Identification of miRNAs involved in pear fruit development and quality

Abstract

Background: MicroRNAs (miRNAs) are a class of small, endogenous RNAs that take part in regulating genes through mediating gene expressions at the post-transcriptional level in plants. Previous studies have reported miRNA identification in various plants ranging from model plants to perennial fruit trees. However, the role of miRNAs in pear (Pyrus bretschneideri) fruit development is not clear. Here, we investigated the miRNA profiles of pear fruits from different time stages during development with Illumina HiSeq 2000 platform and bioinformatics analysis. Quantitative real-time PCR was used to validate the expression levels of miRNAs.

Results: Both conserved and species-specific miRNAs in pear have been identified in this study. Total reads, ranging from 19,030,925 to 25,576,773, were obtained from six small RNA libraries constructed for different stages of fruit development after flowering. Comparative profiling showed that an average of 90 miRNAs was expressed with significant differences between various developmental stages. KEGG pathway analysis on 2,216 target genes of 188 known miRNAs and 1,127 target genes of 184 novel miRNAs showed that miRNAs are widely involved in the regulation of fruit development. Among these, a total of eleven miRNAs putatively participate in the pathway of lignin biosynthesis, nine miRNAs were identified to take part in sugar and acid metabolism, and MiR160 was identified to regulate auxin response factor.

Conclusion: Comparative analysis of miRNAomes during pear fruit development is presented, and miRNAs were proved to be widely involved in the regulation of fruit development and formation of fruit quality, for example through lignin synthesis, sugar and acid metabolism, and hormone signaling. Combined with computational analysis and experimental confirmation, the research contributes valuable information for further functional research of microRNA in fruit development for pear and other species.

Figure 1

The length distribution of small RNAs. Y-axis represents percentages of small RNA identified in the study. X-axis represents the length of small RNA. Six libraries are shown by different colors. The figure shows that 21 nt to 24 nt were highest frequency (nearly 90%) among all of the clean reads in each library, with 24 nt as the most abundant (close to 50%) small RNA length, followed by 23 nt, 21 nt, and 20 nt, in that order.

Figure 2

First base bias at 21 nt of novel pear miRNA. Y-axis represents the frequency of nucleotides and X-axis represents different libraries. Four different colors in the bars represent the four kinds of nucleotide.

Figure 3

Expression pattern confirmed by qRT-PCR and comparison with sequencing data. Relative expression pattern of six different conserved miRNAs among various libraries are confirmed by qRT-PCR results by 2^-∆∆Ct method. For visualization, log₁₀ method is applied to compute the TPM data of sequencing results. The paragraph A to F represent the expression profiles of miR166a, miR4376-5p, miR156k, miR396b-3p, miR164c* and miR159b-3p in sequence. Y-axis represents the expression level and X-axis represents different libraries. Blue lines represent the q-PCR data and red lines represent the sequencing data. The line charts indicate the similar expression patterns with miRNA sequencing.

Figure 4

Nineteen conserved miRNAs from pear fruit and their homologs in 11 other plant species. The color shows the homology of miRNAs among the twelve plants. As the color gets closer to bright red, the relationship of the species gets closer to each other.

Figure 5

Expression profiles of members in miRNA families. Y-axis represents the expression level of miRNAs and X-axis represents different libraries. A-E represent sequencing reads of different members in five conserved miRNA families including miR4414a/4414a*, miR156k/156b*, miR167a/167a*, miR2111a*/2111a and miR164a/164c*). Similar expression patterns of different members in the miRNA family throughout development are shown.

Figure 6

The distribution of target gene numbers for each conserved miRNA among developmental stages. X-axis represents the interval for the target gene number of each conserved miRNA. Y-axis represents the number of miRNA in each interval.

Figure 7

GO annotation of target genes. Y-axis (left) represents percentages of genes identified in this study, Y-axis (right) represents the actual gene number. The genes were annotated in three main categories including biological progress, cellular component, and molecular function (X-axis).

Figure 8

Target gene GO enrichment factor screened by the p-value. A, B and C represent three ontologies: cellular component, molecular function and biological progress respectively.

Figure 9

Expression pattern of miRNAs and target genes. A, B, C, and D represent the comparisons of the expression patterns during pear fruit development for miR397a, miR1132, miR5077 and miR396b and their putative target genes, respectively. Y-axis (left) represents expression level of target genes, Y-axis (right) represents the expression level of miRNAs.

Figure 10

Relationship of miRNAs and target genes in lignin pathway. The triangles, boxes and circles represent miRNAs, pathways and target genes respectively.