Root system architecture (RSA) is the primary predictor of nutrient intake and significantly influences potassium utilization efficiency (KUE). Uncertainty persists regarding the genetic factors governing root growth in rapeseed. The root transcriptome analysis reveals the genetic basis driving crop root growth. In this study, RNA-seq was used to profile the overall transcriptome in the root tissue of 20 Brassica napus accessions with high and low KUE. 71,437 genes in the roots displayed variable expression profiles between the two contrasting genotype groups. The 212 genes that had varied expression levels between the high and low KUE lines were found using a pairwise comparison approach. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification analysis revealed that the DEGs implicated in hormone and signaling pathways, as well as glucose, lipid, and amino acid metabolism, were all differently regulated in the rapeseed root system. Additionally, we discovered 33 transcription factors (TFs) that control root development were differentially expressed. By combining differential expression analysis, weighted gene co-expression network analysis (WGCNA), and recent genome-wide association study (GWAS) results, four candidate genes were identified as essential hub genes. These potential genes were located fewer than 100 kb from the peak SNPs of QTL clusters, and it was hypothesized that they regulated the formation of the root system. Three of the four hub genes’ homologs—BnaC04G0560400ZS, BnaC04G0560400ZS, and BnaA03G0073500ZS—have been shown to control root development in earlier research. The information produced by our transcriptome profiling could be useful in revealing the molecular processes involved in the growth of rapeseed roots in response to KUE.
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