Spring-type Brassica rapa L. is a valuable genetic resource for breeding early-maturing crops, offering advantages such as early flowering and rapid maturation. However, the genetic mechanisms governing flowering time in spring-type B. rapa remain insufficiently understood. In this study, we investigated the flowering-time trait of an extremely early-maturing landrace, ‘Haoyou 11’, originating from the Qinghai-Tibetan Plateau. Initial mapping was conducted using an F₂ population derived from the cross between Haoyou 11 and Dahuang (a late-flowering spring-type landrace of B. rapa). A major quantitative trait locus for flowering time, designated qFTA06, was identified within a 1.7-Mb interval on chromosome A06 using genotyping-by-sequencing and bulked segregant analysis sequencing (BSA-seq). The locus qFTA06 was subsequently fine-mapped to a 75.16-kb region with a set of near-isogenic lines (NILs), and BrCDF3, a gene encoding a Dof transcription factor, was identified as the causal gene underlying qFTA06. Virus-induced gene silencing experiments revealed that BrCDF3 acts as a negative regulator of flowering time under long-day conditions, with sequence variation contributing to the early-flowering phenotype in Haoyou 11. Phenotypic analysis of NILs showed that NIL-E, carrying the BrCDF3 allele from Haoyou 11, flowered ~7 days earlier than NIL-L, which harbors the BrCDF3 allele from Dahuang. By employing CRISPR/Cas9 technology, we further validated that the homologous gene BnCDF3 also functions as a negative regulator of flowering time in Brassica napus L., and analyzed natural variations in the CDF3 gene across natural populations. This study provides new insights into the genetic basis of flowering time in spring-type B. rapa, advancing early-maturity breeding efforts in crops.

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