Transcriptome Of Urediospore Germination Of Puccinia Helianthi

Urediospore stage of sunflower rust as the only stage of infection can repeat many times that plays a decisive role on large-scale outbreak of rust. The aim of this study is to indentify genes related with urediospore germination and understand the molecular regulation mechanism by RNA-Seq technology. In this research, urediospores of race 330 of Puccinia helianthi were used as material for high-throughput analysis by Illumina HiSeq 2500 technology. The main results of this research are as follows:1、Urediospore germination of Puccinia helianthi is closely related to the external environment condition, The results showed the suitable range of temperature of urediospores germination was 10～25℃. The optimal inoculation concentration was 20 mg/L in water. Light had inhibitive effect on germination within 5h after inoculating, but no inhibitive effect at 6h. Fresh spores were used for study to assure a high percentage of germination, it was ascertained that the urediospores had lost their germination abilities after 270 days stored at room temperature. By microscope, we found that there was significant difference in germination rate and length of urediospores germ tubes on the six culture substrates under the optimal temperature and humidity condition. Moreover, we could observe movement of yellow particulates in the germinated tubes and formation of special structure on PVDF film and millipore filter.2、Three cDNA libraries were successfully constructed by Illumina HiSeq 2500 platform. The clean reads were then assembled into 59409 unigenes. A batch of sequences related with process of urediospore germination was obtained, which provided the basis for further research on pathogenicity genes and regulatory mechanism of Puccinia helianthi.3、Approximately 40949 and 22313 unigenes of the urediospores were annotated to homology sequences of NCBI Nt, NCBI Nr, Swiss-Prot, GO, COG and KEGG databases. 19032 unigenes were classified to 24 COG groups. According to GO analysis,17114 unigenes were assigned to three main categories. KEGG pathway analysis showed that 34938 unigenes were classified to 38 pathways.4、Based on the analysis of differential gene expression between different stage of urediospore germination, the number of differentially expressed genes were different in different periods. Between SY0 and SY4, the number of differentially expressed genes was 3936, in addition,2045、609 differentially expressed genes between SY0 and SY8、 SY4 and SY8. Through the function enrichment analysis of differentially expressed genes in KEGG metabolic pathway, pathways that differentially expressed genes participated in at SYO, SY4, SY8 were very different. The number of unigene involved in various pathways at SY4 was much higher than the other two times, and same as the trend of gene expression quanlities of total differencially expressed genes.5、Using Real time PCR detection technology,20 differentially expressed genes were chosen for validation.The results were basically in line with the sequencing result. It indicated that the data from RNA-Seq was accurate and reliable.In the study we got a large numbers gene invoved in the process of urediospore germination of Puccinia helianthi, these genes not only contained a large number of known genes, more important novel genes were also included. These genes will largely contribute to study of the molecular mechanism of urediospore germination process and the development of molecular markers such as SNP, SSR.