Identification Of Chrysanthemum Nankingense Gene Expression Profiles And MicroRNA Responses To Low Nitrogen Stress By RNA-SEQ

The diploid Chrysanthemum L. species Chrysanthemum nankingense is a native plant in China. The species related to the commercially important polyploidy ornamental species C. morifolium （the garden chrysanthemum）. Nitrogen is one of essential mineral nutrients for plants growth. So finding out genes and miRNA responding to low N stress is important for improving the N-use efficiency （NUE） and reducing the loss of nitrogen.In this study, Chrysanthemum nankingense was sequenced to discover the related genes and miRNA using the Illumina HiSeqTM 2000. We constructed four small RNA libraries and four cDNA libraries. The materials were treated as follow:roots treated with normal 4 mmol L-1 NO3- and N-stressed 0.04 mmol L-1 NO3- （R-sample）, leaves treated with normal 4 mmol L-1 NO3- and N-stressed 0.04 mmol L-1 NO3- （L-sample）. Expression profile and miRNA data were analyzed. The main conclusions are as follows:（1） About 7,000,000 clean reads were generated from each cDNA library. Differentially expressed genes are 1510 and 686 in R-sample and L-sample, respectively. There are 458 up-regulated genes and 1052 down-regulated genes in R-sample. There are 419 up-regulated genes and 267 down-regulated genes in L-sample. The R-sample and L-sample have 109 up-regulated genes and 67 down-regulated genes in common. We used gene ontology （GO） assignmentsto classify the functions of the predicted C.nankingense DTGs, and we mapped the DTGs sequences to the reference canonical pathways in Kyoto Encyclopedia of Genes and Genomes （KEGG）. Based on sequence homology,2,855 and 691 sequences were categorized into 42 and 34 functional groups in R-sample and L-sample, respectively. In total,423 sequences were assigned to 94 KEGG pathways in R-sample and 138 sequences were assigned to 67 KEGG pathways in L-samples.（2） The DTGs identified were involved in photosynthesis, transcription factors, nitrogen metabolism genes, and kinases. In particular, we founded two genes （Unigene36710_S2₁, Unigene82641_S2₁） involved in nitrogen transporter. Fifteen selected genes were verified by quantitative RT-PCR. The trends of up-and down-regulation from qRT-PCR were consistent with those from the Solexa sequencing, indicating that the changes detected by DTG analysis were reliable.（3） Roots and leaves of Chrysanthemum nankingense were sequenced to discover the differentially expressed miRNA responding to low nitrogen. Eighty-two miRNA （the log2 ratio≥1） responding to low nitrogen were identified in roots, which contained 29 up-regulated and 53 down-regulated miRNA.102 miRNA （the log2 ratio≥1） responding to low nitrogen were identified in leaves, which contained 40 up-regulated and 62 down-regualted miRNA. We found miR169i-3p and miR398a-5p in roots, miR169r-3p and miR393a-3p in leaves, which the same miRNA species have been reported related to low nitrogen stress.（4） We predicted the targets of known miRNA differentially expressed by Mireap. There are 37 differentially expressed miRNA whose targets can be predicted in roots. The numbers of target gene and target location were 219 and 235, respectively. There are 44 differentially expressed miRNA whose targets can be predicted in leaves. The numbers of targets gene and target location were 219 and 204, respectively. Targets of miRNA differentially expressed were divided into three categories accoding to their function, sa follow:the targets related to metabolism, the targets which edcode transcription factors, and the targets which encode kinases. Selected miRNA and targets were verified by quantitative RT-PCR. The trends of up-and down-regulation from qRT-PCR were consistent with those from the Solexa sequencing.