Study Of Transcriptome And Digital Gene Expression Profiles In Foxtailmillet And Its Gene Expression Analysis During Interaction With Uromyces Setariae-italicae

Foxtail millet originated in China, and is widely planted in arid areas in the north of China. It has many advantages, such as drought resistance, barren resistance, saline-alkaline tolerance, high nutritional value, etc. Therefore, it plays an important role in grain crops planting structure in our country. The foxtail millet rust caused by Uromyces setariae-italicae is a serious disease of foxtail millet all over the world, which often result in millet yield decreased by 10%~30% in the year of rust epidemic. In this paper, millet leaf samples inoculated with rust were administered to Illumina high-throughput sequencing. Comprehensive and systematic understanding of foxtail millet transcriptome features were acquired, and foxtail millet Shilixiang rust resistance related genes and mechanisms of rust-responsive transcription in foxtail millet were found, which provide a new way for rust resistance breeding.Leaf samples of foxtail millet Shilixiang inoculated with rust were collected at 0, 24, and 48 h after inoculation and RNA was isolated. And then Solexa sequencing was performed using Illumina GA II x, a total of 8,439,350 clean reads were obtained. Transcriptome de novo assembly was carried out with short reads assembling program-SOAPdenovo, and 32538 unigenes with a final unigene N50 length of 1021 nt were obtained. The unigenes were functionally annotated according to the protein sequence similarity, KEGG Pathway, COG and Gene Ontology. Out of the 32538 unigenes, 23811 unigenes were annotated to Nr protein databases, 16350 unigenes were annotated to Swiss Prot protein databases, 12223 unigenes were assigned to 124 known metabolic or signaling KEGG pathways, 9250 sequences have a COG classification, and 2240 unigenes were categorized into three main GO categories. According to the annotation information, preliminary identified 228 genes related to the defense mechanism, 448 genes related to secondary metabolites biosynthesis, transport and catabolism, 5 genes related to the immune system process, and 161 genes related to the response to stimulus, which laid a foundation for further analysis of millet-rust interaction gene expression patterns, and revealed the mechanism of interaction between millet and rust.On the basis of the transcriptome sequencing results, digital gene expression profiling analysis of millet Shilixiang leaf samples inoculated with rust was performed using the Illumina Hi Seq? 2000 System. Three millet Shilixiang DGE libraries generated approximately three million clean tags in each library. Foxtail millet Shilixiang leaf transcriptome reference gene database that included 32538 sequences was preprocessed for tag mapping. Among the clean tags, the number of sequences that could be mapped to unigenes ranged from 2.02 to 2.08 million, and the number of mapped unigenes were 13806, 16005, and 15426. The number of unigenes that mapped by unambiguous tag were 13709, 15887, and 15322, and the percentage of these unigenes were 42.13% 、 48.83%, and 47.09% among the reference gene database. To specifically identify genes related to rust inoculation in foxtail millet, the differentially expressed genes between every pair of samples were identified. Comparison of DEGs in Shilixiang at 24 h after inoculation with rust versus Shilixiang inoculated with rust 0 h showed that 4542 genes differed significantly between the two samples. Between Shilixiang at 48 h after inoculation with rust and Shilixiang inoculated with rust 0 h, a total of 5112 DEGs were detected. We identified 968 genes that were expressed differently at 48 h post-inoculation compared to after only 24 h. For the DEGs, we found that ‘ribonucleoprotein complex’ and ‘macromolecular complex’ were the most significantly affected GO terms of cellular component; ‘structural molecule activity’ and ‘organic substance metabolic process’ terms were the most significantly affected GO terms of molecular function; and ‘cellular amino acid metabolic process’ was the most significantly affected GO term of biological process. KEGG pathway analysis of DEGs found that the cluster for ‘Ribosome’ represented the largest group. In order of decreasing group size, this was followed by clusters for: ‘Metabolic pathways’, ‘Biosynthesis of phenylpropanoids’, ‘Phenylalanine, tyrosine and tryptophan biosynthesis’, ‘Arginine and proline metabolism’, ‘Biosynthesis of alkaloids derived from shikimate pathway’, ‘Steroid biosynthesis’, ‘Phenylpropanoid biosynthesis’, ‘Flavonoid biosynthesis’, etc. Through the above analysis, some genes related with rust resistance of foxtail millet were identified and the relatively comprehensive mechanisms of rust-responsive transcription in foxtail millet were revealed.Quantitative real-time polymerase chain reaction(q RT-PCR) analysis was used to analyze the expression of 34 selected genes, and the patterns observed agreed well with DGE analysis. Expression levels of the genes were also compared between a resistant cultivar Shilixiang and a susceptible cultivar Yugu-1, and the result indicated that expression level of Shilixiang is higher than that of Yugu-1. And Unigene5531(GLU), Unigene15666(CHT), Unigene25719(PER1), Unigene734(MKK4), Unigene12730(WRKY7), Unigene30025(WRKY62), Unigene30953(WRKY70), Unigene11273(MYB), Unigene29832(GST24), Unigene7613(GST29), Unigene30834(GST), Unigene25649(SGT), Unigene23762(PAL), Unigene10309(HR), Unigene13691(XA26), Unigene14926(NPR1), Unigene32120(RPS1/RPM2), Unigene16195(DR), and Unigene1811(DR) were the most outstanding differential expression genes of them.