DNA Markers Research Articles

Human diphyllobothriasis in Argentina: assessing the epidemiological significance from historical records and reports of new cases.

Between 1898 and 1940, eight human cases of diphyllobothriasis were reported in Argentina, always in recently arrived European immigrants. In 1982, the first autochthonous case was detected, and since then, 33 other autochthonous cases have been reported, totaling 42 cases of human diphyllobothriasis in Argentina before the present study. Our aim is to update the information on diphyllobothriasis in Argentina by identifying specimens from new cases using morphometrical and/or molecular methods. We also aim to assess the epidemiological relevance of this food-borne disease in the country. Anamnestic data were obtained from patients or professionals, along with 26 worms identified using morphometrical (21 samples) and molecular techniques (5 samples). All the patients acquired the infection by consuming freshwater salmonids caught in Andean lakes in Northern Patagonia. Morphometrics and DNA markers of worms were compatible with Dibothriocephalus latus. In total, 68 human cases have been detected in Argentina, 60 of which were autochthonous. The human population living North-western Patagonia, whose lakes are inhabited by salmonids, is increasing. Similarly, the number of other definitive hosts for Dibothriocephalus dendriticus (gulls) and for D. latus (dogs) is also increasing. In addition, salmonid fishing and the habit of consuming home-prepared raw fish dishes are becoming widespread. Therefore, it is to be expected that diphyllobothriasis in Argentina will increase further.

Rice breeding for higher crop productivity (a review)

The main global trends in the breeding of rice cultivars and hybrids are higher plant productivity, adaptability to cultivation conditions, quality of products, and resistance to environmental stressors, including diseases.Efforts of leading breeding centers in the Philippines and China to further increase the rice yield are associated with a change in the plant morphotype: shorter plants with erectoid leaves and large panicles with a high number of grains.The International Rice Research Institute (IRRI) has developed rice cultivars that under favorable conditions in the tropical zone produce a grain yield of up to 10–11 t/ha, with Kecon = 0.5. A heterotic hybrid of rice was obtained in China with a yield of 13.9 t/ha; it has a new plant morphotype (New Plant Type) and a growing season of 163 days. The hybrid is classified as “super rice”.Modern breeding programs are aimed at producing cultivars of “Green Super Rice” using pedigree methods and backcrossing for introgression of target genes through DNA markers, QTL mapping, pyramiding, and recurrent selection.Russian breeders have traditionally produced rice cultivars adapted to local conditions, with a growing season of no more than 125 days. In the 1980s, a program was launched to develop a high-yielding rice cultivar with erectoid leaves for condensed planting. As a result of multiple stepwise hybridization and targeted selection, new source material with target traits of “super rice” was released. Cv. ‘Polyus-5’ with a new plant morphotype was obtained on its basis. This cultivar was included in the State Register and from 2023 approved for commercial production. During the field trials, ‘Polyus-5’ yielded a maximum of 1380 g/m2 over 120 days of the growing season, under the conditions of condensed sowing and increased mineral nutrition.

Circulating miRNA and circulating tumor DNA application as liquid biopsy markers in gastric cancer.

Liquid biopsy has been investigated as a novel method to overcome the numerous challenges in gastric cancer (GC) management. This non-invasive, feasible, and easy-to-repeat method has been shown to be cost-effective and capable of increasing diagnostic sensitivity and prognostic assessment. Additionally, it is potentially accurate to aid decision-making and personalized treatment planning. MicroRNA (miRNA) and circulating tumor DNA (ctDNA) markers can enhance GC management in various aspects, including diagnosis (mainly earlier diagnosis and the ability to perform population-based screening), prognosis (more precise stratification of prognosis), and treatment (including more accurate prediction of treatment response and earlier detection of resistance to the treatment). Concerning the treatment-related application, miRNAs' mimics and antagonists (by using two main strategies of restoring tumor suppressor miRNAs and inhibiting oncogene miRNAs) have been shown to be effective therapeutic agents. However, these need to be further validated in clinical trials. Furthermore, novel delivery systems, such as lipid-based vectors, polymeric-based vectors, and exosome-based delivery, have been developed to enhance the performance of these agents. Moreover, this paper explores the current detection and measuring methods for these markers. These approaches are categorized into direct methods (e.g., Chem-NAT, HTG EdgeSeq, and Multiplex Circulating Fireplex) and indirect methods (e.g., Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), qPCR, microarray, and NGS) for miRNA detection. For ctDNA measurement, main core technologies like NGS, digital PCR, real-time PCR, and mass spectrometry are suggested.

Characterization of genetic diversity in Curcuma longa L. cultivars using Start Codon Targeted (SCoT) markers

Curcuma longa L. (Family- Zingiberaceae), commonly called turmeric, is one of the most important commercial spices. Being a center of turmeric's diversity, India boasts of more than fifty cultivars with variable agronomic characters and curcumin content. However, the quest for calibrating their genetic variability remains debatable as popular molecular marker-based studies have yielded inconsistent results. A promising PCR-based approach, known as the Start Codon Targeted (SCoT)-PCR system has been established to be more efficient than random DNA marker systems because this technique can generate more information on genetic polymorphism patterns. Hence, the present study proposes to use Start Codon Targeted (SCoT) markers to assess inter-cultivar genetic diversity of turmeric. We report an orthogonal L16 (45) design-based approach to investigate the influence of five crucial factors (dNTPs, Taq DNA polymerase, primer, template DNA and annealing temperature of primer) on the efficiency of the polymerase chain reaction (PCR) for development of an optimized Start Codon Targeted (SCoT) amplification protocol using twenty-seven different cultivars of C. longa. The optimized PCR protocol yields best results with a 20 uL reaction mixture, containing 10×PCR Buffer (with 15 mM MgCl2), 2.5 mM dNTPs, 10 mM SCoT primer, Taq polymerase (2 U), template DNA (50 ng), which was verified by using twenty-five SCoT primers. This standardized rapid protocol of SCoT-PCR reported here was employed to examine genetic diversity among twenty-seven Indian cultivars of turmeric, revealing high range of polymorphism ranging from 81.25 % to 100 %. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data segregated the twenty-seven cultivars into two main clusters. The first cluster comprised of nine cultivars (36 % of the cultivars) which was further divided into three sub-clusters and the second one consisted of eighteen cultivars (64 %) which further divided into four sub-clusters. From the results of this study, it can be inferred that the employment of SCoT markers can be an effective tool for analysing genetic variability, phylogenetic relationships and develop a rich genetic resource management system for C. longa cultivars.

Unveiling genotypic diversity of Theileria orientalis in lethal outbreaks among bovines in Karnataka, India.

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts.

Comparative chloroplast genomes of Argentina species: genome evolution and phylogenomic implications

The genus Argentina Hill belongs to the tribe Potentilleae Sweet and contains approximately 75 species predominantly distributed in the Sino-Himalayan region and the Malesian archipelago. So far we have less knowledge on the phylogenetic relationships within Argentina owing to limited sampling of Argentina taxa or gene fragments in previous studies. Moreover, to date there is no phylogenetic study on Argentina from the perspective of comparative chloroplast (cp) genomics. Here we performed comparative genomic analyses on the cp genomes of 39 accessions representing 18 taxa of Argentina. The Argentina cp genomes presented the typical quadripartite structure, with the sizes ranging from 155 096 bp to 157 166 bp. The 39 Argentina cp genomes contained a set of 112 unique genes, comprising four ribosomal RNA (rRNA) genes, 30 transfer RNA (tRNA) genes, as well as 78 protein-coding genes (PCGs). The cp genome organization, gene content and order in Argentina were highly conserved, but some visible divergences were present in IR/SC boundary regions. Ten regions (trnH-GUG-psbA, trnG-GCC-trnfM-CAU, trnD-GUC-trnY-GUA, rpl32-trnL-UAG, atpH-atpI, rps16-trnQ-UUG, trnS-GCU-trnG-UCC, ndhF-rpl32, trnR-UCU-atpA, and accD-psaI) were identified as excellent candidate DNA markers for future studies on species identification, population genetics and phylogeny of Argentina. Our results indicated that Argentina is monophyletic. In the current sampling, the A. smithiana - A. anserina clade was sister to the remainder of Argentina. Our results corroborated the previous taxonomic treatments to transfer A. phanerophlebia and A. micropetala from the genus Sibbaldia L. to Argentina. Our results showed close relationships among A. stenophylla, A. microphylla, A. taliensis, and A. tatsienluensis, congruent with previous studies based on the morphology of these species. Twenty-six genes (rps3, rps15, rps16, rps19, rpl16, rpl20, rpl22, rpoA, rpoB, rpoC1, rpoC2, atpA, atpF, psbB, psbF, ndhA, ndhB, ndhC, ndhD, ndhF, rbcL, accD, ccsA, matK, ycf1, ycf2) were with sites under positive selection, and adaptive evolution of these genes might have played crucial roles in Argentina species adaptation to the harsh mountain environment. This study will facilitate future work on taxonomy, phylogenetics, and adaptive evolution of Argentina.

Optimizing Bangkaew dog breed identification using DNA technology.

The Bangkaew dog is an indigenous dog breed in the Phitsanulok province of Thailand. This breed is recognized by the Fédération Cynologique Internationale (FCI), a global canine organization. The unique traits of the Bangkaew breed lead to purebred selection for breeding, while only their traits and pedigree from parental history are recorded. Determination of the risk of inbreeding depression and the origin of unknown DNA profiles is essential due to the challenges in predicting puppy characteristics, which are crucial for breed management and conservation. This study aimed to emphasize that current allelic frequency data for the Bangkaew dog breed must be considered for precise individual identification. Approximately 82 Bangkaew dogs from various Thai localities were studied using 15 microsatellite markers for genotypic monitoring and individual identification. Maternal genetic inheritance was assessed via mtDNA D-loop analysis. The results revealed high genetic diversity in the Bangkaew breed, indicating low potential for inbreeding. We also found that using a 15 loci microsatellite panel was effective for the identification of Bangkaew dogs. The optimized 10 loci microsatellite genotyping panel developed in this study presents improved identification testing efficiency, promoting both time- and cost-effectiveness. Analysis of microsatellite DNA markers in Bangkaew dogs using an optimized panel of 10 loci selected from 15 loci effectively facilitated individual identification. This approach not only enhances time and cost efficiency, but also provides accurate allelic frequency estimates, which are crucial for the realistic evaluation of DNA evidence.

Utilizing novel Escherichia coli-specific conserved signature proteins for enhanced monitoring of recreational water quality.

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developedquantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.

Unveiling Genetic Variation in Garlic Genotypes in Response to Rust Disease Using RAPD Markers

Garlic (&lt;em&gt;Allium sativum&lt;/em&gt;), cultivated worldwide for its medicinal and nutritional value, faces challenges due to diseases caused by various pathogens. In this study, eleven garlic genotypes from Iran and one from China were selected and sown under natural infection rendered by the rust fungus (&lt;em&gt;Puccinia alli&lt;/em&gt;) over two consecutive years. Subsequently, disease distribution and severity, percentage of infection and susceptibility of different garlic genotypes to rust disease were investigated. The results showed that genotype Solan was the most susceptible, with disease severity of 30.81%. In comparison, genotypes Aliabad and Bahar were resistant against rust disease with the lowest infection percentages of 13% and 16.37%, respectively. Further, genetic diversity was assessed using random-amplified polymorphic DNA (RAPD) markers. Out of 10 primers used, 95 scorable bands were generated, of which 66 (69.48%) were found to be polymorphic. A dendrogram was constructed based on RAPD polymorphism using the UPGMA method, and the genotypes were separated into six distinct clusters based on Jaccard's coefficient of similarity. Additionally, it was observed that there is no genetic differentiation among the genotypes based on their geographical origin. This study highlights the significant diversity in resistance and susceptibility among garlic genotypes, which can be harnessed in plant breeding programs.

Multiple sources of evidence unravel a complex taxonomic history: the new genus Leonoria of the Spermacoce clade (Spermacoceae-Rubiaceae)

For a long time, the taxonomic status of Denscantia, a genus endemic to the Atlantic Forest along the Brazilian coast, has been a topic of discussion. Phylogenetic analyses using two nuclear and four chloroplast DNA markers from 82 accessions representing 19 genera of the Spermacoce clade (tribe Spermacoceae, Rubiaceae) confirm that the Brazilian genus Denscantia is biphyletic. Together with the recovered phylogenetic relationships, the analyses of reproductive morphological characters, leaf morphology and anatomy, histochemical, geographic distribution ranges, and ecological niche derived from climatic space further demonstrate that Denscantia calcicola is a distinct lineage from the other Denscantia species. Therefore, the above-mentioned species should be taxonomically designated as a new monospecific genus, Leonoria. Significant morphological differences between Leonoria and Denscantia were found in inflorescence organization, style shape, fruit dehiscence and pollen morphology. Morphological and anatomical variation among leaf traits was found in epidermal cells, trichome distribution, mesophyll histochemistry and vascular organization. The analysis of occurrence records of 207 specimens demonstrates a clear ecological distinction between of Denscantia s.s. and Leonoria, which is ecologically confined to limestone outcrops associated with seasonally dry forests. The current study highlights the importance of an integrative taxonomic approach combining multiple sources of evidence to unravel complex taxonomic history within Spermacoceae.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70–80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone.The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains.

Cytochrome b gene as a potential DNA barcoding marker in ecoraces of tropical Tasar silkworm Antheraea mylitta Drury

BackgroundTropical Tasar silkworm Antheraea mylitta Drury plays a very crucial role in the economic development of the country employing around 9.76 million people in rural and semi-urban areas. Since the population of this species has spread over many states with varying environmental factors, the population has been isolated into many pockets, specifically adapted to that niche, and are called as “ecoraces”. Even though phenotypically these ecoraces may look similar to some level but the intra-generic genotypic variability between them is correlated to the economically important yield traits of their cocoons. The purpose of our study was to evaluate the effectiveness of Cytochrome b (Cyt b) gene of mitochondrial genome as a potential DNA barcoding marker for identification of these ecoraces as to are they really ecoraces, and attempts to establish the first country report of DNA barcode reference library of this silkworm species. Methods and resultsDNA was extracted from pupae of pure ecoraces collected from core ecozones. PCR amplicon was bead purified and further subjected to Sanger Sequencing. The parsimony tree was also created using the MEGA 11 software. The genetic distance, gene flow and relation among the ecoraces was evaluated. The conserved regions of Cyt b gene were identified along with the documentation of the mode and tempo of nucleotide variation and evolution. ConclusionThe present study confirms the efficiency of Cyt b gene as a potential DNA marker for identification of ecoraces of Tasar silkworm and is the first country report on barcoding of A. mylitta Drury. Apart from being used as an early-warning system for ecological degradation, this knowledge will help in assessing the evolutionary relationship of these economically important ecoraces and identify their position in the process of evolution aiding in the assessment of conservation priorities.

Development and validation of a new multiplex panel using SNaPshot-based DIP-TriSNP markers for forensic DNA mixtures.

Short tandem repeat analysis is challenging when dealing with unbalanced mixtures in forensic cases due to the presence of stutter peaks and large amplicons. In this research, we propose a novel genetic marker called DIP-TriSNP, which combines deletion/insertion polymorphism (DIP) with tri-allelic single nucleotide polymorphism in less than 230bp length of human genome. Based on multiplex PCR and SNaPShot, a panel, including 14 autosomal DIP-TriSNPs and one Y chromosomal DIP-SNP, had been developed and applied to genotyping 102 unrelated Han Chinese individuals in Sichuan of China and simulated a mixture study. The panel sensitivity can reach as low as 0.1ng DNA template, and the minor contributor of DNA can be detected with the highest ratio of 19:1, as indicated by the obtained results. In the Sichuan Han population, the cumulative probability of informative genotypes reached 0.997092, with a combined power of discrimination of 0.999999998801. The panel was estimated to detect more than two alleles in at least one locus in 99.69% of mixtures of the Sichuan Han population. In conclusion, DIP-TriSNPs have shown promising as an innovative DNA marker for identifying the minor contributor in unbalanced DNA mixtures, offering advantages such as short amplifications, increased polymorphism, and heightened sensitivity.

Discovery and technical validation of high-performance methylated DNA markers for the detection of cervical lesions at risk of malignant progression in low- and middle-income countries

BackgroundCervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy.ResultsMarker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12–100.00], and specificity of 91% [95% CI 62.26–99.53] to 96% [95% CI 79.01–99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00–1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77–70.84] to 89% [95% CI 67.20–98.03], specificity of 93% [95% CI 84.07–97.38] to 96% [95% CI 79.01–99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68–0.89] to 0.99 [95% CI 0.97–1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45–92.69], specificity 95% [95% CI 88.64–98.18], and ROC AUC = 0.925 [95% CI 0.878–0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11–80.44), specificity of 94% (95% CI 88.30–97.40), and ROC AUC = 0.884 (95% CI 0.822–0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers.ConclusionsThis 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.

HO-CR and HOLL-CR: new forms of winter oilseed rape (Brassica napus L.) with altered fatty acid composition and resistance to selected pathotypes of Plasmodiophora brassicae (clubroot).

The priority in oilseed rape (Brassica napus L.) research and breeding programs worldwide is to combine different features to develop cultivars tailored to specific applications of this crop. In this study, forms with a modified fatty acid composition of seed oil were successfully combined with a source of resistance to Plasmodiophora brassicae Wor., a harmful protist-causing clubroot. Three HO-type recombinants in F6-F12 generations with oleic acid content of 80.2-82.1% and one HOLL-type F6 inbred mutant recombinant (HOmut × LLmut), with a high oleic acid content (80.9%) and reduced linolenic acid content (2.3%), were crossed with the cultivar Tosca, resistant to several pathotypes of P. brassicae. The work involved genotyping with the use of DNA markers specific for allelic variants of desaturase genes responsible for the synthesis of oleic and linolenic fatty acids, CAPS (FAD2 desaturase, C18:1), and SNaPshot (FAD3 desaturase, C18:3), respectively. Of 350 progenies in the F3 generation, 192 (55%) were selected for further studies. Among them, 80 HO (≥ 72%) lines were identified, 10 of which showed resistance to at least one up to four P. brassicae pathotypes. Thirty lines in the selected progeny contained high oleic acid and less than 5% linolenic acid; eight of them belonged to the HOLL type conferring resistance to at least one pathotype. Two HO lines and two HOLL lines were resistant to four pathotypes. The resulting HO-CR and HOLL-CR inbred lines with altered seed oil fatty acid composition and resistance to P. brassicae represent unique oilseed rape material with the desired combination of valuable traits.

Associations between mitochondrial copy number, exercise capacity, physiologic cost of walking, and cardiac strain in young adult survivors of childhood cancer.

Childhood cancer survivors are at risk for cardiac dysfunction and impaired physical performance, though underlying cellular mechanisms are not well studied. In this cross-sectional study, we examined the association between peripheral blood mitochondrial DNA copy number (mtDNA-CN, a proxy for mitochondrial function) and markers of performance impairment and cardiac dysfunction. Whole-genome sequencing, validated by quantitative polymerase chain reaction, was used to estimate mtDNA-CN in 1720 adult survivors of childhood cancer (48.5% female; mean age = 30.7years, standard deviation (SD) = 9.0). Multivariable logistic regression was performed to evaluate the associations between mtDNA-CN and exercise intolerance, walking inefficiency, and abnormal global longitudinal strain (GLS), adjusting for treatment exposures, age, sex, and race and ethnicity. The prevalence of exercise intolerance, walking inefficiency, and abnormal GLS among survivors was 25.7%, 10.7%, and 31.7%, respectively. Each SD increase of mtDNA-CN was associated with decreased odds of abnormal GLS (adjusted odds ratio (OR) = 0.88, p = 0.04) but was not associated with exercise intolerance (OR = 1.02, p = 0.76) or walking inefficiency (OR = 1.06, p = 0.46). Alkylating agent exposure was associated with increased odds of exercise intolerance (OR = 2.25, p < 0.0001), walking inefficiency (OR = 2.37, p < 0.0001), and abnormal GLS (OR = 1.78, p = 0.0002). Increased mtDNA-CN is associated with decreased odds of abnormal cardiac function in childhood cancer survivors. These findings demonstrate a potential role for mtDNA-CN as a biomarker of early cardiac dysfunction in this population.

Molecular profiling of Greek native germplasm collection of Rosa canina L. for enhanced fruit extract production: a comprehensive approach utilizing neutral, gene, and exon-based markers

AbstractThe genus Rosa L. is globally distributed and Rosa canina L. is a distinguished member of multifaceted interest. Apart from the traditional uses of R. canina in folk medicine, food and cosmetic industries, or its ornamental applications, its rose hips are renowned for their functional bioactive components. Thus, identifying the genetic diversity within this species is crucial for any plant breeding project. This study employed three molecular markers namely inter simple sequence repeats; (ISSRs), start codon-targeted (SCoTs), and exon-based amplified polymorphisms; (EBAPs) to conduct the first comprehensive genetic analysis of 12 R. canina genotypes. DNA extraction, marker selection, and sequences’ amplification were performed following established protocols. The resulting genetic data were analyzed for polymorphism, diversity indices, and population structure using various statistical methods including principal component analysis (PCA), unweighted pair group method with arithmetic mean (UPGMA) clustering, and STRUCTURE analysis. The ISSR analysis revealed a high level of polymorphism (81.82%) and identified two major clusters in the UPGMA dendrogram. SCoT and EBAP markers also exhibited substantial polymorphism (74.56% and 82.11%, respectively) and formed three distinct clusters. PCA indicated a consistent pattern across markers suggesting reliable genetic grouping. STRUCTURE analysis supported the presence of three genetically uniform subpopulations (K = 3) within the studied R. canina germplasm collection. This study provided a comprehensive genetic characterization of the Greek native R. canina genebank collection. The observed genetic diversity and population structure offered valuable insights for future breeding programs targeting specific R. canina genetic clusters.

Variability of Scots pine (Pinus sylvestris L.) plus trees in the Middle and Upper Volga Region with the use of ISSR markers.

One of the serious issues in forest breeding is how to reduce the variability level in breeding populations of forest tree species that is a set of selected plus trees. The problem is that variability is jeopardized by the risk of losing the genetic diversity of future artificial forests, as well as emerging inbreeding depression in the seed plus trees progeny. DNA markers are an effective tool to study variability, identify features of the genetic structure and degree of plant differentiation. The research focuses on assessing the level of the genetic diversity and the degree of differentiation of plus trees of various geographic origin with the use of ISSR markers. We used six ISSR primers to study 270 plus trees grown in the Penza region, the Chuvash Republic, the Republic of Tatarstan and the Mari El Republic. The samples of plus trees under study were characterized by different levels of genetic diversity. Two hundred fifteen PCR fragments were identified for six ISSR primers in total, while the number of amplified fragments varied from 186 to 201 in different plus trees samples. The genetic variability varied within the following limits: 95.7-96.9 %, polymorphic loci; 1.96-1.97, the number of alleles per locus; 1.31-1.48, the number of effective alleles per locus: finally, 0.291-0.429, Shannon's index; 0.205-0.298, the expected heterozygosity. According to the analysis of molecular variance (AMOVA), 82 % of the variability of ISSR markers is typical for the plus tree samples, while only 18 % is variability among the compared groups of trees from different geographical zones. The dendrogram generated by UPGMA showed that the plus trees grown in the Penza region, the Chuvash Republic and the Republic of Tatarstan are similar in term of the genetic structure of plus trees, while the plus gene pool of Scots pine from the Mari El Republic stands alone. The results of the research prove that the level of genetic diversity, the structure of genetic variability, and the nature of differentiation of plus trees are consistent with those previously elicited for natural populations of Scots pine in the Middle and Upper Volga region.

Study of a genetic collection of strawberry (Fragaria L.) for resistance to powdery mildew.

Powdery mildew (Sphaerotheca macularis Mag. (syn. Podosphaera aphanis Wallr.)) is a dangerous disease of strawberry (Fragaria L.). The resistance of strawberry to powdery mildew is controlled polygenically. Several genetic loci with a large contribution to disease resistance have been identified in various strawberry varieties. Diagnostic DNA markers have been developed for QTL 08 To-f. They showed a high level of reliable gene detection in mapping populations. The purpose of this study was assessment of a strawberry genetic collection for resistance to powdery mildew and identification of promising strawberry forms for breeding for resistance to S. macularis. The objects of the study were wild species of the genus Fragaria L., varieties and selected seedlings of strawberry (Fragaria × ananassa Duch.) created in the I.V. Michurin Federal Scientific Center, and strawberry varieties introduced from various ecological and geographical regions. To identify QTL 08 To-f, DNA markers IB535110 and IB533828 were used. Locus 08 To-f was detected in 23.2 % of the analyzed strawberry genotypes, including wild species F. moschata and F. orientalis, strawberry varieties of Russian breeding (Bylinnaya and Sudarushka) and foreign breeding (Florence, Korona, Malwina, Ostara, Polka and Red Gauntlet). The correlation between the presence of markers IB535110 and IB533828 and phenotypic resistance (powdery mildew effect on strawberry plants is absent) was 0.649. The determination coefficient (R2 ) showing the contribution of the studied locus to the manifestation of the trait was 0.421, that is, in 42.1 % of cases resistance was explained by the presence of QTL 08 To-f, and in 57.9 % of cases, by other genetic factors. All strawberry genotypes with locus 08 To-f were characterized by high field resistance to S. macularis in the conditions of Michurinsk, Tambov region. Thus, locus 08 To-f is promising for conferring resistance on local powdery mildew races, and markers IB535110 and IB533828 can be used in marker-assisted breeding programs to create powdery mildewresistant strawberry genotypes.

Diagnosis of malignant body fluids via cancer-universal methylation in cell-free DNA.

BACKGROUNDDifferentiating malignant from nonmalignant body fluids remains a clinical challenge because of the unsatisfying performance of conventional cytology. We aimed to improve the sensitivity and ubiquity of cancer cell detection by assaying universal cancer-only methylation (UCOM) markers in supernatant cell-free DNA (cfDNA).METHODSAn observational prospective cohort including 1,321 nonmalignant and malignant body fluids of multiple cancers was used to develop and validate a cfDNA UCOM methylation diagnostic assay. All samples were divided into 2 portions for cytology and supernatant cfDNA methylation analysis.RESULTSThe significant hypermethylation of a potentially novel UCOM marker, TAGMe, together with the formerly reported PCDHGB7, was identified in the cfDNA of malignant body fluid samples. The combined model, cell-free cancer-universal methylation (CUE), was developed and validated in a prospective multicancer cohort with markedly elevated sensitivity and specificity, and was further verified in a set containing additional types of malignant body fluids and metastases. In addition, it remained hypersensitive in detecting cancer cells in cytologically negative malignant samples.CONCLUSIONcfDNA methylation markers are robust in detecting tumor cells and are applicable to diverse body fluids and tumor types, providing a feasible complement to current cytology-based diagnostic analyses.TRIAL REGISTRATIONThis study was registered at Chictr.org.cn (ChiCTR2200060532).FUNDINGNational Natural Science Foundation of China (32270645, 31872814, 32000505, 82170088), the National Key R&D Program of Ningxia Hui Autonomous region (2022BEG01003), Shanghai Municipal Key Clinical Specialty (shslczdzk02201), Science and Technology Commission of Shanghai Municipality (20DZ2261200, 20DZ2254400), and Major Special Projects of Basic Research of Shanghai Science and Technology Commission (18JC1411101).