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. 2015 Oct 13;16:776.
doi: 10.1186/s12864-015-1995-1.

Identification of miRNAs Involved in Fruit Ripening in Cavendish Bananas by Deep Sequencing

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Free PMC article

Identification of miRNAs Involved in Fruit Ripening in Cavendish Bananas by Deep Sequencing

Fangcheng Bi et al. BMC Genomics. .
Free PMC article

Abstract

Background: MicroRNAs (miRNAs) are a family of non-coding small RNAs that play an important regulatory role in various biological processes. Previous studies have reported that miRNAs are closely related to the ripening process in model plants. However, the miRNAs that are closely involved in the banana fruit ripening process remain unknown.

Methods: Here, we investigated the miRNA populations from banana fruits in response to ethylene or 1-MCP treatment using a deep sequencing approach and bioinformatics analysis combined with quantitative RT-PCR validation.

Results: A total of 125 known miRNAs and 26 novel miRNAs were identified from three libraries. MiRNA profiling of bananas in response to ethylene treatment compared with 1-MCP treatment showed differential expression of 82 miRNAs. Furthermore, the differentially expressed miRNAs were predicted to target a total of 815 target genes. Interestingly, some targets were annotated as transcription factors and other functional proteins closely involved in the development and the ripening process in other plant species. Analysis by qRT-PCR validated the contrasting expression patterns between several miRNAs and their target genes.

Conclusions: The miRNAome of the banana fruit in response to ethylene or 1-MCP treatment were identified by high-throughput sequencing. A total of 82 differentially expressed miRNAs were found to be closely associated with the ripening process. The miRNA target genes encode transcription factors and other functional proteins, including SPL, APETALA2, EIN3, E3 ubiquitin ligase, β-galactosidase, and β-glucosidase. These findings provide valuable information for further functional research of the miRNAs involved in banana fruit ripening.

Figures

Fig. 1
Fig. 1
Length comparison of small RNAs from different treated banana fruit. CK, ET and 1-MCP stand for sample without any treatment; sample treated with ethylene and sample treated with 1-MCP (1-Methylcyclopropene) respectively. Y-axis represents the numbers of small RNA identified in this study. X-axis represents the length of small RNA. The length of the deep sequencing results were mainly between 18 and 24 nt. The number of 23 nt sequences is greater than the number of other sequence lengths in the control and the ET libraries. However, in the 1-MCP library, 21 nt sequences were the most abundant small RNAs
Fig. 2
Fig. 2
Conserved miRNAs and their family numbers in banana fruit. Y-axis represents the number of member in different miRNA family. Y-axis shows various conserved miRNA family identified in all three libraries. The miR156, miR159, miR166 miR171, miR172 and miR396 had nine members, however, 17 miRNA families had only one member
Fig. 3
Fig. 3
Differentially expressed miRNAs under two comparisons (ET/1-MCP, and ET/CK) in banana fruit. The bar represents the scale of relative miRNA expression (Log2 Fold change). The bottom left shows the color bar. The heatmap was generated by Heml 1.0
Fig. 4
Fig. 4
A comparison of the relative expression of differentially expressed conserved miRNA families. Comparison of ET treatment and CK (a), and comparison of ET treatment and 1-MCP treatment (b)
Fig. 5
Fig. 5
The distribution of target gene numbers for differentially expressed miRNAs among banana ripening process. The differentially expressed miRNAs of ethylene/1-MCP and their targeted genes are analyzed in this figure. The X-axis represents the interval for the target gene number of each miRNA. The Y-axis represents the number of miRNA in each interval
Fig. 6
Fig. 6
Gene ontology of the predicted targets for differentially expressed miRNAs. The targeted genes of differentially expressed miRNAs in the comparison of ethylene/1-MCP is analyzed in this figure. The right-hand-side scale is the targeted gene numbers corresponding to the GO terms. The left-hand-side scale is the percent of the targeted gene numbers corresponding to the GO terms
Fig. 7
Fig. 7
KEGG analysis of the 20 most enriched pathways. The coloring of the q-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 x-axis represents name of enrichment pathway. The Y-axis represents rich factor
Fig. 8
Fig. 8
Quantitative expression analyses of several differentially expressed miRNAs and their target genes. The expression level in day 0 was set as 1. U6 rRNA and CAC (clathrin adaptor complexes gene) gene was used as the internal control for miRNA expression and targeted genes expression respectively. Each bar indicates the mean ± SE of triplicate assays. The different letters indicate significant differences at P < 0.05 using Fisher’s protected least significant difference (PLSD) tests

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