Search Results (536)

Chromosomal rearrangements that lead to the formation of oncogenic gene fusions, such as EML4-ALK, are thought to arise from incorrect repair of double-strand breaks in DNA. However, the mechanisms and factors driving rearrangement formation remain poorly understood, and analysis of these processes is limited by detection methods that are labor-intensive, low-throughput, and not readily quantitative at single-cell resolution. Here, we developed a genetically encoded ALK reporter based on A549 lung adenocarcinoma cells, created by inserting an ALK-P2A-mCherry cassette into the endogenous ALK locus, so that induced EML4-ALK fusion activated mCherry fluorescence. Reporter activation yielded a readily quantifiable mCherry-positive subpopulation that could be measured and enriched by flow cytometry and correlated with EML4-ALK levels. Using this platform, we combined CRISPR-mediated rearrangement induction with knockdown of DNA repair factors using RNA interference. Of the factors involved in base excision repair, homologous recombination-related pathways and canonical non-homologous end joining, knockdown of the APEX1 gene encoding apurinic endonuclease 1 (APE1) selectively increased EML4-ALK levels both in the reporter cell line and in parental A549 cells. Together, this work provides a sensitive, single-cell A549-based ALK reporter platform and a framework for future studies aimed at identifying cellular and environmental factors that modulate oncogenic EML4-ALK rearrangement formation. Full article

The einkorn wheat group, comprising ancient diploid species (2n = 14, AA), including Triticum monococcum, Triticum boeoticum, and Triticum urartu, represents a valuable source of genetic variation for improving disease resistance in wheat. To develop a practical platform for introgressing powdery mildew resistance into bread wheat, we screened 21 einkorn accessions with Blumeria graminis f. sp. tritici (Bgt) race E09 and identified seven resistant donors. Because direct hybridization between diploid einkorn (AA) and hexaploid wheat (AABBDD) is constrained by genomic divergence and poor cross-compatibility, we crossed resistant einkorn accessions with susceptible durum wheat and induced chromosome doubling in the F₁ hybrids to generate synthetic durum–einkorn amphiploids. Nine amphiploids were obtained. Chromosome counts and genomic in situ hybridization confirmed the expected genomic constitution (AABBAA) in most lines, with limited variation in chromosome number in two amphiploids. Phenotyping against Bgt race E09 showed that three amphiploids retained high resistance, four showed moderate resistance, and two were moderately susceptible. Marker analysis identified five einkorn accessions contain known Pm genes such as Pm60, Pm60b, and PmNCA6/Pm37, as well as their derived amphipliods. Two einkorn accessions and their derived amphiploids may harbor novel Pm genes. Field evaluation of the agronomic traits of these amphiploids indicated some improvement in tillering, spike length, and seed weight. Moreover, these amphiploids had better seed-setting rates in crosses and backcrosses with common wheat. These synthetic durum–einkorn amphiploids thus offer a new bridging platform for transferring alien genes from diploid einkorn to hexaploid common wheat, providing valuable genetic resources for wheat-breeding programs. Full article

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches. Full article

Ataxia–telangiectasia is a rare, autosomal recessive primary immunodeficiency caused by mutations in the ATM gene on chromosome 11, which encodes a serine–threonine kinase essential for the recognition and repair of DNA double-strand breaks. The disease is characterized by progressive neurological impairment, immunological dysfunction, and an increased susceptibility to recurrent infections and malignancies. Pulmonary involvement represents a major source of morbidity and frequently arises from chronic infections, aspiration, and impaired airway clearance, ultimately leading to the development of bronchiectasis. The case of a 15-year-old adolescent with a history of recurrent aspiration pneumonias, neuropsychomotor developmental delay, and severe malnutrition is reported, who was admitted for evaluation of chronic productive cough, fever, and dysphagia. Comprehensive clinical assessment and ancillary investigations revealed recurrent respiratory infections, gastroesophageal reflux, and typical features of ataxia–telangiectasia, including cerebellar ataxia, oculomotor apraxia, and conjunctival telangiectasias. Additionally, bronchiectasis was identified as a secondary consequence of the underlying neurological and immunological impairment. This case highlights the diagnostic challenges posed by ataxia–telangiectasia in pediatric patients presenting with chronic respiratory symptoms and emphasizes the importance of early recognition of the underlying systemic disorder. A multidisciplinary approach is essential for accurate diagnosis and optimized management, aiming to address both the primary disease and its pulmonary complications. Full article

Multiple myeloma (MM) is a biologically heterogeneous plasma cell malignancy in which prognosis is strongly influenced by cytogenetic abnormalities. Recent updates from the International Myeloma Working Group (IMWG), along with the European Hematology Association (EHA) and European Myeloma Network (EMN), have refined the definition of high-risk (HR) disease by integrating TP53 alterations, chromosome 1 abnormalities, and specific combinations of cytogenetic lesions. However, validation of these criteria in real-world patient populations remains limited. We conducted a retrospective, single-center study including 738 patients diagnosed with MM between 2017 and 2025, of whom 408 had available fluorescence in situ hybridization (FISH) data at diagnosis. Patients were reclassified according to the latest IMWG/IMS high-risk criteria proposed in international literature. Cytogenetic abnormalities, treatment patterns, and clinical outcomes, including overall survival (OS), progression-free survival (PFS), response rates, and relapse, were analyzed. Survival was estimated using the Kaplan–Meier method. A total of 103 patients (25%) were reclassified as high-risk according to IMWG/IMS high-risk criteria. Cytogenetic HR abnormalities were identified in 17.2% of cases, with del(17p) being the most frequent (14.7%). Median OS and PFS in HR patients were 52.4 months and 16 months, respectively, compared with 68.4 months and 28 months in standard-risk patients (log-rank test p values of 0.0197 and 0.0004, respectively). Although overall response rates were high (83% in HR vs. 91% in standard-risk), relapse remained frequent in HR patients. Outcomes varied significantly according to cytogenetic complexity. Isolated del(17p) was associated with improved survival compared with cases harboring additional abnormalities, while double-hit and triple-hit profiles demonstrated inferior outcomes. The presence of chromosome 1 abnormalities, particularly in combination with IGH translocations, further worsened prognosis. Among HR patients, 44% underwent autologous stem cell transplantation (ASCT), including 10 cases of TANDEM ASCT. No survival benefit was observed for TANDEM compared with single ASCT, with median OS of 52.9 vs. 78.3 months, respectively (log-rank test p values of 0.2516). Our real-world analysis supports the prognostic relevance of the updated IMS/IMWG high-risk criteria in MM. Cytogenetic complexity, rather than individual abnormalities alone, is a key determinant of outcome. Despite high response rates achieved with modern therapies, survival remains inferior in HR patients. TANDEM ASCT did not confer additional benefit in this cohort, supporting a more individualized approach to treatment intensification. Full article

This study aimed to develop an anther culture protocol for triticale (×Triticosecale Wittmack) and generate a doubled haploid (DH) population combining awnlessness and stripe rust resistance. A wheat anther culture protocol was evaluated on nine triticale varieties, with and without Trichostatin A (TSA), and tested on parental genotypes—an awned, stripe rust–resistant breeding line (AT-45) and an awnless variety (‘1143’)—as well as on ten F₁ plants derived from crosses between AT-45 and ‘1143’. Plant regeneration varied widely among varieties, ranging from 0.8 to 39.7 green plants per 30 anthers (1.6–80 per spike), with an overall mean of 9.9 (20 per spike). TSA did not significantly improve green plant production in this study, though further optimisation of the application method may be warranted. An average of 17.4 green plants per spike was obtained from the F₁ plants, and 1130 regenerant plants were grown to maturity, with a mean spontaneous chromosome doubling rate of 42.5%. A total of 480 DH lines were harvested, comprising 250 awned, 60 reduced awn, and 170 awnless lines. Awned and reduced awn lines were discarded, and 114 awnless lines were advanced for field evaluation of stripe rust resistance and agronomic traits. These results establish an effective anther culture system for DH production in triticale and demonstrate the potential of DH technology to accelerate the development of resilient, high-performing varieties. Full article

Cotton, as a globally significant economic crop, is intricately regulated in its growth and development by the key genes for SA (Salicylic acid) biosynthesis. In the present study, a systematic analysis of genes related to SA biosynthesis was conducted across four cotton species, leading to the identification of 70 genes. Specifically, the tetraploid species Gossypium hirsutum and G. barbadense were found to harbor 22 and 23 genes, respectively, representing a substantial expansion compared to the 12 and 13 genes identified in the diploid progenitors G. arboreum and G. raimondii. Comprehensive characterization of chromosomal localization, phylogeny, domain architecture, and promoter cis-elements revealed a uniform distribution of key genes involved in SA biosynthesis across A/D sub-genomes of tetraploids with extensive interspecific collinearity; whole-genome and segmental duplication act as the dominant drivers for the expansion of this gene family, while partial gene loss following polyploidization results in non-doubled gene copy numbers in tetraploids relative to diploids, which reflects the evolutionary selection for genomic dosage balance. The key genes for SA biosynthesis demonstrate a high degree of conservation in protein sequences, protein structures, and conserved motifs, which constitute the structural basis for the stable maintenance of their core functions in the SA biosynthesis pathway during plant evolution. This is closely related to their core function in the salicylic acid (SA) synthesis pathway and serves as the structural basis for the stable maintenance of gene functions during evolution. Analysis of cis-elements revealed that the expression of key genes involved in SA biosynthesis is governed by a complex interplay of phytohormones, stress signals, and transcription factors. Yeast one-hybrid (Y1H) assays confirmed the interaction between the GhPAL and GhICS gene and predicted candidate transcription factors, specifically the binding of GhWRKY21 to GhICS2-1 promoter and GhMYB12 to GhPAL1-2 promoter, thus elucidating their stage-specific regulatory mechanisms in cotton fiber development and reflecting their evolution. This study provides a fundamental basis for investigating the role of the SA signaling pathway in cotton development and offers support for cotton molecular breeding. Full article

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease threatening global wheat production. Thinopyrum intermedium, a wild relative of wheat, harbors valuable resistance genes for wheat improvement. In this study, we characterized a wheat-Th. intermedium double disomic substitution line X482 (4St-J^S (4D) + 6St (6D)) and wheat-Th. intermedium partial amphiploid TA8034 using ND-FISH and Oligo-FISH painting. Importantly, a unique type of translocation between chromosomes 1B and 6B was identified in line X482. Subsequent immunostaining assays revealed the distinct DNA methylation maintained on Thinopyrum and wheat B-genome chromosomes in X482 and TA8034. Genetic analysis combined with ND-FISH of line X482-derived populations demonstrated that chromosome 6St confers adult-stage powdery mildew resistance. Through ⁶⁰Co-γ irradiation of a 6St monosomic addition line, we developed seven homozygous 6St translocation lines with various breakpoints. Physical mapping using 56 6St-specific molecular markers delineated the resistance locus to bin 6StL-3, corresponding to 358.03–398.21 Mb in the Th. intermedium reference genome v2.1. Notably, the wheat-6St translocation line exhibited powdery mildew resistance without significant negative effects on agronomic traits. These results indicated that the distal region of homologous group 6 chromosomes harbors novel powdery mildew resistance genes, and the developed translocation lines provide valuable germplasm for wheat breeding programs. Full article

Gene amplification resulting from double strand breaks (DSBs) is a typical genetic alteration in tumorigenesis and drug-resistant progression. Amplified oncogenes and drug-resistant genes are present on extrachromosomal DNAs (ecDNAs), or chromosomal homogeneously staining regions (HSRs). Considering the role of mismatch repair (MMR) as a sensor of DSBs, we hypothesized that MMR may be involved in gene amplification. We used two MTX-resistant HT-29 colorectal cancer cell lines, which served as models with amplified genes mainly in HSRs or ecDNAs. Expression of MSH2, a key protein in MMR, was increased following the acquisition of MTX-resistant. MMR inhibition was achieved by depleting MSH2. Suppression of MMR led to decreased copy numbers of amplified genes as well as the quantity of ecDNAs and HSR. This was caused by the decreased efficiency of DSBs repair, which resulted from the reduced ability of MMR to recruit DSBs repair proteins. Additionally, it accelerated the formation of micronuclei (MN)/nuclear buds (NBUDs), which functioned to eliminate the amplified genes. Furthermore, the suppression of MMR was capable of inhibiting cell proliferation and enhancing MTX-sensitivity in ecDNA-containing cells. Conversely, suppression of MMR had no effect on gene amplification in HSR-containing cells. Our findings demonstrate that MMR plays a pivotal role in gene amplification through mediating DSBs repair pathways and facilitating the formation of MN/NBUDs in ecDNA-containing cells. MMR is likely to emerge as a prime therapeutic target worthy of in-depth exploration in future clinical investigations. Full article

Grain quality in bread wheat is a complex trait determined by multiple genetic factors and their interaction with environmental conditions. This study investigated the genetic architecture of key grain quality traits in the Avalon × Cadenza double haploid (DH) population under contrasting climatic conditions in Kazakhstan. A set of 101 spring-type DH lines was evaluated over three years in three major wheat-growing regions of Kazakhstan, representing northern, central, and southern environments. Grain yield and nine grain quality traits were assessed, including amylose content (Amc, %), test weight per liter (TWL, g/L), grain protein content (GPC, %), gliadin content (Gli, %), glutenin content (Glu, %), grain hardness (GH, %), grain vitreousness (GV, %), falling number (FN, s), and sedimentation value determined in a 2% acetic acid solution (SV, mL). The objectives were to characterize phenotypic variation, examine trait relationships, and identify major and environmentally stable quantitative trait loci (QTLs) controlling grain quality. QTL mapping identified 89 QTLs associated with the nine studied traits, including 82 major QTLs explaining more than 10% of phenotypic variation and 16 stable QTLs detected in two or more environments. The largest numbers of QTLs were found for GPC, SV, and TWL. Stable QTLs were distributed across all three wheat genomes, with important regions detected on chromosomes 1A, 1B, 2D, 4A, 4D, 5A, 6A, and 7D. Several stable QTLs co-localized with genomic regions previously associated with grain quality and developmental regulation, including loci near Wx-B1, Rht-D1, and Ppd-D1, suggesting biologically meaningful links among gluten composition, starch biosynthesis, plant development, and grain physical properties. These results improve understanding of the genetic control of wheat grain quality across diverse environments in Kazakhstan and provide promising targets for marker-assisted selection to combine improved end-use quality with wide environmental adaptation. Full article

Background: Information on the autopolyploid of Gossypium herbaceum remains limited until now. Previously, the autotetraploid of G. herbaceum was successfully generated via colchicine-induced chromosome doubling from the diploid cultivar ‘Hongxing’ in our lab. Methods: To investigate the drought stress response mechanism of this tetraploid, the autotetraploid S4 was used as the experimental material. The plants were subjected to drought stress during the flowering stage, followed by measurements of physiological and biochemical indicators and transcriptomic sequencing analysis. Results: Under drought stress, MDA content increased, and cell membranes sustained oxidative damage. Photosynthetic parameters, such as net photosynthetic rate (Pn), were significantly suppressed, while the activity of osmotic regulators and key antioxidant enzymes increased significantly. After rehydration, all of the above physiological indicators showed varying degrees of recovery. Transcriptome analysis revealed that, when comparing the treatment group with the control group, a total of 5530 differentially expressed genes (DEGs) were identified, with 2714 up-regulated and 2816 down-regulated. Furthermore, this study investigated the drought resistance mechanism involving the interaction between the MAPK signaling pathway and other metabolic pathways in the autotetraploid. Nine drought-resistant genes, including MAPK3, bHLH47, GaRbohD, RIBA1, PIP1-3, RCA1, RbohD, CYP707A and HSP70, were selected and analyzed using real-time quantitative PCR; the results were generally consistent with the transcriptomic data. Conclusions: These findings substantially enhance our understanding of the molecular mechanisms underlying drought responses in autotetraploids. This novel autotetraploid genotype expands the available cotton germplasm resources and is expected to hold significant value for research on polyploidy evolution. Full article

Haploid and doubled haploid (DH) technologies are important tools for accelerated wheat (Triticum aestivum L.) breeding, enabling the rapid production of fully homozygous lines and increasing the efficiency of genetic analysis of complex traits. This review presents a comprehensive analysis of the main approaches to producing haploid and DH wheat plants, with particular emphasis on androgenesis-mediated and chromosome elimination methods, including wheat-maize hybridisation. The biological basis of androgenesis is discussed in relation to stress-induced microspore reprogramming; however, the primary focus is on the methodological factors determining the efficiency of DH production, including the donor plant genotype, microspore development stage, pretreatment conditions, composition of the induction and regeneration media, and chromosome doubling. However, its widespread application remains limited by pronounced genotypic dependence, low responsiveness of many commercial varieties, albinism, and a lack of universally effective protocols. In contrast, distant hybridisation systems, particularly wheat-maize hybridisation, are generally characterised by greater reproducibility and less genotypic dependence, although they remain labour-intensive and require precise embryo rescue and chromosome doubling procedures. Overall, further progress in producing DH in wheat will be associated with the optimisation of protocols for difficult-to-respond genotypes and the integration of classical haploidisation systems with omics approaches, genomic selection, and genome-editing. Full article

Recombinogenic DNA damage can initiate chromosomal rearrangements that can alter gene expression or accelerate cancer progression in higher eukaryotes. Thus, there is a critical need to identify genes that suppress chromosomal rearrangements and environmental exposures that promote genetic instability. Cell cycle checkpoints modulate the cell cycle so that DNA repair occurs before the replication or segregation of damaged chromosomes. Saccharomyces cerevisiae (budding yeast) RAD9 was the first cell cycle checkpoint gene identified, which initiated intensive research studies into the mechanisms of checkpoint activation and the phenotypes of checkpoint mutants. The budding yeast Rad9 protein serves as both an adaptor and scaffold that facilitates downstream effector activation to orchestrate a DNA damage response at multiple stages of the cell cycle, which facilitates double-strand break (DSB) repair by sister chromatid recombination. However, the role of RAD9 in homologous recombination and in suppressing gross chromosomal rearrangements (GCRs) is not completely understood. In this review we discuss how RAD9 can promote genome instability resulting from aberrant DNA replication intermediates, while suppressing DSB-associated rearrangements. We also discuss possible mechanisms accounting for the synergistic increase in genomic instability in double mutants defective in both RAD9 and recombinational repair. We emphasize that while there is an overlap between checkpoint and recombinational repair pathways, RAD9 and checkpoint pathways can function independently to suppress chromosomal instability. These studies thus elucidate checkpoint mechanisms that control homologous recombination between repeated sequences. Full article

Perilla frutescens(L.) Britt. (P. frutescens) is an important medicinal and aromatic plant, whose leaf color and chemotype strongly influence its medicinal quality and economic value. All the previously discovered perillene (PL)-type P. frutescens are double-sided green, and whether the PL-type trait is tightly linked with the green-leaf trait in genetics remains to be clarified. This study aimed to address this question and attempt to create purple-leaf PL-type germplasm through perillaldehyde (PA) × PL hybridization. Three parallel experiments were conducted using purple-leaf PA-type P. frutescens as male parents and green-leaf PL-type P. frutescens as female parents. Chemotypes were identified by gas chromatography (GC). Association analyses between leaf color and chemotype were performed in segregating F₂ populations. Genes involved in leaf color formation and PL biosynthesis were mapped onto the published Hoko-3 reference genome to provide genomic evidence for the genetic relationship between the two traits. All F₁ individuals were uniformly PA-type. The three F₂ populations exhibited distinct leaf color–chemotype association patterns: Z01 (n = 118) showed a strong association (Fisher’s exact p = 9.13 × 10⁻¹⁰; φ = 0.564), Z02 (n = 117) showed no detectable association (p = 0.9; φ = 0.012), and Z03 (n = 88) showed a moderate association (p = 0.00669; φ = 0.289). Importantly, purple-leaf PL-type recombinants were obtained in F₂ populations and stably maintained through subsequent generations (F₃–F₅), demonstrating that the PL-type trait is not tightly linked with the green-leaf trait in P. frutescens. Genomic mapping genes related to leaf color and PL biosynthesis are distributed across multiple chromosomes and usually present as multiple loci, which is consistent with the pattern of incomplete linkage. The PL-type trait is recessive and not genetically tightly linked to the green-leaf traits in P. frutescens. The successful creation of a purple-leaf PL-type germplasm breaks the historical phenotypic constraint and provides a novel material for further dissection of the molecular mechanisms regulating secondary metabolism and organ coloration in P. frutescens. Full article

Goji, a plant unique to China, is recognized for its dual use as both a food and a medicine and is rich in various nutrients. However, long-term asexual propagation often leads to cultivar degeneration and viral accumulation, which severely impact its yield, quality, and disease resistance. Homozygous seeds can stably produce offspring with uniform traits. Haploid breeding technology, which involves doubling the chromosomes of haploid plants to obtain homozygous diploids, can significantly accelerate the breeding process. The DMP (Domain of Unknown Function 679 Membrane Protein) family is a plant-specific family of membrane proteins involved in various biological functions, including physiological processes, reproductive development, and senescence. Concurrently, loss-of-function of the DMP gene impedes the proper integration of the paternal genome following fertilization. Consequently, the embryo develops with exclusively maternal chromosomes, a mechanism that underlies the induction of haploids. In this study, we conducted a genome-wide identification of the DMP gene family in goji, analyzing the physicochemical properties, chromosomal locations, cis-acting elements, phylogenetic relationships, sequence characteristics, expression patterns, and subcellular localization of its members. The objective was to identify DMP genes capable of inducing haploid production in goji berry for future breeding applications. The results revealed a total of 11 DMP family members in the goji berry genome, distributed across seven chromosomes. The proteins encoded by these members contain 136 to 237 amino acids, with molecular weights ranging from 15,267.96 to 26,141.01 Da and isoelectric points (pI) ranging from 5.14 to 9.32. The LbDMPs were found to contain numerous cis-acting elements that play roles in plant responses to abiotic stresses and various phytohormones. Notably, LbDMP1 and LbDMP11, which contain the typical DUF679 domain, are predominantly expressed in pollen, suggesting their involvement in the reproductive process of goji berry. They were therefore identified as candidate genes for haploid induction. Subcellular localization analysis demonstrated that LbDMP1 is localized to the plasma membrane, while LbDMP11 is localized to membrane systems such as the endoplasmic reticulum. This research provides a fundamental basis for further exploration of the functional roles of the DMP gene family in goji berry and offers valuable genetic resources for haploid induction in its breeding programs. Full article