A Rapid and Reliable qPCR Method for Genetic Purity Testing of BT-Type CMS Rice Seed Lots

Boro II (BT), the first cytoplasmic male sterility (CMS) system in rice, is widely used in three-line japonica hybrid rice production. Accurate detection of maintainer-seed contamination in BT-type CMS seed lots is critical for ensuring genetic purity and hybrid seed quality. In this study, we developed a SYBR Green-based quantitative real-time PCR (qPCR) assay for the detection and quantification of maintainer-seed contamination in BT-type CMS seed lots. Maintainer-specific primers targeting a mitochondrial sequence unique to the maintainer line, together with an endogenous reference targeting a conserved mitochondrial sequence present in both maintainer and CMS lines, were validated for specificity. A standard curve was constructed using defined CMS-maintainer seed mixtures (0.1-5% contamination), and ΔCt values were converted to relative abundance (2^-ΔCt). The assay exhibited high specificity, reproducibility, and sensitivity, with a strong linear relationship between 2^-ΔCt values and actual contamination levels (R² > 0.99). Performance testing using simulated contamination samples (0.2-3.13%) demonstrated accurate quantification with acceptable recovery rates. This method provides a rapid, robust, and reliable tool for routine genetic purity testing and quality control in BT-type CMS hybrid rice seed production.