Screening of tomato germplasm for resistance to yellow leaf curl virus disease

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY � � � UNDERGRADUATION THESIS TITLE: SCREENING OF TOMATO GERMPLASM FOR RESISTANCE TO YELLOW LEAF CURL VIRUS DISEASE HaNoi, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY � � � UNDERGRADUATION THESIS TITLE: SCREENING OF TOMATO GERMPLASM FOR RESISTANCE TO YELLOW LEAF CURL VIRUS DISEASE Student name : Nguyen Thi Ngoc Anh Student code : 620659 Class : K62CNSHE Supervisor : Prof Ph.D PhanHuu Ton Research location Center for Conservation and : Development of Crop Genetic Resources (CCD-CGR) HaNoi, 2022 COMMITMENT I hereby declare that the data and research results in this thesis are true and not copy the results of any previous graduate reports Graduation thesis with references to documents, citation information is indicated in the references section Hanoi, March 15th, 2022 Sincerely, Nguyen Thi Ngoc Anh i AKNOWLEDGEMENTS First of all, with the deepest and most sincere affection, allow me to express my gratitude to Prof Ph.D PhanHuu Ton, who enthusiastically guided and taught me valuable professional knowledge and lessons during the process of completing my undergraduation thesis Besides, I would also like to thank the masters and engineers PhanThanh Tung, PhanHuu Hien and PhamDinh On who are working at the Center for Conservation and Development of Crop Genetic Resources, for helping and creating the most favorable conditions for me in the process internship at the center Second, I would like to express my deep gratitude to all the individuals and organizations that have facilitated and supported me throughout the process of studying and researching In order to successfully complete this undergraduation thesis, in addition to my own efforts, I also received a lot of attention and support from teachers, friends as well as family members Third, I would like to express my deepest gratitude to the teachers of the Faculty of Biotechnology-Vietnam National University of Agriculture With the understanding and dedication of the teachers, they have imparted to me a lot of valuable knowledge during my years of studying at the school so that I can equip myself with the necessary knowledge to be able to successfully complete the undergraduation thesis Finally, I would like to thank my parents and loved ones for their love and support so that I can successfully complete this undergraduation thesis I would like to convey to them my deep gratitude Hanoi, March 15th, 2022 Sincerely, Nguyen Thi Ngoc Anh ii CONTENTS COMMITMENT i AKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii Part INTRODUCTION 1.1 Introduction 1.2 Aim and request 1.2.1 Aim 1.2.2 Request Part LITERATURE REVIEW 2.1 Overview of tomato 2.1.1 Origins of tomato 2.2 Tomato yellow leaf curl virus (TYLCV) 2.2.2 Distinguishing tomato yellow leaf curl virus (TYLCV) from other conditions 2.2.3 Resistance genes to tomato yellow leaf curl virus (TYLCV) Part MATERIAL, CONTENT AND METHOD 23 3.1 Material 23 3.2 Time and research schedule 24 3.3 Research contents 24 3.4 Method 24 3.4.1 Experiment 24 3.4.2 Evaluation of agronomy biological characteristics, yield potention and quality of fruit 24 iii 3.4.3 Evaluation of resistance to tomato yellow leaf curl virus of screening tomato varieties 26 3.4.3.1 Detection of resistance gene to yellow leaf curl virus Ty2 and Ty3 by PCR 26 3.4.3.2 Artificial infection 28 Part RESULTS AND DISCUSSION 30 4.1 Results of evaluation of some agro-biological characteristics 30 4.1.1 Time to complete the growth and development stages 30 4.1.2 Structural, morphological of plant 31 4.1.3 Yield and its componentelements 34 4.1.4 Fruit quality 37 4.2 Results of PCR method to select Ty2 and Ty3 genes 38 4.2.1 Results of total DNA extraction 38 4.2.2 Results of PCR to detect Ty2 and Ty3 genes 39 4.2.2.1 Results of PCR to detect Ty2 39 4.2.2.2 Results of PCR to detect Ty3 42 4.3 Results of artificial infection 45 Part CONCLUSION 48 REFERENCES 49 iv LIST OF TABLES Table 2.1 List of disease and disorder symptoms similar to TYLCV (Walker 2006) Table 2.2 Tomato (Solanum lycopersicum) wild relatives as resistance sources for tomato yellow leaf curl disease 14 Table 4.1 Growth stage of tomato 30 Table 4.2 Structural, morphological of tomatoes 31 Table 4.3 Morphological of fruit 33 Table 4.4 Yield and its component elements 35 Table 4.5 Fruit quality 37 Table 4.6 Characterization of some high yield and high quality varieties compared to control varieties 38 Table 4.7 Result of PCR to detect resistance gene Ty2 41 Table 4.8 Result of PCR to detect resistance gene Ty3 44 Table 4.9 Result of evaluation of the resistance level of each varieties by artificial infection 47 Table 5.1 Evaluation of good varieties 48 v LIST OF FIGURES Figure 2.1 Tomato fruit ripening process at the cell level Figure 2.2 Ty1 expression is elevated in resistant lines 11 Figure 2.3 Schematic representation of the region of interest of chromosome 16 Figure 2.4 Representation of single nucleotide polymorphism (SNP) and indel information for Ty4 closely-linked markers 20 Figure 2.5 SNP information for ty5 (Pelota) gene-based marker development 21 Figure 3.1 Expression levels of tomato yellow leaf curl disease infected by TYLCV (Lapidot and Friedmann, 2002) 29 Figure 4.1 Total DNA of 32 tomato varieties studied 39 Figure 4.2 Electrophoresis of PCR products using primer set T0302F/TY2R1 to detect resistance gene Ty2 (lane 1-32) 40 Figure 4.3 Electrophoresis of PCR products using primer set P6-25-F2/P6-25R5 to detect resistance gene Ty3 (lane 1-32) 43 Figure 4.4 Varieties after 40 days of grafting diseased plants 46 Figure 5.1 Two varieties with high yield, good quality, and the ability to carry resistance genes 48 vi LIST OF ABBREVIATIONS CTAB : Cetyltrimethylammonium bromide DNA : Deoxyribonucleic acid EDTA : Ethylene Diamine Tetraacetic Axit PCR : Polymerase chain reaction SCAR : Sequence characterized amplified regions SNP : Single nucleotide polymorphism TYLCV : Tomato yellow leaf curl virus vii Part INTRODUCTION 1.1 Introduction Tomato (Solanum lycopersicum L.) is an important fruit vegetable in our country It belongs to the family Solanaceae, which includes several other commercially important species Tomato is grown worldwide for local use or as an export crop Tomato has very few competitors in the value addition chain of processing For tomatoes, yellow leaf curl virus causes the most severe effects Tomato yellow leaf curl virus (TYLCV) is caused by several viruses belonging to the genus Begomovirus, family Geminiviridae, first detected in Israel in 1939 (Píco et al., 1996) This disease causes serious 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 (Reynaud et al., 2003) TYLCV is spread by the whitefly Bemisia tabaci, which is a fast-reproducing insect that is very difficult to control Pandemics caused by their outbreaks have been recorded Much effort has been focused on identifying sources of disease resistance by screening wild tomato varieties Currently, there is no effective pesticide to prevent this disease, if infected plants should only be uprooted Therefore, using disease resistant tomato varieties is the most effective measure Disease resistant tomato varieties are not only significant for improving yield and quality, but are also safe for human health, domestic animal and the environment (Phan Huu Ton et al., 2013) To successfully develop tomato varieties resistant to yellow leaf curl virus, the first thing to is to determine the number of resistance genes (Phan Huu Ton et al., 2013) Up till now, scientists around the world have identified different resistance genes to tomato yellow leaf curl virus DNA markers associated with yellow leaf curl resistance genes Ty1, Ty2, Ty3, Ty4 and ty5 have 4.2.2.2 Results of PCR to detect Ty3 Agrama and Scott (2006) reported three regions that contributed to resistance in breeding lines with introgressions from S chilense LA2779 or LA1932 One region corresponded to the region having the Ty1 locus Another region was the Ty3 locus, which was mapped to a region between cLEG-31-P16 (20 cM) and T1079 (27 cM) (Ji and Scott, 2006b; Ji et al., 2007) The third region was near the self-pruning (sp) and potato leaf (c) loci Another begomovirusresistance QTL, derived from an introgression from Solanum pimpinellifolium, was mapped near the marker TG153 (33 cM; Chagué et al., 1997) This is very near the Ty3 locus from LA2779 derived lines Previously, Ji and Scott (2006a, 2006b; Ji et al., 2007) reported that the Ty3 locus mapped to a region that included the FER locus (25 cM, BAC clone 56B23, AY678298) Jensen and Maxwell (Maxwell et al., 2007) found that the sequences for the G8 gene of the BAC clone 56B23 are different for lines derived from LA2779 and LA1932 To differentiate the two introgressions, the one from LA2779 is designated Ty3 and the one from LA1932, Ty3a The co-dominant SCAR marker, FLUW25, will distinguish between the ty3 and Ty3 alleles, but the FLUW25 marker will not detect the Ty3a allele A set of PCR primers that provide co-dominant SCAR markers for detection of the ty3, Ty3 and Ty3a alleles The primer set, P6-25-F2 and P6-25-R5, was designed to include the 143-nt ty3/Ty3 indel and to give smaller fragments than the FLUW25 primer set With begomovirus-resistant breeding lines derived from either the S chilense LA2779 source, Gc9, or the Ih902 line (Vidavsky and Czosnek, 1998), the expected 450bp Ty3 fragment was obtained A 320bp ty3 fragment was amplified from breeding lines lacking the introgression from either of these two begomovirusresistance sources A 630bp Ty3a fragment was obtained from lines derived 42 from S chilense LA1932, such as Gc171 Heterozygous hybrids were easily detected with these primers, which amplified two fragments corresponding to the S lycopersicum ty3 fragment (320bp) and either the Ty3 (450bp) or the Ty3a (630bp) fragment No F1 hybrids were available to test for fragments with the Ty3/Ty3a genotype, but it is expected that this primer pair would also amplify two fragments (450bp and 630bp) with this genotype The three sizes of the P6-25-F2/P6-25-R5 fragments were sequenced The 320bp and the 450bp fragments corresponded to the sequences of S lycopersicum and of the Ty3 locus associated with lines derived from S chilense LA2779, respectively The 660bp fragment from Gc171 had one large insert, when compared with the S lycopersicum sequence This pair of primers detect co-dominant SCAR marker, P6-25, for the ty3, Ty3 and Ty3a alleles at the FER locus, 25 cM It is not known how closely these markers are to the functional Ty3 gene (Ji et al., 2007), so it is possible that some breeding lines would give false negative or false positive results Also, it is not known how this primer pair will function with introgressions from other accessions of wild species Figure 4.3 Electrophoresis of PCR products using primer set P6-25-F2/P625-R5 to detect resistance gene Ty3 (lane 1-32) 43 Table 4.8 Result of PCR to detect resistance gene Ty3 Lane ID of varieties (The mark of Center) Containing the resistance gene Ty3 Size 320bp 125d3 + DV2962F3 + 329F7 + Số 14d1 + Tôn C2 + Samovi F1 + 30 x 1219F4 + 1007VH + 167-1 Da cam + 10 Số 13 + 11 AVTO1420CLN3670B 12 30ĐC2015 + 13 14-1 nhỏ + 14 27(3) + 15 AVTO1H4JY1JY to 16 1004-L + 17 14 x 9802(25111) + 18 AVTO0101VH to + 19 TC + 20 AVTO9803 + 21 VRDi11F2 + 22 Số + 23 165.163.6.1.VH + 24 DV2962F2 + 25 Tôn C1VH + 26 Số 20 vai xanh + 27 CH1 + 28 138.159.6.5.1 + 29 316-1 + 30 AVTO9803-2 + 31 31-1 + 32 Bi LS308 F1 + 450bp 630bp 660bp + + 44 + + + + + + 4.3 Results of artificial infection After 40 days of age from the date of tomato planting, tomato varieties with typical disease symptoms to graft wedges with diseased shoots Use a sharp knife to cut a small piece (length: 3-4 cm, width: 0.5-0.7 cm) of the diseased plant And use a rope to tie the two sections of the two plants to facilitate the movement of the virus from the diseased plant to the good plant Diseased plants in variety 108.141.176.513-VH (Card 2-6): infected by grafting into the following varieties: 165.163.6.1.VH (Card 13-1); 30 x 1219F4 (Card 10-10); DV2962F2 (Card 10-7); Số (Card 10-4) Diseased plants in variety 125d3 (Card 13-4): infected by grafting into the following varieties: Số 14d1 (Card 13-6); Ton C2 (Card 13-7); 31-1 (Card 13-8) Diseased plants in variety 9802 (Card 6-5): infected by grafting into the variety: AVTO9803-2 (Card 3-8) 45 Figure 4.4 Varieties after 40 days of grafting diseased plants 46 Table 4.9 Result of evaluation of the resistance level of each varieties by artificial infection Card ID of varieties (The mark of Center) Level Resistance 13-1 165.163.6.1.VH resistance: leaf edge is slightly yellow 10-10 30 x 1219F4 resistance: no disease symptoms 10-7 DV2962F2 resistance: no disease symptoms 10-4 Số resistance: no disease symptoms 13-6 Số 14d1 resistance: no disease symptoms moderately infected: some terminal 13-7 Ton C2 leaflets are yellowed and a few are curled infected: many leaves turn yellow, 13-8 31-1 curl, and curl upward, the plant continues to grow moderately infected: some terminal 3-8 AVTO9803-2 leaflets are yellowed and a few are curled 47 Part CONCLUSION In the study of some agro-biological characteristics, there are 12 varieties with high yield and high quality compared to the control variety Continue to study the resistance to tomato yellow leaf curl disease and the ability to carry resistance genes Ty2, Ty3 of these varieties, we found that there are varieties with high yield, good quality, and the ability to carry resistance genes Table 5.1 Evaluation of good varieties Theoretical Average weight of fruit (gram) 30 x 1219F4 (Card 10-10) Số (Card 10-4) yield (tons/ha) Degree Containing the Brix resistance gene (%) Ty2 or Ty3 Resistance ability 100.52 73.11 3.86 Ty2 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