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Imputation is a method that supplies missing information about genetic variants that could not be directly genotyped with DNA microarrays or low-coverage sequencing. Imputation plays a critical role in genome-wide association studies (GWAS). It leads to a significant increase in the number of studied variants, which improves the resolution of the method and enhances the comparability of data obtained in different cohorts and/or by using different technologies, which is important for conducting meta-analyses. When performing imputation, genotype information from the study sample, in which only part of the genetic variants are known, is complemented using the standard (reference) sample, which has more complete genotype data (most often the results of whole-genome sequencing). Imputation has become an integral part of human genomic research due to the benefits it provides and the increasing availability of imputation tools and reference sample data. This review focuses on imputation in human genomic research. The first section of the review provides a description of technologies for obtaining information about human genotypes and characteristics of these types of data. The second section describes the imputation methodology, lists the stages of its implementation and the corresponding programs, provides a description of the most popular reference panels and methods for assessing the quality of imputation. The review concludes with examples of the use of imputation in genomic studies of samples from Russia. This review shows the importance of imputation, provides information on how to carry it out, and systematizes the results of its application using Russian samples.

Abstract Hepatitis B virus (HBV) causes hepatitis B (HB) and distinct HBV genotypes can lead to different prognoses. However, HBV genotyping is rarely done in clinics, because the traditional method by PCR-based DNA sequencing is impractical for clinical diagnosis with tedious process and low success rate. Herein, we have established an ELISA-based genotyping method to quickly determine the HBV genotypes of HB patients in China. First, two commercial antibodies, 16D12 and 6H3 specific for HBV genotypes B and C respectively, are chosen as capture antibodies, since these two genotypes dominate in China. Then two home-made genotype-specific antibodies, B19 and C04, are used as the detection antibodies for genotypes B and C in sandwiched ELISA. The ELISA kit shows high sensitivity (> 95%) and specificity (> 95%) in detecting genotypes B and C of Chinese HB patients. Moreover, the ELISA kit has demonstrated higher success rate (98.7%) than PCR-based DNA sequencing (93.5%) and a commercial PCR-based genotyping kit (92.2%) for sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL. Such an advantage is more obvious for the sera with HBV DNA

Abstract Background Toxoplasmosis not only leads to abortion in humans but also in herbivores, which causes significant financial and quality-adjusted life-year losses. The present study aimed to determine the prevalence of toxoplasmosis in aborted fetuses via serological and molecular assays. Moreover, the genotypes of the obtained isolates were detected. Methods Serological and molecular methods were used to study aborted fetuses from Bojnourd City, North Khorasan Province, Iran, which included 52 ovines and 16 bovines. Nested PCR of the B1 gene was used to detect parasite DNA in brain tissues. The PCR-RFLP method for the GRA6 gene was used to determine the genotype of T. gondii. Results Out of 68 aborted fetuses, 16.1% showed the presence of anti-T. gondii IgG. Among these, 11.7% were identified in bovine fetuses and 4.4% in ovine fetuses. Additionally, two (2.94%) samples of ovine tested positive for anti-T. gondii IgM. Our PCR analysis detected parasite DNA in two cases (2.94%) among 11 IgG-positive samples. All obtained isolates belong to type I of T. gondii. Conclusion Infection with Type I of T. gondii during the neonatal period may partly be responsible for abortion and economic losses in livestock farming in our studied region. To understand the molecular epidemiology and genotypes of T. gondii associated with abortion, further evaluation of aborted samples from different geographical locations is necessary.

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, and nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RTPCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

According to the Law “On Seed Production”, all varieties or hybrids included in the state register shall be provided for genetic passports, as well as making a list of plants’ species, the production of which is aimed at ensuring food security. However, the use of morphological characteristics does not allow distinguishing genetically similar samples, identifying hidden genetic variability and ensuring control of the homogeneity of the initial material. In connection with these limitations, the purpose of the current work was to develop approaches for the selection of molecular genetic mic- rosatellite markers (SSR markers) to differentiate common wheat varieties. In accordance with the purpose, there has been conducted a literature review regarding methods for genetic certification and evaluation of the diversity of spring common wheat based on polymorphism of microsatellite loci, and there has been generated a set of Triticum aestivum genome assemblies from the NCBI Gen Bank. Based on the literature data, there have been selected the most polymorphic SSR markers using the invented algorithm. The analysis of databases of SSR markers in the genome of common wheat using bioinformatics methods allowed establishing a minimal discriminatory set of 20 markers that can detect 419 different alleles in Triticum aestivum. The results obtained can become the necessary theoretical foundation for further practical research.

Relevance. Effective responses are required due to the high incidence of cervical cancer (Cc) throughout the World. RT-PCRbased HPV-testing is becoming more prominent in secondary prevention worldwide, replacing cytology. Russian practice still relies on cytology as the primary method mainly because there is a lack of comparative evaluation of the diagnostic characteristics of cytology and HPV-tests. Aim. Evaluation of diagnostic characteristics of laboratory methods and the relevance of extended HPV genotyping for secondary prevention of cervical cancer. Materials & methods. The study included data (liquid cytology, histology, HPV-test results) from a survey of 653 women (M = 33.55 years old, ME = 32.0 years old, IQR: 26-38 years old) infected with 14 HPV types (16, 18, 31, 33, 35, 39, 45, 45, 51, 52, 56, 58, 59, 66, 68), with presence or absence of intraepithelial neoplasia of varying severity. The study analyzed the correlation of cervical cytology - histology, clinical sensitivity for high-grade squamous intraepithelial lesions (HSIL+), the incidence and the role of 14 oncogenic HPV types in the development of cervical intraepithelial pathology, positive predictive value (PPV) and the diagnostic accuracy of the HPV-test. Results. The agreement between cytology and histology is 67.20%. Clinical sensitivity of cytology is 83.78% for HSIL+ and 94.34% for any other than NILM result. The structure of the HPV population varies depending on the degree of neoplasia, with a constant predominance of HPV16. The clinical sensitivity of the laboratory component rises to 99.5% by conducting HPV-test for at least 12 types of virus. HSIL is more commonly associated with viruses of alpha-9 phylogenetic group, than with alpha-7 and alpha-5/6. The positive predictive value for HSIL+ is reduced depending on the HPV type: 16>33>58>35>45>31>18>52>39>59>58>56>68>66. Conclusions. Clinical sensitivity of the HPV-test exceeds that of cytology, so that secondary prevention of cervical cancer can be effectively achieved through HPV testing. The introduction of an extended genotyping provides more complete information about the risk of having HSIL+. The data obtained will form the basis for the development of directions for improving the epidemiological surveillance information subsystem of HPVassociated cervical diseases

Cassava, a crop of importance for subsistence farming in Africa, Asia, and Latin America, has the potential to benefit from global economic integration as a versatile industrial resource. Enhancing cassava productivity is not just a matter of agricultural competitiveness but a crucial step toward ensuring many communities' food security and livelihoods. Given its high performance in marginal environments, where climate change poses threats, ensuring food security and livelihoods relies on rapidly adapting cassava. This study aimed to develop a protocol that swiftly transitions cassava embryogenic short-period liquid suspension cultures, facilitating the regeneration of genetically stable in vitro plants. The resulting protocol, with its potential to be a foundational component in future technologies employing various genome editing or genetic modification techniques, holds promise for the advancement of cassava biotechnology.

Abstract Background This study aimed to design and develop a 5K low-density liquid chip for Hainan cattle utilizing targeted capture sequencing technology. The chip incorporates a substantial number of functional single nucleotide polymorphism (SNP) loci derived from public literature, including SNP loci significantly associated with immunity, heat stress, meat quality, reproduction, and other traits. Additionally, SNPs located in the coding regions of immune-related genes from the Bovine Genome Variation Database (BGVD) and Hainan cattle-specific SNP loci were included. Results A total of 5,293 SNPs were selected, resulting in 9,837 DNA probes with a coverage rate of 85.69%, thereby creating a Hainan cattle-specific 5K Genotyping by Target Sequencing (GBTS) liquid chip. Evaluation with 152 cattle samples demonstrated excellent clustering performance and a detection rate ranging from 96.60 to 99.07%, with 94.5% of SNP sites exhibiting polymorphism. The chip achieved 100% gender coverage and displayed a heterozygosity rate between 14.20% and 29.65%, with a repeatability rate of 99.65–99.85%. Analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed the potential regulatory roles of exonic SNPs in immune response pathways. Conclusion The development and validation of the 5K GBTS liquid chip for Hainan cattle represent a valuable tool for genome analysis and genetic diversity assessment. Furthermore, it facilitates breed identification, gender determination, and kinship analysis, providing a foundation for the efficient utilization and development of local cattle genetic resources.

Abstract Background Cryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China. Results A total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively. Conclusions The present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.

Abstract Pseudomonas aeruginosa, a common pathogen in nosocomial infections, presents significant global health challenges due to its high prevalence and mortality rates. However, the origins and distribution of this bacterium remain unclear, partly due to the lack of effective gene typing methods. This situation necessitates the establishment of trustworthy and high-resolution protocol for differentiating closely related P. aeruginosa strains. In this context, the present study attempted to undertake a comparative genomic analysis of multiple P. aeruginosa strains available in the public database NCBI, with the goal of identifying potential genetic markers for measuring the genetic diversity. The preliminary comparative analysis of 816 P. aeruginosa strains revealed notable variations in two genes—specifically, the CDF family iron/cobalt efflux transporter AitP and the protease modulator HflC—across 44 strains. These variations were associated with single amino acid repeats (SHRs) that responsible for encoding histidine residue. Additionally, comparative gene map analysis revealed differential clustering patterns in the Rsx and TAXI genes among 16 strains. Interestingly, the gene structure pattern observed in TAXI groups displayed a strong correlation with the SHRs pattern in the CDF and HflC groups. In addition, the SHRs pattern of CDF and HflC were strongly correlated with MLST sequence type number. Overall, the study present a novel genetic markers based on SHRs and gene cluster patterns, offering a reliable method for genotyping of P. aeruginosa.

Introduction. Rotavirus infection is the major cause of severe dehydrating diarrhea requiring hospitalization in young children worldwide. Due to their segmented genome, rotaviruses are capable of gene reassortment, which makes the emergence and spread of genetically novel strains possible. The purpose of this study was to search for unusual rotaviruses circulating in Nizhny Novgorod in 2021‒2023 and their molecular genetic characterization based on all genome segments. Materials and methods. Rotavirus-positive stool samples of children were examined by PCR genotyping and electrophoresis in PAAG. cDNA fragments of each of the 11 genes (VP1‒VP4, VP6, VP7, NSP1‒NSP5), 570 to 850 nucleotide pairs in length were sequenced for the selected strains. The phylogenetic analysis was performed in the MEGA X program. Results. In the study period 2021‒2023, 11 G[P] combinations with a predominance of G3P[8] (59.5%) were identified. Six atypical Rotavirus А (RVA) strains were identified: 2 strains of the G2P[4] genotype (G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E2-H3, G2-P[4]-I2-R2-C2-M2-A3-N2-T3-E3-H2) and 4 G3P[9] strains (all strains had the genotype G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3). Phylogenetic analysis based on all genes showed an evolutionary relationship between rotaviruses similar to rotaviruses of cats and dogs (BA222-like) and unusual strains of the G2P[4] genotype, for which a mixed combination of genotypes was identified and characterized for the first time. Discussion. The results obtained expand the understanding of the diversity of reassortant RVAs, as well as complement the data on the genotypic structure of the rotavirus population in Nizhny Novgorod. Conclusion. The wide genetic diversity of reassortant RVA can help rotaviruses overcome the immunological pressure provided by natural and vaccine-induced immunity. In this regard, to control the emergence of new variants and assess changes in the virulence of rotaviruses after reassortment processes, continuous molecular monitoring for circulating RVA is necessary.

Abstract The norovirus (NoV) genome is diverse. Therefore, this study explored the epidemiological characteristics and genetic features of NoV in Ningxia Hui Autonomous Region, China, from 2011 to 2022 to clarify the genetic diversity in this region. Stool samples were screened for NoV and then sequenced and genotyped. In total, 1,788 of 13,083 specimens were NoV -positive (13.67%); 204 (1.56%) and 1,584 (12.11%) cases were GI and GII, respectively. Additionally, 559 were NoV infection with other viruses (4.27%), primarily with rotavirus (277/559, 49.55%). The NoV incidence rate was the highest among children aged 0–2 years (18.09%, 1054/5,828) and lowest among adults aged 45–64 years (110/1,495, 7.36%); it was also higher in the winter and spring than in the other seasons. GI.3[P3] was the dominant GI genotype. The dominant GII genotype changed roughly every two years. In the GII group, GII.4 was the most common genotype (46.79%), followed by GII.3 (21.34%), GII.2 (12.34%), and GII.17 (9.77%). There were three variants of GII.4 Den Haag, GII.4 New Orleans and GII.4 Sydney identified in the detected GII.4 strains, with GII.4 Sydney dominating. The GII.4 (87.36%), GII.3 (86.35%), and GII.2 (72.92%) strains were primarily detected in children, whereas it was the GII.17 (52.63%) strain in adults. Overall, the NoV genotypes in the Ningxia Hui Autonomous Region were diverse. Primarily, GII groups were dominant, but this changed over time.

Abstract Ornithonyssus sylviarum (O. sylviarum) is an obligatory, blood-sucking ectoparasite widely distributed among poultry and other mammals, causing significant economic losses. This study represented the first report of molecular genotypic identification of O. sylviarum from pigeons, Columba livia domestica, in Egypt. PCR and sequencing of the 28S rRNA gene were conducted. The resulting mite sequences were subjected to BLAST analysis, revealing 90–100% similarity to O. sylviarum in all tested samples. The sequences were deposited in GenBank under the accession numbers PP049086 and PP033720. A phylogenetic tree was constructed to compare the obtained species with related species worldwide. Additionally, infected pigeons showed increased expression of IL-1, IL-10, IFN-γ, and TGF-β3 genes and elevated serum levels of stress biomarkers. The increased level of these cytokines indicates there was a disturbance in the immune status of the infected host with parasite compared with control healthy ones. This increases the susceptibility to infection with other pathogens.

Xueya Chen,1,&ast; Zhiling Yan,2,&ast; Weipeng Liu,1 Lili Guo,1 Jinmei Xu,2 Li Shi,3 Yufeng Yao1 1Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, People’s Republic of China; 2Department of Gynaecologic Oncology, The No. 3 Affiliated Hospital of Kunming Medical University, Kunming, People’s Republic of China; 3Department of Immunogenetics, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Li Shi, Department of Immunogenetics, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, Yunnan, 650118, People’s Republic of China, Email shili.imb@gmail.com Yufeng Yao, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, Yunnan, 650118, People’s Republic of China, Email leoyyf@gmail.com; yufeng_yao@imbcams.com.cnPurpose: Cervical cancer (CC) poses a significant threat to women’s health worldwide, and multiple signaling pathways have been confirmed to be involved in its development. The AMPK signaling pathway plays a central role in maintaining energy homeostasis, and its dysregulation is closely associated with the occurrence of CC. Changes in microRNA (miRNA) expression levels might be related to the AMPK signaling pathway. Single nucleotide polymorphisms (SNPs) can affect the function of miRNA and result in the development of CC. To investigate the association between the SNPs of AMPK pathway-associated miRNAs and CC in a Han Chinese population, we selected eight miRNA genes located in the AMPK pathway and analyzed nine SNP loci within these genes to explore whether they are associated with genetic susceptibility to cervical intraepithelial neoplasia (CIN) and CC.Methods: A total of 2,220 subjects were included in this study, including 928 healthy controls, 421 CIN patients, and 871 CC patients. Nine candidate SNPs (rs895819 in miR-27a, rs10061133 in miR-449b, rs41291179 in miR-216a, rs76481776 in miR-182, rs10406069 in miR-5196, rs12803915 and rs550894 in miR-612, rs66683138 in miR-3622b, and rs2620381 in miR-627) were genotyped using the TaqMan method.Results: The results showed significant differences in the allele distribution of rs41291179 and rs12803915 between the control group and the CIN group, as well as between the control group and the CC group (all P values < 0.005). The A allele of rs41291179 and the G allele of rs12803915 were associated with decreased risk of CIN (OR = 0.05, 95% CI: 0.01– 0.39; OR = 0.61, 95% CI: 0.49– 0.76) and CC (OR = 0.08, 95% CI: 0.01– 0.66; OR = 0.71, 95% CI: 0.59– 0.86), respectively.Conclusion: Our results suggest that polymorphisms in miRNA genes of the AMPK signaling pathway are associated with the development of CC.Keywords: genotyping, SNP, gene enrichment, genetic susceptibility

Background: Colorectal cancer (CRC) ranks third in both the incidence and mortality rates among male and female cancers, and it is the leading digestive system cancer. Due to the inter- and intratumor heterogeneity of cancer, the TNM system is insufficient for predicting prognosis, necessitating the use of molecular biomarkers for prognostic prediction. Toll-like receptors (TLRs) have been associated with CRC survival rates. This study focused on the investigation of the role and potential value of TLRs in CRC genotyping to aid in immunotherapy for CRC patients. Methods: Differential gene expression analysis was performed on CRC transcriptomic data from The Cancer Genome Atlas database. TLRs were referred from the literature, and their intersection with differentially expressed genes (DEGs) in CRC yielded TLR-DEGs. The expression patterns of TLR-DEGs were predicted using the STRING website, and copy number variations of TLR-DEGs were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted on TLR-DEGs. ConsensusClusterPlus R package was used for clustering CRC patients, and ESTIMATE and GSEAbase were employed to analyze immune characteristics of different subtypes. Immune phenotyping scores and tumor immune dysfunction and exclusion scores were evaluated. DEGs of different subtypes were analyzed, followed by GO and KEGG enrichment analyses, the protein–protein interaction (PPI) network analysis, and further selection of hub genes. The sensitivity of drugs was assessed using the identified hub genes. Results: We identified 37 TLR-DEGs, and the PPI analysis revealed their coexpression, although they were distributed on different chromosomes. Enrichment analyses indicated that the 37 TLR-DEGs were linked to cancer cell immune response. Based on these TLR-DEGs, CRC patients were classified into three subtypes. Cluster2 exhibited lower survival rates and higher immune infiltration levels and predicted poorer response to immune checkpoint inhibitor therapy. The intersection of DEGs from cluster2 and cluster1 with DEGs from cluster2 and cluster3 yielded a set of 426 commonly shared DEGs. Enrichment analyses revealed that these shared DEGs might regulate immune cell viability. Eight common hub genes for different subtypes were further identified to predict drug-related correlations. Conclusion: The developed TLR genotyping was used to predict the survival status and tumor microenvironment of CRC, providing a foundation for understanding the molecular mechanisms of TLR signaling and deepening its clinical significance.

Objective To study the RHD genotyping of primary screening RhD negative population in Beijing, and analyze the serological characteristics of different genotypes and the distribution of Rh phenotypes. Methods From August 2022 to January 2024, 204 RhD negative samples in our hospital were initially screened by microplate or microcolumn agglutination method. RhD antigen phenotype was identified by saline tube method, and RhD negative confirmation test was performed by microcolumn gel-indirect antiglobulin technology.RHD genotyping was performed by polymerasing chain reaction-sequence specific primmer (PCR-SSP) method and sequencing technology, and the phenotypic frequency and gene frequency were calculated. Results The distribution of Rh serologic phenotypes in 204 RhD negative samples were 112 cases of ccee (54.90%) > 73 cases of Ccee (35.78%) > 9 cases of CCee (4.41%) > 6 cases of ccEe (2.94%) > 4 cases of CcEe (1.96%). A total of 7 RHD genotypes were detected in this study, including 129 cases of RHD* 01N.01 (63.24%), 44 cases of RHD* 01EL.01 (21.57%), 26 cases of RHD* 01N.03 (12.75%), 2 cases of RHD* 01N.16 (0.98%), 1 case of RHD* 06.03.01 (0.49%), 1 case of weak RHD* 15 (0.49%) and 1 case of RHD* 13.01/RHD* 01N.01 (0.49%). The frequency and phenotype distribution of Rh blood group antigen gene were consistent with Hardy-Weinberg equilibrium (P>0.05). Conclusion The RHD gene is polymorphic in the primary screening RhD negative population in Beijing, and the most common RhD negative genotype is RHD* 01N.01, and the RHD variant is RHD* 01EL.01.This study may provide data to support the clinical implementation of genotype-matched blood transfusions.

In modern breeding work, considerable attention is paid to genetic resources of cattle, genetic assessment of which, with proper crossbreeding and selection, can increase efficiency of the entire industry. One way to realize the genetic resources of animals is to use information about polymorphism of milk productivity genes. The aim of the study was to determine polymorphism of -lactoglobulin gene in in Kholmogorsky cows and to identify its effect on economically valuable milk indicators. The experiments were carried out on the breeding stock of Kholmogorsky breed in Kholmogorsky Plemzavod. The genotype of the studied animals for LGB gene was determined by PCR RFLP using Hae III restriction endonuclease. It was established that the allele B of the -lactoglobulin gene was dominant in the genome of the studied sample of cows and its frequency of occurrence was 68%, while the most common genotype was AB (47.4%). To assess the effect of different LGB genotypes on milk productivity and milk quality of Kholmogorsky cows, productivity indicators for the last completed lactation were combined into subgroups depending on the genotypes and compared with each other. Most likely, the uniqueness of the Kholmogorsky breed, expressed not only in terms of milk productivity, exterior and constitution, but also in the gene pool, influenced the discrepancy between the obtained results and general patterns. Firstly, in our case, B allele has a positive effect both on qualitative and technological characteristics of milk, and on quantitative indicators of milk productivity. Secondly, the largest value of milk yield and the mass fraction of dry matter belongs to animals with the AB genotype. We recommended to include the procedure for assessing polymorphism of LGB gene in a set of breeding and production work activities to increase the economic efficiency of the enterprise. Such a step will also contribute to the further comprehensive study of this gene as a breeding marker.

Abstract Background Cannabis sativa is seeing a global resurgence as a food, fiber and medicinal crop for industrial hemp and medicinal Cannabis industries respectively. However, a widespread moratorium on the use and research of C. sativa throughout most of the 20th century has seen the development of improved cultivars for specific end uses lag behind that of conventional crops. While C. sativa research and development has seen significant investments in the recent past, resulting in a suite of publicly available genomic resources and tools, a versatile and cost-effective mid-density genotyping platform for applied purposes in breeding and pre-breeding is lacking. Here we report on a first mid-density fixed-target SNP platform for C. sativa. Results The High-throughput Amplicon-based SNP-platform for medicinal Cannabis and industrial Hemp (HASCH) was designed using a combination of filtering and Integer Linear Programming on publicly available whole-genome sequencing and RNA sequencing data, supplemented with in-house generated genotyping-by-sequencing (GBS) data. HASCH contains 1,504 genome-wide targets of high call rate (97% mean) and even distribution across the genome, designed to be highly informative (> 0.3 minor allele frequency) across both medicinal cannabis and industrial hemp gene pools. Average numbers of mismatch SNP between any two accessions were 251 for medicinal cannabis (N = 116) and 272 for industrial hemp (N = 87). Comparing HASCH data with corresponding GBS data on a collection of diverse C. sativa accessions demonstrated high concordance and resulted in comparable phylogenies and genetic distance matrices. Using HASCH on a segregating F2 population derived from a cross between a tetrahydrocannabinol (THC)-dominant and a cannabidiol (CBD)-dominant accession resulted in a genetic map consisting of 310 markers, comprising 10 linkage groups and a total size of 582.7 cM. Quantitative Trait Locus (QTL) mapping identified a major QTL for CBD content on chromosome 7, consistent with previous findings. Conclusion HASCH constitutes a versatile, easy to use and cost-effective genotyping solution for the rapidly growing Cannabis research community. It provides consistent genetic fingerprints of 1504 SNPs with wide applicability genetic resource management, quantitative genetics and breeding.

Abstract PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants.

Rett syndrome (RTT) is a paediatric neurodevelopmental disorder spanning four developmental stages. This multi-system disorder offers a unique window to explore genotype–phenotype relationships in a disease model. However, genetic prognosticators of RTT have limited clinical value due to the disorder’s heterogeneity on multiple levels. This case report used a precision medicine approach to better understand the clinical phenotype of RTT twins with an identical pathogenic MECP2 mutation and discordant neurodevelopmental profiles. Targeted genotyping, objective physiological monitoring of heart rate variability (HRV) parameters, and clinical severity were assessed in a RTT twin pair (5 years 7 months old) with an identical pathogenic MECP2 mutation. Longitudinal assessment of autonomic HRV parameters was conducted using the Empatica E4 wristband device, and clinical severity was assessed using the RTT-anchored Clinical Global Impression Scale (RTT-CGI) and the Multi-System Profile of Symptoms Scale (MPSS). Genotype data revealed impaired BDNF function for twin A when compared to twin B. Twin A also had poorer autonomic health than twin B, as indicated by lower autonomic metrics (autonomic inflexibility). Hospitalisation, RTT-CGI-S, and MPSS subscale scores were used as measures of clinical severity, and these were worse in twin A. Treatment using buspirone shifted twin A from an inflexible to a flexible autonomic profile. This was mirrored in the MPSS scores, which showed a reduction in autonomic and cardiac symptoms following buspirone treatment. Our findings showed that a combination of a co-occurring rs6265 BDNF polymorphism, and worse autonomic and clinical profiles led to a poorer prognosis for twin A compared to twin B. Buspirone was able to shift a rigid autonomic profile to a more flexible one for twin A and thereby prevent cardiac and autonomic symptoms from worsening. The clinical profile for twin A represents a departure from the disorder trajectory typically observed in RTT and underscores the importance of wider genotype profiling and longitudinal objective physiological monitoring alongside measures of clinical symptoms and severity when assessing genotype–phenotype relationships in RTT patients with identical pathogenic mutations. A precision medicine approach that assesses genetic and physiological risk factors can be extended to other neurodevelopmental disorders to monitor risk when genotype–phenotype relationships are not so obvious.

Since 1998, notifiable bluetongue virus (BTV) serotypes 1–4, 6, 8, 9, 11, and 16 have been reported in Europe. In August 2006, a bluetongue (BT) outbreak caused by BTV serotype 8 began in northwestern Europe. The Netherlands was declared BT-free in February 2012, and annual monitoring continued. On September 3, 2023, typical BT clinical manifestations in sheep were notified to the Netherlands Food and Product Safety Consumer Authority. On September 6, we confirmed BTV infection through laboratory diagnosis; notifications of clinical signs in cattle were also reported. We determined the virus was serotype 3 by whole-genome sequencing. Retrospective analysis did not reveal BTV circulation earlier than September. The virus source and introduction route into the Netherlands remains unknown. Continuous monitoring and molecular diagnostic testing of livestock will be needed to determine virus spread, and new prevention strategies will be required to prevent BTV circulation within the Netherlands and Europe.

Introduction. Enteroviruses (EV) are characterized by: species and type diversity, polymorphism of clinical manifestations, a tendency to epidemic spread, and are often the cause of disease outbreaks, which determines the relevance of monitoring EV strains in various clinical forms of infection, including in conditions of anti-epidemic measures. The aim of the study: to characterize the prevalence and diversity of non-polio enteroviruses (NPEV) types in children with acute intestinal infection (AII) in the period 2018–2023, including the COVID-19 pandemic. Materials and methods. The RT-PCR method was used to study 7302 samples of feces from children hospitalized with a diagnosis of AII in the infectious diseases hospital of Nizhny Novgorod. Genotyping of EV strains was carried out using fragment Sanger sequencing of the genome region encoding capsid protein 1 (VP1) and the online resource BLAST. Results. EVs were found in 5.0 ± 0.3% (1.7–7.8%), both in mono- and mixed infections with other enteric viruses. The long-term dynamics of the frequency of EV detection and the incidence of EV infection in children in the Nizhny Novgorod region was characterized by a sharp decrease in indicators in 2020 against the backdrop of the introduction of anti-epidemic measures. When genotyping 299 strains, 41 types of NPEV of 4 species were identified. The spectrum included the main pathogens of exanthema and neuroinfections and rare types found in “minor” or intestinal forms of infection. During the study period, a redistribution of NPEV species was established. Before the pandemic, the ratio of Enterovirus A : Enterovirus B : Enterovirus C species was as follows — 41.0 : 46.7 : 12.3%; during the 2020 pandemic season the ratio was 0.0 : 37.5 : 62.5%; after the lifting of restrictive measures — 47 : 29 : 23%, which may be due to the different effectiveness of the restrictive measures on the mechanisms of transmission of EVs of different types. Conclusion. The genetic diversity of NPEVs detected in children with AII complements information on the typical composition of the territorial enterovirus population. In children with AII, when the airborne transmission of SARS-CoV-2 was blocked, there was a decrease in the frequency of detection of viruses of the Enterovirus B type, the absence of detection of Enterovirus A and the constant presence of Enterovirus C.

Introduction. Bacillus anthracis, the pathogen of a particularly dangerous zoonotic disease known as anthrax, requires strict epidemiological control and is characterized by high genetic homogeneity, which necessitates the development of genotyping methods. The aim of the study were to to find and characterize the VNTR and INDEL loci of B. anthracis and to develop on their basis a genotyping technique by PCR with electrophoretic detection of the results. Materials and methods. Marker search and phylogenetic analysis were performed on a sample of 388 genomes of B. anthracis strains, 322 from the GenBank collection (RefSeq) and 66 from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor. Phylogenetic analysis was performed on the basis of SNP crustal alignment using the Parsnp program. The search for markers was carried out using the Mauve program and author's scripts in Python. PCR was performed using a ScreenMix-HS kit (CJSC "Eurogen", Russia). Results. Genomic variations of B. anthracis strains (SNP — 25,664, SNR — 14,387, VNTR — 693, INDEL — 14,667) were found, bioinformatic analysis of which revealed nine new VNTR and six INDEL molecular markers most suitable for genotyping. The genetic (allelic) variants of the markers are described. Primers were selected for the found markers and a PCR protocol with detection by electrophoresis in agarose gel was developed. When typing using VNTR markers was applied, the strains were divided into nine clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.005/006, A.Br.008/009 (Tsiankovskii), A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.008/009 (strain 228/269), B.Br.001/002. When typing using INDEL markers, the strains were divided into six clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.008/009(Tsiankovskii), B.Br.001/002(B.Br.014), as well as a cluster comprising several genetic lineages: A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.005/006 и B.Br.001/002. Conclusion. The use of the developed methodology for the identification of variable VNTR and INDEL loci makes it possible to reliably determine the phylogenetic position of B. anthracis strains and is promising for use in the epidemiological investigation of anthrax outbreaks.

Abstract Wheat is an essential food commodity cultivated throughout the world. However, this crop faces continuous threats from fungal pathogens, leaf rust (LR) and stripe rust (YR). To continue feeding the growing population, these major destructors of wheat must be effectively countered by enhancing the genetic diversity of cultivated germplasm. In this study, an introgression line with hexaploid background (IL sp3603 ) carrying resistance against Pt pathotypes 77−5 (121R63-1), 77−9 (121R60-1) and Pst pathotypes 46S119 (46E159), 110S119 (110E159), 238S119 (238E159) was developed from donor wheat wild progenitor, Aegilops speltoides acc pau 3603. To understand the genetic basis of resistance and map these genes (named Lr sp3603 and Yr sp3603 ), inheritance studies were carried out in F6 and F7 mapping population, developed by crossing IL sp3603 with LR and YR susceptible cultivar WL711, which revealed a monogenic (single gene) inheritance pattern for each of these traits. Bulk segregant analysis combined with 35 K Axiom SNP array genotyping mapped both genes as separate entities on the short arm of chromosome 6B. A genetic linkage map, comprising five markers, 1 SNP, 1 PLUG and three gene based SSRs, covered a genetic distance of 12.65 cM. Lr sp3603 was flanked by markers Tag-SSR14 (located proximally at 2.42 cM) and SNP AX-94542331 (at 3.28 cM) while Yr sp3603 was mapped at one end closest to AX-94542331 at 6.62 cM distance. Functional annotation of Lr sp3603 target region (∼ 1 Mbp) revealed 10 gene IDs associated with disease resistance mechanisms including three encoding typical R gene domains.

Abstract Background Genotyping of individuals plays a pivotal role in various biological analyses, with technology choice influenced by multiple factors including genomic constraints, number of targeted loci and individuals, cost considerations, and the ease of sample preparation and data processing. Target enrichment capture of specific polymorphic regions has emerged as a flexible and cost-effective genomic reduction method for genotyping, especially adapted to the case of very large genomes. However, this approach necessitates complex bioinformatics treatment to extract genotyping data from raw reads. Existing workflows predominantly cater to phylogenetic inference, leaving a gap in user-friendly tools for genotyping analysis based on capture methods. In response to these challenges, we have developed GeCKO (Genotyping Complexity Knocked-Out). To assess the effectiveness of combining target enrichment capture with GeCKO, we conducted a case study on durum wheat domestication history, involving sequencing, processing, and analyzing variants in four relevant durum wheat groups. Results GeCKO encompasses four distinct workflows, each designed for specific steps of genomic data processing: (i) read demultiplexing and trimming for data cleaning, (ii) read mapping to align sequences to a reference genome, (iii) variant calling to identify genetic variants, and (iv) variant filtering. Each workflow in GeCKO can be easily configured and is executable across diverse computational environments. The workflows generate comprehensive HTML reports including key summary statistics and illustrative graphs, ensuring traceable, reproducible results and facilitating straightforward quality assessment. A specific innovation within GeCKO is its 'targeted remapping' feature, specifically designed for efficient treatment of targeted enrichment capture data. This process consists of extracting reads mapped to the targeted regions, constructing a smaller sub-reference genome, and remapping the reads to this sub-reference, thereby enhancing the efficiency of subsequent steps. Conclusions The case study results showed the expected intra-group diversity and inter-group differentiation levels, confirming the method's effectiveness for genotyping and analyzing genetic diversity in species with complex genomes. GeCKO streamlined the data processing, significantly improving computational performance and efficiency. The targeted remapping enabled straightforward SNP calling in durum wheat, a task otherwise complicated by the species' large genome size. This illustrates its potential applications in various biological research contexts.

Abstract The study aimed to investigate molecularly the presence of flea-borne viruses in infested small ruminants with fleas. It was carried out in Egypt’s Northern West Coast (NWC) and South Sinai Governorate (SSG). Three specific primers were used targeting genes, ORF103 (for Capripoxvirus and Lumpy skin disease virus), NS3 (for Bluetongue virus), and Rdrp (for Coronavirus), followed by gene sequencing and phylogenetic analyses. The results revealed that 78.94% of sheep and 65.63% of goats were infested in the NWC area, whereas 49.76% of sheep and 77.8% of goats were infested in the SSG region. Sheep were preferable hosts for flea infestations (58.9%) to goats (41.1%) in the two studied areas. Sex and age of the animals had no effects on the infestation rate (p > 0.05). The season and site of infestation on animals were significantly different between the two areas (p

Molecular biology techniques are important tools for studying animal health and production characteristics. Thus, determining the genotypes of animals with high genetic value enables the implementation of genetic improvement programs, especially related to sperm and testicular characteristics in cattle. The objective of this study was to detect polymorphisms in the promoter region of the FSH-β gene using the Tetra-primer-PCR (T-ARMS-PCR) technique and associate these polymorphisms with seminal parameters and scrotal perimeter in Nellore bulls. Semen samples were collected and evaluated for volume, sperm motility, and vigor, and scrotal perimeter was measured in 200 bulls. DNA extractions and genotyping of the FSH-βU1 and FSH-βU2 polymorphisms were performed using the T-ARMS-PCR technique. Population genetics parameters were estimated, and associations between genotypes and semen and testicular characteristics were assessed. Both SNPs showed variability with allele frequencies close to 0.500 and an excess of heterozygotes (above 0.600). No inbreeding was detected (FIS -0.3304 and -0.2511, respectively), and deviations from Hardy-Weinberg equilibrium were observed (P

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Understanding the epidemiology and evolution of Staphylococcus aureus (SA) is crucial for implementing effective infection prevention and control measures. In this study, a short-term (4-month) and local molecular epidemiology of SA in the nosocomial setting was investigated using multiple-locus variable number tandem repeat (VNTR) analysis (MLVA). The study included 31 clinical SA isolates. The analysis was based on two complimentary panels encompassing 14 VNTR loci wherein a first-line assay comprised a subset of 10 loci (Panel 1), followed by a second subset of four loci (Panel 2). Thirty MLVA profiles were generated. Small amplicons were obtained for three out of the four loci included in Panel 2 for more than half of the isolates, making the calculation of the number of repeats impossible. Therefore, phylogenetic clustering was based on the analysis of basic loci included in Panel 1 (which identified two major genetic clusters). Cluster I included 26 (84%) isolates, among which 14 were classified into five sub-clusters (cutoff value ≥65%). Only one pair of the isolates shared an identical MLVA pattern. Our study indicates the usefulness of a MLVA tool comprised of a primary subset of 10 loci to track the possibility of SA intra-hospital transmission. We report a high genetic diversity of the isolates and suggest a limited degree of intra- and inter-ward SA transmission. Further genetic studies are necessary to investigate the nature of the small alleles yielded in Panel, as their subset can provide even higher resolution of the assay.

Abstract Background Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. Methods In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). Results Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in İzmir in our previous study. Conclusions High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.

Abstract In January 2020, a different cervical cancer screening program started in Germany. Women above the age of 35 are recommended to have a combined HPV and cytology swab every three years. Showing persistent high-risk human papillomavirus (hrHPV), cytologic negative cervical samples at baseline and after 12 months, patients are referred to colposcopy. Entailing considerable additional workload due to the required colposcopies, we analyzed the risk of high-grade cervical intraepithelial neoplasia (CIN 3) in cytologic negative and persistent hrHPV women according to their hrHPV genotypes. Methods In this single center retrospective study, patients with persistent hrHPV, cytology negative cervical samples from our certified Colposcopy Unit in 2020 and 2021 were analyzed. Patient demographics, hrHPV types, biopsy rates and histological reports were collected. Results During the study, 69 patients were enrolled. Most frequent hrHPV genotypes were: hrHPV other 72.5%; HPV 16, 20.3% and HPV 18, 7.2%. Colposcopy showed no or minor changes in 92.7% and major changes in 7.2%. CIN 3 was found in 7 patients (10.1%). Prevalence of CIN 3 by hrHPV genotypes was 27.3% for HPV16, 20.0% for HPV18 and 7.1% for HPVO. A statistically significant dependency between hrHPV and cervical intraepithelial neoplasia was demonstrated (p = 0.048). Conclusion Within this single center study of persistent hrHPV, cytologic negative samples, patients with HPV 16 were more likely to have high-grade disease compared to other hrHPV subtypes. Larger prospective randomized trials are needed to substantiate our results and obtain adjusted cervical cancer screening time intervals according to the hrHPV genotypes.

Abstract Toxoplasma gondii is an obligate intracellular parasite associated with severe disease, especially in the immunosuppressed. It is also a cause of congenital malformation and abortion in both animals and humans and is considered one of the most important foodborne pathogens worldwide with different strains showing variable distribution and differing pathogenicity. Thus, strain-level differentiation of T. gondii isolates is an essential asset in the understanding of parasite’s diversity, geographical distribution, epidemiology and health risk. Here, we designed and implemented an Oxford Nanopore MinION protocol to analyse genomic sequence variation including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDel’s) of four different genomic loci, part of protein coding genes SAG2, SAG3, ROP17 and ROP21. This method provided results with the sequencing depth necessary for accurate differentiation of T. gondii strains and represents a rapid approach compared to conventional techniques which we further validated against environmental samples isolated from wild wood mice. In summary, multi-locus sequence typing (MLST) of both highly conserved and more polymorphic areas of the genome, provided robust data for strain classification in a platform ready for further adaption for other strains and pathogens.

BackgroundMetformin is the first-line antidiabetic therapy for type 2 diabetes in Mexico, despite recent recommendations highlighting alternatives like GLP-1 receptor agonists for individuals with obesity. Metformin elimination is reliant on liver and kidney function, and variants in transport proteins such as Multidrug and Toxin Extrusion Protein 1 (MATE1), MATE2, and Organic Cation Transporter 2 (OCT2) can influence its pharmacokinetics. Understanding these variants’ frequencies in the Mexican population is crucial for tailoring personalized treatment strategies.ObjectiveThis study aimed to determine the genotypic and allelic frequencies of key variants in metformin transporters within a Mexican population, addressing the interindividual variability in drug response.MethodologyGenetic analysis was conducted on 101 healthy, unrelated Mexican subjects who were genotyped for the MATE1, MATE2, and OCT2 variants using allele-specific real-time PCR assays.ResultsThe allele frequencies were 0.07 for OCT2, 0.23 for MATE1, and 0.67 for MATE2. The g.–66T→C variant was found only in wild-type and heterozygous forms. Comparative analysis indicated significant differences in allele frequencies between this Mexican population and other ethnic groups, highlighting potential implications for metformin efficacy and safety.ConclusionThis study provides crucial insights into the genetic variability of metformin transporter genes in a Mexican population, offering a foundation for personalized therapeutic approaches in type 2 diabetes management.

Genotyping is the process of determining the genetic makeup of an organism by examining its DNA sequences using various genetic markers. It has been widely used in various fields, such as agriculture, biomedical and conservation research, to study genetic diversity, inheritance, the genetic basis of disease-associated traits, evolution, adaptation, etc., Genotyping markers have evolved immensely and are broadly classified as random markers (RFLP, RAPD, AFLP, etc.) and functional markers (SCoT, CDDP, SRAP, etc.). However, functional markers are very limited in genotype studies, especially in animal science, despite their advantages in overcoming the limitations of random markers, which are directly linked with phenotypic traits, high specificity, and similar logistic requirements. The current review surveyed the available random and functional markers for genotyping applications, focusing on livestock including plant and microbe domains. This review article summarises the application, advantages, and limitations of developed markers and methods for genotyping applications. This review aims to make the reader aware of all available markers, their design principles, and methods, and we discuss the marker inheritance patterns of RLFP and AFLP. The review further outlines the marker selection for particular applications and endorses the application of functional markers in genotyping research.

BackgroundMalaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria.MethodsDried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology.ResultIn total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32).Conclusion and recommendationsHere a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

IntroductionNeisseria meningitidis is a gram-negative bacterium responsible for life-threatening invasive infections known as invasive meningococcal disease and is associated with high fatality rates and serious lifelong disabilities among survivors.MethodsThis study aimed to characterize N. meningitidis isolates cultured from blood and cerebrospinal fluid collected between 2009 and 2021 in Lithuania, assess their genomic relationships with European strains, and evaluate the possibility of using a cost-effective method for strain characterization, thus improving the national molecular surveillance of invasive meningococcal disease. In total, 321 N. meningitidis isolates were collected and analyzed using multilocus restriction typing (MLRT). Amplification of the penA gene and restriction fragment length polymorphism analysis were performed to identify the modified penA genes. Based on the MLRT genotyping results, we selected 10 strains for additional analysis using whole-genome sequencing. The sequenced genomes were incorporated into a dataset of publicly available N. meningitidis genomes to evaluate genomic diversity and establish phylogenetic relationships within the Lithuanian and European circulating strains.ResultsWe identified 83 different strains using MLRT genotyping. Genomic diversity of N. meningitidis genomes analysed revealed 21 different sequence types (STs) circulating in Lithuania. Among these, ST34 was the most prevalent. Notably, three isolates displayed unique combinations of seven housekeeping genes and were identified as novel STs: ST16969, ST16901, and ST16959. The analyzed strains were found to possess virulence factors not commonly found in N. meningitidis. Six distinct penA profiles were identified, each with different frequencies. In the present study, we also identified N. meningitidis strains with new penA, NEIS0123, NEIS1320, NEIS1525, NEIS1600, and NEIS1753 loci variants. In our study, using the cgMLST scheme, Minimum Spanning Tree (MST) analysis did not identify significant geographic relationships between Lithuanian N. meningitidis isolates and strains from Europe.DiscussionDiscussion: To our knowledge, this is the first study to employ whole genome sequencing (WGS) method for a comprehensive genetic characterization of invasive N. meningitidis isolates from Lithuania. This approach provides a more detailed and precise analysis of genomic relationships and diversity compared to prior studies relying on traditional molecular typing methods and antigen analysis.

Clostridium perfringens is a causative agent of various human and animal enteric diseases including food poisoning. In this study, we describe an interesting case of a persistent food poisoning outbreak among Finnish peacekeepers in Eritrea, possibly caused by Clostridium perfringens carrying a new variant of the chromosomally encoded enterotoxin gene. C. perfringens strains causing food poisoning carry the enterotoxin gene, cpe, in its chromosome (c-cpe) or on a plasmid (p-cpe). PCR assays are widely used for toxinotype C. perfringens strains. The integration sites for the cpe gene are highly conserved, and PCR assays targeting the cpe gene and the adjacent IS elements (the IS1470 in c-cpe and the IS1470-like or IS1151 in p-cpe strains) are used to further determine the genetic location of the cpe gene. We sequenced nine enteropathogenic C. perfringens strains related to a persistent food poisoning outbreak among Finnish peacekeepers in Eritrea. Six of these strains produced non-typeable cpe results in the standard PCR assay due to divergence in the enterotoxin integration site. The gene order of the new variant of the chromosomal cpe insertion site with an additional IS1470 element impairing genotyping PCR assay for the location of cpe is described. In addition, variant c-cpe strains carried 58–81 copies of IS1470 in their genomes, compared to 9–23 copies found in previously described c-cpe strains. Thus, the present study represents an untraditional type of C. perfringens food poisoning caused by variant c-cpe strains, and the sequenced strains bring geographic variation to the existing strain collection of sequenced C. perfringens.

Nucleic acid amplification assays represent a pivotal category of methodologies for targeted sequence detection within contemporary biological research, boasting diverse utility in diagnostics, identification, and DNA sequencing. The foundational principles of these assays have been extrapolated to various simple and intricate nucleic acid amplification technologies. Concurrently, a burgeoning trend toward computational or virtual methodologies is exemplified by in silico PCR analysis. In silico PCR analysis is a valuable and productive adjunctive approach for ensuring primer or probe specificity across a broad spectrum of PCR applications encompassing gene discovery through homology analysis, molecular diagnostics, DNA profiling, and repeat sequence identification. The prediction of primer and probe sensitivity and specificity necessitates thorough database searches, accounting for an optimal balance of mismatch tolerance, sequence similarity, and thermal stability. This software facilitates in silico PCR analyses of both linear and circular DNA templates, including bisulfited treatment DNA, enabling multiple primer or probe searches within databases of varying scales alongside advanced search functionalities. This tool is suitable for processing batch files and is essential for automation when working with large amounts of data.

Coxsackievirus A16 (CV-A16) is one of the major pathogens of Hand, Foot and Mouth disease. Here, we analyzed 287 full-length genome sequences of CV-A16 found worldwide from 1994 to 2019 to see the genomic evolution characteristics. Full-length genome-based phylogenetic tree divided the viruses into five different genotypes, G-a to G-e. The CV-A16 strains circulating in China dominate G-a and G-c, but can also be found in other genotypes including G-b and G-e. Phylogeographic analysis showed a high diversity of CV-A16 distribution. In addition, recombination was shown to drive the genomic evolution of CV-A16 during past decades. However, the structural proteins still remain relative conserved while there is extensive genomic recombination. This study updates the phylogenetic and phylogeographic information of CV-A16 and provides insights into the genetic characteristics of CV-A16.

Abstract Premise The prompt categorization of Phytophthora infestans isolates into described clonal lineages is a key tool for the management of its associated disease, potato late blight. New isolates of this pathogen are currently classified by comparing their microsatellite genotypes with characterized clonal lineages, but an automated classification tool would greatly improve this process. Here, we developed a flexible machine learning–based classifier for P. infestans genotypes. Methods The performance of different machine learning algorithms in classifying P. infestans genotypes into its clonal lineages was preliminarily evaluated with decreasing amounts of training data. The four best algorithms were then evaluated using all collected genotypes. Results mlpML, cforest, nnet, and AdaBag performed best in the preliminary test, correctly classifying almost 100% of the genotypes. AdaBag performed significantly better than the others when tested using the complete data set (Tukey HSD P

IntroductionThe epidemic spread of the harmful bacterium Xylella fastidiosa causing the “olive quick decline syndrome”, decimating olive trees in southern Italy, in the region of Apulia, prompted investigations to search for olive genotypes harbouring traits of resistance.MethodsA prospecting survey was carried out to identify, in the heavily infected area of Apulia, olive genotypes bearing resistance. Given the limited genetic diversity in the commercial olive groves with few cultivars widely cultivated, surveys targeted predominantly spontaneous olive genotypes in natural and uncultivated areas. Trees, selected for the absence of symptoms, were subjected to diagnostic tests and parentage analysis to disclose their genetic background. Transcriptomic analyses were also employed to decipher the molecular pathways in resistant genotypes. Artificial inoculations were carried out to confirm the resistant phenotypes of four open-pollinated seedlings of the cultivar Leccino.ResultsAmong the 171 olive collected genotypes, 139 had unique simple sequence repeat (SSR) profiles, with the cultivars Leccino, Cellina di Nardò, and Ogliarola salentina being the most frequent candidate parents. Among the Leccino progeny (n. 61), 67% showed a highly resistant (HR), resistant (R), or tolerant (T) phenotype to infection by X. fastidiosa. The occurrence of such phenotypes among those deriving from Cellina di Nardò and Ogliarola salentina was 32% and 49%, respectively. Analyses of the transcriptomic profiles of three Leccino-bearing genotypes, naturally infected and not showing symptoms, unravelled that a total of 17,227, 13,031, and 4,513 genes were found altered in the expression, including genes involved in photosynthesis, cell wall, or primary and secondary metabolism.DiscussionIndeed, transcriptomic analyses showed that one of these genotypes (S105) was more resilient to changes induced by the natural bacterial infection than the remaining two (S215 and S234). This study consolidates the evidence on the presence and heritage of resistance traits associated with the cv. Leccino. Moreover, valuable insights were gathered when analysing their transcriptomic profiles, i.e., genes involved in mechanisms of response to the bacterium, which can be used in functional genetic approaches to introduce resistance in susceptible cultivars and initiate strategies in olive-breeding programs through marker-assisted selection.

Background: Scrub typhus, an acute febrile disease caused by Orientia tsutsugamushi, is transmitted to humans through infected chigger mites. We present a case of scrub typhus in a previously healthy man from Shandong Province diagnosed using next-generation sequencing (NGS) and PCR and review recent literature on NGS for scrub typhus diagnosis. Methods: NGS was utilized for testing whole blood collected on admission. Confirmatory testing was done by detecting IgM and IgG antibodies to Orientia in acute and convalescent sera by ELISA. Orientia 47-kDa protein gene TaqMan and standard PCR of the 56-kDa protein gene and Sanger sequencing were performed on eschar scab DNA. Results: The NGS diagnosis was confirmed by 47-kDa protein gene TaqMan and sequencing of a fragment of the O. tsutsugamushi 56-kDa protein gene from the eschar scab. Analysis of this sequence and the NGS data indicated O. tsutsugamushi strain Cheeloo2020 is a novel genotype. Mapping of the NGS data against the O. tsutsugamushi Gilliam strain genome sequence identified 304 reads with high similarity. Conclusions: NGS is not only useful for multiplex diagnosis of scrub typhus, but also provides insight into the genetic diversity of O. tsutsugamushi. The common failure to submit sequences to databases makes it difficult to determine the minimal quantity and quality of NGS data being used for the positive identification of Orientia DNA in clinical specimens.

IntroductionPiscirickettsia salmonis, the causative agent of Piscirickettsiosis, poses a significant threat to the Chilean aquaculture industry, resulting in substantial economic losses annually. The pathogen, first identified as specie in 1992, this pathogen was divided into two genogroups: LF-89 and EM-90, associated with different phenotypic mortality and pathogenicity. Traditional genotyping methods, such as multiplex PCR, are effective but limited by their cost, equipment requirements, and the need for specialized expertise.MethodsThis study validates Loop-mediated Isothermal Amplification (LAMP) as a rapid and specific alternative for diagnosing P. salmonis infections. We developed the first qPCR and LAMP assay targeting the species-conserved tonB receptor gene (tonB-r, WP_016210144.1) for the specific species-level identification of P. salmonis. Additionally, we designed two genotyping LAMP assays to differentiate between the LF-89 and EM-90 genogroups, utilizing the unique coding sequences Nitronate monooxygenase (WP_144420689.1) for LF-89 and Acid phosphatase (WP_016210154.1) for EM-90.ResultsThe LAMP assays demonstrated sensitivity and specificity comparable to real-time PCR, with additional benefits including rapid results, lower costs, and simplified operation, making them particularly suitable for field use. Specificity was confirmed by testing against other salmonid pathogens, such as Renibacterium salmoninarum, Vibrio ordalii, Flavobacterium psychrophilum, Tenacibaculum maritimum, and Aeromonas salmonicida, with no cross-reactivity observed.DiscussionThe visual detection method and precise differentiation between genogroups underscore LAMP's potential as a robust diagnostic tool for aquaculture. This advancement in the specie detection (qPCR and LAMP) and genotyping of P. salmonis represents a significant step forward in disease management within the aquaculture industry. The implementation of LAMP promises enhanced disease surveillance, early detection, and improved management strategies, ultimately benefiting the salmonid aquaculture sector.

ABSTRACT Faba bean (Vicia faba L.) is an important pulse crop with a wide range of agroecological adaptations. The development of genomic tools and a comprehensive catalog of extant genetic diversity are crucial for developing improved faba bean cultivars. The lack of a cost‐effective genotyping platform limits the characterization of large germplasm collections, understanding of genetic diversity across populations, and implementing breeder's tools like genomic selection. Genotyping‐by‐sequencing (GBS) offers high‐resolution genotyping for both model and crop plant species, even without a reference genome sequence. The genome fragments targeted by GBS depend substantially on the restriction enzyme (RE) used for the complexity reduction step. Species with complex genomic architecture require optimization of GBS with proper RE to realize the full potential of GBS. Here, we evaluated various REs in the GBS method and identified that the combination of ApeKI/MseI proved to be the most appropriate for faba bean based on the best library quality, a high number of genomic loci spread across chromosomes, and high enrichment loci associated with the gene space. With the new optimized protocol, we constructed a genetic map using a recombinant inbred line (RIL) population and identified a QTL for seed hilum color on Chromosome 1. In addition, we also genotyped a diversity panel and performed a genome‐wide association studies (GWAS) for important agronomic traits, including plant height (PH), flowering time (FT), and number of pods per plant (PPP). We identified six SNP markers significantly associated with these traits and listed potential candidate genes. The optimized faba bean‐specific GBS procedure will facilitate access to the untapped genetic diversity for genetic research and breeding and may facilitate functional genomics.

PurposeWe aimed to retrospectively investigate an outbreak of linezolid-resistant Staphylococcus epidermidis (LRSE), at Tours University Hospital between 2017 and 2021.MethodsTwenty of the 34 LRSE isolates were included in the study. Antimicrobial susceptibility testing was performed using the disk diffusion method and MICs of last-resort antibiotics were determined using broth microdilution or Etest®. Seventeen of the 20 resistant strains were sent to the French National Reference Centre for Staphylococci to determine the mechanism of resistance to linezolid. The clonal relationship between LRSE strains was assessed by PFGE and the sequence type determined by MLST. We retrospectively evaluated a new typing tool, IR-Biotyper®, and compared its results to PFGE to evaluate its relevance for S. epidermidis typing. Medical records were reviewed, and antibiotic consumption was determined. Search for a cross transmission was performed.ResultsAll LRSE strains showed high levels of resistance to linezolid (MICs ≥ 256 mg/L) and were multi-drug resistant. Linezolid resistance was associated with the 23S rRNA G2576T mutation and none of the 17 strains analyzed carried the cfr gene. Ninety-five percent of the 20 LRSE studied strains were genetically related and belonged to sequence-type ST2. The dendrogram obtained from IR-Biotyper® showed 87% congruence with the PFGE analysis. Prior to isolation of the LRSE strain, 70% of patients received linezolid. No patients stayed successively in the same room.ConclusionLinezolid exposure may promote the survival and spread of LRSE strains. At Tours University Hospital, acquisition of the resistant clone may also have been triggered by hand-to-hand transmission by healthcare workers. In addition, IR-Biotyper® is a promising typing tool for the study of clonal outbreaks due to its low cost and short turnaround time, although further studies are needed to assess the optimal analytical parameters for routine use.

Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein (eHRG) with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of eHRG in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed eHRG genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed eHRG genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for eHRG genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.

Cultivated and wild species of the genus rye (Secale) are important but underexploited gene sources for increasing the genetic diversity of bread wheat. Gene transfer is possible via bridge genetic materials derived from intergeneric hybrids. During this process, it is essential to precisely identify the rye chromatin in the wheat genetic background. In the present study, backcross generation BC2F8 from a cross between Triticum aestivum (Mv9kr1) and S. cereanum (‘Kriszta,’ a cultivar from the artificial hybrid of S. cereale and S. strictum) was screened using in-situ hybridization (GISH and FISH) and analyzed by DArTseq genotyping in order to select potentially agronomically useful genotypes for prebreeding purposes. Of the 329,267 high-quality short sequence reads generated, 27,822 SilicoDArT and 8,842 SNP markers specific to S. cereanum 1R–7R chromosomes were identified. Heatmaps of the marker densities along the ‘Lo7’ rye reference pseudomolecules revealed subtle differences between the FISH- and DArTseq-based results. This study demonstrates that the “exotic” rye chromatin of S. cereanum introgressed into wheat can be reliably identified by high-throughput DArTseq genotyping. The Mv9kr1-’Kriszta’ addition and translocation lines presented here may serve as valuable prebreeding genetic materials for the development of stress-tolerant or disease-resistant wheat varieties.

Abstract In South America, Tambaqui (Colossoma macropomum) stands as the primary target for aquaculture, yet breeding programs for this Amazon native species are in their early stages. While high-density single nucleotide polymorphism (SNP) arrays are pivotal for aquaculture breeding, their costs can be prohibitive for non- or semi-industrial species. To overcome this, a cost-effective approach involves developing low-density SNP arrays followed by genotype imputation to higher densities. In this study, a 1K SNP array for tambaqui was created and validated, offering a balance between SNP quantity and genome representativity. The imputation accuracy from various SNP densities to a medium-density array was evaluated, with the 1K density demonstrating the best trade-off (accuracy of 0.93). This subset was further utilized to construct a commercial array through Agriseq™ targeted genotyping-by-sequencing, validated in 192 DNA samples, affirming its high quality for genotyping tambaqui. The low-density SNP array, with genome-wide coverage and high polymorphism, emerges as an effective tool for exploring genetic variation within diverse populations. Population analyses using the 1K panel proved to be an efficient tool for genetic characterization of sampled broodstocks, making it a valuable resource for genetic improvement programs targeting this Amazon native species.

Cats, being the definitive host of Toxoplasma gondii, have a significant impact on the spread and outbreaks of the parasite. An essential factor in comprehending the transmission pattern of this parasite is an analysis of the genetic diversity distribution in cats infected with T. gondii. This study was aimed at determining the prevalence rate and genotyping of T. gondii in stray cat feces from Khorramabad, West Iran. In the years 2016–2017, 200 cats were sampled to get fresh feces specimens. Parasitological methods were utilized for the identification of oocysts. The DNA was isolated from the feces using a commercially available Genomic Mini Kit. In order to identify the genetic composition of T. gondii, we employed PCR-RFLP, sequencing, and phylogenetic analysis of the GRA6 target gene. No one of the samples tested positive for parasitology techniques. A total of 6.5 % (13/200) samples were positive when using the GRA6-PCR method. Based on PCR-RFLP results, all 13 samples were of T. gondii type III genotype. The nucleotide sequences of two samples from this study were found to be 5 % different from those of 12 references of T. gondii and one strain of Hammondia hamondi that was used as an external control. Based on the findings, molecular tests are more sensitive than parasitological methods. The RFLP approach revealed that type III of T. gondii is the prevailing and important genotype in Khorramabad, West Iran.