Genotyping analysis of over 130,000 CIMMYT bread wheat breeding lines: A decade‐long effort in optimizing wheat genotyping

The International Maize and Wheat Improvement Center (CIMMYT) spring bread wheat ( Triticum aestivum L.) program represents the largest and most diverse set of elite wheat germplasm globally. From 2013 to 2023, genotyping was conducted on 130,247 bread wheat lines advanced through CIMMYT's bread wheat breeding program with the objective to perform genomic prediction and identify genomic regions associated with important traits such as yield and disease resistance. We constructed and sequenced 636 genotyping‐by‐sequencing (GBS) libraries, multiplexed at 96‐ to 384‐plex, and generated 30.7 terabases of sequence. Using an optimized TASSEL pipeline, we identified 24,125 high‐quality single nucleotide polymorphism on 21 chromosomes. Population genetic clustering of 444 selected lines within 10 pedigrees supported the accuracy of the GBS approach. Genome‐wide analysis of nucleotide diversity (π) and minor allele frequency across the entire dataset revealed significantly reduced genetic variation in pericentromeric regions of all chromosomes, which was confirmed by comparison to a genetic outgroup of diverse winter wheat lines. This pattern of low genetic diversity indicates fixation of large centromeric haplotype blocks. The limited diversity in non‐recombining regions has critical implications for future genetic gains in wheat breeding. Temporal pairwise F ST analyses further demonstrated signatures of selection that aligned with previously published genome‐wide association studies for agronomic traits such as grain yield and disease resistance. These datasets have been implemented for the selection of superior breeding lines and are distributed as a publicly available resource for global wheat breeding efforts and genetic studies.