Exchangeable aluminum (Al) ions released from acidic soils with pH < 5.5 inhibit root elongation of crops, ultimately leading to yield reduced. It is necessary to identify the quantitative trait locus (QTLs) and candidate genes that confer toxicity resistance to understand the mechanism and improve tolerance of rapeseed. In this study, an F₂ segregating population was derived from a cross between Al-tolerance inbred line FDH188 (R178) and -sensitive inbred line FDH152 (S169), and the F_2:3 were used as materials to map QTLs associated with the relative elongation of taproot (RET) under Al toxicity stress. Based on bulked segregant analysis sequencing (BSA-seq), three QTLs (qAT-A07-1, qAT-A07-2, and qAT-A09-1) were detected as significantly associated with RET, and 656 candidate genes were screened. By combined BSA and RNA-seq analysis, 55 candidate genes showed differentially expressed, including genes encoding ABC transporter G (ABCG), zinc finger protein, NAC, ethylene-responsive transcription factor (ERF), etc. These genes were probably positive factors in coping with Al toxicity stress in rapeseed. This study provides new insight into exploring the QTLs and candidate genes’ response to Al toxicity stress by combined BSA-seq and RNA-seq and is helpful to further research on the mechanism of Al resistance in rapeseed.

The multidrug and toxic compound extrusion (MATE) protein plays a crucial role in mediating plant responses to aluminum (Al) toxicity. The key candidate gene BnaMATE43b related to Al toxicity stress in rapeseed was identified using GWAS and transcriptome analysis. In this study, the BnaMATE43b gene was cloned and functionally characterized in rapeseed. Compared with wild-type rapeseed (WT), the BnaMATE43b overexpression lines (OE) demonstrated stronger aluminum tolerance, specifically manifested in higher relative elongation of taproots (RETs) and relative total root length (RTRL); under Al toxicity stress, the enzyme activities (SOD and POD) and root activity were significantly increased in the OE lines, whereas the MDA content and relative electrical conductivity were reduced in rapeseed root. Further transcriptome analysis of OE-3 showed that the differentially expressed genes (DEGs) were mainly enriched in zeatin biosynthesis (map00908), glucosinolate biosynthesis (map00966), phenylpropanoid biosynthesis (map00940), and ascorbate and aldarate metabolism (map00053). In addition, the yeast cDNA library of rapeseed was constructed, and twenty-two candidate upstream transcription factors (UTFs) of BnaMATE43b were screened; furthermore, four candidate UTFs were obtained through one-on-one interaction validation and luciferase assays, comprising three bHLH transcription factors (BnaA02g28220D, BnaA06g07840D, and BnaA08g24520D) and one ERF transcription factor (BnaA05g23130D). Collectively, these results suggest that BnaMATE43b could improve Al tolerance in rapeseed by mediating antioxidant enzyme activities and the related metabolic pathway, while the obtained UTFs lay the foundation for further analysis of the gene regulatory network under Al toxicity stress.