Transcriptome Analysis Of Two Different Pathotypes Of Puccinia Triticinia With Different Virulence

Wheat leaf rust caused by Puccinia triticina（PT） is an airborne diseases, and hasoccurred worldwide, causing significant yield losses in wheat production when thediesease developes seriously. PT is an obligate parasite and is distributed all over the world.The interaction between avirulence（Avr） genes from the pathogen and resistance（R） genesfrom the host is in line with gene-for-gene theory. In the past, most studies at the molecularlevel were focus on the host resistance gene expression mechanisms and wheat leaf rustresistance gene mining, etc., but the molecular mechanisms and gene expression studies ofparasitic and pathogenic PT are less. In this study, Chinese PT races were as the researchmaterials, and latest research means transcriptome sequencing based Illumina sequencingwas employed to study PT development and pathogenic related genes on transcriptomelevel. RNA analyses of different virulence strains revealed the differences effectively, andon this basis the speculate pathogenicity-related genes will be deduced. This providedproof for revealing the interaction between wheat and PT. Also the results will contributeto a better future in looking for antimicrobial drug targets, and promote research anddevelopment of antimicrobial drugs, having important theoretical significance and majorinnovations. The main results are as follows:1. The de novo transcriptome sequencing on PT was carried out on the platform ofIllumina HiSeqTM2000.4153708620nt bases were generated totally. In the results ofassembly,46008unigenes were detected, total length for unigenes was31256810nt,average length was679nt，N50was1064nt. For function annotation analysis, we got25960,17884,14822,14384,11004,11241unigenes which annotate to the Nr, Nt,Swiss-Prot, KEGG, COG, GO database, respectively. The total annotation unigenes were27030. For protein coding region prediction analysis, the number of CDS that mapped tothe protein database was25834, the number of predicted CDS was6646. The total numberof CDS was32480.7804SSR was harvested and on the base of SSR fragments3065SSRprimers were developed.2.Total RNA from wheat leaves6days after inoculated with09-12-284-1and09-19-727-1, and urediospores of this two PT races, were sequenced using IlluminaHiSeqTM2000paired-end sequencing. The sequencing samples were named as Tc284₂, Tc727₂, PT284₃and PT727₃, respectively. The sequencing results compared totranscriptome of PT,7206771,7070710,7316832and7273551total mapped reads wereobtained. In sample Tc284₂, total mapped reads matched to reference genome locationsand reference genes locations were33.04%and40.74%, respectively, in sample Tc727₂,the two data were27.52%and33.94%, respectively, in sample PT284₃, the two datawere46.49%and56.26%, respectively, and in sample PT727₃, the two data were47.66%and57.29%, respectively.3.Based on RPKM method, a total of10077up-regulated genes were detected, and10699genes were down-regulated in Tc727₂-vs-Tc284₂. The significantly differentiallyexpressed genes had63, which had34genes up-regulated and29genes down-regulated inTc727₂-vs-Tc284₂. A total of9645genes were up-regulated and7044genes weredown-regulated in PT727₃-vs-PT284₃.64significantly differentially expressed geneswere detected, of which62genes were up-regulated and2genes were down-regulated inPT727₃-vs-PT284₃. A total of15798genes were up-regulated and5323genes weredown-regulated in PT284₃-vs-Tc284₂. The significantly differentially expressed geneswere up to7706, which contained6384up-regulated genes and1322down-regulatedgenes in PT284₃-vs-Tc284₂. A total of15541genes were up-regulated and5331geneswere down-regulated in PT727₃-vs-Tc727₂.7814significantly differentially expressedgenes had been detected, of which6572genes were up-regulated and1242genes weredown-regulated in PT727₃-vs-Tc727₂. Functional annotation of genes with GO mainlyinvolved in binding, hydrolytic, catalytic activity, transfer of substances, signaltransduction, biosynthesis of substance and substance metabolism.4.Twele genes selected from the differentially expressed genes inTc727₂-vs-Tc284₂and PT727₃-vs-PT284₃, were suggested be associated with thewheat leaf rust development or pathogenicity. The annotation information includedtyrosinase （Unigene13386_Tc15₂）, Hsp20（Unigene19506_Tc15₂andUnigene11663_Tc15₂）, thaumatin （Unigene4298_Tc15₂）, subtilisin-like serineproteases （Unigene14052_Tc15₂）, glycosyl hydrolase family18（Unigene4195_Tc15₂）,cytochrome P450（Unigene4769_Tc15₂）, Pheromone A receptor （Unigene9410_Tc15₂）,pectinesterase （Unigene21335_Tc15₂）, major facilitator superfamily（Unigene9911_Tc15₂）, PAZ domain/Piwi domain （Unigene11491_Tc15₂） and majorintrinsic proteins （Unigene8781_Tc15₂）.The signal peptide predicted showed that5of theprotein encoded by the12genes had signal peptide, and4of them had transmembranedomains. Predicted results of all of the protein that had annotated functional wereconsistent with their respective functions. The five libraries and the analysis based on thelibraries built a good platform for the researches on mechanism of pathogenicity of PT.