow leaf curl virus 122 Begomovirus is the largest genus in the geminivirus family The begomoviruses have single-stranded circular DNA genomes of 2.6-2.8 kb in size They have a double genome consisting of DNA molecules called DNA-A and DNA-B or have a single genome equivalent of DNA-A 72 and the disease vector is the whitefly 1.2.1.4 Disease transmission vectors All begomoviruses are spread in the wild by the whitefly (Bemisia tabaci) in a persistent cyclic 45 Currently, only three whitefly species are considered vectors of plant viruses: Bemisia tabaci, Trialeurodes vaporariorum, and T abutilonia Among them, B tabaci is the most critical vector of more than 100 different viral diseases (mainly viruses of the genus begomovirus) in the tropics and subtropics 87, 116 1.2.1.5 Virus transmission mechanism TYLCV enters plants by the whitefly (Bemisia tabaci) When infecting plant cells, the single-stranded viral DNA enters the host cell's nucleus and uses the host's materials to replicate and synthesize envelope proteins, completing the viral structure 1.2.2 Research on late blight disease 1.2.2.1 Origin and effects of late blight Late blight of tomato tomatoes, caused by the fungus Phytophthora infestans, is one of the most devastating diseases in most tomato regions worldwide In Vietnam, for many years now, the disease has often caused damage in tomato growing areas, with an average loss of 63.7%, sometimes up to 100%, not harvested 14 1.2.2.2 Symptom Symptoms of late blight are very diverse, it depends on the severity of the disease and environmental conditions, when the early tomato leaves to infection, it is only a tiny spot (2-10 mm), there is no apparent limit, the underside of the leaves where the disease is affected has a white spongy layer like hoarfrost, most evident when it is wet; it is a cluster of spores of the fungus causing the disease, the disease continues to spread on the surface of leaves, petioles, stems, and fruits 1.2.2.3 Classification and biological characteristics of late blight disease P infestans belongs to the class Oomycetes, the order Peronosporales, this class belongs to a different kingdom from the true fungi Some authors believe that the class Oomycetes belongs to the kingdom Protoctista, while others think it belongs to the kingdom Chromista 7 The main feature of P infestans is the relatively well-developed unicellular mycelium, a fungus with a complete growth cycle Still, in tropical conditions, only the asexual stage occurs; therefore, they are forced to parasite on the host 1.2.2.4 Geographic distribution and host range of late blight P infestans is a heterozygous fungus with two forms (matin types) A1 and A2 depending on the ecological region in the world's tomato and potato growing regions Pathogenic fungi are considered specialized parasites with a narrow host spectrum, but P infestans has been reported to cause disease in many plant species 1.3 Research on resistance genes to yellow leaf curl disease, late blight disease and associated molecular DNA markers 1.3.1 Research on resistance genes to yellow leaf curl and markers So far, scientists have discovered genes for resistance to leaf curl and tomato leaf disease, named Ty1, Ty2, Ty3, Ty4, ty5 and Ty6, respectively Ty1, Ty2, Ty3 are the primary genes used extensively in tomatoes resistant to yellow leaf curl disease breeding programs 125 The Ty1 gene is a dominant gene, identified by Zamir et al on chromosome 139 Later, scientists identified molecular markers associated with this gene Castro et al (2007) identified the JB-1 marker associated with the Ty1 gene However, the JB1 marker is dominant, so it cannot distinguish homozygous and heterozygous resistance genotypes Next, Han et al (2012) successfully developed the CAPS TG97 co- dominant marker that distinguishes homozygous and heterozygous resistant genotypes The Ty2 gene was identified by Hanson et al (2006), located on the long branch of chromosome 11, located between the markers TG36 and TG26 61 Then Garcia et al (2007) developed the SCAR T0302 marker associated with this gene Using primer pair T0302F/TY2R1, allows the detection of the Ty2 gene in three different states, homozygous dominant, heterozygous and homozygous recessive 53 The Ty3 gene is a dominant gene located on chromosome 73, 78 According to Ji et al (2007), the Ty3 gene is located in a region containing the FER locus (25 cM, vector line BAC56B23, AY678298) The DNA markers associated with the Ty3 gene, T0507, C2_At3g11210 and P6-25, all indicated the presence of the Ty3 gene However, markers T0507 and C2_At3g11210 are dominant, P6-25 is co-dominant In addition, many Ty3 markers have also been identified, namely P169C, TG118, TG590 and FERG8, but irrespective of the Ty3-carrying lines in different allele states The Ty4 gene is a dominant gene discovered by Ji & cs (2008) The author detected a 14cM S Chilense translocation region on the long branch of chromosome in some resistant cultivars derived from LA1932 A novel begomovirus resistance locus, Ty4 is mapped with markers at about 2.3 cM between C2_At4g17300 and C2_At5g60160 in a 550 kb translocation on chromosome 74, 77 The marker C2_AT5g51110 was identified as binding to this gene 74 The ty5 gene is a recessive gene located on chromosome 31 Gene ty5 is recessive, so it does not have much meaning in selecting hybrid tomato varieties, but it has great significance in selecting pure tomato varieties A DNA marker that binds to the ty5 gene has also been detected, which is the TM719 marker 42 1.3.2 Research on late blight resistance genes and associated markers Up to now, scientists around the world have identified different resistance genes to late blight: Ph1 gene on chromosome 7, Ph2 on chromosome 10 101, Ph3 on chromosome number 143, Ph4 on chromosomes 22 Ph5-1, Ph5-2 on chromosomes and 10 96 , 97 Ph1, Ph2 and Ph3 genes have been commonly used for breeding resistant tomato varieties Along with discovering resistance genes, the scientists also studied the DNA molecular markers associated with those resistance genes However, there are no published markers associated with the Ph1 gene, while the UF-Ph2-1 marker associated with the Ph2 gene 117, the SCARSCU602 marker associated with the Ph3 gene have been published.132 1.4 Breeding applied MAS varieties 1.4.1 Concept Marker assisted selection (MAS) is “smart variety selection” or the technology of fast and accurate plant breeding It is a tool used by research institutes and companies to rapidly develop improved varieties that can select desired traits using DNA markers 1.4.2 MAS in breeding disease resistant varieties 1.4.3 MAS in breeding tomato varieties resistant to yellow leaf curl and late blight Among the plants of the tomato family, the tomato is one of the most studied plants based on genetic studies MAS is a method of indirect trait selection based on the genotype of an associated marker rather than the trait of interest 108 Over the past few decades, MAS has been the most commonly used technique by breeders to develop new cultivars In addition, MAS is a constructive way to cluster genes or identify quantitative trait loci (QTLs), especially for traits with low heritability 71 1.4.3.1 Studies on MAS in breeding tomato varieties resistant to leaf curl disease The discovery of yellow leaf curl resistance genes and associated markers is a prerequisite for breeding tomato varieties resistant to the disease Although many TYLCV resistance genes have been studied, most commercial tomato varieties have a single TYLCV resistance gene, usually the Ty1 or Ty3 gene The emergence of new TYLCV strains resistant to Ty1/3, or outbreaks of Ty1/3 resistance due to specific environmental conditions, has been repeatedly reported 107 Therefore, clustering multiple resistance genes into a single tomato variety is necessary to achieve reliable and durable TYLCV resistance 118 Using molecular markers for targeted resistance gene selection (MAS) is essential for programs that combine multiple resistance genes into an efficient variety 88 1.4.3.2 Studies on MAS in breeding tomato varieties resistant to late blight disease Five late blight resistance genes have been transferred from S pimpinellifolium into cultivated tomatoes 106, the Ph2 and Ph3 resistance genes have been widely used in commercial cultivars 141 F1 hybrid with combined Ph2 and Ph3 genes commercialized in the US 46 Similar to the selection of genes for resistance to yellow leaf curl disease by MAS, molecular markers are a prerequisite for selecting tomato varieties resistant to late blight The application of MAS to bring together many genes into a 12 scale of - 83: 2.4.3.2 Artificial infection of late blight diseases Samples of P infestans were collected at Soc Son - Hanoi, Quynh Phu - Thai Binh, Vinh Bao - Hai Phong, Tu Ky - Hai Duong, Dong Son Thanh Hoa, Moc Chau - Son La on typical late blight tomato plants The fungus was isolated according to Sobkowiak and Śliwka 132 Late blight is transmitted on detached leaves After sowing 30-35 days, the plants appear 5-6 true leaves Cut off the 4th true leaf (fully grown), disease-free, and mark with a tag corresponding to the studied variety, keep in the cool, damp paper Place tomato leaves face down on damp paper in a Petri dish, then use a small micropipette between each leaflet 30 µl of spore solution (104 - 105 spores/ml) of powdery mildew For each plant was repeated times After infection, the petry box was tightly closed and kept in a 17℃ incubator Assessment of disease after days of infection is based on disease index 2.4.4 Method of hybrid, breeding new varieties 2.4.4.1 Method of hybridization and breeding new lines and varieties of tomato The F1 hybrid population was bred by single crossing method, the mother line is the good tomato varieties that have been evaluated and screened, the father line is the samples carrying the Ty resistance gene or the Ph gene The F1 population was screened using a DNA marker From the F2 population, pedigree selection and DNA markers were used to select individuals with homozygous genes 2.4.4.2 Comparison, basic test and ecology experiments The comparison experiments, testing of good lines were arranged according to the method of complete randomization block (RCBD), replications, plot area of 10m2/plot and planting of 30 plants/plot The control variety is tomato variety C155 Monitoring and evaluation criteria according to QCVN01-63: 2011/BNNPTNT Prospective tomato lines were surveyed in different ecological regions: Hanoi, Hai Phong, Son La 2.5 Methods and techniques to care for seedlings Care according to the process of the Field Crops Research Institute 2.6 Methods of processing experimental data - Biostatistics in the field were processed on the Excell 2011 program on the computer - Analysis of variance ANOVA, coefficient of variation Cv(%), significant difference LSD0.05, comparison Duncal, using software IRRISTAT ver 5.0 13 CHAPTER III RESULTS AND DISCUSSION 3.1 Evaluation of genetic resources for breeding of tomato varieties To serve the breeding of tomatoes, within the framework of the thesis, 230 samples of tomato varieties were evaluated for agro-biological characteristics, yield and yield components, from which to choose good variety samples used as a source of breeding material 3.1.1 Growth type The results of observing the growth and flowering characteristics of 230 samples of the studied varieties showed that there were 112 samples of semi-determinant varieties, 49 samples of determinant growth varieties, the remaining 69 samples were of in-determinant growth type 3.1.2 Growth stages Monitoring of the growth stages found: 104 variety samples had the time from planting to flowering in the range of 26-30 days and 68 samples had the time from planting to flowering over 30 days Time from planting to fruit collection in the first phase: 51 samples of early ripening, 112 samples of medium ripening, 67 samples of late ripening The time from planting to the end of harvest or when all the fruits are harvested are found: Growth time ranges from 81 to 130 days In which, 52 varieties have short growing period ( 0.7 cm thick, ripe fruit color is dark red or red, average fruit size is over 70 g, individual yield is over 2000 g/plant or more, actual yield is over 55 tons/tree (Table 3.5) Table 3.5 Some agro-biological characteristics of good variety samples were screened from 230 variety samples under winter crop conditions 3.2 Detection of genes for resistance to yellow leaf curl disease and late blight disease by molecular markers 3.2.1 Detecting resistance gene for yellow leaf curl disease 3.2.1.1 Detecting resistance gene for yellow leaf curl disease Ty1 17 The CAPS marker TG97 was used to detect the Ty1 resistance gene PCR product with primer pair TG97F/R was a DNA fragment about 400 bp long in all studied cultivars After cutting the product with TaqI enzyme, the Ty1 gene-carrying samples showed bands about 300bp and 100bp long 60 Ty1 resistance gene detection in 230 seed samples found that 11 cultivars contained genes, which were AVRDC139, AVRDC154, AVRDC188, AVRDC189, AVRDC193, AVRDC198, Fr28, Fr34, Is23, Is34 and Ru07 3.2.1.2 Detecting resistance gene for yellow leaf curl disease Ty2 The SCAR marker T0302 was used to detect the Ty2 gene PCR products with primer pair T0302F/TY2R1 upon electrophoresis, if a band of 600bp size appears, are the same samples carrying the Ty2 53 Thereby, varieties were detected out of a total of 230 varieties containing the Ty2 yellow leaf curl resistance gene, namely AVRDC135, AVRDC138, AVRDC139, AVRDC142 and AVRDC144 3.2.1.3 Detection of resistance gene Ty3 The P6-25 marker was used to detect the Ty3 resistance gene The P6-25 F/R primer pair amplifies the product as a 660bp band for the Ty3b allele and a 630bp band for the Ty3a allele, the ty3 susceptibility allele for a 320bp band Using the marker SCAR P6-25 with primer pair P6-25F/R to detect Ty3 resistance in 230 tomato varieties, samples containing this gene were detected: AVRDC154, AVRDC165, AVRDC166, AVRDC192, AVRDC195, Is11, Is12 and Is22 3.2.1.4 Detection of resistance gene Ty4 In this study, the marker C2_AT5g51110 was used to detect the Ty4 gene Variety samples containing the Ty4 gene had a band of about 325 bp in size, and without the gene, no PCR product was replicated Four samples containing Ty4 gene were detected, namely AVRDC102, AVRDC115, AVRDC122 and AVRDC123 3.2.1.5 Detection of resistance gene ty5 Using the primer pair, multiply TM719 by PCR to detect the ty5 gene The detection results revealed that cultivars containing ty5 resistance genes were Is4 and Is5, Fr13, AVRDC139, AVRDC140 and AVRDC151 Thus, by DNA molecular markers, 11 variety samples containing Ty1 gene were detected, variety samples had Ty2 gene, variety samples contained Ty3 gene, variety samples contained Ty4 gene and variety samples contained ty5 gene Especially, there are varieties containing 02 resistance genes and 03 resistance genes, namely AVRDC154 containing Ty1 and Ty3 genes and AVRDC139 containing Ty1, Ty2 and ty5 genes 3.2.2 Detection of genes for resistance to late blight disease 3.2.2.1 Detection of resistance gene Ph2 18 In this study, the UF-Ph2-1 marker was selected to detect Ph2 PCR products using the primer pair multiply the UF-Ph2-1 marker were about 500 bp in size in both resistant and infected cultivars Using the restriction enzyme Hinf I to cut the above PCR product can distinguish the states of alleles If it is the resistance allele (dominant homozygous form), bands of size 355, 125 and 27 bp appear Investigating 230 variety samples with UF-Ph2-1 marker, 11 samples were found carrying Ph2 resistance genes, which are: AVRDC113, AVRDC114, AVRDC150, AVRDC181; AVRDC182; Us03, Us12; Us13; Fr20 and Fr23 3.2.2.2 Detection of resistance gene Ph3 To detect late blight resistance gene Ph3, in this study the marker SCU602 was used The primer pair SCU602F3/R3 multiplies the marker SCU602, with a 400 bp PCR product resistance allele Out of a total of 230 variety samples investigated, 17 samples were found to carry the Ph3 resistance gene, which are: AVRDC124, AVRDC125, AVRDC130, AVRDC131, AVRDC139, AVRDC140, AVRDC152, AVRDC157, AVRDC181, AVRDC182 and AVRDC198; Fr18, Fr23; Fr33; Is04, Is05 and Is14 3.3 Artificial infection identifies effective resistance genes 3.3.1 Artificial infection identifies effective resistance genes Four yellow leaf curl disease sources were collected in Hai Phong, Hung Yen, Bac Giang and ToLCHnV infected structures used for artificial infection The infection results found that the genotypes carrying Ty1 and Ty3 had the best resistance to all pathogens Especially, variety samples containing two genes Ty1 and Ty3 simultaneously or three genes Ty1, Ty2 and ty5 are entirely resistant to all infectious diseases 3.3.2 Identification of effective late blight resistance genes Evaluation of disease resistance of two genes Ph2 and Ph3 by artificial infection of isolates of late blight by the method of Nelson and Truong Van Du The infection results showed that all the cultivars carrying the Ph2 gene were highly resistant (HR) to the isolate collected in Thai Binh and Hai Phong and resistant (R) to the isolate collected in Hai Duong Infection (S) with isolates collected in Hanoi, Son La and Thanh Hoa Varieties carrying the high resistance (HR) Ph3 gene to isolates were collected in Son La and Thanh Hoa, resistance (R) to isolates collected in Hanoi, Thai Binh and Hai Phong and infected with isolates collected in Hai Phong Positive Thus, the Ph3 gene is more resistant than the Ph2 gene Samples carrying two genes for resistance to Ph2 and Ph3 were resistant to all late blight isolates 3.4 Hybrid, breeding new varieties 3.4.1 Crossing between genetic tomato varieties with good varieties From good variety samples and varieties carrying resistance genes Ty1, Ty3, Ph2 and Ph3 were crossed Thereby, 271 hybrid combinations 19 were bred, of which 135 hybrids between good samples and varieties carrying resistance genes Ty1 and Ty3 and 136 combinations between good samples and varieties carrying genes for resistance to Ph2 and Ph3 Evaluation of 135 F1 hybrid combinations containing resistance to yellow leaf curl disease, selected 19 good hybrid combinations In which, there are hybrid combinations with father carrying the gene for Ty1 resistance, 08 hybrids with father carrying the gene for resistance Ty3 and 03 hybrid combinations with father carrying two genes for resistance Ty1 and Ty3 Evaluation of 136 F1 hybrid combinations containing late blight resistance genes selected 29 good hybrid combinations, giving high hybrid advantage Of which, there are 19 hybrids with fathers carrying Ph3 resistance genes, 02 hybrids with fathers carrying Ph2 resistance genes and 08 hybrids with fathers carrying two genes for resistance to Ph2 and Ph3 simultaneously (Table 3.11a and 3.11b) To make sure that the selected hybrids are all true hybrids (correct parents as intended) a DNA marker was applied to test the F1 hybrid The combinations containing the resistance genes Ty1, Ty3, Ph2 and Ph3 were tested by TG97, P6-25, UF-Ph2-1 and SCU60 markers, respectively Test results show that 100% of F1 hybrids are true hybrids Table 3.11a List of 19 good F1 hybrid combinations, with high hybrid advantage and tested by DNA molecular markers 20 Table 3.11b List of 29 good F1 hybrid combinations, with high hybridization advantage and tested by DNA markers 3.4.2 Selection of individuals in the F2 population that carry homozygous resistance genes 3.4.2.1 Selection of individuals carrying genes for resistance to TYLCV In the F2 population, the TG97 marker continued to be used to select the homozygous dominant Ty1 resistance gene and the P6-25 marker to select the homozygous dominant Ty3 resistance gene Approximately 50 best F2 individuals of each combination were tagged to determine the 21 homozygous resistance genotype From 19 F2 populations, 59 homozygous Ty1 carriers, 49 homozygous Ty3 carriers and concurrent carriers of Ty1 and Ty3 resistance genes were selected 3.4.2.2 Selection of individuals carrying genes for resistance to late blight In the F2 population of 29 combinations, the UF-Ph2-1 marker was used to select the homozygous dominant Ph2 resistance gene and the SCU60 marker to select the homozygous dominant Ph3 resistance gene As a result, 65 homozygous Ph2 resistant carriers, 175 homozygous Ph3 resistant carriers and concurrent carriers of two resistance genes were selected F2 individuals were mixed and self-pollinated continuously for many generations, then cloned in the F6 generation 3.4.3 Split lines On the basis of F6 populations, each population is selected the best individual with high yield, at least the individual yield is from 1.5 kg or more Thereby, 20 lines carrying genes for resistance to yellow leaf curl Ty1 and Ty3 and 24 lines carrying genes for resistance to late blight Ph2 and Ph3 were selected The origin and characteristics of the selected lines are described in tables 3.14a and 3.14b Table 3.14a Origin and characteristics of 20 F6-generation tomato lines carrying genes for resistance to yellow leaf curl Ty1 and Ty3 22 Table 3.14b Origin and characteristics of 24 lines of F6 generation tomato carrying genes for resistance to late blight Ph2 and Ph3 To ensure that the Ty1, Ty3, Ph2 and Ph3 genes were immobilized in the selected lines, DNA markers were further applied for testing Selected lines carrying resistance genes Ty1, Ty3, Ph2 and Ph3 were tested using markers TG97, P6-25, UF-Ph2-1 and SCU60, respectively The test results showed that 100% of the selected lines carried targeted diseaseresistance genes 3.4.4 Evaluation and comparison of selected lines Up to the F6 generation, 44 good tomato lines were selected, in which 20 tomato lines carried the yellow leaf curl genes Ty1, Ty3 and 24 lines carried the late blight resistance genes Ph2 and Ph3 To select the most elite lines with both disease resistance and high yield for comparison and testing, the selected lines were evaluated under winter crop conditions in 2018 23 Through evaluation of agro-biological characteristics, yield and yield components, elite lines were selected They have a beautiful phenotype and especially give a higher yield than the control C155 Among them, lines carrying genes for resistance to yellow leaf curl disease are lines TP48-1; TP88, TP90, TP130, P135 and lines carrying late blight resistance genes are P1, P7, P15 and P24 The characteristics of the lines are summarized in Table 3.16 Table 3.16 Characteristics of elite tomatoes 3.4.5 Testing the elite tomato lines 3.4.5.1 Basic testing of elite tomato lines The basic test was conducted at Gia Lam Hanoi in two crops, SpringSummer and Winter in 2019 to assess the adaptability of the abovementioned elite tomato lines in the North of Vietnam Evaluation criteria include growth, development, morphological characteristics, resistance to pests and diseases, yield and yield components, and quality criteria In both basic trials, it was found that the elite lines showed superiority over the control C155, especially lines that gave higher yields than the control C155 were TP130, TP135, P7 In addition to the assessment and agronomic and yield characteristics, we also evaluated the resistance to leaf curl and late blight in the field and by artificial infection The results showed that the lines carrying the Ty1 and Ty3 resistance genes were well resistant to leaf curl both in the field and in artificial infection The lines carrying the gene for resistance to Ph2 or Ph3 also showed very good resistance In the field there are almost no symptoms and by artificial infection the results are similar to the previously evaluated resistance gene samples 3.4.5.2 Ecological testing of elite tomato lines 24 In order to evaluate the adaptability of the elite tomato lines that have been tested above, we continue to plant ecological trials in regions: Moc Chau - Son La, Soc Son - Hanoi and Vinh Bao - Hai Room, these are high tomato production areas Testing in crops including: Winter 2019 crop, Spring Summer 2020 crop and Winter 2020 crop, sowing spring on January 20 and Winter sowing on September 10 Particularly in Son La, the winter crop is sown month earlier than the other two locations Through the trial of consecutive crops at trial sites, three lines with high and stable yield were selected, which are the lines TP130 (carrying the gene for Ty1 resistance), TP130 (carrying the gene for resistance Ty3) and P7 (carrying the gene for resistance to Ph3) These lines all gave higher yield than control C155 in all crops and at all sites In addition to high yield, they also have good disease resistance The lines with good resistance to tomato yellow leaf curl disease are TP130 carrying the Ty1 gene and TP135 carrying the Ty3 gene The P7 line with good resistance to late blight carries the Ph3 gene These lines need to be recognized to develop in practice to bring high economic efficiency to farmers and businesses CONCLUSION AND SUGGESTION Conclusion 1) 230 tomato varieties were evaluated for agro-biological characteristics such as: growth phenotype, growth stages, leaf morphological characteristics and plant structure, flower structure, blooming characteristics, yield and yield components, some morphological characteristics of fruit quality Thereby, 13 varieties with beautiful phenotypes and high yield of over 2.0 kg/plant were selected as a source of materials for breeding new varieties 2) Application of DNA molecular markers to detect genes for resistance to yellow leaf curl and late blight of 230 tomato cultivars Detected 11 varieties carrying the Ty1 gene, samples carrying the Ty2 gene, samples carrying the Ty3 gene, carrying the Ty4 gene, carrying the ty5 gene, 11 carrying the Ph2 gene and 17 carrying the gene Ph3 In particular, there are 02 samples of AVRDC139 and AVRDC 140 varieties carrying two genes for resistance Ty and Ph These varieties are an extremely valuable source of material in the program to select tomato varieties resistant to yellow leaf curl and late blight 3) Artificial infection of 04 sources of yellow leaf curl disease on the varieties carrying the yellow leaf curl disease resistance gene and 06 isolates of late blight disease on the varieties carrying the late blight resistance gene Two genes that are effective against yellow leaf curl 25 disease have been identified in the North of Vietnam, Ty1 and Ty3, and Ph2 and Ph3 resistance genes are well resistant to late blight, however, Ph3 genes are better resistant than Ph2 4) Cross 13 good varieties with varieties carrying genes for resistance to yellow leaf curl Ty1 and Ty3 and genes for resistance to late blight Ph2 and Ph3 Application of DNA molecular markers to select individuals carrying homozygous disease resistance genes from F2 segregation generation Maintenance, selection, basic testing and ecological testing have selected best tomato lines with high yield, resistance to yellow leaf curl and late blight The selected good lines were TP130 carrying the Ty1 gene, TP135 carrying the Ty3 gene and P7 carrying the Ph3 resistance gene for stable yield and a yield of over 50 tons/ha in the Spring-Summer crop and over 60 tons/ha in the Winter crop, higher than the control variety C155 across different crops and ecological regions Suggestion 1) Continue to use the evaluated genetic resources, especially the good, high yielding varieties and the varieties containing the resistance genes to yellow leaf curl and late blight to create new varieties resistant to both yellow left curl and late blight diseases for the northern provinces 2) Continue to evaluate and expand the production of good, high yielding tomato varieties, resistant to yellow leaf curl and late blight, TP130, TP135 and P7 to bring economic benefits to farmers and businesses 3) Continue to study farming techniques for the above pure lines, in order to exploit their full potential to bring about the highest economic efficiency for producers and businesses 26 LIST OF PUBLISHED WORKS RELATED THE THESIS Tong Van Hai, Phan Thi Hien, Trinh Thi Thu Thuy, Phan Huu Ton, Nguyen Quoc Trung (2020), Detection of effective resistance genes for tomato leaf curl virus by dna molecular markers and artificial infection, Vietnam National Conference on Biotechnology 2020, p.607613 Tong Van Hai, Phan Huu Ton, Phan Thi Hien, Nguyen Quoc Trung (2021), Breeding Inbred Tomato for Resistance to Yellow Leaf Curl Virus by Marker-Assisted Selection, Vietnam J Agri Sci 2021, Vol 19, No 3, p 399-409 Tong Van Hai, Phan Huu Ton, Phan Thi Hien, Nguyen Quoc Trung, Trinh Thi Thu Thuy (2021), Detecting resistance genes to tomato late blight by DNA molecular marker and evaluation of resistance genes by artificial inoculation, Vietnam National Conference on Biotechnology 2021, p.740-746