Map-based cloning and functional characterization reveal <i>CDF3</i> as the causal gene for the flowering time phenotype in <i>Brassica rapa</i> and <i>Brassica napus</i>

Spring-type <i>Brassica rapa</i> 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 <i>B. rapa</i> 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 <i>B. rapa</i>). A major quantitative trait locus for flowering time, designated <i>qFTA06</i>, was identified within a 1.7-Mb interval on chromosome A06 using genotyping-by-sequencing and bulked segregant analysis sequencing (BSA-seq). The locus <i>qFTA06</i> was subsequently fine-mapped to a 75.16-kb region with a set of near-isogenic lines (NILs), and <i>BrCDF3</i>, a gene encoding a Dof transcription factor, was identified as the causal gene underlying <i>qFTA06</i>. Virus-induced gene silencing experiments revealed that <i>BrCDF3</i> 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 <i>BrCDF3</i> allele from Haoyou 11, flowered ~7 days earlier than NIL-L, which harbors the <i>BrCDF3</i> allele from Dahuang. By employing CRISPR/Cas9 technology, we further validated that the homologous gene <i>BnCDF3</i> also functions as a negative regulator of flowering time in <i>Brassica napus</i> L., and analyzed natural variations in the <i>CDF3</i> gene across natural populations. This study provides new insights into the genetic basis of flowering time in spring-type <i>B. rapa</i>, advancing early-maturity breeding efforts in crops.