Search Results (302)

Aspergillus fijiensis is an industrially important filamentous fungus, whose genetic analysis has been limited by the absence of species-specific tools. This study establishes an optimized CRISPR–Cas9 genome editing platform for A. fijiensis, from protoplast preparation to DNA repair pathway engineering. Antibiotic screening first identified hygromycin B and 5-FOA (5-fluoroorotic acid) as effective positive and counter-selection markers. A high-efficiency protoplast regeneration protocol was developed depending on specific osmotic stabilization and mycelial competence. Evaluation of a plasmid-based CRISPR system revealed that while autonomous replication was feasible, gene editing was constrained by low efficiency and a predominant bias toward NHEJ (non-homologous end joining). We implemented a Cas9–sgRNA RNP (ribonucleoprotein) delivery approach, with RNP delivery alone producing frequent indels. However, targeted integration remained inefficient when using conventional MMEJ (Microhomology-mediated end joining) donors. By employing donors containing short (5 bp) microhomology arms between cleavage sites, we effectively engaged the MMEJ pathway, enabling precise insertions and large-fragment deletions in 92% of the analyzed transformants. Donor templates containing minimal 5 bp microhomology sequences could effectively shift the predominant repair pathway from NHEJ to MMEJ. These findings demonstrate that MMEJ is the superior pathway with a unique mechanism for genome engineering in A. fijiensis, providing a versatile toolkit for unlocking the biotechnological potential of this recalcitrant species and a successful paradigm for establishing genetic systems in other species. Full article

Forensic DNA profiling commonly relies on polymerase chain reaction (PCR) amplification followed by capillary electrophoresis (CE) or massively parallel sequencing (MPS), which requires expensive, laboratory-based equipment that depends on a stable power supply and is unsuitable for field applications. Here, we present a proof-of-concept assay that uses recombinase polymerase amplification (RPA) combined with exo probe detection for rapid, isothermal genotyping of insertion–deletion (InDel) markers. To the best of our knowledge, this study represents the first demonstration of forensic DNA typing using RPA coupled with exo probes. The reaction proceeds at 39 °C and combines amplification and detection in a single 20 min step. Thirteen DNA samples were genotyped in triplicate across eight InDel loci using allele-specific fluorescent probes. Genotypes were derived from differential endpoint fluorescence between matched and mismatched probes. Compared with benchmark genotyping, 97.07% of genotypes (n = 307) were correct at 1 ng DNA input. Accurate profiles were reliably obtained for DNA inputs as low as 250 pg, and partial profiles were still detectable at 31 pg. The results demonstrate that RPA-based InDel genotyping is fast, sensitive, and reproducible. With further optimization, such as refined probe design and selection of robust loci, the assay has clear potential to achieve complete accuracy and to be integrated into portable lab-on-a-chip platforms for rapid, field-deployable forensic identification. Full article

Accurate genotyping of small insertions and deletions (InDels; <5 bp) remains technically challenging in routine molecular breeding, largely due to the limited resolution of agarose gel electrophoresis and the labor-intensive nature of polyacrylamide-based assays. Here, we present the Tri-Primer Amplification Refractory Mutation System (TP-ARMS), a simple and cost-effective PCR-based strategy that enables high-resolution genotyping of small InDels using standard agarose gels. The TP-ARMS employs a universal reverse primer in combination with two allele-specific forward primers targeting insertion and deletion alleles, respectively. This design allows clear discrimination of homozygous and heterozygous genotypes using a two-tube PCR workflow. The method showed complete concordance with Sanger sequencing in detecting 1–5 bp InDels across multiple crop species, including rice (Oryza sativa) and quinoa (Chenopodium quinoa). In addition, using a TP-ARMS reduced experimental time by approximately 90% compared with PAGE-based approaches and avoided the high equipment and DNA quality requirements of fluorescence-based assays. The practical applicability of the TP-ARMS was demonstrated in breeding populations, including efficient genotyping of a 3-bp InDel in OsNRAMP5 associated with cadmium accumulation and a 6-bp promoter InDel in OsSPL10 underlying natural variation in rice trichome density across 370 accessions. Collectively, the TP-ARMS provides a robust, scalable, and low-cost solution for precise small InDel genotyping, with broad applicability in marker-assisted breeding and functional genetic studies. Full article

Salmonella Typhimurium is a major zoonotic pathogen, with pigs and chickens serving as key reservoirs for human infection, yet the genomic determinants of its host adaptation remain incompletely understood. This study integrated comparative genomics, genome-wide association studies (GWASs), and interpretable machine learning on 1654 high-quality genomes of swine- and chicken-origin S. Typhimurium isolates to identify host-associated genetic features. Phylogenetic analysis revealed host-preferred lineages and significantly lower genetic diversity within chicken-adapted subpopulations. Meta-analysis identified distinct host-associated profiles of antimicrobial resistance genes (e.g., higher prevalence of floR and bla_TEM-1 in swine) and virulence factors (e.g., enrichment of allB and the yersiniabactin system in chickens). GWASs pinpointed 1878 host-associated genes and multiple SNPs/indels, functionally enriched in metabolism, regulation, and cell processes. A two-stage Random Forest model, built using the most contributory features, accurately discriminated between swine and chicken origins (AUC = 0.974). These findings systematically revealed the genomic signatures of host adaptation in S. Typhimurium, providing a prioritized set of candidate markers for experimental validation. Full article

Hollowness of the cucumber fruit, caused by carpel separation during growth, severely impacts fruit quality. Several Sikkim cucumber accessions originating from the India–Pakistan region exhibit pronounced internal cavities. We previously identified the QTL mfh2.1 as a key contributor to this phenotype. In this study, we investigated the genetic and physiological basis of fruit hollowness in the Sikkim cucumber line WI7120 through an integrative analysis combining histological staining, HPLC for hormonal profiling, and fine mapping using a large F2 segregation population. Comparative analysis between the hollow-fruited WI7120 and the non-hollow line 9930 revealed distinct growth dynamics: WI7120 displayed accelerated radial expansion and aberrant cell patterning at carpel junctions. Histological examination using paraffin sectioning uncovered disorganized endocarp cell arrangements in WI7120 occurring as early as pre-anthesis (0 days post-pollination), with enlarged suture cells that likely facilitate tissue separation during fruit enlargement. Hormonal assays indicated elevated levels of gibberellin (GA) and zeatin (ZT), along with reduced indole-butyric acid (IBA) in WI7120, suggesting that a hormonal imbalance and mechanical stress contribute to compromised cell adhesion. By screening ~2000 F₂ individuals with SSR and InDel markers, we refined the mfh2.1 locus to a 50.92 kb interval on chromosome 2, pinpointing CsRPT4Bb—encoding a 26S proteasome subunit—as the candidate gene. A non-synonymous SNP (I135V) in CsRPT4Bb was associated with tissue-specific expression patterns during cavity formation, implicating proteasome-mediated cellular remodeling in carpel cohesion. Spatial-temporal expression analysis further revealed upregulation of CsRPT4Bb in the WI7120 exocarp during fruit expansion, potentially influencing cell wall dynamics. This study demonstrates a coordinated interplay among genetic, hormonal, and mechanical factors underlying cucumber fruit hollowness, offering new avenues for breeding cultivars with improved fruit integrity and postharvest quality. Full article

Gastrointestinal nematode (GIN) infections are the most prevalent parasitic diseases in grazing sheep worldwide, causing significant productivity losses, high mortality and, as a result, economic losses and emerging animal welfare concerns. Conventional control strategies, primarily relying on anthelmintic treatments, face limitations due to rising drug resistance and environmental concerns, underscoring the need for sustainable alternatives. Selective breeding for host genetic resistance has emerged as a promising strategy, while recent advances in transcriptomics and integrative omics research are providing deeper insights into the immune pathways and molecular and genetic mechanisms that underpin host–parasite interactions. This review summarizes current evidence on transcriptomic signatures associated with resistance and susceptibility to H. contortus and T. circumcincta GIN infections, highlighting candidate genes, functional genetic markers, key immune pathways, and regulatory networks. Furthermore, we discuss how other omics approaches, including genomics, proteomics, metabolomics, microbiome, and multi-omics integrations, provide perspectives that enhance the understanding of the complexity of the GIN resistance trait. Transcriptomic studies, particularly using RNA-Sequencing technology, have revealed differential gene expression, functional genetic variants, such as SNPs and INDELs, in expressed regions and splice junctions, and regulatory long non-coding RNAs that distinguish resistance from susceptible sheep, highlighting pathways related to Th2 immunity, antigen presentation, tissue repair, and stress signaling. Genomic analyses have identified SNPs, QTL, and candidate genes linked to immune regulation and parasite resistance. Proteomic and metabolomic profiling further elucidates breed- and tissue-specific alterations in protein abundance and metabolic pathways, while microbiome studies demonstrate distinct microbial signatures in resistant sheep, suggesting a role in modulating host immunity. In conclusion, emerging multi-omics approaches and their integration strategies provide a comprehensive framework for understanding the complex host–parasite interactions that govern GIN resistance, offering potential candidate biomarkers for genomic selection and breeding programs aimed at developing sustainable, parasite-resistant sheep populations. Full article

Since the molecular mechanisms underlying sex determination in Procambarus clarkii are still unclear, it is important to investigate the genetic basis of sex determination in crustaceans. Currently, the molecular mechanisms of sex determination and the gender-specific markers in this species remain poorly understood. In this study, a total of 14,046,984 SNPs and 2,160,652 InDels were identified through genome-wide resequencing of 89 individuals (45 females and 44 males). Further analysis confirmed that the candidate chromosome was Chr38, the sex determination system was identified as XY, and the sex determination region was located at Chr38: 6,000,000–21,100,000 bp. A pair of sex-specific molecular markers has been identified based on a 21 bp female-specific insertion within the candidate sex-determining region. Additionally, SOAT, NPC1, PTGS2, FANCD1, and VAlRS were identified as candidate sex-determining genes through the screening of candidate genes and RT-qPCR validation analysis. These findings provide a robust foundation for investigating sex-determining mechanisms in crustaceans. Through the integration of genome-wide association studies (GWAS), selection signals, and transcriptome analysis, we identified, for the first time, genes associated with sex determination, growth, and immunity. These genes represent promising candidates for further functional studies and genetic improvement in Procambarus clarkii. Full article

Background: Several genes and genomic regions have been implicated in COVID-19 susceptibility and severity, but their clinical relevance remains uncertain. We comprehensively assessed both copy number variants (CNVs) and single-nucleotide variants (SNVs) disrupting genes implicated in COVID-19 in a Swedish cohort of ICU-treated COVID-19 patients with detailed phenotype data. Methods: Patients (n = 301) with severe COVID-19 treated in intensive care units (ICU) between March 2020 and January 2021 at two large Swedish university hospitals were included. Whole exome sequencing (WES) was performed to identify both large copy number variations (CNVs) and single-nucleotide variants (SNVs), including small indels, using the Genome Analysis Toolkit (GATK) pipelines. We focused our analyses on variants disrupting coding genes implicated in severe COVID-19, but also assessed variants known to cause human disease. Results: We identified 11 rare CNVs and several SNVs potentially linked to severe COVID-19. Patients carrying a CNV spanning a COVID-19-implicated gene had higher levels of the heart failure marker NT-proBNP (median 4440 [1558–8160] vs. 1170 [329–3152], p = 0.017), worse renal function at ICU admission (p = 0.0026), and a higher need for continuous renal replacement therapy (CRRT) (28% vs. 10%, p = 0.045) compared to patients without a potentially damaging CNV. Conclusions: Although patients with a potentially damaging CNV or SNV exhibited some differences in cardiac and renal markers, our findings do not support broad genetic screening as a predictive tool for COVID-19 severity. Full article

The large and hard olive pit adversely affects oil quality during traditional crushing, as seed- and pit-derived enzymes modify phenolic profiles and volatile compounds. Polyploid breeding offers a potential means to reduce pit size and improve processing traits, yet cultivated olive (Olea europaea L. subsp. europaea) is a strictly diploid species, and natural polyploids have not been previously documented. To evaluate the potential of triploids in olive improvement, we screened seed-derived progeny from multiple cultivars for polyploidy using flow cytometry and chromosome observation. One naturally occurring triploid seedling (‘Olive-3x’) was identified from a mixed lot of open-pollinated seeds. Whole-genome resequencing was used to develop 64 polymorphic InDel markers, and three markers indicated ‘Koroneiki’ as one putative parent of the triploid. Morphological and cytological analyses showed that the triploid exhibited typical polyploid characteristics, including thicker leaves and enlarged epidermal and palisade mesophyll cells compared with diploid controls. These findings provide the first evidence of a naturally occurring triploid in cultivated olive and show that triploids can arise within seed-derived progeny. The identified triploid plant and the developed markers offer useful resources for future studies on olive polyploidy and provide foundational resources for future research on olive polyploidy and cultivar improvement. Full article

Shanlan upland rice is a unique genetic resource from the mountainous regions of Hainan, China, yet its genetic diversity and agronomic potential remain poorly characterized. This study systematically evaluated 114 Shanlan upland rice landraces using phenotypic assessment and 38 genome-wide Insertion/Deletion (InDel) markers. Significant phenotypic variability was observed in key agronomic traits, including plant height, tiller number, and yield components. The molecular analysis revealed a moderate level of genetic diversity (average PIC = 0.43) and consistently grouped the landraces into three distinct genetic subpopulations. To facilitate efficient germplasm management, we developed a DNA fingerprinting system using a reduced set of 19 core InDel markers, which was integrated with a phenotypic QR code database. Furthermore, a network-based strategy identified a core collection of 54 accessions, streamlining the resource for future breeding and conservation efforts. These findings provide a robust molecular framework for the conservation and genetic improvement of Shanlan upland rice. Full article

Background: Grapevine breeding increasingly relies on molecular tools to introduce durable resistance to downy and powdery mildew. However, the reproducibility of simple sequence repeat (SSR) markers across platforms remains a challenge for marker-assisted selection (MAS). This study aimed to evaluate the performance of SSR markers associated with key resistance loci (Run1/Rpv1, Ren3/Ren9, Rpv3, Rpv10, Rpv12) using the Qsep100 system and to validate selected markers on the ABI platform. Methods: A panel of grapevine cultivars and breeding genotypes was analyzed for SSR markers linked to resistance loci. PCR amplicons were separated on the Qsep100 BioFragment Analyzer, and a subset of markers was cross-validated using ABI capillary electrophoresis. Results: Only a limited subset of markers displayed consistent performance across genotypes. Sc34-8 and Sc35-2 were most reliable for Run1/Rpv1, Indel-27 and Indel-20 for Ren3/Ren9, UDV737 for all Rpv3 sub-loci, GF-09-44 and GF-09-57 for Rpv10, and UDV340 and UDV343 for Rpv12. ABI validation of UDV737 and Indel-27 confirmed high concordance with Qsep100 results, with allele size differences typically ≤2 bp. Conclusions: The study identifies a core set of robust SSR markers suitable for routine MAS in grapevine breeding. Results demonstrate that the Qsep100 system is a reliable alternative to ABI for large-scale genotyping, supporting its broader implementation in resistance breeding programs. Full article

Melon (Cucumis melo L.) is a significant horticultural crop valued for its aroma and health-promoting compounds. However, the genetic similarity among numerous varieties poses challenges for identification and breeding. ‘Dongfangmi No.4’ is an F₁ hybrid derived from a cross between two Hami melon inbred lines, ‘M06-1-3’ and ‘M15-3’. This study utilized resequencing data derived from the bi-parents of ‘Dongfangmi No.4’ to identify 557,878 insertion and deletion (InDel) variations across the entire genome. Thirty-nine highly polymorphic InDel markers were screened to conduct a genetic analysis of 40 representative cultivated varieties, with marker MS108 specifically distinguishing ‘Dongfangmi No.4’ from the other 39 cultivated varieties. Genetic analysis revealed a high level of genetic diversity within the population (average observed heterozygosity Ho = 0.313, Shannon index I = 0.528), and polymorphic information content (PIC) analysis indicated that 54% of the markers (21/39) were highly polymorphic. Principal component analysis (PCA) and clustering demonstrated significant genetic differentiation between cantaloupe and Hami melons, as well as between cantaloupe and honeydew. In contrast, the genetic boundaries between Hami melons and honeydew were obscured due to frequent germplasm exchange. Ultimately, seven core InDel markers were selected to construct the DNA fingerprinting map, successfully achieving complete differentiation of 40 varieties. This marker system provides an effective molecular tool for melon variety identification, intellectual property protection, and breeding. Full article

Hypoxia is a critical environmental stressor in aquaculture, significantly affecting the survival and growth performance of cultured fish. To explore the genetic basis of hypoxia tolerance in grass carp (Ctenopharyngodon idella), we integrated genome-wide association analysis (GWAS) and multi-tissue transcriptome profiling. A total of 2000 grass carp were subjected to hypoxic stress, from which the 150 most hypoxia-intolerant (HI) and 150 most hypoxia-tolerant (HT) individuals were selected based on the time to loss of equilibrium (LOE). GWAS using 3,730,919 SNPs and 851,595 InDels identified 21 SNPs and 6 InDels associated with hypoxia tolerance. Two SNPs on chromosomes 10 and 13 reached genome-wide significance, accounting for 2.7% and 4.8% of the phenotypic variance explained (PVE), respectively. Validation of identified SNPs was performed using kompetitive allele-specific PCR (KASP) analysis. Candidate genes within ±50 kb of these variants were enriched in steroid biosynthesis, insulin signaling, and glycosphingolipid biosynthesis pathways. Transcriptomic analysis of six tissues (brain, gill, intestine, kidney, liver, and spleen) revealed 1620, 1221, 796, 246, 210, and 58 differentially expressed genes (DEGs) in the HT group compared to the HI group, respectively. DEGs in the brain were primarily enriched in steroid metabolic processes and angiogenesis regulation, while those in kidney and spleen DEGs were associated with oxygen transport and erythrocyte development. Integrated analysis of GWAS and transcriptome data identified 16 shared genes, including usf1 and trpv4. These findings reveal key genomic loci and molecular pathways underlying hypoxia tolerance in grass carp, providing valuable markers for future selective breeding programs. Full article

The genetic improvement of Phalaenopsis-type Dendrobium, a valuable ornamental and medicinal orchid, is hindered by the lack of a complete reference genome. In this study, a transcriptome-based approach was employed to develop and validate insertion–deletion (InDel) markers for genetic analysis and variety identification. RNA-seq was performed on two distinct varieties, resulting in the de novo assembly of 156,108 unigenes. A bioinformatics pipeline was developed to identify 5083 high-quality InDel loci, from which 1029 potential markers were designed. Fifty primer pairs were selected and validated experimentally, with 84% successfully amplifying clear products, and 76% showing polymorphism. The polymorphism information content (PIC) of the markers ranged from 0.25 to 0.78, indicating their high potential for use in genetic diversity studies. These markers were used to classify 24 Phalaenopsis-type Dendrobium varieties into distinct genetic clusters. This work provides a scalable and robust platform for molecular breeding, DNA fingerprinting, and germplasm management in non-model species that lack a reference genome. By leveraging transcriptome data, these markers will contribute to the efficient genetic improvement of Dendrobium and other similar crops. Full article

Accurate species identification is essential for the quality control and standardization of herbal medicines. Agrimonia species, the authentic sources of Agrimoniae Herba, have long been used in traditional medicine, yet limited genomic resources have hindered the establishment of reliable molecular approaches for accurate species discrimination within this genus. Here, we report the newly assembled complete chloroplast genomes (155,156–155,302 bp) of four Agrimonia species, which exhibit the typical quadripartite structure and contain 112 unique genes. Comparative analysis revealed 684 variable sites, including 497 single nucleotide polymorphisms (SNPs) and 187 insertions/deletions (InDels), predominantly located in the single-copy regions. Based on these species-specific variations, we developed nine PCR-based molecular markers that distinguished the four species. The markers were validated using herbarium specimens and commercial herbal products, demonstrating reproducibility and practical applicability. Phylogenetic analysis supported the monophyly of the genus Agrimonia and resolved each species into distinct clusters within the subtribe Agrimoniinae. These results showed that chloroplast genome sequences of the genus Agrimonia can serve as effective super DNA barcodes for species identification. Our study provides fundamental genomic resources for Agrimonia and reliable molecular tools for species authentication, providing a basis for ensuring the authenticity and safety of Agrimoniae Herba. Full article