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.

Direct seeding of winter rapeseed in the Yangtze River Basin often coincides with low temperatures during establishment. The aim of this study was to test whether low-temperature (LT) germination performance predicts overwintering survival and yield under delayed sowing. Thirty accessions were evaluated in controlled germination at 20/14 °C (CK) and 12/6 °C (LT) and in two field seasons (2020–2021 and 2021–2022), with six sowing dates from 15 October to 4 December. Mean germination rate was 97.6% in CK and 88.0% in LT. Germination potential (GP) averaged 95.7% in CK and 41.9% in LT. Root and shoot length decreased from 7.63 and 5.02 cm in CK to 1.47 and 0.48 cm in LT. Overwintering survival declined with later sowing. In the colder season (2020–2021), survival for sowings after November fell below 20% for most accessions, whereas S1–S2 averaged above 80%. Yield decreased with delay. In 2021–2022, yield under S1 exceeded S2–S6 by 5.5%, 8.5%, 13.9%, 14.0%, and 23.3%. In 2020–2021, S1 was similar to S2, but 6.3–22.8% higher than S3–S6. Thousand-seed weight followed the same trend. LT GP and LT root length were positively correlated with yield at several sowing dates in the colder season, indicating that LT germination traits are predictive of late-sown performance under harsher winters. Seven accessions (3409, M417, Zheza0903, 86155, 3445, Zheyou50, and 3462) showed superior LT germination and comparatively better field performance. For the lower Yangtze site, a practical latest safe sowing window is late October, based on two seasons; November sowing substantially increases winter mortality and yield risk. Selecting genotypes with strong LT germination and managing for rapid autumn establishment can stabilize 1000-seed weight and yield when sowing is delayed.

Low-temperature stress inhibits seed germination in rapeseed. Nonetheless, the continuous dynamic changes in seed germination under low-temperature stress, particularly at the transcriptome level, remain poorly understood. In this study, two rapeseed lines with contrasting LTG phenotypes—HY7201 (cold-tolerant) and HY3404 (cold-sensitive)—were subjected to transcriptome analysis. In total, we identified 76,996 DEGs across 18 groups, with a greater number of DEGs detected in HY7201 compared to HY3404. Additionally, genes related to antioxidative metabolism were specifically upregulated in HY7201. Furthermore, WGCNA identified 29 hub candidate genes associated with specific time points. Gene expression changes during LTG were most pronounced between 18 and 96 h. The average fold change relative to the control was 4.74. Among these genes, some exhibited particularly high fold changes, such as LOC106407757 (HERK1) and LOC106437922 (FER), which were upregulated 11.6-fold at 24 h and 35.4-fold at 18 h, respectively. Finally, 17 key candidate genes specifically expressed in the two lines were identified. Among these, BnaRGL2 was of particular interest, as it is predicted to interact with ABI3 to modulate LTG through GA and ABA signaling pathways. The findings provide valuable insights for breeders aiming to utilize rapeseed germplasm resources to advance low-temperature resistance breeding.

To develop innovative, high-value utilization models for green manure rapeseed, this study investigated the effects of planting density (PD) and nitrogen (N) application rate on the yield and quality of rapeseed flowering stalks harvested at different plant heights. A factorial experimental design was employed, incorporating three factors, PD, N application, and harvesting height, each at three levels. Morphological traits, including stem diameter, fresh weight per plant, flowering stalk yield, and fresh biomass yield after stalk harvesting, were evaluated, together with biochemical traits comprising cellulose, soluble protein, soluble sugar, and vitamin C concentrations in the flowering stalks. The results showed that stem diameter, individual plant fresh weight, and flowering stalk yield generally declined with increasing PD and delayed harvesting height, whereas responses to N application varied depending on PD level. Flowering stalk yield ranged from low to high values across treatments, with the highest yields consistently recorded at a PD of 202,000 plants ha⁻¹ (PD2) across all N levels and at 303,000 plants ha⁻¹ (PD3) combined with 17.25 kg N ha⁻¹ (N1). Variations in PD and N application significantly influenced both yield performance and biochemical composition of the flowering stalks. Elevated concentrations of soluble protein, soluble sugar, and vitamin C were observed at a harvesting height of 25 cm under PD3N1 and at 30 cm under PD2N1 and PD2N2, whereas lower cellulose concentrations were detected at 30 cm under PD2N1 and PD2N2. Overall, these findings demonstrate that coordinated optimization of planting density, nitrogen input, and harvesting height can effectively balance yield and quality traits, providing a practical dual-purpose production strategy for rapeseed cultivated as both a vegetable and green manure in the Shanghai region.