A two-stage screening approach integrated with GWAS for rare phenotypes such as spontaneous haploid genome doubling in maize.

Spontaneous haploid genome doubling (SHGD) is a valuable trait in maize breeding, enabling the development of doubled haploid (DH) lines without chemical chromosome doubling. However, SHGD is a rare phenotype, expressed in only a small fraction of maize germplasm, making its identification resource-intensive. This study evaluated the efficiency of a two-stage field screening approach designed to identify maize genotypes with high haploid male fertility (HMF), a key indicator of SHGD potential. Simulation analyses showed that evaluating 50 haploid plants per genotype, combined with a 25% HMF threshold, provides an optimal balance between detection accuracy and resource efficiency. Across three growing seasons, HMF exhibited a highly skewed distribution, with most genotypes showing low HMF and a small subset exceeding 30% HMF. Field evaluations conducted in 2022, 2023, and 2024 consistently identified high-performing genotypes, including A427, N525, N516, and NK778, which maintained stable HMF expression across years. A genome-wide association analysis identified genomic regions associated with HMF. Our two-stage screening approach identified both SHGD donor lines and genomic loci and candidate genes for HMF.