Thousand-kernel weight (TKW) is a measure of grain weight, a target of wheat breeding. The object of this study was to fine-map a stable quantitative trait loci (QTL) for TKW and identify its candidate gene in a recombinant inbred line (RIL) population derived from the cross of Kenong 9204 (KN9204) and Jing 411 (J411). On a high-density genetic linkage map, 24, 26 and 25 QTL were associated with TKW, kernel length (KL), and kernel width (KW), respectively. A major and stable QTL, QTkw-2D, was mapped to an 8.3 cM interval on chromosome arm 2DL. By saturation of polymorphic markers in its target region, QTkw-2D was confined to a 9.13 Mb physical interval using a secondary mapping population derived from a residually heterozygous line (F_6:7). This interval was further narrowed to 2.52 Mb using QTkw-2D near-isogenic lines (NILs). NILs^KN9204 had higher fresh and dry weights than NILs^J411 at various grain-filling stages. The TKW and KW of NILs^KN9204 were much higher than those of NILs^J411 in field trials. By comparison of both DNA sequence and expression between KN9204 and J411, TraesCS2D02G460300.1 (TraesKN2D01HG49350) was assigned as a candidate gene for QTkw-2D. This was confirmed by RNA sequencing (RNA-seq) of QTkw-2D NILs. These results provide the basis of map-based cloning of QTkw-2D, and DNA markers linked to the candidate gene may be used in marker-assisted selection.

The TaGS3 homoeologous genes (homoeologs) located on chromosomes 7A, 4A, and 7D in hexaploid wheat were cloned. Relative expression analysis of the three TaGS3 homoeologs revealed that the expression levels of TaGS3-4A and TaGS3-7D in developing grains were higher than that of TaGS3-7A. Genetic evidence showed that TaGS3 was a negative regulator of grain weight and grain size. Fifteen polymorphic sites and five haplotypes were detected in TaGS3-4A. Two molecular markers were developed to distinguish the five haplotypes. Association analysis using 260 accessions from Chinese wheat mini-core collection (MCC) indicated that TaGS3-4A affected thousand grain weight (TGW) and grain length (GL). HAP-4A-1 and HAP-4A-2 were favorable haplotypes that increased TGW and GL and had undergone strong selection during domestication of wheat. In addition, interaction of the TaGS3-4A and TaGS3-7D homoeologs had significant additive effects on the grain traits. Hap-4A-1/Hap-7D-2 was the best haplotype combination in increasing TGW and GL. The frequencies and geographic distributions of favorable TaGS3 haplotypes among 1388 wheat accessions from worldwide sources provided clues for selection of yield-related traits. Our findings demonstrated that TaGS3-4A had significant effects on TGW and GL. Marker-assisted selection of HAP-4A-1/2 combined with HAP-7D-2 has potential to increase wheat yields.