Pectin methylesterase inhibitors GhPMEI53 and AtPMEI19 improve seed germination by modulating cell wall plasticity in cotton and Arabidopsis

The germination process of seeds is influenced by the interplay between two opposing factors,pectin methylesterase(PME)and pectin methylesterase inhibitor(PMEI),which collectively regulate patterns of pectin methylesterification.Despite the recognized importance of pectin methylesterification in seed germination,the specific mechanisms that govern this process remain unclear.In this study,we demonstrated that the overexpression of GhPMEI53is associated with a decrease in PME activity and an increase in pectin methylesterification.This leads to seed cell wall softening,which positively regulates cotton seed germination.AtPMEI19,the homologue in Arabidopsis thaliana,plays a similar role in seed germination to GhPMEI53,indicating a conserved function and mechanism of PMEI in seed germination regulation.Further studies revealed that GhPMEI53 and AtPMEI19 directly contribute to promoting radicle protrusion and seed germination by inducing cell wall softening and reducing mechanical strength.Additionally,the pathways of abscicic acid(ABA)and gibberellin(GA)in the transgenic materials showed significant changes,suggesting that GhPMEI53/AtPMEI19-mediated pectin methylesterification serves as a regulatory signal for the related phytohormones involved in seed germination.In summary,GhPMEI53 and its homologs alter the mechanical properties of cell walls,which influence the mechanical resistance of the endosperm or testa.Moreover,they impact cellular phytohormone pathways(e.g.,ABA and GA)to regulate seed germination.These findings enhance our understanding of pectin methylesterification in cellular morphological dynamics and signaling transduction,and contribute to a more comprehensive understanding of the PME/PMEI gene superfamily in plants.

Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

The morphological development of rice(Oryza sativa L.)leaves is closely related to plant architecture,physiological activities,and resistance.However,it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment.In this study,a drought-sensitive,roll-enhanced,and narrow-leaf mutant(renl1)was induced from a semi-rolled leaf mutant(srl1)by ethyl methane sulfonate(EMS),which was obtained from Nipponbare(NPB)through EMS.Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase,allelic to NRL1/OsCLSD4.The RENL1 mutation resulted in reduced vascular bundles,vesicular cells,cellulose,and hemicellulose contents in cell walls,diminishing the water-holding capacity of leaves.In addition,the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species(ROS)was decreased,leading to an increase in ROS after drought stress.Meanwhile,genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components.Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance,and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance,thereby realizing simultaneous improvement of multiple traits in rice.

EPSPS regulates cell elongation by disrupting the balance of lignin and flavonoid biosynthesis in cotton

EPSPS is a key gene in the shikimic acid synthesis pathway that has been widely used in breeding crops with herbicide resistance.However,its role in regulating cell elongation is poorly understood.Through the overexpression of EPSPS genes,we generated lines resistant to glyphosate that exhibit an unexpected dwarf phenotype.A representative line,DHR1,exhibits a stable dwarf phenotype throughout its entire growth period.Except for plant height,the other agronomic traits of DHR1 are similar to its transgenic explants ZM24.Paraffin section observations showed that DHR1 internodes are shortened due to reduced elongation and division of the internode cells.Exogenous hormones confirmed that DHR1 is not a classical brassinolide(BR)-or gibberellin(GA)-related dwarfing mutant.Hybridization analysis and fine mapping confirmed that the EPSPS gene is the causal gene for dwarfism,and the phenotype can be inherited in different genotypes.Transcriptome and metabolome analyses showed that genes associated with the phenylpropanoid synthesis pathway are enriched in DHR1 compared with ZM24.Flavonoid metabolites are enriched in DHR1,whereas lignin metabolites are reduced.The enhancement of flavonoids likely results in differential expression of auxin signal pathway genes and alters the auxin response,subsequently affecting cell elongation.This study provides a new strategy for generating dwarfs and will accelerate advancements in light simplification in the cultivation and mechanized harvesting of cotton.

Natural variation in the cytochrome c oxidase subunit 5B OsCOX5B regulates seed vigor by altering energy production in rice

Seed vigor is a crucial trait for the direct seeding of rice.Here we examined the genetic regulation of seed vigor traits in rice,including germination index(GI)and germination potential(GP),using a genome-wide association study approach.One major quantitative trait locus,qGI6/qGP6,was identified simultaneously for both GI and GP.The candidate gene encoding the cytochrome c oxidase subunit 5B(OsCOX5B)was validated for qGI6/qGP6.The disruption of OsCOX5B caused the vigor traits to be significantly lower in Oscox5b mutants than in the japonica Nipponbare wild type(WT).Gene co-expression analysis revealed that OsCOX5B influences seed vigor mainly by modulating the tricarboxylic acid cycle process.The glucose levels were significantly higher while the pyruvic acid and adenosine triphosphate levels were significantly lower in Oscox5b mutants than in WT during seed germination.The elite haplotype of OsCOX5B facilitates seed vigor by increasing its expression during seed germination.Thus,we propose that OsCOX5B is a potential target for the breeding of rice varieties with enhanced seed vigor for direct seeding.

Rice AGL1 determines grain size and sterile lemma identity

The grass spikelet is a unique inflorescence structure that determines grain size.Although many genetic factors have been well characterized for grain size and glume development,the underlying molecular mechanisms in rice are far from established.Here,we isolated rice gene,AGL1 that controlled grain size and determines the fate of the sterile lemma.Loss of function of AGL1 produced larger grains and reduced the size of the sterile lemma.Larger grains in the agl1 mutant were caused by a larger number of cells that were longer and wider than in the wild type.The sterile lemma in the mutant spikelet was converted to a rudimentary glume-like organ.Our findings showed that the AGL1(also named LAX1)protein positively regulated G1 expression,and negatively regulated NSG1 expression,thereby affecting the fate of the sterile lemma.Taken together,our results revealed that AGL1 played a key role in negative regulation of grain size by controlling cell proliferation and expansion,and supported the opinion that rudimentary glume and sterile lemma in rice are homologous organs.

Identification of the BTA8 gene reveals the contribution of natural variation to tiller angle in rice

Plant architecture is a collection of major agronomic traits that determines rice grain production,and it is mainly influenced by tillering,tiller angle,plant height and panicle morphology(Wang and Li 2006).Tiller angle is one of the critical components that determines rice plant architecture,which in turn influences grain yield mainly due to its large impact on plant density(Wang et al.2022).

A novel Effective Panicle Number per Plant 4 haplotype enhances grain yield by coordinating panicle number and grain number in rice

Increasing effective panicle number per plant(EPN)is one approach to increase yield potential in rice.However,molecular mechanisms underlying EPN remain unclear.In this study,we integrated mapbased cloning and genome-wide association analysis to identify the EPN4 gene,which is allelic to NARROW LEAF1(NAL1).Overexpression lines containing the Teqing allele(TQ)of EPN4 had significantly increased EPN.NIL-EPN4^(TQ) in japonica(geng)cultivar Lemont(LT)exhibited significantly improved EPN but decreased grain number and flag leaf size relative to LT.Haplotype analysis indicated that accessions with EPN4-1 had medium EPN,medium grain number,and medium grain weight,but had the highest grain yield among seven haplotypes,indicating that EPN4-1 is an elite haplotype of EPN4 for positive coordination of the three components of grain yield.Furthermore,accessions carrying the combination of EPN4-1 and haplotype GNP1-6 of GNP1 for grain number per panicle showed higher grain yield than those with other allele combinations.Therefore,pyramiding of EPN4-1 and GNP1-6 could be a preferred approach to obtain high yield potential in breeding.

A gap-free and haplotype-resolved lemon genome provides insights into flavor synthesis and huanglongbing (HLB) tolerance

The lemon(Citrus limon;family Rutaceae)is one of the most important and popular fruits worldwide.Lemon also tolerates huan-glongbing(HLB)disease,which is a devastating citrus disease.Here we produced a gap-free and haplotype-resolved chromosome-scale genome assembly of the lemon by combining Pacific Biosciences circular consensus sequencing,Oxford Nanopore 50-kb ultra-long,and high-throughput chromatin conformation capture technologies.The assembly contained nine-pair chromosomes with a contig N50 of 35.6 Mb and zero gaps,while a total of 633.0 Mb genomic sequences were generated.The origination analysis identified 338.5Mb genomic sequences originating from citron(53.5%),147.4Mb frommandarin(23.3%),and 147.1Mb frompummelo(23.2%).The genome included 30528 protein-coding genes,and most of the assembled sequences were found to be repetitive sequences.Several significantly expanded gene families were associated with plant-pathogen interactions,plant hormone signal transduction,and the biosynthesis of major active components,such as terpenoids and f lavor compounds.Most HLB-tolerant genes were expanded in the lemon genome,such as 2-oxoglutarate(2OG)/Fe(II)-dependent oxygenase and constitutive disease resistance 1,cell wall-related genes,and lignin synthesis genes.Comparative transcriptomic analysis showed that phloem regeneration and lower levels of phloem plugging are the elements that contribute to HLB tolerance in lemon.Our results provide insight into lemon genome evolution,active component biosynthesis,and genes associated with HLB tolerance.

Visual learning graph convolution for multi-grained orange quality grading

The quality of oranges is grounded on their appearance and diameter.Appearance refers to the skin’s smoothness and surface cleanliness;diameter refers to the transverse diameter size.They are visual attributes that visual perception technologies can automatically identify.Nonetheless,the current orange quality assessment needs to address two issues:1)There are no image datasets for orange quality grading;2)It is challenging to effectively learn the fine-grained and distinct visual semantics of oranges from diverse angles.This study collected 12522 images from 2087 oranges for multi-grained grading tasks.In addition,it presented a visual learning graph convolution approach for multi-grained orange quality grading,including a backbone network and a graph convolutional network(GCN).The backbone network’s object detection,data augmentation,and feature extraction can remove extraneous visual information.GCN was utilized to learn the topological semantics of orange feature maps.Finally,evaluation results proved that the recognition accuracy of diameter size,appearance,and fine-grained orange quality were 99.50,97.27,and 97.99%,respectively,indicating that the proposed approach is superior to others.

Rice3K56 is a high-quality SNP array for genome-based genetic studies and breeding in rice(Oryza sativa L.)

Single nucleotide polymorphism(SNP)genotyping arrays provide an optimal high-throughput platform for genetic research and molecular breeding programs in both animals and plants.In this study,a highquality and custom-designed Rice3K56 SNP array was developed with the resequencing data of 3024 rice accessions worldwide,which was then tested extensively in 192 representative rice samples.Printed on the Gene Titan chips of Affymetrix Axiom each containing 56,606 SNP markers,the Rice3K56 array has a high genotyping reliability(99.6%),high and uniform genome coverage(an average of 6.7-kb between adjacent SNPs),abundant polymorphic information and easy automation,compared with previously developed rice SNP arrays.When applied in rice varietal differentiation,population diversity analysis,gene mapping of 13 complex traits by a genome-wide association study analysis(GWAS),and genome selection experiments in a recombinant inbred line and a multi-parent advanced generation inter-cross populations,these properties of the Rice3K56 array were well demonstrated for its power and great potential to be a highly efficient tool for rice genetic research and genomic breeding.

The morphological diversity of pollen in the genus Gossypium

Background Plant pollen has diverse morphological characteristics that can be consistently passed down from generation to generation.Information on pollen morphology is thus immensely important for plant classification and identification.In the genus Gossypium,however,in-depth research on pollen morphology is lacking,with only few reports on limited cotton species.To evaluate the diversity of pollen in Gossypium,we therefore conducted a comprehensive analysis of the pollen morphology of 33 cotton species and varieties using scanning electron microscopy.Results The 33 analyzed cotton samples exhibited common pollen morphological features,including spherical shapes,radial symmetry,echination,panporation,and operculation,while the pollen size,spine shape,spine density and length showed distinctive features.Pollen size varied significantly among species,with diameters ranging from62.43 μm in G.harknessii to 103.41 μm in G.barbadense.The exine had an echinate sculptural texture,and spines were mostly conical or sharply conical but occasionally rod-like.Spine density varied from 173 in G.incanum to 54 in G.gossypioides,while spine length ranged from 3.53 μm in G.herbaceum to 9.47 μm in G.barbadense.In addition,the 33cotton species and varieties were grouped at a genetic distance of 3.83 into three clusters.Cluster Ⅰ comprised five allotetraploid AD-genome cotton species,four D-genome species,and one K-genome species.Cluster Ⅱ included 13diploid species from A,B,D,E,and G genomes,whereas Cluster Ⅲ only consisted one E-genome species G.incanum.Conclusions Although pollen characteristics alone are not enough to resolve taxonomic and systematic relationships within the genus Gossypium,our results add to knowledge on palynomorphology and contribute to phenological information on these taxa.Our findings should aid future systematic and phylogenetic studies of the Gossypium genus.

The haplotype-resolved T2T reference genome highlights structural variation underlying agronomic traits of melon

Melon(Cucumis melo L.)is an important vegetable crop that has an extensive history of cultivation.However,the genome of wild and semi-wild melon types that can be used for the analysis of agronomic traits is not yet available.Here we report a chromosome-level T2T genome assembly for 821(C.melo ssp.agrestis var.acidulus),a semi-wild melon with two haplotypes of∼373 Mb and∼364 Mb,respectively.Comparative genome analysis discovered a significant number of structural variants(SVs)between melo(C.melo ssp.melo)and agrestis(C.melo ssp.agrestis)genomes,including a copy number variation located in the ToLCNDV resistance locus on chromosome 11.Genome-wide association studies detected a significant signal associated with climacteric ripening and identified one candidate gene CM_ac12g14720.1(CmABA2),encoding a cytoplasmic short chain dehydrogenase/reductase,which controls the biosynthesis of abscisic acid.This study provides valuable genetic resources for future research on melon breeding.

Cloning of a new allele of ZmAMP1 and evaluation of its breeding value in hybrid maize

Gene resources associated with plant stature and flowering time are invaluable for maize breeding.In this study,using an F2:3population derived from a natural semi-dwarf mutant grmm and a normal inbred line Si 273,we identified a major pleiotropic QTL on the distal long arm of chromosome 1(qPH1_dla),and found that qPH1_dla controlled plant height,flowering time,ear and yield traits.qPH1_dla was finemapped to a 16 kb interval containing ZmAMP1,which was annotated as a glutamate carboxypeptidase.Allelism tests using two independent allelic mutants confirmed that ZmAMP1 was the causal gene.Realtime quantitative PCR and genomic sequence analysis suggested that a nonsynonymous mutation at the598th base of ZmAMP1 gene was the causal sequence variant for the dwarfism of grmm.This novel ZmAMP1 allele was named ZmAMP1_grmm.RNA sequencing using two pairs of near isogenic lines(NILs)showed that 84 up-regulated and 68 down-regulated genes in dwarf NILs were enriched in 15metabolic pathways.Finally,introgression of ZmAMP1_grmm into Zhengdan 958 and Xianyu 335 generated two improved F1lines.In field tests,they were semi-dwarf,early-flowering,lodging-resistant,and high-yielding under high-density planting conditions,suggesting that ZmAMP1_grmm is a promising Green Revolution gene for maize hybrid breeding.

Breeding design in wheat by combining the QTL information in a GWAS panel with a general genetic map and computer simulation

A large amount of genome-wide association study(GWAS)panels together with quantitative-trait locus(QTL)information associated with breeding-targeted traits have been described in wheat(Triticum aestivum L.).However,the application of mapping results from a GWAS panel to conventional wheat breeding remains a challenge.In this study,we first report a general genetic map which was constructed from 44 published linkage maps.It permits the estimation of genetic distances between any two genetic loci with physical map positions,thereby unifying the linkage relationships between QTL,genes,and genomic markers from multiple genetic populations.Second,we describe QTL mapping in a wheat GWAS panel of 688 accessions,identifying 77 QTL associated with 12 yield and grain-quality traits.Because these QTL have known physical map positions,they could be mapped onto the general map.Finally,we present a design approach to wheat breeding by using known QTL information and computer simulation.Potential crosses between parents in the GWAS panel may be evaluated by the relative frequency of the target genotype,trait correlations in simulated progeny populations,and genetic gain of selected progenies.It is possible to simultaneously improve yield and grain quality by suitable parental selection,progeny population size,and progeny selection scheme.Applying the design approach will allow identifying the most promising crosses and selection schemes in advance of the field experiment,increasing predictability and efficiency in wheat breeding.

A KNOX II transcription factor suppresses the NLR immune receptor BRG8-mediated immunity in rice

Nucleotide-binding site and leucine-rich repeat(NLR)proteins are activated by detecting pathogen effectors,which in turn trigger host defenses and cell death.Although many NLRs have been identified,the mechanisms responsible for NLR-triggered defense responses are still poorly understood.In this study,through a genome-wide association study approach,we identified a novel NLR gene,Blast Resistance Gene 8(BRG8),which confers resistance to rice blast and bacterial blight diseases.BRG8 overexpression and complementation lines exhibit enhanced resistance to both pathogens.Subcellular localization assays showed that BRG8 is localized in both the cytoplasm and the nucleus.Additional evidence revealed that nuclear-localized BRG8 can enhance rice immunity without a hypersensitive response(HR)-like phenotype.We also demonstrated that the coiled-coil domain of BRG8 not only physically interacts with itself but also interacts with the KNOX II protein HOMEOBOX ORYZA SATIVA59(HOS59).Knockout mutants of HOS59 in the BRG8 background show enhanced resistance to Magnaporthe oryzae strain CH171 and Xoo strain CR4,similar to that of the BRG8 background.By contrast,overexpression of HOS59 in the BRG8 background will compromise the HR-like phenotype and resistance response.Further analysis revealed that HOS59 promotes the degradation of BRG8 via the 26S proteasome pathway.Collectively,our study highlights HOS59 as an NLR immune regulator that fine-tunes BRG8-mediated immune responses against pathogens,providing new insights into NLR associations and functions in plant immunity.

BTA2 regulates tiller angle and the shoot gravity response through controlling auxin content and distribution in rice

Tiller angle is a key agricultural trait that establishes plant architecture,which in turn strongly affects grain yield by influencing planting density in rice.The shoot gravity response plays a crucial role in the regulation of tiller angle in rice,but the underlying molecular mechanism is largely unknown.Here,we report the identification of the BIG TILLER ANGLE2(BTA2),which regulates tiller angle by controlling the shoot gravity response in rice.Loss-of-function mutation of BTA2 dramatically reduced auxin content and affected auxin distribution in rice shoot base,leading to impaired gravitropism and therefore a big tiller angle.BTA2 interacted with AUXIN RESPONSE FACTOR7(ARF7)to modulate rice tiller angle through the gravity signaling pathway.The BTA2 protein was highly conserved during evolution.Sequence variation in the BTA2 promoter of indica cultivars harboring a less expressed BTA2 allele caused lower BTA2 expression in shoot base and thus wide tiller angle during rice domestication.Overexpression of BTA2 significantly increased grain yield in the elite rice cultivar Huanghuazhan under appropriate dense planting conditions.Our findings thus uncovered the BTA2-ARF7 module that regulates tiller angle by mediating the shoot gravity response.Our work offers a target for genetic manipulation of plant architecture and valuable information for crop improvement by producing the ideal plant type.

Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-toK base editing in rice plants

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement.Here,we isolated a rice endogenous hypoxanthine excision protein,N-methylpurine DNA glycosylase(OsMPG),and engineered two plant A-to-K(K=G or T)base editors,rAKBE01 and rAKBE02,for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor,ABE8e.We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04,respectively.Testing these four rAKBEs,at five endogenous loci in rice protoplasts,indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64.Furthermore,whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus,in comparison with rAKBE02 and rAKBE03,rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57-and 1.75-fold in the rice protoplasts,respectively.Moreover,although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04,rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing,at all the five target loci,with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31%and 1.67 to 4.84%in rice stable lines,respectively.Together,these rAKBEs enable different portfolios of editing products and,thus,now expands the potential of base editing in diverse application scenario for crop improvement.

Strigolactone-gibberellin crosstalk mediated by a distant silencer fine-tunes plant height in upland cotton

Optimal plant height is crucial in modern agriculture, influencing lodging resistance and facilitating mechanized crop production. Upland cotton (Gossypium hirsutum) is the most important fiber crop globally;however, the genetic basis underlying plant height remains largely unexplored. In this study, we conducted a genome-wide association study to identify a major locus controlling plant height (PH1) in upland cotton. This locus encodes gibberellin 2-oxidase 1A (GhPH1) and features a 1133-bp structural variation (PAVPH1) located approximately 16 kb upstream. The presence or absence of PAVPH1 influences the expression of GhPH1, thereby affecting plant height. Further analysis revealed that a gibberellin-regulating transcription factor (GhGARF) recognizes and binds to a specific CATTTG motif in both the GhPH1 promoter and PAVPH1. This interaction downregulates GhPH1, indicating that PAVPH1 functions as a distant upstream silencer. Intriguingly, we found that DWARF53 (D53), a key repressor of the strigolactone (SL) signaling pathway, directly interacts with GhGARF to inhibit its binding to targets. Moreover, we identified a previously unrecognized gibberellin-SL crosstalk mechanism mediated by the GhD53-GhGARF-GhPH1/PAVPH1 module, which is crucial for regulating plant height in upland cotton. These findings shed light on the genetic basis and gene interaction network underlying plant height, providing valuable insights for the development of semi-dwarf cotton varieties through precise modulation of GhPH1 expression.

Widespread incomplete lineage sorting and introgression shaped adaptive radiation in the Gossypium genus

Cotton(Gossypium)stands as a crucial economic crop,serving as the primary source of naturalfiber for the textile sector.However,the evolutionary mechanisms driving speciation within the Gossypium genus remain unresolved.In this investigation,we leveraged 25 Gossypium genomes and introduced four novel assem-blies—G.harknessii,G.gossypioides,G.trilobum,and G.klotzschianum(Gklo)—to delve into the speciation history of this genus.Notably,we encountered intricate phylogenies potentially stemming from introgres-sion.These complexities are further compounded by incomplete lineage sorting(ILS),a factor likely to have been instrumental in shaping the swift diversification of cotton.Our focus subsequently shifted to the rapid radiation episode during a concise period in Gossypium evolution.For a recently diverged lineage comprising G.davidsonii,Gklo,and G.raimondii,we constructed afinely detailed ILS map.Intriguingly,this analysis revealed the non-random distribution of ILS regions across the reference Gklo genome.Moreover,we identified signs of robust natural selection influencing specific ILS regions.Noteworthy variations per-taining to speciation emerged between the closely related sister species Gklo and G.davidsonii.Approxi-mately 15.74%of speciation structural variation genes and 12.04%of speciation-associated genes were esti-mated to intersect with ILS signatures.Thesefindings enrich our understanding of the role of ILS in adaptive radiation,shedding fresh light on the intricate speciation history of the Gossypium genus.

TWAS facilitates gene-scale trait genetic dissection through gene expression,structural variations,and alternative splicing in soybean

A genome-wide association study(GWAS)identifies trait-associated loci,but identifying the causal genes can be a bottleneck,due in part to slow decay of linkage disequilibrium(LD).A transcriptome-wide association study(TWAS)addresses this issue by identifying gene expression-phenotype associations or integrating gene expression quantitative trait loci with GWAS results.Here,we used self-pollinated soybean(Glycine max[L.]Merr.)as a model to evaluate the application of TWAS to the genetic dissection of traits in plant species with slow LD decay.We generated RNA sequencing data for a soybean diversity panel and identified the genetic expression regulation of 29286 soybean genes.Different TWAS solutions were less affected by LD and were robust to the source of expression,identifing known genes related to traits from different tissues and developmental stages.The novel pod-color gene L2 was identified via TWAS and functionally validated by genome editing.By introducing a new exon proportion feature,we significantly improved the detection of expression variations that resulted from structural variations and alternative splicing.As a result,the genes identified through our TWAS approach exhibited a diverse range of causal variations,including SNPs,insertions or deletions,gene fusion,copy number variations,and alternative splicing.Using this approach,we identified genes associated with flowering time,including both previously known genes and novel genes that had not previously been linked to this trait,providing insights complementary to those from GWAS.In summary,this study supports the application of TWAS for candidate gene identification in species with low rates of LD decay.