QTL-Seq Identifies Extra QTLs and Candidate Genes Controlling High Haploid Induction Rate in Maize

Double-haploid (DH) technology is a well-established method for speeding up the development of inbred lines in breeding programs. The major loci qhir1 and qhir8 are widely used in marker-assisted selection (MAS) to increase the haploid induction rate (HIR) in maize. However, previous studies have shown that HIR can be unstable within populations, even in the presence of these two loci. To identify novel loci associated with HIR, we performed QTL-seq analysis on 337 S₂ haploid inducers (qhir1+/qhir8+) derived from crossing K8 with BHI306. The population exhibited HIR ranging from 0% to 31.16%. We sequence-bulked DNA from 30 extremely high-HIR lines (15.72-31.16%) and 30 extremely low-HIR lines (0-3.84%), identifying candidate intervals on chromosomes 2 (qHI2), 3 (qHI3), 6 (qHI6), and 8 (qHI8). Based on the QTL-seq results, 147 high-confidence SNPs/InDels (R² > 0.3) led to the analysis of 58 genes across three QTLs. We retrieved ten missense mutation SNPs from three genes (GRMZM2G359746 (qHI2), AC198725.4 (qHI3), and GRMZM2G091276 (qHI8)), which are located on chromosomes 2, 3, and 8. Regression analysis of these SNPs showed an R² range of 0.27 to 0.72. The two most highly associated SNPs were located in exon 2 of GRMZM2G359746 (qHI2) and in exon 5 of GRMZM2G091276 (qHI8), respectively. Marker-trait association analysis revealed that lines carrying favorable alleles at both loci, together with qhir1+ and qhir8+, exhibited significantly higher average HIR (12.77%) compared to those with unfavorable alleles (6.66%). These findings provide valuable markers for enhancing maternal haploid inducer breeding programs in maize.