Comparative Transcriptome Analysis Of The Regulation Of Responding To Low Temperature In Dendranthema Morifolium By RNA-Seq

Chrysanthemum(Dendranthema morifolium)is a type of herbaceous perennials,which is a popular ornamental plant worldwide.During Chrysanthemum bloom,low temperature is the primary constraint affecting the quality and productivity.Therefore,the research on hardiness has great importance to production and application of Chrysanthemum.In this study,RNA-seq technology was used to compare the transcript profiles of Chrysanthemum’s leaves under two different temperatures(20°C and-8°C).The main research results are as follows:1)The total data size of two read libraries was 13.54 Gb.The present sequencing data was completely saturated and sufficient for subsequent analysis.the T01 and T02 group c DNA libraries with Q30>80% were selected as high quality reads for further analysis.All contigs were further assembled into 71,917 unigenes in order to reduce sequence redundancy.Searching against Nr,Swiss-Prot,KEGG,GO and COG databases,33,282 unigenes were annotated,among them 32,981 unigenes showed high homology with sequences in the Nr database.Through the analysis of Unigene sequences using MISA software,seven types of SSR were identified.2)The number of unigenes with significant similarity to sequences in GO,Swiss-Prot,COG,and KEGG databases was 24,252,22,554,9,579,and 7,123,respectively.The annotated unigenes were assigned to 24 COG functional classes.‘General function prediction only’ concluded the highest proportion,which had2,664 unigenes;The second was ‘Replication,recombination and repair’,which had 1,569 unigenes;The ‘Transcription’ and ‘Signal transduction mechanisms’ were followed by ‘Replication,recombination and repair’,which had 1,433 and1,259 unigenes,respectively.There are 24,252 unigenes was annotated to GO databases.Based on sequence homology,the annotated sequences were grouped into the three main GO categories of biological process,cellular component,and molecular function,GO annotation of unigenes showed that these genes were assigned to a total of 56 subgroups,16 subgroups in ‘cellular component’ group,the unigenes that mapped to ‘cell part’,‘cell’ and ‘organelle’ constituted a high proportion.16 in ‘molecular function’,the unigenes that mapped to ‘catalytic activity’ and ‘binding’ constituted a high proportion.24 in ‘biological process’ group,the unigenes that mapped to ‘metabolic process’ and ‘cellular process’ constituted a high proportion.3)In order to identify differential expression genes under low temperature conditions,the transcriptome profiles were compared between T01 and T02 sample using FPKM method.A total of 2,310 DEGs were identified between the two groups.Among these DEGs,1,592 were up-regulated and 718 were down-regulated.GO annotation of DEGs showed that 1,354 DEGs were assigned to a total of 49 subgroups,13 subgroups in ‘cellular component’ group,13 in ‘molecular function’ and 23 in ‘biological process’.GO analysis indicated that significant differences in the DEGs between the two groups in biological Process was ‘oxidation-reduction process’,‘protein phosphorylation’,‘regulation of transcription,DNA-templated’ and ‘phosphorylation’.According to cellular components,the DEGs that mapped to ‘nucleus’,‘plasma membrane’,‘mitochondrion’,‘chloroplast’,‘cytosol’ constituted a high proportion.According to molecular function,the DEGs that mapped to ‘ATP binding’,‘nucleotide binding’,‘DNA binding’ constituted a high proportion.Additionally,annotated unigenes were assigned to 22 COG functional classes.‘General function prediction only’ concluded the highest proportion,which had 159 DEGs;The second was ‘Signal transduction mechanisms’ and ‘Transcription’,which had 103 and 102 DEGs,respectively.KEGG annotation of DEGs indicated that these genes were involved in 71 pathways.The clusters for ‘Plant hormone signal transduction’,‘Phenylpropanoid biosynthesis’,‘Photosynthesis-antenna proteins’,‘Plant-pathogen interaction’,‘Phenylalanine metabolism’,‘Biosynthesis of unsaturated fatty acids’ and ‘Starch and sucrose metabolism’were significantly enriched metabolic pathways.4)12 DEGs were selected randomly to compare with Illumina sequencing analysis using q RT-PCR.The results indicated that the changes of gene expression and transcriptome sequencing are basically identical.Among these selected DEGs,a few genes have no specific annotation.In annotated DEGs,‘MADS-box transcription factor’ was up-regulated,the expression quantity under-8 ℃ was3.12 times than that at room temperature.‘Flavine-containing monoxygenase’was down-regulated,it was 0.13 times than that at room temperature.‘Probable calcium-binding protein CML31-like’ was down-regulated,it was only 0.067 times than that at room temperature.‘Proline-rich protein’ was up-regulated,the expression quantity under-8 ℃ was 1.72 times than that at room temperature.‘Chlorophyll a-b binding protein CP26,chloroplastic’ was 0.10 times than that at room temperature.‘U-box domain-containing protein’ was 2.02 times than that at room temperature.In this study,an overview of the many changes to the Chrysanthemum ‘Jin Long Teng Yun’ transcriptome induced by exposure to sub-zero temperature has been provided using Illumina technology.The research analyzed the expression profiles of these genes during cold treatment stage and our results facilitated further studies on genes discovery of chrysanthemum and provided the theory reference on the resistance breeding and molecular mechanisms in the process of responding to low temperature.