Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation

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-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start 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, qP CR 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-1 initiation 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-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.

In silico curation of QTL-rich clusters and candidate gene identification for plant height of bread wheat

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 GA2ox genes 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 mapbased cloning of wheat dwarfing genes and application in breeding.

Identification and validation of stable quantitative trait loci for yield component traits 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-5A and QTGW.caas-7A.2 for TGW co-located with those for grain size.QTGW.caas-2D also 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 TaGL3-5A and WAPO-A1 were candidate genes of QTGW.caas-5A and 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.1 is likely a new QTL.These findings provide not only valuable genetic information for yield improvement but also useful tools for marker-assisted selection.

QTL mapping for pre-harvest sprouting in a recombinant inbred line population of elite wheat varieties Zhongmai 578 and Jimai 22

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-3A and 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-3A was contributed by JM22,whereas that of QGI.caas.5A was from ZM578.The two QTL did not correspond to any previously identified genes or genetic loci for PHSrelated 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-109402944flanking QGI.caas-5A,were developed and validated in a natural population of 100 wheat cultivars.The distribution frequency of resistance alleles at Qphs.caas-3A and Qphs.caas-5A loci were 82.7%and57.1%,respectively,in the natural population.These findings provide new QTL and tightly linked KASP markers for improvement of PHS resistance in wheat.

Orchestrating seed storage protein and starch accumulation toward overcoming yield–quality trade-off in cereal crops

Achieving high yield and good quality in crops is essential for human food security and health.However,there is usually disharmony between yield and quality.Seed storage protein(SSP)and starch,the predominant components in cereal grains,determine yield and quality,and their coupled synthesis causes a yield–quality trade-off.Therefore,dissection of the underlying regulatory mechanism facilitates simultaneous improvement of yield and quality.Here,we summarize current findings about the synergistic molecular machinery underpinning SSP and starch synthesis in the leading staple cereal crops,including maize,rice and wheat.We further evaluate the functional conservation and differentiation of key regulators and specify feasible research approaches to identify additional regulators and expand insights.We also present major strategies to leverage resultant information for simultaneous improvement of yield and quality by molecular breeding.Finally,future perspectives on major challenges are proposed.

Wheat genomic study for genetic improvement of traits in China

Bread wheat(Triticum aestivum L.)is a major crop that feeds 40%of the world’s population.Over the past several decades,advances in genomics have led to tremendous achievements in understanding the origin and domestication of wheat,and the genetic basis of agronomically important traits,which promote the breeding of elite varieties.In this review,we focus on progress that has been made in genomic research and genetic improvement of traits such as grain yield,end-use traits,flowering regulation,nutrient use efficiency,and biotic and abiotic stress responses,and various breeding strategies that contributed mainly by Chinese scientists.Functional genomic research in wheat is entering a new era with the availability of multiple reference wheat genome assemblies and the development of cutting-edge technologies such as precise genome editing tools,highthroughput phenotyping platforms,sequencing-based cloning strategies,high-efficiency genetic transformation systems,and speed-breeding facilities.These insights will further extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process,ultimately contributing to more sustainable agriculture in China and throughout the world.