Total spikelet number per spike (TSS) is a crucial yield component in wheat. Dissecting and characterizing major stable quantitative trait loci (QTL) associated with TSS can significantly enhance the genetic improvement of yield potential. In a previous study, we identified a stable major QTL for TSS, named QTss.cas-3D. In the present study, we conducted fine mapping of QTss.cas-3D, interval to approximately 6.35 Mb, ranging from 105.03 to 111.38 Mb, based on the IWGSC RefSeq v2.1. Through genome re-sequencing and gene function annotation, we identified TraesCS3D03G0308000 (TaFT-D2) as the candidate gene. Phenotypic evaluation with paired near-isogenic lines revealed that this locus predominantly increases kernel number per spike by enhancing TSS and fertile spikelet number per spike, without significantly affecting thousand-kernel weight or tiller number. The presence of the TaFT-D2 allele in the parent P3228, which is rare in nature populations, highlights its potential value. This study provides a valuable gene resource and functional marker for wheat molecular breeding aimed at improving TSS and establishes a foundation for gene functional analysis of TaFT-D2.
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