Integrated genomic and transcriptomic analysis reveals candidate genes underlying herbicide resistance in Sorghum

Herbicide-resistant germplasms provide critical genetic resources for improving weed control and understanding resistance mechanisms in crops. To screen sorghum accessions for tolerance to ACCase and ALS inhibitor herbicides at the seedling stage, identify the major locus and strong candidate gene associated with feproxydim resistance, and verify the gene expression pattern and genetic variation by quantitative real‑time PCR (qRT‑PCR) and KASP genotyping. A total of 316 sorghum accessions were screened for seedling-stage herbicide tolerance using gradient herbicide treatments. Bulked segregant analysis sequencing (BSA-Seq) was performed on resistant and susceptible gene pools constructed from the F₂ population derived from IS1219 × RTx430. Transcriptome sequencing (RNA-Seq) was conducted on leaf tissues after feproxydim treatment to identify candidate genes within the mapped interval. KASP markers were developed for the functional variation site of the key candidate gene for genotyping validation. Quantitative real‑time PCR (qRT-PCR) was used to measure the relative expression level of the target gene and compare it with the susceptible control line. Protein sequence comparison was used to detect variations in the key candidate gene between resistant and susceptible lines. In the screening with the ACCase inhibitor 10% feproxydim, IS1219 exhibited high-level resistance. Preliminary screening under the ALS inhibitor mesosulfuron-methyl treatment identified only SJ304 with visible tolerance, which was not subjected to further mapping or validation. BSA-Seq identified a major feproxydim resistance QTL on chromosome 1. RNA-Seq revealed five co-expressed candidate genes in the target interval, among which Sobic.001G431500 (encoding carboxylesterase 17, an α/β‑hydrolase) was markedly upregulated in the resistant line IS1219 but not in the susceptible line RTx430. Quantitative real‑time PCR (qRT-PCR) analysis confirmed that Sobic.001G431500 was significantly upregulated in the resistant line IS1219 compared with the susceptible control. KASP genotyping demonstrated that the IS1219 allele cosegregated with feproxydim resistance. Protein sequence comparison showed that the IS1219 allele carried a missense mutation V300A and a deletion P301_P303 at and after position 300. These findings identify a major QTL and a strong candidate gene Sobic.001G431500 associated with feproxydim resistance in the sorghum line IS1219, based on differential expression, genetic variation, and genotype–phenotype cosegregation. This study provides valuable genetic resources and functional markers for marker-assisted selection and breeding of feproxydim-tolerant sorghum varieties.