Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes

Abstract

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

Figure 1.

Nucleotide preferences of small RNAs. A, Percentage of adenosine at the last position of 21- to 24-nucleotide (nt) small RNAs. The asterisk indicates a difference of P < 0.01 (Student’s single-sample two-sided t test). B, Nucleotide preferences of the 24-nucleotide-independent 23-nucleotide small RNAs at each position. C, Percentage of adenosine at each position of the 23-nucleotide small RNAs of 162 Arabidopsis deep sequencing data sets deposited in the NCBI GEO database.

Figure 2.

Expression of several miRNAs and their corresponding miRNA*s. A to C, Normalized sequence reads of miR160a (A), miR171b (B), and miR408 (C) and their miRNA*s in the four libraries. D, Northern blot hybridization of miR408 and miR408*.

Figure 3.

Expression profile of miRNAs between the H and L cultivars. A, Comparison of miRNA abundance between the two cultivars. B and C, Quantitative RT-PCR (B) and northern-blot hybridization (C) analyses of selected differentially expressed miRNAs between the cultivars. Mixed samples containing all examined time points were used. Error bars indicate the sd of three replicates.

Figure 4.

Expression changes of miRNAs at different silique developmental stages. A, Quantitative RT-PCR analysis for the relative expression of miR156, miR390, miR2111, and miR6028 during early silique development. The expression level of each high-oil-content cultivar miRNA at 3 DAF is set to 1. B, Northern-blot hybridization of miR156, miR390, miR167, and miR6029 during early silique development. Error bars indicate the sd of three replicates.

Figure 5.

Validation and expression of selected miRNA target genes. A, 5′-RLM-RACE analysis of the cleavage on target mRNAs by corresponding miRNAs. B, Quantitative RT-PCR analysis of miR156 target gene TC98126 at different developmental stages and miR6028 target gene CD814980 in different cultivars. Error bars indicate the sd of three replicates.

Figure 6.

MiRNA-derived ta-siRNAs in B. napus. A, Expression abundance of ta-siRNAs generated from TAS3 gene TC65909. The locations of two miR390 target sites and TAS3 5′ D7(+) are labeled by short bars. B and C, Quantitative RT-PCR (B) and northern-blot hybridization (C) analyses of TAS3 5′ D7(+) during early silique development. D, Validation of cleavage on B. napus TC110025 (ARF3) mRNAs by TAS3 5′ D7(+) using 5′-RLM-RACE. Error bars indicate the sd of three replicates.