Search Results (114)

This study represents the first comprehensive characterization of fish biodiversity in the Sonche Canyon (northeastern Peru) using environmental DNA (eDNA), revealing a community of 19 taxa whose detection depended critically on a multi-marker approach. The analysis demonstrated that the use of both Metafish and Mifish-U PCR primer pairs generated a high complementarity index (0.53), allowing the capture of divergent biological signals; Metafish was essential for identifying characiformes; while Mifish-U revealed the presence of specific gymnotiformes and loricariids. The findings, supported by rarification curves confirming sampling saturation. Principal Component Analysis (PCA) revealed co-variation between the biodiversity “hotspot” in Tingorbamba and elevated nitrate concentration (6.1 mg/L), as well as the ubiquitous presence of the invasive species Oncorhynchus mykiss. In conclusion, the utility of the molecular markers and the observed co-variation between physicochemical variables and species distributions underscore the need for standardized protocols for the monitoring and conservation of fauna in the vulnerable lotic systems of the Peruvian Andes. Full article

Stilbocrea banihashemiana Bolboli, Tavakolian & Mostowf. is an emerging pathogen causing canker and dieback in a broad range of fruit and ornamental trees in Iran, and its distribution is expanding across the country. Extensive surveys conducted over five consecutive years (2019–2023) yielded 88 isolates of S. banihashemiana from multiple hosts, including different fig (Ficus caricae L.) cultivars, as well as loquat (Eryobotria japonica (Thunb.) Lindl.), pomegranate (Punica granatum L.), and walnut (Juglans regia L.) trees, across eight distinct regions of southern Iran. Species identification was performed morphologically and molecularly by employing the S. banihashemiana-specific primer pair TEF-Sb1 and TEF-Sb3. The genetic diversity of the S. banihashemiana population of isolates was assessed using eight inter-simple sequence repeats (ISSRs) markers. The UPGMA dendrogram demonstrated broad genetic variability among the isolates, with similarity coefficient values spanning from 0.46 to 1.00. This wide range indicates the presence of multiple divergent genotypes within the population, rather than a single dominant lineage. Principal coordinate analysis (PCoA) grouped the 88 isolates into three distinct genetic clusters that partially corresponded to geographic origin and host species. Pathogenicity assessment of 53 selected isolates from various hosts and geographic origins on detached fig shoots demonstrated highly significant variability in aggressiveness among isolates originating from different host species and geographically distinct regions. Multivariate analysis using principal component analysis (PCA) combined with heatmap-based clustering of the aggressiveness dataset clearly separated the isolates into four distinct groups, ranging from highly to less aggressive. A susceptibility assessment of 10 fig cultivars using the ex-type-isolate of S. banihashemiana revealed that the pathogen caused internal lesions and wood discoloration in all cultivars. Based on statistical analysis, the cultivars were classified into three groups: susceptible (cv. ‘Siah’), moderately susceptible (‘Brown Turkey’, ‘C8-M’, ‘C8-F’, ‘Dehdez’, ‘Gilasi’, ‘Payves’, ‘Shah-Anjeer’ and ‘Sabz’), and less susceptible (‘Matti’). High genetic variability, multiple-host association, and partial geographic structure indicate that in Fars Province S. banihashemiana’s population structure and epidemiology are complex, with high adaptive potential. This complexity may influence disease spread, management strategies, and long-term evolutionary trajectories. Full article

The Fusobacterium nucleatum complex, which comprises oral lineage 1 (L1) strains, is strongly associated with colorectal cancer (CRC). NusG (N-utilisation substance G) is a transcription elongation factor that is universally conserved. This study aimed to develop and validate a novel nusG-directed polymerase chain reaction (PCR) assay to specifically and sensitively detect ectopic Fusobacterium L1 colonisation in clinical CRC patient samples. Four L1-specific primer pairs targeting the nusG gene were designed using MEGA11 software (Molecular Evolutionary Genetics Analysis, version 11.0.13) and successfully employed in 40 stool samples from CRC patients and healthy controls (HC). Additionally, five species-specific primer pairs were designed for the L1 species F. animalis clades 1 and 2, F. nucleatum, F. polymorphum, and F. vincentii, and were successfully applied to stool and saliva samples. Their specificity was verified via Sanger sequencing. Two L1-specific primer pairs (NusG5-F/NusG6-R and NusG2a-F/NusG5-R) demonstrated robust performance in our cohort, showing statistical significance (padj < 0.05) and a large effect size (|r| ≥ 0.5) in the difference in Ct values and absolute cell counts between CRC patients and the HC group. These primer pairs also exhibited promising preliminary diagnostic potential, with respective area under the curve (AUC) values of 0.909 and 0.883. However, Fusobacterium L1 abundance in saliva samples did not differ significantly between groups, indicating that definitive conclusions cannot be drawn due to the limited power of the salivary sub-cohort. The data indicates that nusG-based PCR primers could be used as reliable, non-invasive biomarkers as a complementary tool for early CRC diagnostics. While potentially applicable in the context of other Fusobacterium-implicated diseases, further validation in larger, ethnically diverse cohorts remains essential. Full article

Reliable identification of seafood species is critical for fisheries management and product authentication, especially when morphological characteristics are lost during processing. In this study, a multiplex PCR system was developed to distinguish seven cuttlefish species (six Sepia spp. and Sepiella inermis) commercially distributed in the Korean seafood market. Species identity was first confirmed by amplifying a mitochondrial cytochrome c oxidase subunit I (COI) fragment (~658 bp) using universal primers (LCO1490/HCO2198), showing 99–100% sequence similarity to corresponding GenBank reference sequences. Analysis of genetic variation based on a 530 bp aligned region demonstrated complete interspecific differentiation without shared haplotypes among species. The number of haplotypes per species ranged from 5 to 21, with haplotype diversity values between 0.667 and 1.000. An extended COI fragment (~1200 bp) was further analyzed to identify diagnostic interspecific variation for marker development. Seven diagnostic single-nucleotide polymorphism (SNP) sites were identified and used to design species-specific forward primers with diagnostic nucleotides positioned at the 3′ termini. Distinct amplicons (220–1099 bp) were generated and clearly resolved by agarose gel electrophoresis. Because simultaneous amplification of all seven primer pairs reduced amplification efficiency, the assay was divided into two multiplex sets. Under optimized conditions (56 °C), each species produced a single expected band without cross-amplification. This multiplex PCR system provides a rapid and sequencing-free approach for reliable species discrimination and can be effectively applied to fisheries monitoring and seafood authentication in commercial supply chains. Full article

Abies georgei var. smithii (Viguie & Gaussen) is a dominant conifer along the southeastern margin of the Qinghai–Tibet Plateau, where heart rot often develops covertly, complicating forest health monitoring and disease management. Fomitopsis subpinicola B.K. Cui, M.L. Han & Shun Liu is an important causal agent of heart rot affecting A. georgei var. smithii in this region, yet rapid, field-deployable molecular diagnostics of this pathogen remain limited. Here, we developed and evaluated two TEF1α-based isothermal platforms for specific detection of F. subpinicola: RAA and LAMP. To reduce potential cross-reactivity, TEF1α sequences from representative taxa within the F. pinicola species complex and closely related non-complex species were aligned for primer/probe design. Candidate RAA primers were screened by gel electrophoresis to select an optimal pair, and two LAMP primer sets were compared by specificity testing to identify the best-performing set. Both assays specifically detected F. subpinicola with no cross-amplification in the tested non-target fungi. Limits of detection were 9.97 copies/μL for fluorescent RAA (25 min), 9.97 × 10² copies/μL for RAA-LFD (15 min), and 9.97 × 10³ copies/μL for LAMP (35 min). In 30 increment core samples from A. georgei var. smithii, all methods consistently detected samples with obvious decay, while fluorescent RAA additionally yielded positives in some apparently asymptomatic samples, indicating promise for early or low-abundance screening. Together, these assays constitute a tiered and application-oriented detection system, enabling flexible selection of diagnostic approaches according to sensitivity requirements, operational conditions, and field surveillance needs for heart rot of A. georgei var. smithii. Full article

Background: Odontogenic sinusitis (ODS) represents a distinct form of maxillary sinus inflammation arising from dental pathology and is most commonly unilateral. Despite its polymicrobial nature and predominance of anaerobic organisms, molecular characterization of the bacterial profile and its relationship to clinical severity remains limited. This study aimed to evaluate associations between targeted quantitative PCR (qPCR) findings from paired maxillary sinus and periapical lesion samples and clinical, endoscopic, and radiological features of disease. Additionally, the influence of oroantral communication on microbial concordance between odontogenic and sinus sites was examined. Methods: Twenty-eight patients with confirmed ODS were included for analytical cross-sectional study and underwent combined otolaryngological and dental assessment. During endoscopic sinus surgery with extraction of the causative tooth, paired specimens were collected from sinus mucosa and periapical lesions under sterile conditions and preserved for molecular analysis. Targeted qPCR assays using 16S rRNA–based primers were performed to detect predefined odontogenic pathogens. Associations between bacterial detection patterns and clinical, endoscopic, and imaging variables were analyzed. Results: Detection of Streptococcus anginosus group organisms was significantly associated with complete maxillary sinus opacification. Fusobacterium nucleatum and Porphyromonas endodontalis demonstrated higher detection rates in patients with more advanced radiological disease, although statistical significance was not reached. Purulent nasal discharge correlated with detection of Fusobacterium nucleatum, Porphyromonas endodontalis, and streptococcal species. Cases with intraoperative oroantral communication exhibited greater concordance between sinus and dental microbial profiles. Conclusions: ODS is characterized by a polymicrobial environment dominated by anaerobic bacteria, with specific organisms associated with markers of disease severity such as purulent secretion and extensive sinus opacification. Targeted molecular profiling may improve recognition of odontogenic origin and support individualized therapeutic strategies, although larger studies integrating clinical outcomes are required to clarify prognostic implications. Full article

The flowers of Panax ginseng C. A. Mey. (PG) and Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow (PN) are morphologically indistinguishable after drying, leading to prevalent adulteration that compromises product quality and consumer safety. To address this issue, we developed a rapid, closed-tube molecular authentication method based on high-resolution melting (HRM) analysis. Species-specific primer pairs were designed to target the conserved ITS and rbcL-accD regions, with PNG-2 selected as the optimal candidate owing to its stable genotyping performance and moderate GC content. Our results established GC content, rather than amplicon length, as the primary determinant of the melting temperature (Tm). Notably, the experimentally measured Tm values were consistently 0.7–1.5 °C higher than theoretical predictions, a discrepancy attributable to the stabilizing effect of the saturated fluorescent dye. To ensure maximum diagnostic reliability, the HRM results were cross-validated through a three-tier system comprising ITS2 phylogenetic analysis, agarose gel electrophoresis, and Sanger sequencing. The practical utility and matrix robustness of the assay were further verified using a diversified validation cohort of 30 commercial samples, including 24 floral batches and 6 root-derived products (root slices and ultramicro powders). The HRM profiles demonstrated 100% concordance with DNA barcoding results, effectively identifying mislabeled products across different botanical matrices and processing forms. This methodology, which can be completed within 3 h, provides a significantly more cost-effective and rapid alternative to traditional sequencing-based methods for large-scale market surveillance and industrial quality control. Full article

Thrips are cosmopolitan agricultural pests and important vectors of plant viruses, and the increasing coexistence of multiple morphologically similar species has intensified the demand for species-specific molecular identification. However, traditional morphological identification and PCR assays using universal primers are often inadequate for mixed-species samples and field-adaptable application. In this study, we developed a species-specific molecular identification framework targeting a polymorphism-rich region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which is more time-efficient than sequencing-based COI DNA barcoding, for four economically important thrips species in southern China, including the globally invasive Frankliniella occidentalis. By aligning COI sequences, polymorphism-rich regions were identified and used to design four species-specific primer pairs, each containing a diagnostic 3′-terminal nucleotide. These primers were combined with a PBS-based DNA extraction workflow optimized for single-insect samples that minimizes dependence on column-based purification. The assay achieved a practical detection limit of 1 ng per reaction, demonstrated species-specific amplification, and maintained reproducible amplification at DNA inputs of ≥1 ng per reaction. Notably, PCR inhibition caused by crude extracts was effectively alleviated by fivefold dilution. Although the chemical identities of the inhibitors remain unknown, interspecific variation in inhibition strength was observed, with T. hawaiiensis exhibiting the strongest suppression, possibly due to differences in lysate composition. This integrated framework balances target specificity, operational simplicity, and dilution-mitigated inhibition, providing a field-adaptable tool for thrips species identification and invasive species monitoring. Moreover, it provides a species-specific molecular foundation for downstream integration with visual nucleic acid detection platforms, such as the CRISPR/Cas12a system, thereby facilitating the future development of portable molecular identification workflows for small agricultural pests. Full article

Quantitative real-time PCR (qPCR) remains a cornerstone method for analyzing gene expression due to its high sensitivity, specificity, and reproducibility. However, for reliable results in relative quantification studies, the choice of an appropriate reference gene is critical to ensure accurate normalization. The expression of commonly used reference genes can vary depending on developmental stage and experimental conditions, making their validation essential. To date, no validated reference genes have been reported for Agrostemma githago L. (corn cockle, Caryophyllaceae). To facilitate research on genes involved in natural product biosynthesis and specialized metabolism regulation, we aimed to identify the most stable reference genes across various plant organs and cultivation conditions of this species. Drawing on previous literature, we have selected seven housekeeping genes widely used for evaluation: actin, β-tubulin, elongation factor 1α, glyceraldehyde-3-phosphate dehydrogenase, histone H3, translation elongation factor 1, and eukaryotic translation initiation factor 5A1 (for which two primer sets were tested). The nucleotide sequences of these potential reference genes were identified from the A. githago transcriptome. Using qRT-PCR, transcript levels of seven potential reference genes were estimated in 40 different A. githago samples, including 25 in vitro samples under various treatment conditions and 15 soil-grown samples representing A. githago organs in different developmental stages. Expression stability of candidate reference genes was assessed using the RefFinder platform, which combines four commonly applied statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ-Ct method. The results revealed that the selection of optimal reference genes varied based on the particular organ, developmental stage and condition being examined. TIF5A1-2 (one of the two primer pairs tested) and GAPHD consistently exhibited the most stable expression under various conditions in vitro. EF1α and H3 exhibited superior performance across different organs of soil-grown plants. Moreover, our integrated analysis enabled the identification of the two most stable, universal reference genes suitable for normalization in A. githago under all tested conditions—H3 and TIF5A1-2. Our work provides a robust foundation for future transcriptomic and functional studies of the specialized metabolism of A. githago and other related species. Full article

Modern sequencing technologies have transformed the identification of medicinal plant species and varieties, overcoming the limitations of traditional approaches. To address the challenge of discriminating Toona sinensis varieties, we sequenced and compared 15 complete chloroplast genomes from five varieties in northern China. Although these genomes exhibited a highly conserved structure, we identified eight variety-specific simple sequence repeats (SSRs), two unique tandem repeats, and several hypervariable regions with elevated nucleotide diversity. Phylogenetic analysis demonstrated that whole chloroplast genomes provided the highest resolution for variety identification, outperforming conventional barcodes. Furthermore, we developed 13 specific primer pairs targeting variable regions, and PCR validation confirmed their reliable amplification across varieties. In addition, sequence-level validation by Sanger sequencing of representative SSR and tandem repeat markers revealed stable, variety-specific repeat copy number differences. These results demonstrate that the identified chloroplast markers can effectively discriminate closely related T. sinensis varieties. This study confirms that despite overall conservation, the T. sinensis plastome contains sufficient variation for reliable identification, providing a robust framework for future germplasm conservation and molecular breeding. Full article

Rivomarginella morrisoni (Gastropoda: Marginellidae) is a narrowly distributed freshwater snail inhabiting drainage basins of central and southern Thailand. To clarify patterns of genetic differentiation across its range, 45 individuals from 11 sites across eight river basins were analyzed using two dominant molecular markers: sequence-related amplified polymorphism (SRAP) and inter-simple sequence repeats (ISSR). SRAP primers produced higher polymorphic information content (PIC) values than ISSR primers (0.35 vs. 0.27). Analysis of molecular variance (AMOVA) revealed strong population structure, with 80.29% of the genetic variation occurring among populations and 19.71% within populations Population differentiation statistic (PhiPT) = 0.803, p < 0.001). Unweighted Pair Group Method with Arithmetic mean (UPGMA) and principal coordinate analysis (PCoA) consistently separated central and southern populations, and STRUCTURE supported K = 2 as the most likely number of clusters. Similarly, principal component analysis (PCA) of morphological traits also distinguished specimens into two groups corresponding to these geographic regions, confirming region-specific divergence. Overall, the genetic and morphological patterns indicate restricted gene flow among basins and a clear separation between central and southern lineages of R. morrisoni. This study provides the first molecular evidence of population structure in this species and offers important baseline information for future taxonomic, ecological, and conservation research on freshwater marginellid snails. Full article

Heterocapsa bohaiensis is an emerging harmful dinoflagellate increasingly reported from coastal regions of the Pacific. However, an available molecular assay offering rapid and sensitive detection is still lacking. This study developed a SYBR Green real-time quantitative PCR (qPCR) assay for the identification and quantification of H. bohaiensis. Species-specific primers (F: 5′-CCATCGAACCAGAACTCCGT-3′; R: 5′-AGTGTAGTGCACCGCATGTC-3′) were designed and the assay was optimized and evaluated using laboratory cultures for specificity, sensitivity, and quantitative performance. Primer screening and melt-curve analysis confirmed that the selected primer pair produced a single, specific amplification peak for H. bohaiensis, with no cross-reactivity observed in non-target species (Chlorella pyrenoidosa, Phaeocystis globosa, Skeletonema costatum, Alexandrium tamarense) or mixed algal communities. The standard curve displayed strong linearity (R² = 0.9868) and a high amplification efficiency (102.5%). The limit of detection (LOD) was approximately 2–3 cells per reaction, as determined from 24 replicates of 5-cell equivalents and verified at ~2.7-cell equivalents. This sensitivity was comparable to or exceeded that reported for assays targeting other HABs forming dinoflagellates. Quantitative results derived from the qPCR assay closely matched microscopic cell counts, with a relative error of 10.79%, falling within the acceptable threshold for phytoplankton surveys. In summary, this study established and validates a species-specific qPCR assay for H. bohaiensis under controlled laboratory conditions. The method shows strong potential for incorporation into HAB monitoring programs, early-warning systems, and future ecological investigations of this emerging species. Full article

Background: Kenaf (Hibiscus cannabinus L.) is an important fiber crop belonging to the genus Hibiscus in the Malvaceae family. Research on its chloroplast genome holds significant importance for deciphering the evolutionary relationships of the Hibiscus species, developing genetic markers, and promoting kenaf (H. cannabinus) genetic breeding. Methods: Based on high-throughput sequencing technology, this study completed the sequencing and assembly of the kenaf (H. cannabinus) chloroplast genome. Results: (1) The kenaf (H. cannabinus) chloroplast genome exhibits a typical circular quadripartite structure with a total length of 163,019 bp, including a large single-copy region (LSC) of 90,467 bp, a small single-copy region (SSC) of 19,486 bp, and a pair of inverted repeat regions (IRa/IRb) of 26,533 bp each. The total GC content is 36.62%, among which, the IR region has the highest GC content (42.61%) and the SSC region the lowest (30.87%). (2) A total of 131 genes were annotated, including 85 mRNAs, 37 tRNAs, 8 rRNAs, and 1 pseudogene. Their functions cover photosynthesis (e.g., pet and atp family genes), self-replication (e.g., rpl, rps, and rpo family genes), and genes with unknown functions (e.g., ycf1 and ycf2). A codon usage bias analysis revealed that the relative synonymous codon usage (RSCU) value of the stop codon UAA is the highest (1.6329), and codons ending with A/U are preferentially used (e.g., GCU for alanine with RSCU = 1.778). (3) A repeat sequence analysis identified various interspersed repeat sequences (predominantly 30~31 bp in length, with a relatively high proportion in the 30~40 bp range, including forward and palindromic types) and simple sequence repeats (cpSSRs). Among them, single-base repeat SSRs account for the highest proportion (e.g., (A)8 and (T)9), and specific SSR primers were designed. (4) A comparative evolutionary analysis indicated that the Ka/Ks ratios (nonsynonymous substitution rate/synonymous substitution rate) of core chloroplast genes (e.g., rps2 and rpoC2) in kenaf (H. cannabinus) are all less than 1 (0.145~0.415), suggesting that they are under purifying selection. The collinearity similarity of chloroplast genomes between kenaf (H. cannabinus) and its closely related species reaches over 99.97%, and the IR region boundaries are relatively conserved. The phylogenetic tree shows that kenaf (H. cannabinus) clusters with closely related Hibiscus species with a 100% bootstrap value, indicating a close genetic relationship. Conclusions: This study provides basic data for the functional analysis of the kenaf (H. cannabinus) chloroplast genome, the phylogeny of Hibiscus, and the utilization of genetic resources. Full article

Helichrysum italicum (Roth) G. Don (Asteraceae) is a valuable medicinal and aromatic plant native to a variety of habitats across the Mediterranean region. However, genetic studies of this morphologically diverse species have been limited by the scarcity of species-specific DNA markers. To address this limitation, we generated the first de novo transcriptome assembly comprising 24,806 transcripts from young shoots containing leaves and flowers, developed EST-SSR markers, and evaluated their utility in population genetic analysis. Seventy-eight primer pairs were designed, of which 23 showed successful amplification, polymorphism, and transferability to Helichrysum litoreum Guss. and Helichrysum arenarium (L.) Moench. A subset of 12 EST-SSRs was used to genotype 270 individuals from 12 natural populations of H. italicum in Corsica (France), along with one outgroup population from Croatia. The polymorphic information content ranged from 0.250 to 0.796, and Shannon’s information index ranged from 0.588 to 1.843, indicating the markers’ suitability for population genetic studies. Analysis of molecular variance revealed that 15% of the total genetic variation was attributable to differences among populations. Discriminant analysis of principal components and Bayesian clustering in STRUCTURE identified distinct population clusters corresponding to geographic locations. Notably, the southernmost coastal populations were clearly differentiated from the others. Full article

Environmental DNA (eDNA) has become a promising tool for phytoplankton surveys. However, the accuracy of eDNA-based detection is related to primer selection across diverse environments, and optimal primer pairs selection on phytoplankton community in human impacted ecosystems is still lacking. The aim of this study is to evaluate how primer selection shapes phytoplankton community profiles by eDNA biomonitoring diverse anthropogenically disturbed aquatic systems (rivers, reservoirs, and seas). Four primer pairs targeting the 18S rRNA (V9-1 and V9-2), chloroplast rbcL, and ITS regions, were explored and our results revealed that primer choice critically governed the accuracy of phytoplankton profiling. Significant variations in annotated phytoplankton eDNA sequences in different groups of primer pairs were observed, where the primers 18SV9-1 and rbcL demonstrated superior specificity, amplifying >90% of phytoplankton OTUs. 18S-targeted primers detected the highest species richness, while the ITS primer showed the lowest. Alpha diversity was highest and most consistent for 18S primers. Beta diversity ordination (nMDS/Bray–Curtis) further highlighted primer-dependent community structuring in which 18S primers effectively clustered reservoir and marine samples separately, whereas primer rbcL discriminated habitat-specific signatures across three ecosystems. The primer ITS failed to distinguish among different habitats. Overall, our data demonstrated the critical role of primer optimization in eDNA-based phytoplankton studies, and could provide methodological guidelines for the design of effective monitoring protocols in rapidly urbanizing aquatic systems. Full article