Assessment of doubled haploid production efficiency in maize using sequential phenotypic marker screening

Abstract

Doubled haploid (DH) technology has become an important tool for accelerating maize breeding by enabling the rapid development of completely homozygous lines. However, breeding decisions depend not only on haploid induction rate or chromosome doubling success, but also on the cumulative efficiency of the entire DH pipeline. Despite its practical importance, information on material losses from induced seed to recovered DH line remains limited under routine breeding conditions. The aim of this study was to assess the efficiency of doubled haploid production in maize by quantifying material losses across successive stages of haploid induction, phenotypic selection, chromosome doubling, and final recovery of fertile DH lines. Fifty genetically diverse maize donor populations were pollinated with a maternal haploid inducer carrying the dominant phenotypic markers R1-nj and Pl1. Putative haploid seeds were initially identified using the R1-nj anthocyanin marker, followed by secondary validation using the Pl1 root pigmentation marker. A total of 38,916 seeds were evaluated, of which 5,870 putative haploids were identified at the first screening stage, corresponding to a putative haploid selection rate of 15.08%. Secondary screening reduced this number to 3,316 putative haploids (8.52%). Following chromosome doubling, field evaluation, fertility screening, and the removal of non-uniform and electrophoretically segregating ears, 213 fertile putative DH0 ears were retained, resulting in a final operational recovery efficiency of 0.55%. Seed storage protein electrophoresis of well-filled ears confirmed the genetic uniformity of selected DH lines, while low-seed DH0 ears were retained as putative lines requiring further progeny evaluation for full confirmation. The results demonstrate that sequential phenotypic marker screening improves haploid identification accuracy and provide practical estimates of operational losses across the DH pipeline under applied breeding conditions.