Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing

Abstract

Background: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing.

Results: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions.

Conclusions: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development.

Figure 1

Early developmental stages of potato tuber. Samples were collected from field-grown potato plants during the period of tuberization. PT0 - unhooked stolon tip (0 stage), PT1 - hooked stolon tip (1^st stage), PT2 - sub-apical stolon swelling (2^nd stage), PT3 - tuber initiation (3^rd stage). See methods for details of physiological and environmental conditions in which potato plants were grown.

Figure 2

Length distribution of small RNA sequences identified in all the small RNA libraries. A) Size distribution of total reads. 24 and 21 nt length are most abundant reads among the small RNAs. B) Size distribution of unique small RNA sequences where 24 and 23 nt are the most abundant. nt - nucleotide, PT0 - 0 stage of tuberization, PT1 - 1st stage of tuberization, PT2 - 2^nd stage of tuberization, PT3 - 3^rd stage of tuberization.

Figure 3

Genomic locus distribution of predicted miRNA precursors. Most of the miRNA precursors originate from intergenic regions, and in genic regions majority of them are encoded by introns.

Figure 4

Conserved miRNA families and their members identified in potato. Graphical representation of the different members of conserved miRNA families identified by deep sequencing of small RNA libraries prepared from tissues of leaf, root, stem and four developmental stages of potato tuber.

Figure 5

Predicted secondary structure of precursors (Mfold) of few potato-specific miRNAs. The mature miRNA sequences are highlighted in light red colour while the star sequences are highlighted in light green colour. Hairpin structure of all pre-miRNAs of potato-specific miRNAs, predicted using RNAfold, is given in Additional file 4.

Figure 6

Expression analysis of miRNAs in different tissues and different development stages of tuberization in potato by qRT-PCR. A) The expression profile of 15 conserved miRNA identified in this study in potato (miR164_1, miR172_1, miR394_1, miR396_1, miR399_1, miR171_2, miR159_1, miR157_1, miR172_5, miR171_9, miR6149_1, miR482_1, miR171_10, miR398_1, miR8044_1). B) The expression profile of 6 potato-specific miRNAs identified in this study (miRNA72, miRNA111, miRNA53, miRNA193, miRNA34, miRNA152). The expression level of each miRNA in PT0 was set as control and taken as 1, and expression level in all other tissues was quantified relative to it. 18S rRNA was taken as an endogenous control. L - Leaf, S - Stem, R - Root, PT0 - 0 stage of tuberization, PT1 - 1^st stage of tuberization, PT2 - 2^nd stage of tuberization, PT3 - 3rd stage of tuberization (for details of the tuberization stages see Figure 1).

Figure 7

GO analysis and categorization of putative targets of potato-specific miRNAs. According to GO annotation, the putative targets of potato-specific miRNAs are categorized into biological process, molecular function and cellular component.

Figure 8

Target validation of conserved miRNAs by 5′ RLM-RACE. Cleavage site of four conserved miRNAs targets were experimentally verified. The arrow indicates the cleavage site with the frequency of cloned RACE products shown above it. The vertical lines indicates matched base pairs and dot denotes G:U pairs, ‘:’ symbolize mismatches.