Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.
BACKGROUND
MicroRNAs (miRNAs) are small, non-coding RNAs that are important regulators of gene expression, and play major roles in plant development and their response to the environment. Root extracts from Panax notoginseng contain triterpene saponins as their principal bioactive constituent, and demonstrate medicinal properties. To investigate the novel and conserved miRNAs in P. notoginseng, three small RNA libraries constructed from 1-, 2-, and 3-year-old roots in which root saponin levels vary underwent high-throughput sequencing.


METHODS
P. notoginseng roots, purified from 1-, 2-, and 3-year-old roots, were extracted for RNA, respectively. Three small libraries were constructed and subjected to next generation sequencing.


RESULTS
Sequencing of the three libraries generated 67,217,124 clean reads from P. notoginseng roots. A total of 316 conserved miRNAs (belonging to 67 miRNA families and one unclassified family) and 52 novel miRNAs were identified. MIR156 and MIR166 were the largest miRNA families, while miR156i and miR156g showed the highest abundance of miRNA species. Potential miRNA target genes were predicted and annotated using Cluster of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Comparing these miRNAs between root samples revealed 33 that were differentially expressed between 2- and 1-year-old roots (8 increased, 25 decreased), 27 differentially expressed between 3- and 1-year-old roots (7 increased, 20 decreased), and 29 differentially expressed between 3- and 2-year-old roots (8 increased, 21 decreased). Two significantly differentially expressed miRNAs and four miRNAs predicted to target genes involved in the terpenoid backbone biosynthesis pathway were selected and validated by quantitative reverse transcription PCR. Furthermore, the expression patterns of these six miRNAs were analyzed in P. notoginseng roots, stems, and leaves at different developmental stages.


CONCLUSIONS
This study identified a large number of P. notoginseng miRNAs and their target genes, functional annotations, and gene expression patterns. It provides the first known miRNA profiles of the P. notoginseng root development cycle.
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China. wrc830612@163.com.|Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China. wrc830612@163.com.
Rongchang
R
Wei
Department of Molecular Biology, The Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, China. qiudy@caf.ac.cn.
Deyou
D
Qiu
CSIRO Agriculture, PO Box 1600, Canberra, ACT 2001, Australia. Iain.wilson@csiro.au.
Iain W
IW
Wilson
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China. 53522722@163.com.
Huan
H
Zhao
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China. sflu@implad.ac.cn.
Shanfa
S
Lu
Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China. mjh1962@vip.163.com.
Jianhua
J
Miao
Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China. shixin1996@126.com.
Shixin
S
Feng
Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China. whitefh2008@126.com.
Longhua
L
Bai
Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China. wqh196501@163.com.
Qinghua
Q
Wu
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China. fairytdp@163.com.
Dongping
D
Tu
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, China. mayixuan10@163.com.
Xiaojun
X
Ma
Guangxi Botanical Garden of Medicinal Plants, Nanning, 530023, China. tangqi423@sina.com.|Hunan Provincial Key Laboratory of Crop Germplasm innovation and Utilization and National Chinese Medicinal Herbs (Hunan) Technology Center, Hunan Agricultural University, Changsha, 410128, China. tangqi423@sina.com.
Qi
Q
Tang
D019295:Computational Biology / Q000379:methods; D017124:Conserved Sequence; D019143:Evolution, Molecular; D018506:Gene Expression Regulation, Plant; D059014:High-Throughput Nucleotide Sequencing; D035683:MicroRNAs / Q000235:genetics*; D053723:Panax notoginseng / Q000737:chemistry / Q000235:genetics*; D018517:Plant Roots / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics*; D015203:Reproducibility of Results; D012503:Saponins / Q000737:chemistry; D014315:Triterpenes / Q000737:chemistry
D035683:MicroRNAs; D012333:RNA, Messenger; D018749:RNA, Plant; D012503:Saponins; D014315:Triterpenes