Transcriptome And Digital Gene Expression Analysis Of Chrysanthemum Responses To White Rust

Chrysanthemum is one of the most important ornamental crops in the world and widely used in cut flowers, potted plant and landscape. Diseases stress seriously limits its production and distribution. Chrysanthemum white rust (CWR), one of the primary diseases of chrysanthemum, has been considered as a quarantine disease in many countries. At present, there have been few studies on the molecular mechanism of white rust resistance in chrysanthemum. In this study, we perform transcriptome and digital gene expression profiling analysis of chrysanthemum response to white rust infection using the RNA-Seq technology, to screen the differential expression genes (DEGs) for function annotation and metabolic pathway analysis.The main results are listed as follow:The leaves of Chrysanthemum were used as material, total RNA was extracted by four different methods (CTAB method, Trizol method, modified Trizol method and RNA prep pure kit method) and we analyzed the quality of the extraction. The results showed that total RNA which extracted by the CTAB method had DNA contamination. The stripe of total RNA which extracted by Trizol method was complex, has the impurities such as protein pollution. Using modified Trizol method to extract total RNA, the stripe was clear, but the RNA was degradated. Using RNA prep pure kit method to extract total RNA, the stripe was clear and the RNA had higher purity, the OD260/OD280 ratio was 1.889, the concentration was 144.000 μg/mL, which was an easy and efficient method for total RNA extracting from chrysanthemum leaves.The resistant (C029) and susceptible (C008) varieties were sequenced by Illumina HiSeqTM 2000.20597533,19519160,20570705 and 20301157 total reads were obtained from four libraries, respectively, and for all samples, clean reads were over 97.34% of the total. We obtained 392137 unigenes through sequencing assembly and gained 213531 complete protein coding sequences by annotation (account for 54.45%). Based on KOG analysis,119750 unigenes aligned successfully and were classified into 26 categories, and 56.08% of the sequences were attributed to ’general Function Prediction Only’(1378, account for 1.15%). A total of 103633,12650 unigenes were annotated into the GO and KEGG databases. According to SSR analysis,32636 SSR sites were obtained (account for 8.33%), with an occurrence frequency of 1/5.55 kb, wherein the number of single nucleotide repeat type was the most, and the five nucleotide repeat type was the least.Four samples (R0, R1, S0, S1) were analyzed in digital gene expression profiling, statistical evaluation of fragment size, PCR amplification, sequencing error rate distribution and equencing quality, the sequencing results are high quality, can be used for subsequent analysis.433 significant DEGs were obtained between RO and R1, and 787 DEGs were obtained between SO and S1 through expression quantity analysis and functional annotation in 194179 genes, screening of differentially expressed genes. There were 14 genes which had differential expressions in both two comparison groups, wherein those of 13 genes had opposite variation tendencies between the two comparison groups.These DEGs were involved in the pentose phosphate pathway, phenylpropanoid biosynthesis pathway, carbon metabolism pathway and phenylalanine metabolism pathway. The results of the qRT-PCR indicated that the expression tendency of DEGs was consistent with that of the transcriptome sequencing results.Our transcriptome sequencing can help to fill the gaps in the literature on CWR transcriptome and enrich sequence information of chrysanthemum transcriptome. It also offers candidate genes that can be used to guide future studies attempting to breed resistant cultivars, and provides a theoretical basis for exploring the mechanism of response of chrysanthemum to white rust.