Preliminary Study On Molecular Identification Of Cherry Cultivars And Resistance Mechanism Of Two Cherry Rootstocks To Crown Gall Disease

Cherry industry is promising planting industry with high quality and added value.However,in recent years,with the continuous expansion of cherry cultivation area,the problems of variety confusion and cherry disease are more and more serious.To solve the problem of synonyms and homonyms that occurred in cherry production,63 cherry cultivars(varieties and rootstocks)planted in Shaanxi province,China were used to conduct molecular identification,establish DNA fingerprinting and do cluster analysis,which has important guiding significance to the identification of cherry varieties.Crown gall disease caused by Agrobacterium tumefaciens results in great economic losses in cherry orchard.In this study,‘Gisela 6’ and Mahaleb ‘CDR-1’ were used to explore the defense response to crown gall disease.Mahaleb ‘CDR-1’ with higher resistance was used to carry out the resistance study of crown gall disease.It provides a new resistance gene for crown gall research field,and lays a theoretical foundation for further research and development of crown gall agents,which is of great significance for speeding up the solution of crown gall and reducing the economic loss in cherry orchard.Below are the key research results:(1)Establishment of cherry DNA fingerprinting.The capillary electrophoresis with fluorescent-labeled simple sequence repeat(SSR)primers was used to identify 63 cherry cultivars(varieties and rootstocks).A total of 146 alleles were amplified by 10 SSR primer pairs,ranging from 10 to 20 per locus(mean: 14);among the SSR primer pairs,genotype number ranged from 12 to 26(mean: 18).The mean values of gene diversity,heterozygosity,and polymorphism information content(PIC)were 0.7549(range: 0.4011 to 0.8782),0.5952(range: 0.3810 to 0.9683),and 0.7355(range: 0.3937 to 0.8697),respectively.An unweighted pair-group method with arithmetic average(UPGMA)cluster analysis was used to separate the cherry cultivars.A model-based structure analysis separated the cultivars into three populations,which was consistent with the results of a phylogenic and principal component analysis(PCA).Based on Bayes’ Rule,the cultivars were further subdivided into seven populations.Some of the 63 cherry cultivars that are often confused in production were distinguished,and DNA fingerprinting of cherry cultivars was established.(2)The physiological changes induced by A.tumefaciens in cherry rootstock ‘Gisela 6’.The morphology of the infected tissue surface was examined.We determined the activity of defense-related enzymes and the content of phytohormones,salicylic acid(SA)and jasmonic acid(JA).It was found that infection with A.tumefaciens increased the activity of superoxide dismutase(SOD),peroxidase(POD),polyphenol oxidase(PPO),ascorbate peroxidase(APX),monodehydroascorbate reductase(MDHAR),and glutathione reductase(GR).It also elevated the JA content of cherry plants.No significant difference was noted in catalase(CAT)and phenylalanine ammonialyase(PAL)activity between the infected and control groups.In the treatment group,a slight increase in lipoxygenase(LOX)activity was observed at 15 days post-infection(dpi),whereas dehydroascorbate reductase(DHAR)activity declined by almost 50% at 10 dpi.The total SA content showed a general upward trend in infected plants but did not show a clear difference compared with the control.Moreover,we assayed the expression of genes encoding these enzymes and SA and JA biosynthesis genes using quantitative real-time PCR,the expression of the corresponding genes was upregulated,which was consistant with the changes of enzymes activity and phytohormones content.The results suggested that Agrobacterium infection did not elicit a hypersensitive response in ‘Gisela 6’ but the development of tumor altered the expression level of genes involved in defense responses and phytohormone biosynthesis.(3)The resistance mechanism of ‘CDR-1’ to crown gall disease.The morphology of the infected tissue surface was examined.We determined the activity of ten defense-related enzymes and the content of SA and JA.Transcriptome analysis was further used to screen the target gene,and finally,the function of the target gene in ’CDR-1’ was verified through transient expression and transgenic test in tobacco.The results showed POD increased in the first 10 days,while PAL and LOX increased in the first 15 days post-infection.Four key enzymes in the AsA-GSH cycle also responded,to a certain extent;although JA content increased significantly after the treatment,the SA content did not.In a follow-up transcriptome analysis,the differentially expressed genes Pm4CL2,PmCYP450,PmHCT1,PmHCT2,and PmCAD were up-regulated.We further measured lignin content,and found it increased significantly.The Pm4CL2 gene screened from the differentially expressed genes was used to conduct transient expression and transgenic experiments in tobacco to verify its function in crown gall disease resistance.It showed the relative expression of the treatment group was almost 14-fold that of the control group at 12 h post-treatment.After the infection treatment,clear signs of resistance were found in the transgenic lines;this indicated that under the higher expression level and earlier activation of Pm4CL2,plant resistance was enhanced.This study indicated that the crown gall resistance is likely related to the lignin synthesis pathway,in which Pm4CL2 functions crucially during the plant defense response to the pathogen A.tumefaciens.