Identification of Candidate Genes for Alkaline Tolerance in Rice Seedlings Using BSA-Seq and RNA-Seq

Alkaline stress is one of the most critical limiting factors affecting high and stable rice yields. Therefore, analyzing the impact of alkaline stress on rice productivity and identifying salt- and alkali-tolerant genes are crucial for breeding rice varieties with strong stress resistance. In this study, the alkali-sensitive variety QJ74 and the alkali-tolerant variety QJ22 were used as parental lines. Under alkaline conditions (pH 9.0), 30 alkali-sensitive and 30 alkali-tolerant F₂ individuals from the segregating population were selected for BSA-Seq. Transcriptomic analysis was performed on the parental lines under the same alkaline treatment. The results showed that under alkali stress, QJ22 exhibited significantly greater plant height and chlorophyll content, but significantly lower relative electrical conductivity and malondialdehyde content than QJ74, indicating less severe cell membrane damage. Meanwhile, QJ22 showed significantly higher proline content and superoxide dismutase and peroxidase activities, together with significantly lower Na⁺ and higher K⁺ contents compared with QJ74. Overall, QJ22 outperformed QJ74 in cell membrane stability, photosynthetic pigment maintenance, antioxidant enzyme activity, and osmotic regulation, demonstrating strong alkali tolerance. Based on these findings, under alkali stress (pH 9.0), 30 alkali-sensitive and 30 alkali-tolerant F₂ individuals from the cross between the two parents were selected for BSA-Seq, and transcriptomic sequencing was performed on the parental lines under the same conditions. A combined BSA-Seq and RNA-Seq analysis identified five candidate genes (Os10g0376200, Os10g0389000, Os10g0389500, Os10g0390500 and Os10g0401000). Subsequent cis-acting element analysis, phylogenetic analysis, and qRT-PCR validation revealed that Os10g0389000 is a promising alkali stress-responsive candidate gene potentially contributing to the differential alkali tolerance between QJ74 and QJ22. This study integrated BSA-Seq and RNA-Seq to systematically identify candidate genes involved in the alkali stress response in rice, providing key genetic targets and a molecular basis for developing new alkali-tolerant varieties.