SNP Mining And Effectors Screening In Puccinia Helianthi Transcriptome

Sunflower rust（Puccinia helianthi Schw.）is one of the major diseases of sunflower,which seriously affects the yield and quality of sunflower.At present,it happens all over the world.In this study,sunflower rust transcriptome data obtained using the new generation of high-throughput Illumina sequencing technology were used to perform SNP locus excavation and effector screening using software.The polymorphism analysis of sunflower rust was performed and the relative expression levels of candidate effector proteins were determined.1.To understand the function and pathogenicity of the SNP gene in the P.helianthi.,we used SOAPsnp to search for the single nucleotide polymorphisms（SNP）of 59409 unigenes sequence in the transcriptome data of different germination stages（0,4,8h）of race 330 of P.helianthi.The frequency of sequences with SNP was calculated.Nr,Nt annotation,GO classification,COG classification,KEGG metabolic pathway annotation,and PHI（Pathogen Host Interaction）alignment were performed.A total of 29966 SNPs were identified in 8321 unigenes,the frequency of SNPs was 1/2764 bp,and the frequency of transitions was 65.40%.The annotation results showed that there were 79.64% and 43.00% unigenes annotated to Nr and Nt libraries.Genes involved in genetic information process pathways were mostly of translation,and some of them were lethal genes.Therefore,we believed that the pathogenicity of the rust was related to fungal cell wall modification proteins and potential effector protein.2.We amplified 48 samples of sunflower rust by screening eight pairs of primers,and then digested the PCR products of these 8 primers with 10 commonly used restriction enzymes,In the primer enzyme combination,only35 pairs could obtain a clear and stable PCR-RFLP marker.Only 10 pairs detected polymorphism,accounting for 28.60%.A total of 115 DNA fragments were detected in 35 pairs combination,of which 19 were polymorphic.The genetic similarity coefficient GS = 0.81 was the threshold,48 samples based on geographical location can be divided into two groups,the group 1 samples are mainly from Alashan,Bayannaoer,Ordos and other places in InnerMongolia.Group 2 were mainly from Hohhot,Wulanchabu,Datong in Shanxi Province,and Zhangjiakou in Hebei Province.3.Our team has analyzed 35286 protein sequences of the sunflower rust transcriptome,yielding a total of 908 secreted proteins.We used Excel to further screen out 656 candidate effector proteins that were pathogen-related but did not contain the PFAM domain.Among them,17 contained PFAM domains related to pathogenicity;There were 192 [Y/F/W]x C motifs similar to effector of the wheat powdery mildew of.（Blumeria graminis f.sp.tritici）,of which there were 78 Yx C motifs,96 Fx C motifs and 18 Wx C motifs;There are 13 Rx LR motifs similar to Phytophthora in the（Phytophthora）;20[L/I]x AR motifs similar to the effector of Magnaporthe grisea;eight G[I/F/Y][A/L/S/T]R similar to the Melampsora lini effector.27NLS-containing candidate effector proteins,304 SCR proteins,and 94R-proteins were screened by T-REKS algorithm.It indicated that the candidate effector proteins contained in sunflower rust had certain sequence homology with known effector proteins.4.To further identify candidate effector proteins,we used real-time quantitative PCR to analyze the gene expression level of 11 genes at the initial infection stage（3d,4d,5d）,to confirmed whether the expression level of these genes was two times to pure spores.The results showed that the relative expression of the two genes Cluster₂237,Cluster₂9163 were more than twice as much as pure spores of rust on the third day;The Cluster₃6818 were more than twice as much as pure spores of rust on the fifth day;The expression levels of the four genes Cluster 32378,Cluster 38252,Cluster39758,and Cluster 40966 at 3d,4d,and 5d were all more than twice as much as pure spores of rust.Only the expression of Cluster 32378 on the fifth day had the highest ratio of 160.50-fold to the pure spores of rust.