Small RNA and degradome profiling reveals miRNA regulation in the seed germination of ancient eudicot Nelumbo nucifera

Abstract

Background: MicroRNAs (miRNAs) play important roles in plant growth and development. MiRNAs and their targets have been widely studied in model plants, but limited knowledge is available concerning this small RNA population and their targets in sacred lotus (Nelumbo nucifera Gaertn.).

Results: In this study, a total of 145 known miRNAs belonging to 47 families and 78 novel miRNAs were identified during seed germination using high-throughput small RNA sequencing. Furthermore, some miRNA families which have not yet been reported in monocot or eudicot species were detected in N. nucifera, indicating that these miRNAs was divergence from monocots and core eudicots during evolution. Using degradome sequencing, 2580 targets were detected for all the miRNAs. GO (Gene Ontology) and KEGG pathway analyses showed that many target genes enriched in "regulation of transcription" and involved in "carbohydrate", "amino acid and energy metabolism". Nine miRNAs and three corresponding targets of them were further validated by quantitative RT-PCR.

Conclusions: The results present here suggested that many miRNAs were involved in the regulation of seed germination of sacred lotus, providing a foundation for future studies of sacred lotus seed longevity. Comparative analysis of miRNAs from different plants also provided insight into the evolutionary gains and losses of miRNAs in plants.

Keywords: Degradome sequencing; Nelumbo nucifera; Quantitative qRT-PCR; Target genes; miRNA.

Fig. 1

miRNA distribution in different plant species. The miRNA data were obtained from the miRbase 20 and the present study

Fig. 2

Potential novel miRNAs identified in this study. a The secondary structure of novel_ miR_36 precursor was predicted by MFOLD. Mature miRNA is highlighted in red. b Stem-loop RT-PCR analysis of the identified novel miRNAs. Six novel miRNAs (novel_miR_2, novel_miR_13, novel_miR_29, novel_miR_32, novel_miR_36, novel_miR_66) were confirmed via stem-loop RT-PCR. The sizes of the obtained PCR products were approximately 60 bp. Marker indicates a 50 bp DNA Ladder Marker

Fig. 3

Small RNAs and their expression patterns at each of the five stages during the seed germination of sacred lotus. a Heatmap for the clustering analysis of differentially expressed known miRNAs. The bar represents the scale of the expression levels of the miRNAs. b Venn diagram of the common and specific known miRNAs at four different stages (12 h, 24 h, 36 h and 72 h) compared with 0 h

Fig. 4

Target plots (t-plots) of six identified known miRNA targets using degradome sequencing. The red lines indicate signatures consistent with miRNA-directed cleavage. a Nnu-miR156a slicing NNU_02903-RA at nt 1107(SPL17: Squamosa promoter-binding-like protein 17). b Nnu-miR159b slicing NNU_10896-RA at nt 1284 (GAM1: Transcription factor GAMYB). c Nnu-miR160a slicing NNU_12564-RA at nt 1549 (ARF18: Auxin response factor 18). d Nnu-miR169a slicing NNU_09768-RA at nt 1642 (NFYA10: Nuclear transcription factor Y subunit A-10). e Nnu-miR319c slicing NNU_00026-RA at nt 2081(TCP2: Transcription factor TCP2). f Nnu-miR393a-5p slicing NNU_01291-RA at nt 1689 (TIR1: Protein TRANSPORT INHIBITOR RESPONSE 1). "alignment score" is the score for mismatch. Score = 0 represents perfect match and G:U = 0.5

Fig. 5

GO analysis and KEGG pathways of the target genes. a GO analysis of the target genes for differentially expressed miRNAs. The colouring of the p-values indicates the significance of the rich factor. The circle indicates the target genes that are involved, and the size is proportional to the gene numbers. The Y-axis represents the name of enrichment GO terms. The X-axis represents rich factor. The "Rich factor" was calculated by the number of genes mapped to the GO term divided by the number of all genes in the input list. b KEGG pathways of the target genes for differentially expressed miRNAs

Fig. 6

Validation of differentially expressed miRNAs and some of their targets during the seed germination in sacred lotus by qRT-PCR a Nnu-miR157a, b Nnu-miR168a-5p, c Nnu-miR393A-5p, d Nnu-miR396b-5p, e Nnu-miR397, f Nnu-miR2111a,  g Nnu-miR156a, h Nnu-miR319c, i Nnu-miR394a,  j Target of Nnu-miR156a (NNU_02036-RA, SPL16), k Target of Nnu-miR319c (NNU_00026-RA, TCP2), l Target of Nnu-miR394a (NNU_22183-RA, FBX)

Fig. 7

Analysis of miRNA editing events. a Summary of the nucleotide substitutions positions among miRNAs. b Summary of the nucleotide substitution types among miRNAs. c Validation of the editing sites in miRNAs obtained from deep sequencing by Sanger sequencing. The edited sites are highlighted with black frames. The upper is sequences of genomic DNA (gDNA), and the lower is sequences of cDNA

Fig. 8

The potential regulatory network for miRNAs in the seed germination of sacred lotus