Insertional Mutations in <scp><i>RsFLC2</i></scp> Disrupt Vernalization‐Induced Intragenic Looping and <scp>H3K27me3</scp> Enrichment in Radish

The floral transition of many winter-annual, biennial, and perennial plants accelerates upon prolonged exposure to winter temperatures, a phenomenon known as vernalization. This study aimed to elucidate the molecular mechanisms underlying vernalization-induced floral transition in radish (Raphanus sativus L.). We performed a comparative transcriptomic analysis between the normal-flowering cultivar "Jinjudaepyung (JD)" and the very-late-flowering inbred line, "Simu (SM)." Notably, the radish FLC homolog RsFLC2 was significantly upregulated in "SM," regardless of vernalization treatment. When the RsFLC2 transgene was introduced into the winter-annual Arabidopsis FLC knockout mutant flc-2FRI, the transgenic plants exhibited a significantly delayed floral transition. Furthermore, two DNA insertions, 1037 and 1627 bp in length, were detected in the proximal promoter and first intron region of RsFLC2, respectively, in "SM". Arabidopsis transgenic plants expressing the RsFLC2 transgene from "SM," which harbors these insertions, exhibited a substantially delayed flowering phenotype compared to those expressing the RsFLC2 transgene from "JD." Additionally, polycomb repressive complex 2 (PRC2)-mediated H3K27me3 deposition was significantly impaired in "SM" compared to "JD." We also found that an intragenic loop formed within the RsFLC2 region in radish upon vernalization. However, compared to "JD," intragenic looping was substantially impaired in "SM." Collectively, this study revealed that insertional DNA mutations in RsFLC2 disrupted the vernalization response by impairing both intragenic loop formation and PRC2-mediated H3K27me3 deposition in radish.