The Transcriptome Analysis Of Apple Resistance To Diplocarpon Mali

The Marssonina leaf blotch of apple,caused by Diplocarpon mali,is one of the world’s early defoliation diseases,which could result in severe etiolation and abscission of leaves during the growing season then lead to serious economic losses.At present,most of the main apple cultivars in the production are susceptible to Diplocarpon mali,and using the disease resistance is the most effective way to solve and control this disease.Therefore,it is a great significance to excavate the resistance-germplasm resources of Marssonina and to clear the disease resistance mechanism in apple breeding.It has been found that the Malus baccata has resistance to Marssonina leaf blotch of apple,but studies on interaction between Malus baccata and Diplocarpon mali are few,and the resistance mechanism of the pathogen is not clear yet.In this research,the resistance material（Malus baccata）and the susceptible material（Malus domestica）infected with Diplocarpon mali were studied by the high-throughput sequencing technology of transcriptome sequencing,and bioinformatic methods were used to study the mechanism of Malus baccata resistant to Diplocarpon mali,which will provide theoretical foundation and guidance for clearing the resistance mechanism.The main results were as follows:1.Transcript expression profiles of two materials with reference transcriptome have been obtained,which infected with Diplocarpon mali.GO（Gene Ontology）enrichment analysis found that many of genes about the response to external biological stimuli,stress response and defense response were significantly enriched in the resistance material of Malus baccata.And a lot of differential expression genes related to biological metabolic processes were also enriched in Malus domestica when infected with Diplocarpon mali.KEGG（Kyoto Encyclopedia of Genes and Genomes）enrichment showed that the carotenoid biosynthetic pathway and the biosynthetic metabolic pathway of unsaturated fatty acids were enriched in the leaves of Malus baccata.Some differential expression genes about the flavonoid biosynthetic pathway and some amino acids metabolic pathways were actived when infected with Diplocarpon mali in Malus domestica.Malus baccata and Malus domestica could both stimulate the expression of resistance gene to resist Diplocarpon mali infection.But the percentages of Malus domestica’s R gene and LRR（leucine-rich repeats）structure were significantly smaller than that of Malus baccata.Quantitative Real-time PCR（q RT-PCR）showed consistent results with transcriptome analysis.The potential resistance genes in Rosaceae apple found in this study would provide a basis theoretical for the future study of the Diplocarpon mali.2.Using non-reference transcriptome analysis to study the resistance material of Malus baccata infected with Diplocarpon mali.48 868 unigenes were obtained.Using NR（Non-Redundant）,Swiss-port（Swiss-Prot Protein Sequence Database）,KOG（Eukaryotic Orthologous Groups of Proteins）,GO and KEGG etc.to predict the homology of the 48 868 unigenes,25 797 unigenes have been directly aligned onto the genes of Rosaceae Malus Apple.Aligning the unigenes with KOG database,according to their function,they can be divided into 25 categories.Taking KEGG database as a reference,according to the metabolic pathways,unigenes of Malus baccata infected with Diplocarpon mali have been targeted to 145 branched metabolic pathways,including ribosome metabolic pathways,carbohydrate metabolism,and so on.A total of 12 517SSR（simple repeat sequence）sites were searched by SSR site analysis.The FPKM of genes expression were calculated by RSEM,and the differential expression genes were identified by edge R wich FDR（False Discovery Rate）<0.05 and log₂Fold Change>2 or log₂Fold Change<-2.1 230 were up-regulated and 1869 were down-regulated.The number of identified differential expression genes was similar in Malus baccata by using the reference transcriptome and the non-reference transcriptome,and both were down-regulated more than up-regulated.In addition,GO enrichment analysis showed that partial of down-regulation genes were involved in carboxylic acid biosynthesis process,aromatic amino acid family biosynthesis process,jasmonic acid biosynthesis process,reaction to chitin and acid-stimulating reactions.The KEGG Pathway enrichment results showed that 94 differential genes were significantly enriched in plant-pathogen interactions,plant signal transduction pathways and amino acid biosynthesis,etc.Some regulated genes were validated by q RT-PCR and showed consistent results with transcriptome analysis.The results of these studies would provide theoretical foundation for clearing the resistance mechanism of the Malus baccata aganist Diplocarpon mali.