Thousand-grain weight (TGW), a key determinant of final wheat yield, is of great importance for genetic dissection. Precise identification of stable loci and key candidate genes controlling TGW provides theoretical foundations and genetic resources for marker-assisted breeding of high-TGW and high-yield wheat varieties.

A total of 291 wheat accessions from diverse origins were genotyped using a 100K SNP array. TGW phenotypic data collected over two consecutive years and their best linear unbiased predictions (BLUPs) were analyzed using a genome-wide association study (GWAS) based on a mixed linear model (MLM) incorporating both population structure (P) and kinship (K). Significant loci were further subjected to haplotype analysis.

TGW showed broad variation across years and BLUP values (mean: 38.24-38.82 g; coefficient of variation: 17.62%-19.93%). The correlation between years was 0.88 (P＜0.01), and correlations with BLUP values reached 0.97 (P＜0.01). Phenotypic data displayed normal distributions under different environments, meeting the basic requirements for GWAS. A total of 19 SNPs significantly associated with TGW were detected on chromosomes 3B, 5A, and 7A, explaining 6.85%-9.68% of the phenotypic variation; 16 of them were repeatedly detected across multiple environments, indicating stability. Haplotype analysis at locus 7A_145980808 revealed four haplotypes (Hap1-Hap4), of which Hap4 was associated with high TGW (P＜0.01) and Hap2 with low TGW (P＜0.01). The frequencies of Hap1-Hap4 were 72.36%, 14.55%, 8.73%, and 4.36%, respectively. Domestic accessions were enriched for Hap3 (95.83%) and Hap4 (83.33%), with Hap4 predominantly distributed in the Northwestern winter wheat region, especially in germplasm from Gansu. Candidate gene mining within 3.6 Mb regions flanking significant loci on chromosomes 3B, 5A, and 7A identified 95 genes, among which four were highlighted based on gene annotation and expression profiles.

GWAS identified 16 stable SNP loci significantly associated with TGW, four distinct haplotypes, and four key candidate genes. These genes are mainly involved in carbohydrate synthesis and transport, cell wall polysaccharide assembly, protein homeostasis, and transcriptional regulation of starch biosynthesis, providing valuable targets for molecular breeding of high-yield wheat.