Fine Mapping Of Tomato Leaf Mould Resistance Gene Cf-10 And Analysis Of Resistance Response Mechanism

Tomato(Solanum lycoperslcum)is a worldwide vegetable crop,which plays an important role in people's daily life.According to the United Nations Food and Agriculture Organization(FAO)statistics,the output of tomatoes in 2012 was 16.2 million tons,which would bring a net profit of 55 billion dollars.Tomato leaf mould is one of the most important tomato diseases caused by Cladosporium fulvum.The tomato yield is usually reduced by 20~30%,and it can reach 50% when it is serious.Therefore,it is very important to find the resistance genes of tomato leaf mould and improve the molecular breeding.Currently,the cloned resistant genes of tomato leaf mould include Cf-2?Cf-4?Cf-5?Cf-9 and Cf-9DC,among which such disease resistant genes have been applied in the actual breeding work.Among them,Cf-4,Cf-9 and other resistance genes have been applied to the actual breeding production.However,the physiological races of Cladsoporium fulvum feature such traits as a big number of races and rapid speed of differentiation,and some of the species with contents of disease-resistant genes are gradually overcome by the newly generated physiological races;therefore,it is of an urgent need for us to explore new resistant genes and reduce the economic losses.The interactions between tomato and the leaf mould are the model for the research of the interactions between the plants and the pathogenic,and meanwhile it complies with the gene to gene hypothesis.The key for the interactions between the disease-resistant gene in the disease-resistant species and the physiological races in the pathogenic is that the receptor-like protein of the Cf gene can specifically identify the effect factors of the non-poisonous gene coding in the pathogenic,thus causing Hypertensive Response,HR.With the incident of HR,the protective enzyme system and the contents of active oxygen within the plants all incur changes,thus further stimulating the defensive response of the lower stream of the disease resistant mechanism.The exploration of new disease resistant genes and through some resorts of molecular biology,and the application of the disease-resistant genes in the breeding of new species features important roles.Through long-term field surveys and character identifications,the resistant material Ontario 792 of the tomato leaf mould resistant gene Cf-10 exhibits effective resistance on most physiological races.Cf-10 gene was initially located on the long arm of chromosome 8 by Kanwar in 1980 using the analytical method of genetics;there had been scarce studies in the successive periods,thus affecting the applications of Cf-10 gene in breeding.This research regards the range of the physiological races resistant of tomato leaf mould gene Cf-10,genetic regularities of the resistance,the process of the incompatiable interaction between the Cf-10 gene and C.fulvum,as well as the tomato leaf mould resistance gene Cf-10 mediate the resistance respose as the key points,this provides a theoretical basis for the cloning of the Cf-10 gene and the comprehensive disclosure of the resistance mechanism of Cf genes.The main results of this study are as follows:(1)Through artificial inoculation,the resistance range of thee tomato leaf mould resistant gene Cf-10,and the survey results indicated that the disease resistant material Ontario 792 with contents of Cf-10 genes exhibits disease resistance to the physiological races 1.2.3.4.5 and physiological races 1.2.3.4.9.In combination with the basis of the previous studies,this material exhibits disease resistance to a total of 8 known physiological races,and 2 physiological races exhibits immunity.(2)Upon inoculations of physiological traces of 1.2.3.4 on the resistance and infection parents,the differences and similarities between the compatible interaction and incompatible interactions.It is discovered through the trypan blue staining and electron microscopic observation that there are occurrences of allergic necrosis in the incompatible interactions(resistance);And the growth of the mycelium is inhibited in the resistant species;during the compatible interactions(susceptible),there are no occurrences of obvious necrosis,and the mycelium proliferation is more rapid;Upon inoculation of the pathogenic bacteria,the activities of the protective enzymes(SOD?POD?CAT)within the resistant parents and the contents of the active oxygen ROS all present upward trends;however,the activities of CAT and POD in the resistant parent Ontario 792 has been constantly higher than the susceptible parent Money Maker;LC-MS is used to verify the variations in the contents of salicylic acid SA and jasmine acid JA,and in the resistant species Ontario 792,it is discovered that the JA content reaches the peak value on Day 9 after inoculation,and the range of changes of JA contents in the susceptible parent Money Maker are smaller.(3)Inoculation verifications are conducted on the F2 generation group constructed by the resistant parent,and the statistical results indicate that the disease resistant parent and the F1 generation all have disease resistance,while the susceptible parents all expressed disease infection,and the F1 generation group separation ratio conforms to the Mendelian genetic law of 3:1,and the resistance ratio of the backcross community conforms to 1:1;it is re-verified that the genetic regulation of the leaf mould resistant gene Cf-10 is single dominance inheritance.(4)Through the re-sequencing analysis of the parents and the resistant and infective pools,the Cf-10 gene is positioned within the 3.29 Mb interval of the sort arm of No.1 chromosome,and within candidate zone,there are 23 mutations of shift codes whose non-synonymous mutations have 204 Indel of the SNP sites.Based on the typical structural characteristics of the coding protein of the clones tomato leaf mould disease gene,i.e.the e LRR-TM structure,and in combination with the function notes of all the genes within the candidate zone,within the candidate zone,16 genes(Solyc01g005720.3,Solyc01g005715.1,Solyc01g005865.1,Solyc01g005760.3,Solyc01 g 005710.2,Solyc01g007130.3,Solyc01g008140.3,Solyc01g005730.3,Solyc01g008390.2,Solyc01g006545.1,Solyc01g008410.2,Solyc01g006550.3,Solyc01g005775.1,Solyc01g005870.2,Solyc01g005755.1,Solyc01g008800.2)with similar structures of the cloned lead mould-resistant structures serve as the candidate genes,including 9 non-synonymous mutations and mutation genes of shift codes(Solyc01g005730.3,Solyc01g008390.2,Solyc01g006545.1,Solyc01g008410.2,Solyc01g006550.3,Solyc01g005775.1,Solyc01g005870.2,Solyc01g005755.1,Solyc01 g 008800.2).(5)Using KASP markers,and based on the SNP non-synonymous mutations within the candidate interval and the Indel shift code mutation,16 pair of KASP primers,in which 5 pairs effectively typed the F2 generation,and successfully narrow the candidate intervals in tomato No.1 chromosome 1224466~2014004,within about 790 kb.Similarly,based on the special structure of the Cf gene' coding protein,the genes are screened within that interval,and o1 candidate gene Solyc01g007130 is screened.(6)After the resistant materials Ontario 792 and susceptible material Money Maker are inoculated the physiological races 1.2.3.4 of leaf mould,sequencing transcription analysis is conducted;during the resistance response process of the Cf-10 gene regulations,a total of 2242 differentiation expression genes are detected,among which 1501 genes have upward expressions,while 741 genes downward expressions.Most differentiation expression genes are associated with the defense Signal transduction pathways,mainly including the interactions between the plants-pathogenic,oxidation reduction process and the plant hormone conduction.These differentiation expression genes contain 192 transcription factors,and these transcription factors are distributed in 42 families.When q RTPCR is conducted the transcription group data,the Solyc12g094620 gene on the signal conduction pathway(ko04146)related to the plant protective enzymes,the Solyc12g094620 is noted to be related to CAT synthesis in the database;meanwhile,the expression volume of the results of the gene's transcription sequencing have up-regulation,which is consistent with the previous assay results of CAT activity.(7)In the comparison of the transcription group data of the disease resistant responses regulated by the Cf-10 gene and Cf-12 gene,there are 1193 differentiation expression genes are completely consistent.