The content of grain protein (GPC) in cereals is an important part of total protein in human food. Exploring and utilizing new GPC genes is one of the most effective approaches for wheat quality breeding. Three homoeologues of TaAAP6(−3A, 3B, 3D)were cloned by homology cloning from OsAAP6.Temporal and spatial expression analysis showed that TaAAP6homoeologues were preferentially expressed in developing grains, and TaAAP6-3B may play a major role in regulating GPC in wheat. Association analysis indicated thatTaAAP6-3B-I is significantly correlated with higher GPC than that of TaAAP6-3B-II for 115 wheat lines in all five environments. TaAAP6-3B-I, the favored allele of TaAAP6-3B, was preferentially expressed in preliminary developing grain stage. Two functional markers were developed to discriminate 197F₂populations and the result showed that TaAAP6-3B-I (high-protein content) was completely dominant. Two cis-regulatory elements appear to be associated with high GPC were found in the 5’UTR of TaAAP6-3B-I.The change of the TaAAP6-3B locus types indicated that the gene was subjected to selection pressures during long process of artificial selection. TaAAP6-3B is a regulator of GPC and its favored allele TaAAP6-3B-I exhibits an obvious potential application in wheat high-GPC breeding.

Colored wheat is a valuable resource that is rich in anthocyanins and minerals and thus contributes additional nutritional value to a healthy human diet. However, the effects of nitrogen fertilization on anthocyanin content (AC) and the balance between quality and yield still merit discussion. In this study, blue, purple, and common-colored wheat genotypes were used to investigate three nutrient quality traits, seven processing quality traits, three yield traits and seven grain morphology traits at three nitrogen levels in two years to excavate their possible plasticity under low-nitrogen stress and the tradeoffs among these traits. The highest AC was found in the blue genotypes followed by the purple genotypes. Analysis of variance (ANOVA) showed that AC could be significantly increased by reducing N application, especially in the purple genotypes. Therefore, growing colored wheat with low nitrogen input could allow efficient harvesting of grain with higher AC. However, the other nutrient quality traits and most processing quality traits were observed to decrease under low-nitrogen (LN) stress. Additionally, a correlation analysis indicated that the nutrient quality traits had stable tradeoffs with thousand kernel weight at all N levels because of the significantly negative correlations among them. Therefore, the additive main effect and multiplicative interaction (AMMI) model was used to further identify the most suitable colored genotypes with the best yield potential and also nutrient quality relative characteristics under LN stress. The blue lines Lanmai2999 and purple varieties Zhongkezinuomai 168 were found to be specifically adapted to LN stress with the highest AC values and showed stable performance in the other nutrient quality- and yield-related features. To further investigate the possible mechanism of anthocyanin accumulation in response to reduced N application, the expression of four genes (TaCHS, TaFDR, TaCHI and TaANS) involved in the anthocyanin synthesis pathway was evaluated. All four genes were downregulated under high nitrogen fertilizer application, indicating that anthocyanin synthesis in colored wheat might be inhibited by nitrogen fertilizer. Therefore, this research provided information for optimizing nitrogen fertilizer management in producing colored wheat and also demonstrated that it is efficient and economical to plant colored wheat genotypes in nitrogen-poor areas for use in a healthy human diet, improving the benefits of wheat planting and facilitating nitrogen pollution control.

Mining novel and less utilized thousand grain weight (TGW) related genes are useful for improving wheat yield. In this study, a recombinant inbred line population from a cross between Zhongkemai 138 (ZKM138, high TGW) and Chuanmai 44 (CM44, low TGW) was used to construct a new Wheat 50K SNP array-derived genetic map that spanned 1,936.59 cM and contained 4, 139 markers. Based on this map, ninety-one quantitative trait loci (QTL) were detected for eight grain-related traits in six environments. Among 58 QTLs, whose superior alleles were contributed by ZKM138, QTgw.cib-6A was a noticeable major stable QTL and was also highlighted by bulked segregant analysis with RNA sequencing (BSR-Seq). It had a pyramiding effect on TGW enhancement but no significant trade-off effect on grain number per spike or tiller number, with two other QTLs (QTgw.cib-2A.2 and QTgw.cib-6D), possibly explaining the excellent grain performance of ZKM138. After comparison with known loci, QTgw.cib-6A was deduced to be a novel locus that differed from nearby TaGW2 and TaBT1. Seven simple sequence repeat (SSR) and thirty-nine kompetitive allele-specific PCR (KASP) markers were finally developed to narrow the candidate interval of QTgw.cib-6A to 4.1 Mb. Only six genes in this interval were regarded as the most likely candidate genes. QTgw.cib-6A was further validated in different genetic backgrounds and presented 88.6% transmissibility of the ZKM138-genotype and a 16.4% increase of TGW in ZKM138 derivatives. And the geographic pattern of this locus revealed that its superior allele is present in only 6.47% of 433 Chinese modern wheat varieties, indicating its potential contribution to further high-yield breeding.

Grain hardness (Gh) is important for wheat processing and end-product quality. Puroindolines polymorphism explains over 60% of Gh variation and the novel genetic factors remain to be exploited. In this study, a total of 153 quantitative trait loci (QTLs), clustered into 12 genomic intervals (C1-C12), for 13 quality-related traits were identified using a recombinant inbred line population derived from the cross of Zhongkemai138 (ZKM138) and Chuanmai44 (CM44). Among them, C7 (harboring eight QTLs for different quality-related traits) and C8 (mainly harboring QGh.cib-5D.1 for Gh) were attributed to the famous genes, Rht-D1 and Pina, respectively, indicating that the correlation of involved traits was supported by the pleotropic or linked genes. Notably, a novel major stable QTL for Gh was detected in the C12, QGh.cib-7D, with ZKM138-derived allele increasing grain hardness, which was simultaneously mapped by the BSE-Seq method. The geographic pattern and transmissibility of this locus revealed that the increasing-Gh allele is highly frequently present in 85.79% of 373 worldwide wheat varieties and presented 99.31% transmissibility in 144 ZKM138-derivatives, indicating the non-negative effect on yield performance and that its indirect passive selection has happened during the actual breeding process. Thus, the contribution of this new Gh-related locus was highlighted in consideration of improving the efficiency and accuracy of the soft/hard material selection in the molecular marker-assisted process. Further, TraesCS7D02G099400, TraesCS7D02G098000, and TraesCS7D02G099500 were initially deduced to be the most potential candidate genes of QGh.cib-7D. Collectively, this study provided valuable information of elucidating the genetic architecture of Gh for wheat quality improvement.