Common wheat (Triticum aestivum L.) is the most important crop in the world and a typical allopolyploid with a large and complex genome. Pre-harvest sprouting (PHS) leads to a significant reduction in grain quality worldwide. PHS is a complex trait with related QTL located on different chromosomes. However, the study of markers and genes related to PHS resistance is limited especially for white-grained wheat. Four pairs of near isogenic lines (NILs) from a white-grained wheat cross of Chara × DM5637B*8 targeting a major QTL for PHS resistance (Qphs.ccsu-3A.1) on wheat chromosme 3AL were genotyped using the 90K SNP Illumina iSelect array. Ten SNPs were identified, with a 75%–100% consistency between genotype and phenotype in the resistant or susceptible isolines. The 10 SNPs were converted to cost-effective kompetitive allele-specific PCR (KASP) markers. Screening of 48 wheat cultivars with different phenotypes of PHS identified four KASP markers with 81.3%–85.4% conformity between genotype and phenotype. Further investigation revealed that the four SNPs (BS00022245_51, Kukri_c49927_151, BS00022884_51 and BS00110550_51) corresponding to the four validated KASP markers are residing in three independent genes (TraesCS3A03G1072800, TraesCS3A03G1072400, TraesCS3A03G1071800) close to each other with a distance of 4.28–4.48 Mb to the targeted QTL. These three annotated genes have potential functions related to PHS resistance. Our study revealed that combined use of NILs and the 90K SNP chip is a powerful approach for developing KASP markers and mining functional genes in wheat. The KASP markers for PHS resistance on chromosome 3AL are useful for high-throughput evaluation and marker-assisted selection, and the three identified genes could lead to a better understanding of the genetic pathways controlling PHS.

ADP-glucose pyrophosphorylase (AGPase) influences cereal productivity. There are few reports on the function of cytosolic AGPase small subunit in bread wheat (TaAGPS). In the present study, TaAGPS was preferentially expressed in developing endosperm during grain-filling stages in bread wheat. TaAGPS allelic variations were characterized in 143 wheat accessions by PacBio RS Ⅱ sequencing. Two haplotypes (TaAGPS-7A-TG and TaAGPS-7A-CT) of TaAGPS-7A were identified and corresponding functional markers were developed, whereas no variants of TaAGPS-7B and TaAGPS-7D were detected. TaAGPS-7A was associated with thousand-kernel weight (TKW) by haplotype–trait association analysis in two populations. Near-isogenic lines (NILs) with TaAGPS-7A-TG showed higher TKW and total kernel starch content than those with TaAGPS-7A-CT, owing to the higher AGPase activity of TaAGPS-7A-TG than TaAGPS-7A-CT both in vitro and in vivo. Overexpression of TaAGPS-7A-TG in bread wheat doubled the transcription levels of TaAGPS and increased AGPase activity by 55.7%, resulting in a 3.0-g higher TKW than in the wild type (WT). Knockdown of TaAGPS led to reduced expression of TaAGPS, AGPase activity, and TKW than in the WT. Thus, owing to the 218th amino acid change of Ser to Ala in TaAGPS-7A, the favorable haplotype TaAGPS-7A-TG showed higher AGPase activity, resulting in higher kernel starch content and grain weight. This finding could be applied to increasing starch content and grain weight in bread wheat.