Mapping Of Tomato Leaf Mold Resistance Gene Cf-16 And Analysis Of Resistance Response Mechanism

Tomato leaf mold is caused by Cladosporium fulvum?C.fulvum?,which is a serious fungal disease in production.The spread of leaf mold is very rapid.Leaf mold can cause a serious reduction in tomato yield at the worst stage,especially for tomatoes in protected areas.The most effective method for resistance to leaf mold is still to breed disease-resistant varieties containing disease-resistant genes.At present,the leaf mold resistance genes used in breeding production are mainly Cf-5 and Cf-9.However,their application has been restricted with the continuous differentiation of physiological races.The excavation of new leaf mold resistance genes has become particularly important in order to cultivate new leaf mold resistance varieties,reduce losses in tomato production,and increase tomato yield.The leaf mold resistant material Ontario7816 containing the Cf-16 gene has shown good resistance in years of field identification,but there is less research on Cf-16 and it has not yet been cloned.In this study,a comprehensive analysis of the Cf-16 gene was performed,including the resistance range and genetic regularity,the interaction process,related physiological indicators,mapping and transcriptome sequencing.Our research provides a theoretical basis for the cloning and breeding of the Cf-16 gene and the comprehensive disclosure of the resistance mechanism of Cf gene to leaf mold.The main results of this study are as follows:?1?The resistance range of tomato leaf mold resistance gene Cf-16 was confirmed through artificial inoculation.The results showed that the resistant material Ontario7816 containing the Cf-16 gene had good resistance to C.fulvum.In combination with the results of previous research in our laboratory,this material exhibits disease resistance to a total of 10 known physiological races.?2?We performed inoculation identification on the F₁,F₂,and BC₁P₂ generations constructed by the parents Ontario7816 and Moneymaker.The results showed that the F₁ generation showed full resistance to C.fulvum.The segregation ratio between resistant and susceptible plants in the F₂population was 3:1.The segregation ratio between resistant and susceptible plants in BC₁P₂ plants was 1:1.They all conformed to Mendel's genetic laws.It is clear that the resistance of Cf-16 to C.fulvum is a dominant inheritance controlled by a single gene.?3?The interaction process between the resistance,susceptible materials and C.fulvum was observed by trypan blue staining.Obviously,plants carrying the Cf-16 resistance gene showed a strong HR after infection with C.fulvum,whereas the susceptible plants?i.e.,Moneymaker?showed continuous hyphal growth.?4?We identified two important time points?4 and 8 dpi?for transcriptome sequencing analysis based on trypan blue staining observations.?5?The physiological indicators of tomato plants at different time points after C.fulvum infection were determined.The content of ROS and the activity of three protective enzymes?SOD,POD,and CAT?in resistant and susceptible materials were increased after C.fulvum infection,and the increasing trend of resistant materials was more significant.The contents of SA and JA in Ontario7816 showed an upward trend after infection by C.fulvum,and the JA content increased rapidly in the early stage of inoculation.?6?The Cf-16 gene was mapped to tomato chromosome 6 by parental resequencing combined with BSA association analysis.Subsequently,two SSR markers TGS447 and TES312 which closely linked to the Cf-16 gene were obtained by?SNP-index and the traditional SSR molecular markers.The genetic distances between the two markers and the Cf-16 gene were both 1.3 c M.The physical distance of this interval was 3.63Mb.Two candidate genes XM?004240667.3 and XM?010323727.1were obtained which may be similar in structure to the cloned Cf genes.?7?To obtain transcriptome profiles of Cf-16 tomato and Moneymaker following C.fulvum infection,we performed RNA-Seq analysis at 4 and 8 dpi.Drastic transcriptional changes occurred at 4 dpi in both tomato cultivars.The most highly enriched GO terms were those associated with the organization of the cell wall or the metabolism of its components.The most highly enriched pathways,“Plant hormone signal transduction”and“Plant-pathogen interaction”,may be the major metabolic pathways involved in the Cf-16-mediated resistance response to C.fulvum.Map Man results showed that the upregulated genes were classified as R genes,MAPKs,PR proteins,TFs and genes associated with the hormones ethylene,ABA,SA and JA.The genes 109120689,101253178,and BGI?novel?G001591 encoding RPM1 in the plant-pathogen interaction pathway are most likely candidate genes for leaf mold resistance genes.In this preliminary comparison,more up-regulated DEGs were detected in Cf-16 compared to Cf-10,Cf-12 and Cf-19 tomatoes during the early stage of C.fulvum infection.