MicroRNAs and their putative targets in Brassica napus seed maturation

Abstract

Background: MicroRNAs (miRNAs) are 20-21 nucleotide RNA molecules that suppress the transcription of target genes and may also inhibit translation. Despite the thousands of miRNAs identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant Brassica napus (canola) - especially in seeds.

Results: Using next-generation sequencing technologies, we performed a comprehensive analysis of miRNAs during seed maturation at 9 time points from 10 days after flowering (DAF) to 50 DAF using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at 4 selected time points. We identified more than 500 conserved miRNA or variant unique sequences with >300 sequence reads and also found 10 novel miRNAs. Only 27 of the conserved miRNA sequences had been previously identified in B. napus (miRBase Release 18). More than 180 MIRNA loci were identified/annotated using the B. rapa genome as a surrogate for the B.napus A genome. Numerous miRNAs were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. miRNA targets in B. napus were predicted and their expression patterns profiled using microarray analyses. Global correlation analysis of the expression patterns of miRNAs and their targets revealed complex miRNA-target gene regulatory networks during seed development. The miR156 family was the most abundant and the majority of the family members were primarily expressed in the embryo.

Conclusions: Large numbers of miRNAs with diverse expression patterns, multiple-targeting and co-targeting of many miRNAs, and complex relationships between expression of miRNAs and targets were identified in this study. Several key miRNA-target expression patterns were identified and new roles of miRNAs in regulating seed development are suggested. miR156, miR159, miR172, miR167, miR158 and miR166 are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. miR156 may regulate the developmental transition to germination.

Figure 1

Size distribution of small RNA (sRNA). A. Changes in sRNA size abundance (bp) in flower buds and seeds at 9 developmental stages. B. Changes in abundance of sRNA species in various seed tissues at 25DAF.

Figure 2

Total sequencing reads in each library (normalized to 10 M). A. Total reads in flower buds and at 9 seed developmental stages. B. Total reads in embryo, endosperm and seed coat at 4 seed developmental stages. C. Total reads in radicle, hypocotyl and cotyledon of embryo at 25DAF and 45DAF.

Figure 3

Proportion of miRNA variants in each miRNA family. 42 miRNA families with total reads more than 300 are shown in order of decreasing abundance (only variants with reads >300 are included in the analysis).

Figure 4

Temporal expression patterns of conserved miRNAs and variants. Hierarchical clustering of the 80 most abundant miRNA/variants during seed development and at flowering (FL). The color indicates the relative expression level: Blue-low, yellow-medium and red-high. Four major expression patterns (A1 – A4) are identified.

Figure 5

Tissue-specific expression of most of the conserved miRNA/variants. Hierarchical clustering of the most abundant 80 miRNA/variants in seed tissues during seed development. The color indicates the relative expression level: Blue-low, yellow-medium and red-high. Four major expression patterns (B1 – B4) were identified.

Figure 6

Comparison of miRNA expression measured by qPCR (TaqMan MicroRNA Assays) and Solexa sequencing (NGS). For 6 selected miRNAs, the miRNA expression changes at different tissues/stages were calculated relative to the embryo at 15 DAF(15EM). 15EM: embryo at 15 DAF, 25EM: embryo at 25DAF, 35EM: embryo at 35DAF, 15EN: endosperm at 15 DAF, 25EN: endosperm at 25 DAF, 15SC: seed coat at 15 DAF, 25SC: seed coat at 25 DAF.

Figure 7

Typical mapping patterns of miRNAs. Sequence reads are mapped to the putative precursor regions of the B. rapa genome. Four examples of miRNA patterns are shown including miR158 and miR408 and two novel putative miRNAs (miR5802 and miR5804), each exhibiting two well-defined regions of alignment. The unique reads are mapped to the stem-loop sequence (the secondary structure with folding energy value is shown immediately below the genome sequence). Sequences with <5 reads are not shown. The sum of the read counts of each sequence are incorporated into the sequence name as _x read count. The green color represents the forward reads, red represents reverse reads. Mature miR sequences and miR* sequences are highlighted in orange and blue respectively.

Figure 8

Overview of gene expression during seed development (in blue). The predicted miRNA targets are highlighted in orange. Each line represents one unigene. The Y-axis shows the normalized signal intensity (with baseline transformation to the median of all samples).