Genetic Dissection Research Articles (Page 1)

Coronaviruses induce the formation of double-membrane vesicles (DMVs) to facilitate viral RNA replication and transcription by the replication-transcription complexes (RTCs), comprising non-structural proteins (nsps) 2-16 and nucleocapsid protein. Nsp3 and nsp4 are the minimal components necessary for DMV formation and assemble a molecular pore that spans the DMV double membrane, connecting the DMV interior to the cytosol. However, the recruitment mechanisms of additional RTC components and the roles of other viral proteins in DMV assembly remain poorly understood. To dissect these processes independently of viral replication, we sought to establish a surrogate expression system using severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) polypeptides to recapitulate DMV formation and RTC recruitment. We characterized DMVs formed in cells expressing various nsp combinations and assessed the localization of RTC components using proteinase K (PK) protection assays following cell permeabilization. Compared to nsp3-4, expression of nsp3-8 and nsp3-10 resulted in larger and more morphologically heterogeneous DMVs. Portions of nsp5, nsp7, and nsp8 are associated with DMV-enriched membrane fractions, with nsp5 and nsp8 showing partial resistance to proteinase K digestion, suggesting that these proteins are at least partially localized within the DMV interior or protected by the DMV membrane architecture. Notably, mutations in the membrane-associated element (MAE) of nsp6 impaired nsp5-mediated proteolytic processing, abrogated DMV formation, and induced a cross-linked endoplasmic reticulum (ER) phenotype. These results highlight the essential role of nsp6 in the DMV biogenesis and demonstrate the utility of this surrogate system for mechanistic studies of coronavirus-induced membrane remodeling.IMPORTANCECoronaviruses remodel host membranes through the action of non-structural proteins to generate double-membrane vesicles (DMVs), which serve as platforms for viral replication-transcription complexes (RTCs). Deciphering the molecular mechanisms governing DMV assembly and RTC recruitment is critical for understanding coronavirus replication and identifying novel antiviral targets. Here, we developed a surrogate system that recapitulates DMV formation in the absence of viral replication, enabling genetic manipulation and functional dissection of individual proteins. Using this system, we demonstrate that expression of the SARS-CoV-2 nsp3-10 polyprotein is sufficient to drive DMV formation and reveal a pivotal role for the membrane-associated element (MAE) of nsp6 in this process. These findings establish a tractable model for investigating coronavirus-induced membrane remodeling and underscore the essential contributions of nsp6 to DMV biogenesis.

Genetic dissection of heat stress tolerance in soybean through genome-wide association studies and use of genomic prediction to enhance breeding applications

Integrative genetic, single-cell and mechanistic dissection identifies bisphenol a as a causal driver of preeclampsia via fibroblast-derived IL-6/STAT3 signaling.

Seven co-localized QTLs that control low-sulfur tolerance in soybean seedlings were identified. Two putative candidate genes and 3 promising parental cross combinations were further predicted. Low-sulfur nutrient stress severely affects yield and quality in soybean production. However, genetic studies related to soybean tolerance to low-sulfur conditions are insufficient. Here, soybean tolerance to low-sulfur conditions was evaluated according to ten traits at the seedling stage. A total of 72 quantitative trait loci (QTLs) and 103 quantitative trait nucleotides (QTNs) related to low-sulfur tolerance in soybean seedlings were detected via linkage analysis and genome-wide association analysis (GWAS) in a recombinant inbred line (RIL) population and a natural population, respectively. Among these loci, 7 co-localized QTLs were identified via two methods across chromosomes 1, 6, 8, 9, 14, and 17. Glyma.17G167100, which includes two significant SNPs (AX-93862060 andAX-93862061), and Glyma.14G169300 were suggested as putative candidate genes on the basis of transcriptome data, haplotype analysis and real-time quantitative PCR. In addition, 3 promising parental cross combinations with the aim of improving low-sulfur tolerance have been designed across favorable alleles, which were determined on the basis of the co-localized QTLs and relative values of trait phenotypes in three environments. These results provide important evidence for understanding the genetic basis of low-sulfur tolerance in soybean and may be helpful in the breeding of new soybean varieties with high tolerance to low-sulfur soil.

The 66 stable QTLs related to fiber quality and yield components were identified using Gh-Gb introgression population. A keratin-associated gene GbKAP from qFU-D04-1b is verified to modulate cell elongation and seed yield. Chromosome segment substitution lines (CSSLs) are a powerful tool for genetic dissection of complex quantitative traits. Here, we re-sequenced 117 CSSLs introgressed from Gossypium barbadense acc. Hai7124 into G. hirsutum acc. TM-1. A total of substitution segment length of 4572Mb, with a total genome coverage of 68.78%, were identified. Utilizing both regression stepwise-likelihood ratio test (RSTEP-LRT) and composite interval mapping (CIM) methods, 145 common QTLs (co-QTLs) were detected simultaneously across four environments/years, including 65 associated with fiber quality traits and 80 related to yield components. Among these, 66 co-QTLs were detected in two or more environments/years as stable QTLs. We further verified a stable fiber uniformity (FU)-related QTL qFU-D04-1b using F2 and F2:3 secondary segregating populations derived from a cross between the elite introgression line CSSL25 and TM-1 and fine-mapped the QTL to the region of 762kb on D04 chromosome. Within this interval, GB_D04G0512 named as GbKAP, encoding a keratin-associated protein, exhibited higher transcript level during fiber elongation stages in CSSL25 than in TM-1. There was a non-synonymous SNP in the coding region and three specific transcription factor binding sites in the promoter of GbKAP in CSSL25 compared to its homolog in TM-1. Heterologous expression of GbKAP in Arabidopsis resulted in increased root length, root cell length, hypocotyl length, rosette leaf growth, plant height, and seed size and weight, indicating GbKAP plays an important role in cell elongation and seed yield. This study provides valuable resources for the improvement of upland cotton fiber quality and yield traits.

Huntington's disease (HD) is driven by somatic expansion of the HTT CAG repeat, with onset modified by genetic factors. One such modifier, 8AM1, maps to chromosome 8 near RRM2B, a gene not directly involved in the machinery that lengthens the repeat. To investigate this locus, we performed capture sequencing and identified variants at both the 5' and 3' ends of RRM2B with expected minor allele frequencies. A polymorphic frameshift variant (rs1037699) in an alternate exon 1 disrupts expression of a previously uncharacterized RRM2B isoform 2, but not isoform 1. Functional analyses in RRM2B knock-out cells and 8AM1 heterozygous LCLs suggest that isoform 2 may function at mitochondria. Several 3' variants, including a 21bp 3'UTR deletion (rs200678743) and peak tag-SNV (rs79136984), act as cis expression quantitative trait loci. Analysis of HD onset data (n= 12,982) revealed that 5' and 3' variants contribute independently to the 8AM1 modifier effect, with full impact observed only in the absence of the frameshift variant. Knockdown of both isoforms increased neurodegeneration in HD neurons derived from pre-symptomatic patient fibroblasts, supporting an intersection of RRM2B biology and HD pathogenesis. We conclude that the 8AM1 haplotype, present in ~ 14% of Europeans, modifies RRM2B expression in a cell- and context-dependent manner, thereby accelerating HD onset in mutation carriers.

The online version contains supplementary material available at 10.1007/s11032-025-01596-8.

Pre-mRNA splicing is essential for eukaryotic gene expression and is achieved through the accurate recognition of exon-intron boundaries. Although nonconventional introns, which do not follow the conventional GT-AG splicing rule, have been identified in several species, these introns are typically rare in any given genome. Here, we demonstrate the widespread occurrence of nonconventional introns (71.8% of all introns) in the Euglena agilis genome and identify consensus motifs at these nonconventional exon-intron boundaries. We assessed the splicing efficiency of nonconventional introns and variants with point mutations via genomic knock-in within the second exon of Glucan synthase-like 2 in Euglena gracilis and genetically defined the sequence signature (5'-N3CDG-/-CH'GN5-6|Rexon-3') required for their proper splicing. This signature is present in 61.2% of all nonconventional introns detected in the E. agilis genome. Accordingly, we present a noncanonical splicing code for Euglena introns, highlighting the global coexistence of dual splicing rules for conventional and nonconventional introns.

Biofilm formation is a highly regulated process that contributes to the environmental fitness of microorganisms, including pathogenic bacteria. The second messenger c-di-GMP is a critical regulator of biofilm formation whose cellular levels are tightly regulated by the abundance and activity of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). These enzymes synthesize and degrade c-di-GMP, respectively. The Vibrio cholerae VpvABC system encodes a DGC and is critical for biofilm formation; however, much remains unknown about its regulation. Here we demonstrate that the vpvABC system is transcriptionally regulated by c-di-GMP and the master biofilm regulators VpsT and VpsR. However, we also identify the alternative sigma factor RpoS as a positive regulator of vpvABC. RpoS is involved in the regulation of many c-di-GMP metabolism genes and plays a role in biofilm architecture, likely mediated in part through vpvC. In mature biofilms, vpvA transcription was highest near the biofilm substratum and VpsT, VpsR, and RpoS were critical for vpvABC transcription. Overall, our genetic dissection reveals the vpvABC system is regulated by two parallel circuits: a c-di-GMP sensing-circuit acting through VpsT and VpsR and a stationary growth phase circuit via RpoS. These findings underscore the multilayered regulatory mechanisms that precisely govern biofilm formation by a pathogen.

Background/Objectives: Global maize production is considerably affected by drought aggravated by climate change. No genome-wide association study (GWAS) or candidate gene analysis has been performed using chlorophyll fluorescence (ChlF) and hyperspectral (HS) indices measured in young plants challenged by a water deficit. Our objective was to conduct a GWAS of nine ChlF and HS indices measured in a diversity panel of drought-stressed young plants grown in a controlled environment using a maize single nucleotide polymorphism (SNP) 50k chip. Methods: A total of 165 inbred lines were genotyped using the Infinium Maize50K SNP array and association mapping was carried out using a mixed linear model. Results: The GWAS detected 37 respective SNP markers significantly associated with the maximum quantum yield of the primary photochemistry of a dark-adapted leaf (Phi_Po), the probability that a trapped exciton moves an electron into the electron transport chain further than QA (Psi_o), the normalized difference vegetation index (NDVI), the Zarco–Tejada and Miller Index (ZMI), greenness, modified chlorophyll absorption in reflectance (MCARI), modified chlorophyll absorption in reflectance 1 (MCARI1), and Gitelson and Merzlyak indices 1 and 2 (GM1 and GM2). Conclusions: Our results contribute to a better understanding of the genetic dissection of the ChlF and HS indices, which is directly or indirectly related to physiological processes in maize, supporting the use of HS imaging in the context of maize breeding.

Poultry egg production is shaped by the intertwined action of multiple physiological systems, greatly magnifying the complexity of its underlying genetic regulation. Although multitissue mapping of regulatory variants offers a powerful route to untangle this complexity, comprehensive data sets in ducks remain scarce. Meanwhile, the contributions of peripheral systems beyond neuroendocrine regulation on poultry egg production are still largely unexplored. Here, we generate 979 RNA-seq samples from the liver, ovary, oviduct shell gland, and spleen, along with matched whole-genome sequencing data from 307 egg-laying ducks. We map cis-regulatory variants associated with gene expression (eQTL), alternative splicing (sQTL), and 3' alternative polyadenylation (apaQTL), yielding 14,074, 6267, and 4994 genes with at least one significant eQTL, sQTL, and apaQTL, respectively. By integrating this resource and GWAS results, we confirm that ABCG2 expression in the shell gland specifically regulates eggshell color, with additional involvement of ENOPH1's 3'APA sites in both the shell gland and liver. In addition, expression of LOC101800576 and LOC101790890 in the shell gland, of LOC119713219 in the ovary, and of GLP2R in the spleen is causally linked to declining egg production at peak laying. Last, we delineate a cross-tissue regulatory landscape underlying duck egg production and identify liver-derived modules, particularly Liver_ME1, which is mainly involved in cell cycle regulation, as central hubs coordinating with peripheral tissues affecting duck egg production. This work delivers a key resource and fresh perspectives for the genetic mechanism dissection of duck egg production and for future studies on cross-tissue regulation of reproduction.

SummaryIn symbiotic plant–microbe interactions, the host invests considerable amounts of resources in the microbial partner. If the microbe does not reciprocate with a comparable symbiotic benefit, it is regarded as a cheater. The host responds to cheaters with negative feedback mechanisms (sanctions) to prevent fitness deficits resulting from being exploited. We study sanctioning in the symbiosis between Medicago truncatula and the nitrogen‐fixing rhizobium Sinorhizobium meliloti.We manipulated the exchange of resources between the partners in three ways: by using mutant rhizobia defective in nitrogenase; replacing nitrogen in the atmosphere with argon gas; and supplying rich nitrogen fertilizer to the host. We follow the consequences of simulated cheating by examining the metabolome and proteome of both partners.We find that sanctioning occurs at multiple levels. In particular, we observe repression of essential symbiotic functions and changes in central metabolism that are likely to be relevant for microbial fitness and that could therefore contribute to sanctioning. In addition, sanctioning triggers a broad panel of defense markers.A thorough understanding of the multilevel phenomenon of sanctioning will be essential for its genetic dissection and for the breeding of elite legume crops with efficient symbiosis.

The 14-3-3 proteins are highly conserved and widely distributed across eukaryotes. Some 14-3-3 proteins have been identified as regulators of phosphorus (Pi) deficiency tolerance in rice, but their diverse functions remain largely unexplored. In this study, we characterized the role of rice plant-specific non-ε group 14-3-3 proteins (OsGF14a-f) in response to Pi starvation by mutating these genes. We found that the expression of OsGF14a decreased in response to Pi starvation, while the expression of other non-ε group genes was induced. Subcellular localization studies transiently expressing them in tobacco leaves showed that OsGF14a was present in both the cytoplasm and nucleus, whereas the other proteins were predominantly localized in the cytoplasm. By developing single and multiple mutants, we demonstrated that OsGF14a plays a negative role in Pi homeostasis and root growth, while OsGF14b, OsGF14c and OsGF14f may act as positive regulators of Pi homeostasis and root growth in rice. However, all non-ε group 14-3-3 genes positively regulated rhizosphere acidification. Furthermore, the mutation of OsGF14a enhanced Pi accumulation and plant growth under various Pi supply conditions, likely due to the induction of OsPHR3, OsPT2 and OsPHO1;2 in the roots. Overall, this study highlights the diverse functions of plant-specific non-ε group 14-3-3 proteins in response to Pi starvation in rice and identifies the mutation of OsGF14a as a potential strategy to improve rice tolerance to Pi deficiency.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12284-025-00840-1.

Genetic dissection of two elite japonica varieties reveals founder transmission and selection in breeding.

Comprehensive genetic dissection of yield-related traits utilizing quantitative trait loci sequencing approach in mungbean.

Genetic dissection of plant architecture reveals haplotypes controlling sink-related traits in oilseed rape under limited nitrogen fertilization

A critical challenge in crop breeding is the trade-off between improving the mean of an important trait and maintaining its phenotypic plasticity. Grain chalkiness is a key cereal grain-quality trait highly susceptible to environments. However, the genetic and molecular mechanisms controlling the trade-off between phenotypic mean and plasticity of grain chalkiness remain unknown. Here, utilizing comprehensive genome-wide association studies on ten grain chalkiness traits over five years in a mini-core collection, substantial phenotypic plasticity of grain chanlkiness is found, whichdeclines during modern breeding. A general trade-off between phenotypic mean and plasticity of grain chalkiness is demonstrated, which are controlled by distinct genetic architectures revealed through a detailed QTL atlas. High temperature and wide grain significantly increase grain chalkiness mean but decrease its plasticity and genetic dissection, representing two major external drivers for the trade-off. Two key quantitative trait genes MPC5 and GCP6 are identified to control this trade-off. The transcription factor GCP6 biochemically and genetically inhibits MPC5 expression, forming a key module that confers the mean-plasticity trade-off of both grain chalkiness and width. Finally, minimal marker sets for molecular breeding accounted for two thirds of grain chalkiness variation. These findings elucidate the genetic architecture of the mean-plasticity trade-off in grain chalkiness and offer a proof-of-concept breeding strategy to simultaneously optimize both phenotypic mean and plasticity in crop improvement.

BackgroundGenome-scale mutagenesis integrated with high-throughput phenotypic screening and causal mutation mapping serves as a robust paradigm for systemic genetic dissection. Despite the application of non-homologous end joining (NHEJ)-mediated genome editing in Yarrowia lipolytica, the development of alternative genome-wide mutagenesis strategies remains unexplored in this industrially relevant oleaginous yeast.ResultsWe developed the Helicase-Assisted (Helicase-CDA) system, a genome-wide mutagenesis platform integrating the helicase domain of Yarrowia MCM5 (Encoded by YALI1_A01766g) with cytidine deaminase (CDA). This system enables continuous C-to-T specific mutations at random genomic loci. Applied to an industrial β-carotene-producing Y. lipolytica strain, Helicase-CDA system generated a mutagenized library through 7-day subculturing. Through high-throughput screening, we successfully isolated the mutant strain CDA-14, which demonstrated a 25% enhancement in β-carotene production (448.1 mg/L) compared to the wild-type strain. Notably, its productivity of β-carotene reached 6.15 g/L in fed-batch fermentation. Whole-genome sequencing revealed a G1637A substitution in YALI1_B16239g, which encodes a membrane protein showing homology to sterol biosynthesis regulator MGA2. This mutation led to reduced ERG1 expression level and redirected central carbon flux toward carotenoid synthesis by perturbing isoprenoid precursor partitioning.ConclusionHelicase-CDA system circumvents the dependency on NHEJ-mediated whole-genome mutation approach, offering a robust tool for continuous genome evolution in pre-engineered industrial strains. This study not only enhances genome editing in Y. lipolytica but also identifies a practical target for optimizing terpenoid biosynthesis, demonstrating significant potential for applications in metabolic engineering and synthetic biology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-025-02819-5.

We identified Rht28, a novel dwarf gene on the wheat chromosome arm 2AL, and investigated its effect on important agronomic traits. The genetic dissection of wheat plant height (PH) and the identification of new dwarf or semi-dwarf genes will help breeders to improve yield potential. In this study, QPh-2A.2, a newly discovered locus controlling PH and explaining 7.29%-14.61% of the phenotypic variation across ten environments, was identified using a recombinant inbred line (RIL) population derived from the cross Yanda 1817 × Beinong 6 through a previously constructed single nucleotide polymorphism (SNP) genetic linkage map. To confirm the stability and effect of QPh-2A.2, 22 tightly linked markers were developed based on the released Chinese Spring (CS) reference genome sequence, and a high-density genetic linkage map of the target locus was constructed. QPh-2A.2 is a newly discovered locus controlling PH in wheat, which is temporarily designated Rht28. Additionally, Rht28 was found to have significant effects on thousand-grain weight, grain width, grain thickness, and grain weight per spike, as evaluated in the RIL population, while other agronomic traits related to flag leaf and spike traits were not significantly affected. Moreover, using a large segregating population and extensive progeny tests, we mapped Rht28 within a 3.17-Mb physical region between the markers WBUA4036 and WBUA4413 on the long arm of chromosome 2A. This targeted region contains 51 annotated genes according to CS v2.1 reference genome sequence, and two of these were speculated to be candidates for Rht28. Exploring new dwarf genes is a vital route to breed lodging-resistant wheat cultivars. This study also provides important information for Rht28 marker-assisted selection in wheat breeding programs.

Studies of globin gene clusters have established many paradigms of gene regulation. This review focuses on the α- and β-globin gene clusters of humans and mice, summarizing important insights from high-throughput biochemical assays and directed genetic dissections and emphasizing similarities across the types of gene clusters and between species. The overall arrangements and architectures are similar, with each gene cluster being localized within a topologically constrained unit of chromatin containing a multicomponent enhancer (i.e., a locus control region) and other regulatory elements bound by a similar set of transcription factors and coactivators. Differential expression of the globin genes within each cluster during ontogeny is associated with changes in contacts with the locus control region and involves the action of gene-specific repressors. Detailed study of the fetal β-like HBG1 and HBG2 globin genes has revealed a remarkable diversity of regulatory pathways that provide candidates for therapeutic approaches to reactivate these genes for β-hemoglobinopathies.