High-throughput sequencing and characterization of the small RNA transcriptome reveal features of novel and conserved microRNAs in Panax ginseng

Abstract

microRNAs (miRNAs) play vital regulatory roles in many organisms through direct cleavage of transcripts, translational repression, or chromatin modification. Identification of miRNAs has been carried out in various plant species. However, no information is available for miRNAs from Panax ginseng, an economically significant medicinal plant species. Using the next generation high-throughput sequencing technology, we obtained 13,326,328 small RNA reads from the roots, stems, leaves and flowers of P. ginseng. Analysis of these small RNAs revealed the existence of a large, diverse and highly complicated small RNA population in P. ginseng. We identified 73 conserved miRNAs, which could be grouped into 33 families, and 28 non-conserved ones belonging to 9 families. Characterization of P. ginseng miRNA precursors revealed many features, such as production of two miRNAs from distinct regions of a precursor, clusters of two precursors in a transcript, and generation of miRNAs from both sense and antisense transcripts. It suggests the complexity of miRNA production in P. ginseng. Using a computational approach, we predicted for the conserved and non-conserved miRNA families 99 and 31 target genes, respectively, of which eight were experimentally validated. Among all predicted targets, only about 20% are conserved among various plant species, whereas the others appear to be non-conserved, indicating the diversity of miRNA functions. Consistently, many miRNAs exhibited tissue-specific expression patterns. Moreover, we identified five dehydration- and ten heat-responsive miRNAs and found the existence of a crosstalk among some of the stress-responsive miRNAs. Our results provide the first clue to the elucidation of miRNA functions in P. ginseng.

Figure 1. Analysis of P. ginseng small RNA population.

(A) Size distribution of clean reads. (B) Electrophoretic analysis of small RNAs. Low molecular weight (LMW) RNA was resolved on a 15% denaturing polyacrylamide gel and stained with ethidium bromide. The sizes of RNA ladder are indicated. (C) Size distribution of unique small RNA sequences. (D) Percentage of unique sequences starting with an A, C, G, and U in each size group.

Figure 2. Number of reads for the conserved miRNA families.

The read numbers of MIR159 and MIR166 are shown.

Figure 3. The MIR482a and MIR2118 precursors cluster in a unigene.

(A) The secondary structure of PUT-183a-Panax_Ginseng-17125 was predicted by the mfold program using the default parameters . Arrows indicate the position and orientation of miRNAs and miRNA*s. (B) Alignment of the mature miR482a and miR2118 sequences. Vertical dashes indicate the Watson-Crick pairing between miR482a and miR2118 sequences.

Figure 4. Validation of the predicted mRNA targets.

The cleavage sites were determined by 5′ RLM-RACE. The nucleotide positions of the miRNA complementary sites of unigenes are indicated. The unigene sequence of each complementary site from 5′ to 3′ and the cloned miRNA sequence from 3′ to 5′ are shown. Watson-Crick pairing (vertical dashes) and G:U wobble pairing (circles) are indicated. Vertical arrows indicate the 5′ termini of miRNA-guided cleavage products, as identified by 5′ RLM-RACE, with the frequency of clones shown.

Figure 5. Tissue-specific expression of miRNAs in P. ginseng.

Fold changes of miRNAs in leaves (L), stems (S), flowers (F), roots (R) of five-year-old P. ginseng plants and embryogenic calli (C) induced from the cotyledons of P. ginseng seeds are shown. miRNAs were analyzed using the poly(T) adaptor RT-PCR method. miRNA levels in leaves were arbitrarily set to 1.

Figure 6. Responses of non-conserved miRNAs to dehydration stress.

Fold changes of non-conserved miRNAs in ginseng embryogenic calli treated with dehydration for 0, 1, 3, 6, 12 and 24 h are shown. miRNAs were analyzed using the poly(T) adaptor RT-PCR method. miRNA levels in calli treated for 0 h were arbitrarily set to 1.

Figure 7. Responses of non-conserved miRNAs to heat stress.

Fold changes of non-conserved miRNAs in ginseng embryogenic calli treated with heat (37°C) for 0, 1, 3, 6, 12 and 24 h are shown. miRNAs were analyzed using the poly(T) adaptor RT-PCR method (A) and the stem-loop RT-PCR method (B). miRNA levels in calli treated for 0 h were arbitrarily set to 1.

Figure 8. Analysis of chloroplast-derived small RNAs.

(A) Size distribution of clean reads. (B) Size distribution of unique sequences. (C) The number of small RNA sequences mapped onto P. ginseng chloroplast genome. X-axis shows nucleotide positions of P. ginseng chloroplast genome. Y-axis shows the number of sRNAs mapped at each position. Black represents sense small RNAs, Red represents antisense small RNAs. The number of antisense sRNAs is shown as negative numbers.