Developmental staging and genetic control of male reproductive traits for advancing hybrid wheat breeding

Hybrid wheat breeding is constrained by the predominantly self-pollinating nature of wheat florets, which limits cross-pollination efficiency and increases grain production costs. Enhancing hybridization requires a better understanding of male reproductive traits, particularly pollen quantity and viability. Here, we combined developmental staging, environmental assays, and genetic mapping to dissect these traits in European winter bread wheat. Anther and pollen maturation, characterized using an enhanced high-resolution Waddington scale, followed a proximo-distal gradient across the spike and an acropetal gradient within spikelets, with W10 marking as peak pollen maturation and viability stage. Time-lapse imaging revealed that anthesis follows the same coordinated spatial pattern, indicating tightly synchronized reproductive development across the spike. Importantly, pollen viability was best maintained at room temperature (22 °C) for up to 8 h, but, unexpectedly, declined more rapidly under cooler storage conditions (15 °C and 8 °C). To test the genetic basis of pollen viability and pollen number we performed Quantitative Trait Locus (QTL) analysis and identified seven loci, including a major QTL for pollen number on chromosome 2A and a distinct QTL for pollen viability on 5A. In both cases alleles from cultivar Piko consistently conferred beneficial trait effects. These findings refine anther staging, highlight environmental sensitivity of wheat pollen, and identify genomic regions that can improve reproductive efficiency for hybrid wheat production and breeding.