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The total number of spikelets (TSPN) and the number of fertile spikelets (FSPN) affect the final number of grains per spikelet in wheat. This study constructed a high-density genetic map using 55K single nucleotide polymorphism (SNP) arrays from a population of 152 recombinant inbred lines (RIL) from crossing the wheat accessions 10-A and B39. Twenty-four quantitative trait loci (QTLs) for TSPN and 18 QTLs for FSPN were localized based on the phenotype in 10 environments in 2019–2021. Two major QTLs, QTSPN/QFSPN.sicau-2D.4 (34.43–47.43 Mb) and QTSPN/QFSPN.sicau-2D.5(32.97–34.43 Mb), explained 13.97%–45.90% of phenotypic variation. Linked kompetitive allele-specific PCR (KASP) markers further validated these two QTLs and revealed that QTSPN.sicau-2D.4 had less effect on TSPN than QTSPN.sicau-2D.5 in 10-A×BE89 (134 RILs) and 10-A×Chuannong 16 (192 RILs) populations, and one population of Sichuan wheat (233 accessions). The alleles combination haplotype 3 with the allele from 10-A of QTSPN/QFSPN.sicau-2D.5 and the allele from B39 of QTSPN.sicau-2D.4 resulted in the highest number of spikelets. In contrast, the allele from B39 for both loci resulted in the lowest number of spikelets. Using bulk-segregant analysis–exon capture sequencing, six SNP hot spots that included 31 candidate genes were identified in the two QTLs. We identified Ppd-D1a from B39 and Ppd-D1d from 10-A and further analyzed Ppd-D1 variation in wheat. These results identified loci and molecular markers with potential utility for wheat breeding and laid a foundation for further fine mapping and cloning of the two loci.

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