Identification of stable QTLs for spike length and density and development of KASP markers for wheat breeding.

UNLABELLED: Spike length (SL) and spikelet density (SD) are core agronomic traits determining spike development and grain yield. Dissecting their genetic basis is crucial for high-yield wheat breeding. In this study, a population of 209 recombinant inbred lines (RILs) derived from the cross 'Lantian 26' × 'Luohan 6' was evaluated. Genetic loci controlling SL and SD were systematically explored using multi-environment phenotypic data and a high-density genetic map based on a 16 K SNP array. Consequently, two major, stable, and pleiotropic QTLs (QSl/Sd.LT-4A.2 and QSl/Sd.LT-5A) were identified, consistently co-regulating both SL and SD across multiple environments. To facilitate marker-assisted selection (MAS), we successfully developed a functional KASP marker for the QSl/Sd.LT-4A.2 interval, alongside gene-specific markers targeting sequence variations within the putative candidate genes for QSl/Sd.LT-5A. Validation in a diverse natural population confirmed that the favorable alleles at both loci significantly improve spike architecture and thousand-grain weight (TGW). Furthermore, pyramiding these two major loci exerted a significant additive effect, providing an effective genetic strategy to enhance wheat yield potential. The identified pleiotropic QTLs and the newly developed KASP markers offer useful genetic loci and practical diagnostic tools for high-yielding wheat breeding. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s11032-026-01713-1.