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Some haplotypes of the sucrose synthase gene TaSus1are associated with thousand-grain weight (TGW) in wheat (Triticum aestivumL.). However, no mutations have been identified within the gene to test this association. The effects of TaSus1on grain number per spike (GNS) also are largely unknown. Our previous genome-wide association study identified TaSus-A1as a candidate gene controlling fertile spikelet number per spike (FSN). In the present study, we generated two independent mutants for the three TaSus1homoeologs by CRISPR/Cas9-mediated genome editing. The triple mutants displayed lower FSN, GNS, grain number per spikelet (GNST), and TGW than wild-type plants. In 306 hexaploid wheat accessions, two single-nucleotide polymorphisms in TaSus-A1contributed differently to GNS. Introgression of the two alleles into a wheat genetic background confirmed their effects. The alleles differed in geographical distribution among the accessions.

Pre-harvest sprouting (PHS) is one of the serious global issues in wheat production. Identification of quantitative trait loci (QTL) and closely-linked markers is greatly helpful for wheat improvement. In the present study, a recombinant inbred line (RIL) population derived from the cross of Zhongmai 578 (ZM578)/Jimai 22 (JM22) and parents were phenotyped in five environments and genotyped by the wheat 50 K single-nucleotide polymorphism (SNP) array. Two QTL of germination index (GI), QGI.caas-3Aand QGI.caas-5A, were detected, explaining 4.33%–5.58% and 4.43%–8.02% of the phenotypic variances, respectively. The resistant effect of QGI.caas-3Awas contributed by JM22, whereas that of QGI.caas.5Awas from ZM578. The two QTL did not correspond to any previously identified genes or genetic loci for PHS-related traits according to their locations in the Chinese Spring reference genome, indicating that they are likely to be new loci for PHS resistance. Four kompetitive allele-specific PCR (KASP) markers K_AX-109605367and K_AX-179559687 flanking QGI.caas-3A, and K_AX-111258240 and K_AX-109402944 flanking QGI.caas-5A, were developed and validated in a natural population of 100 wheat cultivars. The distribution frequency of resistance alleles at Qphs.caas-3Aand Qphs.caas-5Aloci were 82.7% and 57.1%, respectively, in the natural population. These findings provide new QTL and tightly linked KASP markers for improvement of PHS resistance in wheat.

Grain weight and grain number are important yield component traits in wheat and identification of underlying genetic loci is helpful for improving yield. Here, we identified eight stable quantitative trait loci (QTL) for yield component traits, including five loci for thousand grain weight (TGW) and three for grain number per spike (GNS) in a recombinant inbred line population derived from cross Yangxiaomai/Zhongyou 9507 across four environments. Since grain size is a major determinant of grain weight, we also mapped QTL for grain length (GL) and grain width (GW). QTGW.caas-2D, QTGW.caas-3B, QTGW.caas-5Aand QTGW.caas-7A.2for TGW co-located with those for grain size. QTGW.caas-2Dalso had a consistent genetic position with QGNS.caas-2D, suggesting that the pleiotropic locus is a modulator of trade-off effect between TGW and GNS. Sequencing and linkage mapping showed that TaGL3-5Aand WAPO-A1were candidate genes of QTGW.caas-5Aand QTGW.caas-7A.2, respectively.We developed Kompetitive allele specific PCR (KASP) markers linked with the stable QTL for yield component traits and validated their genetic effects in a diverse panel of wheat cultivars from the Huang-Huai River Valley region. KASP-based genotyping analysis further revealed that the superior alleles of all stable QTL for TGW but not GNS were subject to positive selection, indicating that yield improvement in the region largely depends on increased TGW. Comparative analyses with previous studies showed that most of the QTL could be detected in different genetic backgrounds, and QTGW.caas-7A.1is likely a new QTL. These findings provide not only valuable genetic information for yield improvement but also useful tools for marker-assisted selection.

Wheat cultivar Zhongmai 895 was earlier found to carry YR86in an 11.6 Mb recombination-suppressed region on chromosome 2AL when crossed with Yangmai 16. To fine-map the YR86locus, we developed two large F2populations from crosses Emai 580/Zhongmai 895 and Avocet S/Zhongmai 895. Remarkably, both populations exhibited suppressed recombination in the same 2AL region. Collinearity analysis across Chinese Spring, Aikang 58, and 10 + wheat genomes revealed a 4.1 Mb chromosomal inversion spanning 708.5–712.6 Mb in the Chinese Spring reference genome. Molecular markers were developed in the breakpoint and were used to assess a wheat cultivar panel, revealing that Chinese Spring, Zhongmai 895, and Jimai 22 shared a common sequence named InvCS, whereas Aikang 58, Yangmai 16, Emai 580, and Avocet S shared the sequence named InvAK58. The inverted configuration explained the suppressed recombination observed in all three bi-parental populations. Normal recombination was observed in a Jimai 22/Zhongmai 895 F2population, facilitating mapping of YR86to a genetic interval of 0.15 cM corresponding to 710.27–712.56 Mb falling within the inverted region. Thirty-three high-confidence genes were annotated in the interval using the Chinese Spring reference genome, with six identified as potential candidates for YR86based on genome and transcriptome analyses. These results will accelerate map-based cloning of YR86and its deployment in wheat breeding.

Wheat high-molecular-weight glutenin subunits (HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1start codon (−1000 to −1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules (CCRMs), CCRM1 (−300 to −101), CCRM2 (−650 to −400), and CCRM3 (−950 to −750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qPCR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter (−300 to −1), spanning CCRM1 and core region (−100 to −1), was enough to ensure accurate Glu-1initiation at 7 days after flowering (DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2 (−300 to −209) containing Complete HMW Enhancer (−246 to −209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1 (−208 to −101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1transcription regulatory machinery but also lay foundations for modifying Glu-1expression.

Many genetic loci for wheat plant height (PH) have been reported, and 26 dwarfing genes have been catalogued. To identify major and stable genetic loci for PH, here we thoroughly summarized these functionally or genetic verified dwarfing loci from QTL linkage analysis and genome-wide association study published from 2003 to 2022. A total of 332 QTL, 270 GWAS loci and 83 genes for PH were integrated onto chromosomes according to their locations in the IWGSC RefSeq v2.1 and 65 QTL-rich clusters (QRC) were defined. Candidate genes in each QRC were predicted based on IWGSC Annotation v2.1 and the information on functional validation of homologous genes in other species. A total of 38 candidate genes were predicted for 65 QRC including three GA2oxgenes in QRC-4B-IV, QRC-5A-VIII and QRC-6A-II (Rht24) as well as GA 20-oxidase 2(TaSD1-3A) in QRC-3A-IV. These outcomes lay concrete foundations for map-based cloning of wheat dwarfing genes and application in breeding.