Genome Size Variation Is Associated with Hybrid Vigor in Near-Isogenic Backgrounds in Brassica napus

Although heterosis plays a crucial role in enhancing crop yield and stress resistance, its underlying genetic mechanism remains not yet fully understood. Previous studies have shown that heterosis tends to increase with greater genetic distance in the absence of reproductive isolation barriers. However, whether variation in parental genome size alone can generate heterosis under near-isogenic backgrounds has not been thoroughly explored. Here, we used a rapeseed double haploid (DH) inducer line to generate progeny from the <i>Pol</i> CMS three-line hybrid Rongyou 18 (RY18). Although the progeny maintained the same ploidy level as the parents, their genome sizes showed notable variation (818.99-1024.88 Mb). To eliminate genetic distance effects, multiple DH progeny carrying restorer genes were crossed as paternal parents with the female parent 0068A of RY18, creating novel F₁ hybrids. Using RY18 as the control, we observed a marked reduction in the genetic distance between the newly induced restorer line and the female parent (0068A). Correlation analysis further revealed a significant negative correlation (r = -0.310 *) between the paternal genome size and heterosis for thousand-seed weight (TSW). Furthermore, the genomic expansion in hybrid offspring relative to the male parent showed that significant correlations were observed between paternal genome size and heterosis over the standard for both TSW (r = 0.300, <i>p</i> < 0.05) and plot yield (r = 0.326, <i>p</i> < 0.05). Resequencing of high-and low-yielding F₁ hybrids identified SNP sites, indicating that under an identical genetic background, heterosis for yield was more pronounced on chromosome A and chromosome C04. The doubled haploid (DH) induction line facilitates the generation of parental lines with distinct genome sizes, potentially providing a potential novel approach for studying heterosis research in <i>Brassica napus</i>.