An efficient screening system at seedling-stage for genic male-sterile lines in pepper hybrid breeding

1 A novel pepper mutant (aa) with a recessive CaDFR mutation lacks anthocyanins. 2 CaDFR is tightly linked to the male-sterile gene CaDYT1 , enabling hypocotyl color as a visible morphological marker for early selection of male-sterile plants. 3 A non-GMO two-line hybrid seed production system utilizing CaDYT1-CaDFR linkage in pepper. Heterosis is a highly effective strategy for increasing yield and quality of pepper ( Capsicum annuum L.). Although genic male sterile (GMS) lines offer advantages for seed production by avoiding the limitations associated with cytoplasmic male sterile lines, their use typically requires the labor-intensive removal of 50% of fertile plants during seed production. To address this challenge, we identified an anthocyanidin-absent (aa) mutant characterized by green hypocotyls at the seedling stage and yellow anthers. We identified dihydroflavonol 4-reductase ( CaDFR) as a key gene regulating anthocyanin biosynthesis in pepper through fine mapping and virus-induced gene silencing. Genetic segregation analysis revealed that CaDFR and CaDYT1 (a GMS gene) were closely linked. Based on this finding, we developed an efficient hybrid seed production strategy for screening sterile plants at the seedling-stage by combining the green hypocotyl morphological marker from the aa mutant with the male sterile line gms1 (CaDYT1 locus). In conclusion, we successfully cloned CaDFR , a key gene controlling hypocotyl and anther color in pepper. In addition, we proposed an efficient seed propagation strategy to accelerate hybrid seed production and facilitate the utilization of heterosis. This study not only deepens our understanding of the genetic regulation of pepper pigmentation but also establishes a practical framework for optimizing hybrid breeding protocols, thereby simplifying the pepper breeding process.