Background: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few. Results: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, “6251AB” and “6284AB”. At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, “4001AB” and “4006AB”. Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, four differentially expressed miRNAs between “6251A” and “6251B”, and ten differentially expressed miRNAs between “6284A” and “6284B”. The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5′ modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed. Conclusions: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

Plant cuticular wax is the covering of the outer layer of the plant. It forms a protective barrier on the epidermis of plants and plays a vital role like a safeguard from abiotic and biotic stresses. In the present study, Brassica napus L. materials with and without wax powder were observed. Genetic analysis showed that the separation ratio of waxy plants to waxless plants was 15:1 in the F₂ population, which indicated that the wax powder formation was controlled by two pairs of genes. In order to identify the candidate genes associated with the wax powder trait of B. napus L., bulked segregant analysis (BSA) was performed. The homozygous waxy plants, the homozygous waxless plants, and plants from three parents were selected for establishing five DNA pools for genome-wide resequencing. The results of the resequencing showed that the site associated with wax powder trait was located in the region of 590,663–1,657,546 bp on chromosome A08. And 48 single nucleotide polymorphisms (SNPs) were found between the DNA sequences of waxy plants and waxless plants in this region. These SNPs were distributed across 16 gene loci. qRT-PCR analysis was conducted for the 16 candidate genes and three genes (BnaA08g01070D, BnaA08g02130D, and BnaA08g00890D) showed significantly differential expression between waxy and waxless parents. BnaA08g01070D and BnaA08g02130D were significantly down-regulated in the waxless parent, while BnaA08g00890D was significantly up-regulated in the waxless parent. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the BnaA08g02130D gene was enriched in lipid biosynthetic or metabolic processes. All the results in our study would provide valuable clues for exploring the genes involved in wax powder development.