Twenty-one years of three-way hybrid breeding shaped diversity and complementarity of male and female gene pools in sugar beet.

key messageThree-way hybrid breeding in sugar beet is associated with strong genomic differentiation between male and female parental pools and persistent haplotypic differentiation at agronomically relevant loci over the last two decades. Three-way hybrid breeding has been used in several major crops to increase seed yield and manage heterosis. Sugar beet (Beta vulgaris ssp. vulgaris) breeding relies exclusively on it and combines cytoplasmic male sterility (CMS) and monogermity to exploit heterosis by combining male and female parental pools. The genomic consequences of this breeding scheme remain poorly characterized. We assembled a unique dataset comprising 1285 accessions spanning 21 years of selection within a breeding program, genotyped for 10,033 SNPs, to investigate genetic structuring and selection dynamics. Principal component and admixture analyses revealed a clear differentiation between parental pools. Over 21 years, males displayed a significant decline in within-pool diversity, accompanied by a rise in inter-pool differentiation. Linkage disequilibrium decreased in recent male lines, reflecting breeding practices that increase allele shuffling. Selection scans identified 174 outlier SNPs differentiating pools. Outliers clustered into seven haploblocks, including a region encompassing rhizomania-resistance gene (Rz1) and, more surprisingly, a region encompassing the domestication bolting locus B (BvBTC1). These results demonstrate that over the last 21 years, three-way hybrid breeding has increased differentiation between heterotic pools and shaped haplotypic variation at agronomically relevant loci, providing a genomic basis for future evaluation of their effects on agronomic performance.