Thyasirid species composition (Bivalvia: Thyasiridae) and genetic connectivity of Parathyasira equalis (A. E. Verrill & K. J. Bush, 1898) in deep basins of sub-Arctic fjords.

BACKGROUND: Thyasirid bivalves are often recorded as a dominant component of macrobenthic infaunal communities in depositional environments such as fjord basins. Fjord basins comprise patchy soft-bottom habitats bounded by steep walls and sills; however, little is known how this semi-isolated nature of fjords affects benthic populations. Accordingly, data on the composition and population connectivity of thyasirids can provide valuable information on the ecology of these ecosystems. RESULTS: The species composition of thyasirid bivalves has been studied in the basins of three sub-Arctic fjords (Nordland, Northern Norway). Overall, six thyasirid species were recorded: Parathyasira equalis, Parathyasira dunbari, Mendicula ferruginosa, Genaxinus eumyarius, Thyasira sarsii, and Thyasira obsoleta. The species composition remained stable within the basins during the sampling period (2013-2020) and suggested the importance of local reproduction over advection of individuals for population dynamics. Only one species, Parathyasira equalis, was common in all fjords. We have further investigated the population genetics of this species by combining two types of genetic markers: a 579 bp fragment of the cytochrome c oxidase subunit I (COI) gene and 4043 single-nucleotide polymorphisms (SNPs) generated by genotyping-by-sequencing. The latter provided a more in-depth resolution on the population genetics of this species and revealed a weak but significant differentiation of populations within fjords, further indicating limited connectivity between basins. CONCLUSION: Based on our findings, we conclude that limited dispersal between the basin communities results in weakly connected populations and might be an important structuring factor for macrobenthic communities.

Genetic diversity of the PvMSP-3α gene in Plasmodium vivax isolates circulating in the National Capital Region (NCR) of India.

Malaria is still a public health problem in tropical countries like India; major malaria parasite species are Plasmodium falciparum and P. vivax. Of which, P. vivax is responsible for ∼40% of the malaria burden at least in the Indian scenario. Unfortunately, there is limited data on the population structure and genetic diversity of P. vivax parasites in India. In this study, we investigated the genetic diversity of P. vivax strains in the South-west district, Delhi and, Nuh district, Haryana [National Capital Region (NCR)], using a polymorphic marker- P. vivax merozoite surface protein-3α (PvMSP-3α) gene. Dried blood spots from microscopically confirmed P. vivax patients were used for investigation of the PvMSP-3α gene. PCR-RFLP was performed on the PvMSP-3α gene to investigate the genotypes and allelic variability with HhaI and AluI restriction enzymes. In total, 40 successfully PCR amplified PvMSP-3α gene segments were subjected to RFLP analysis. Amplified products showed three different base pair size variations viz. genotype A in 31(77.5%), genotype B in 4(10%) and genotype C in 5(12.5%) P. vivax specimens. RFLP with HhaI and AluI revealed 17 (H1-H17) and 25 (A1-A25) allelic variants, respectively. Interestingly, two similar sub-allelic variants, ie. H8 (with HhaI), and A4 (with AluI) clustered within the rural area of Nuh district, Haryana in two samples. With this study, we propose to commission such type of genetic diversity analysis of P. vivax to investigate the circulating genotypes of the parasites from distinct geographical locations across India, that can have significant implications in understanding the population structures of P. vivax.

Predictive accuracy of genetic variants for eye color in a Kazakh population using the IrisPlex system.

OBJECTIVE: This study assesses the accuracy of the IrisPlex system, a genetic eye color prediction tool for forensic analysis, in the Kazakh population. The study compares previously published genotypes of 515 Kazakh individuals from varied geographical and ethnohistorical contexts with phenotypic data on their eye color, introduced for the first time in this research. RESULTS: The IrisPlex panel's effectiveness in predicting eye color in the Kazakh population was validated. It exhibited slightly lower accuracy than in Western European populations but was higher than in Siberian populations. The sensitivity was notably high for brown-eyed individuals (0.99), but further research is needed for blue and intermediate eye colors. This study establishes IrisPlex as a useful predictive tool in the Kazakh population and provides a basis for future investigations into the genetic basis of phenotypic variations in this diverse population.

Evaluation of early fluoropyrimidine toxicity in solid organ cancer patients: a retrospective observational study in Australia.

BACKGROUND: Despite common global usage, fluoropyrimidine (FP; 5-flurouracil and capecitabine)-related chemotherapy toxicity is poorly reported in the literature, with serious toxicity ranging from 10% to 40% and early toxicity (within 60 days of exposure) quoted at 14%. Data reflecting the incidence of Grades 3-5 FP-related toxicity in Australian cancer patients is scant, despite the significant impact of toxicity on patients (hospitalisations, intensive care unit (ICU) admissions and even death). AIMS: This retrospective audit evaluated Grades 3-5 toxicities in a contemporaneous cohort of 500 patients receiving FP chemotherapies within the Hunter-New England Local Health District from June 2020 to June 2022. Data were extracted from public hospital records and oncology-specific e-records to determine rates of toxicity and associated hospitalisations, intensive care admissions and deaths that occurred within 60 days of first exposure to FP chemotherapy-containing regimens. RESULTS: One hundred and fifty incidents of Grades 3-4 toxicity in the first 60 days led to 87 patients presenting to hospital (87/500, 17.4%). The most common serious toxicities were diarrhoea (39.3%), nausea and vomiting (22.7%) and febrile neutropaenia (10%). Four patients were admitted to the ICU, and four patients died of toxicity. Within the first 60 days, 22.2% of patients required treatment delays, 21.4% required dose reductions, and 7.8% of patients ceased treatment because of toxicities. DISCUSSION AND CONCLUSION: Our experience reflects international reports and is likely generalisable to the Australian population. These data are a basis to understand the potential benefits of precision medicine strategies such as pharmacogenomic screening to improve patient tolerability and the cost-effectiveness of FP chemotherapy prescribing.

Epidemiological exploration of fleas and molecular identification of flea-borne viruses in Egyptian small ruminants.

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 < 0.05). Ctenocephalides felis predominated in NWC and Ctenocephalides canis in SSG, and males of both flea species were more prevalent than females. Molecular analysis of flea DNA revealed the presence of Capripoxvirus in all tested samples, while other viral infections were absent. Gene sequencing identified three isolates as sheeppox viruses, and one as goatpox virus. The findings suggest that Capripoxvirus is adapted to fleas and may be transmitted to animals through infestation. This underscores the need for ongoing surveillance of other pathogens in different regions of Egypt.

Clustered Regularly Interspaced Short Palindromic Repeat-1 (CRISPR-1) Locus as a Tool for Tracing the Zoonotic History of Salmonella enterica Strains.

Background Salmonella enterica is a significant foodborne pathogen that causes considerable illness and death in humans and animals. The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system in bacteria acts as an adaptive immune defense against invasive genetic elements by incorporating short intergenic spacers (IGSs) into CRISPR loci. These loci serve as molecular records of past interactions with phages and plasmids, providing insights into the transmission and evolution of bacterial strains across different hosts. Aim This study aimed to investigate the diversity of IGSs in the CRISPR-1 locus of S. enterica isolates from humans and camels. The objective was to assess the potential of IGSs to distinguish strains, track sources, and understand patterns of zoonotic transmission. Materials and methods Genomic DNA was extracted from multiple strains of S. enterica, and the CRISPR-1 locus was polymerase chain reaction (PCR) amplified and sequenced. The sequences were compared to identify distinct patterns of IGSs and potential host-specific characteristics. Sanger sequencing and bioinformatics tools were used to classify the IGSs and determine their similarity to known sequences in the National Center for Biotechnology Information (NCBI) database. Results Sequence analysis revealed five distinct CRISPR-1 types among S. enterica isolates from humans and three among camel isolates. The presence of shared IGSs between human and camel S. enterica isolates suggested zoonotic or reverse-zoonotic transmission events. Additionally, host-specific unknown IGSs (UIGS) were identified. Importantly, camel isolates initially identified as S. enterica subspecies enterica serovar Enteritidis based on rrnH gene sequencing were reclassified as S. enterica serovar Enteritidis based on CRISPR-1 profiling, demonstrating the higher resolution of CRISPR-based genotyping. Conclusion The diversity of IGSs in the CRISPR-1 locus effectively differentiated S. enterica strains and provided insights into their evolutionary origins and transmission dynamics. CRISPR-based genotyping proves to be a promising tool to complement traditional serotyping methods, enhancing the molecular epidemiology of salmonellosis and potentially leading to better management and control strategies for this pathogen.

Identification of the new allele HLA-C*04:01:01:186 in a Greek individual using next generation sequencing.

The newly discovered HLA-C*04:01:01:186 allele differs from HLA-C*04:01:01:01 by a single nucleotide substitution in intron 3.

Mapping of Aegilops speltoides derived leaf rust and stripe rust resistance genes using 35K SNP array.

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 (ILsp3603) 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 Lrsp3603 and Yrsp3603), inheritance studies were carried out in F6 and F7 mapping population, developed by crossing ILsp3603 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. Lrsp3603 was flanked by markers Tag-SSR14 (located proximally at 2.42 cM) and SNP AX-94542331 (at 3.28 cM) while Yrsp3603 was mapped at one end closest to AX-94542331 at 6.62 cM distance. Functional annotation of Lrsp3603 target region (∼ 1 Mbp) revealed 10 gene IDs associated with disease resistance mechanisms including three encoding typical R gene domains.

Investigation of Mitochondrial Cytb Gene Region of Both Echinococcus granulosus Eggs from Dogs and Cystic Echinococcosis Isolates Obtained from Sheep and Cattle by Molecular Methods.

Background: We aimed to determine the common Echinococcus granulosus genotypes in Agri, Türkiye and to obtain information on the transmission of this parasite. Methods: Cystic echinococcosis samples from 100 slaughtered cattle and 100 slaughtered sheep and faecal samples from 200 stray dogs were included in 2021. Collected cyst fluid samples and faces were examined microscopically. DNA was isolated from the germinal membrane of the cysts and from the parasite eggs in the stool samples. The mitochondrial cytb gene region of the parasite was amplified by PCR. Genotypes were determined using the Basic Local Alignment Search Tool (BLAST) after sequence analysis of PCR amplicons. Results: The highest percentage of cysts was found in the lungs of sheep and the liver of cattle. In addition, 75% of sheep cysts and 25.6% of cattle cysts were fertile. Taenia spp./Echinococcus spp. eggs were found in 6% of the faeces of 200 dogs examined microscopically. E. granulosus eggs were detected in 4 out of 50 stool samples analysed by PCR. All samples analysed by sequence analysis were identified as E. granulosus s.s. G1 genotype. Sequence comparison revealed revealed one or more-point mutations in different regions of the five samples. Conclusion: E. granulosus s.s. G1 genotype, known as sheep strain, is common in the Agri, Türkiye. The controlled slaughter of livestock, especially sheep, and the avoidance of feeding hydatid cyst organs to dogs, together with public education, were necessary to prevent the spread of the disease.

Further screening of SNP loci of eggshell translucency related genes and evaluation of genetic effects.

Eggshell translucency is a widespread issue in the field of egg quality. Previous research has established that the heritability of eggshell translucency is relatively low or moderate. Scientists have also successfully identified SNP loci related to eggshell translucency on different chromosomes by using gene chips and single-variant GWAS. However, the specific impact of single or multiple genes on the trait of eggshell translucency remains unknown. In an effort to investigate this, we examined 170 SNPs associated with eggshell translucency obtained by our research group. We selected 966 half-sibling laying hens from 2 generations in 3 pure lines: Dwarf Layer-White, Rhode Island Red-White Strain, and Rhode Island Red. Eggs were collected from each hen over a period of 5 consecutive days, and eggshell translucency was measured using a grading method in which the hens were divided into 2 groups: an opaque group and a translucent group. We collected blood samples from the laying hens and extracted DNA. Time of flight mass spectrometry (TOF-MS) was used for genotyping to identify SNP loci that influence the trait of eggshell translucency. The results of our analysis revealed that using TOF-MS in 3 chicken strains, we were able to eliminate loci with low gene polymorphism, genetic effect contribution less than 1%, and deviation from Hardy-Weinberg equilibrium. Ultimately, 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were identified on chromosomes 1, 5, and 19. Additionally, nine candidate genes (DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A) were found to be associated with these SNPs. No linkage disequilibrium relationship was observed between the 2 pairs of SNP loci on chromosomes 1 and 19. Based on previous studies on the formation mechanism of eggshell translucency, we hypothesize that NUCB2, FTL, and ACACA genes may be affecting the eggshell structure through different mechanisms, such as increase the water permeability or make thin of eggshell membrane, which promote moisture or part of other egg contents and ultimately lead to the formation of eggshell translucency. These findings validate and identify five SNP loci that regulate the translucency trait, and provide molecular markers for breeding non-translucent populations. Furthermore, this study serves as a reference for further investigation of the genetic regulatory mechanisms underlying eggshell translucency.

The impact of geographic isolation and host shifts on population divergence of the rare cicada Subpsaltria yangi.

The contributions of divergent selection and spatial isolation to population divergence are among the main focuses of evolutionary biology. Here we employed integrated methods to explore genomic divergence, demographic history and calling-song differentiation in the cicada Subpsaltria yangi, and compared the genotype and calling-song phenotype of different populations occurring in distinct habitats. Our results indicate that this species comprises four main lineages with unique sets of haplotypes and calling-song structure, which are distinctly associated with geographic isolation and habitats. The populations occurring on the Loess Plateau underwent substantial expansion at âˆ¼0.130-0.115 Ma during the Last Interglacial. Geographic distance and host shift between pairs of populations predict genomic divergence, with geographic distance and acoustical signal together explaining > 60% of the divergence among populations. Differences in calling songs could reflect adaptation of populations to novel environments with different host plants, habitats and predators, which may have resulted from neutral divergence at the molecular level followed by natural selection. Geomorphic barriers and climate oscillations associated with Pleistocene glaciation may have been primary factors in shaping the population genetic structure of this species. Ultimately this may couple with a host shift in leading toward allopatric speciation in S. yangi, i.e., isolation by distance. Our findings improve understanding of divergence in allopatry of herbivorous insects, and may inform future studies on the molecular mechanisms underlying the association between genetic/phenotypic changes and adaptation of insects to novel niches and host plants.

GeCKO: user-friendly workflows for genotyping complex genomes using target enrichment capture. A use case on the large tetraploid durum wheat genome.

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.

Genetic analysis of feline parvovirus reveals predominance of feline parvovirus -G1 group among cats in China.

Feline parvovirus (FPV) or feline panleukopenia virus is a highly contagious, life-threatening infectious virus in cats. Although FPV vaccination is routinely practiced in China, clinical diseases continue to occur. The investigation of genotypes and viral evolution can contribute to the prevention, diagnosis, and treatment of FPV. Therefore, this study aimed to provide an up-to-date understanding of the epidemiological, genotypic, and phylogenetic characteristics of FPV. In total, 152 rectal swabs were collected from diseased cats. All swab samples were tested for FPV using molecular methods. Amplification of the complete viral protein 2 (VP2) gene was performed for further analysis and to infer the genotypic and evolutionary characteristics of FPV. Of the 152 samples, FPV DNA was detected in 17 (17/152, 11.18%). Cats with FPV showed variable clinical signs such as dehydration, anorexia, fever, vomiting, and blood-stained diarrhea. Furthermore, VP2 sequences were identified in 17 PCR-positive cats, confirming the presence of FPV. Phylogenetic and nucleotide pairwise identity analyses revealed high genetic similarity among FPV sequences (99.6-100%) and clustered them into the FPV-G1 group. Amino acid analysis indicated a novel mutation (Ala91Ser) in all VP2 gene sequences amplified in this study. Our study provides baseline epidemiological data for the better prevention of FPV with respect to vaccination strategies. Genotypic and phylogenetic analyses confirm that FPV-G1 was the predominant FPV group in infected cats in Kunshan. Therefore, a rigorous countrywide investigation of the genotypic and evolutionary characteristics of FPV is warranted.

Comparison of serological and molecular methods for differentiation between genotype A and genotype B strains of small ruminant lentiviruses.

Introduction: Small ruminant lentiviruses (SRLV) cause multisystemic, degenerative and chronic disease in sheep and goats. There are five genotypes (A, B, C, D and E), of which A and B are the most widespread. The purpose of this study was to evaluate the serotyping efficiency of the Eradikit SRLV Genotyping ELISA and the molecular typing efficiency of a newly developed nested real-time PCR targeting the long terminal repeat-gag (LTR-gag) region using samples from animals infected with subtypes of SRLV known to circulate in Poland. Material and Methods: A total of 97 sera samples taken from 34 sheep and 63 goats were immunoassayed, and 86 DNA samples from 31 sheep and 55 goats were tested with the PCR. All ruminants were infected with known SRLV strains of the A1, A5, A12, A13, A16, A17, A18, A23, A24, A27, B1 and B2 subtypes. Results: A total of 69 (80.2%, 95% confidence interval 71.6%-88.8%) out of 86 tested samples gave positive results in the PCR. In 17 out of the 86 (19.8%) samples, no proviral DNA of SRLV was detected. The differentiation between MVV (genotype A) and CAEV (genotype B) by PCR matched the predating phylogenetic analysis invariably. No cross-reactivity was observed. On the other hand, the proportion of samples genotyped the same by the older phylogenetic analysis and the Eradikit SRLV Genotyping ELISA was 42.3%. The test was unable to classify 40.2% of samples, and 17.5% of sera were incorrectly classified. Conclusion: Our results showed that the Eradikit SRLV genotyping kit is not a reliable method for predicting SRLV genotype, while the nested real-time PCR based on the LTR-gag region did prove to be, at least for genotypes A and B.

Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces.

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.

Population genetics of Todarodes pacificus (Cephalopoda: Ommastrephidae) in the northwest Pacific Ocean via GBS sequencing.

The common squid, Todarodes pacificus, is an important commercial species that inhabits the northwest Pacific Ocean, particularly the East Japan Sea, the Pacific coast of Japan, and the East China Sea. In this study, we chose 29 individuals from three areas: one type from the Sea of Japan and two types from the East China Sea. A total of 43,529 SNPs were obtained using genotyping-by-sequencing (GBS). Our analyses revealed low genetic diversity and genetic differentiation in each type. Heterozygote deficiency and inbreeding have caused this low level of genetic diversity. Population structure analysis indicated that the three types were genetically similar, which may be attributed to strong gene flow combined with the demographic history events during the last 2 million years. This new GBS application technique provides valuable information for the conservation of marine species genetics and could be useful for the effective management of this resource.

Allele-specific PCR with fluorescently labeled probes: criteria for selecting primers for genotyping.

Single-nucleotide polymorphisms (SNPs) can serve as reliable markers in genetic engineering, selection, screening examinations, and other fields of science, medicine, and manufacturing. Whole-genome sequencing and genotyping by sequencing can detect SNPs with high specificity and identify novel variants. Nonetheless, in situations where the interest of researchers is individual specific loci, these methods become redundant, and their cost, the proportion of false positive and false negative results, and labor costs for sample preparation and analysis do not justify their use. Accordingly, accurate and rapid methods for genotyping individual alleles are still in demand, especially for verification of candidate polymorphisms in analyses of association with a given phenotype. One of these techniques is genotyping using TaqMan allele-specific probes (TaqMan dual labeled probes). The method consists of real-time PCR with a pair of primers and two oligonucleotide probes that are complementary to a sequence near a given locus in such a way that one probe is complementary to the wild-type allele, and the other to a mutant one. Advantages of this approach are its specificity, sensitivity, low cost, and quick results. It makes it possible to distinguish alleles in a genome with high accuracy without additional manipulations with DNA samples or PCR products; hence the popularity of this method in genetic association studies in molecular genetics and medicine. Due to advancements in technologies for the synthesis of oligonucleotides and improvements in techniques for designing primers and probes, we can expect expansion of the possibilities of this approach in terms of the diagnosis of hereditary diseases. In this article, we discuss in detail basic principles of the method, the processes that influence the result of genotyping, criteria for selecting optimal primers and probes, and the use of locked nucleic acid modifications in oligonucleotides as well as provide a protocol for the selection of primers and probes and for PCR by means of rs11121704 as an example. We hope that the presented protocol will allow research groups to independently design their own effective assays for testing for polymorphisms of interest.

Bidirectional transfer of human cytomegalovirus strains in donor and recipient seropositive lung transplant patients.

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post-transplantation (post-Tx) strain population is unknown. Here, we deciphered the D and R origin of the post-Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin-fixed paraffin-embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/- alveolar macrophages. In three LTRs, we demonstrate both rapid D-strain dissemination and persistence in the transplanted lung for >1 year post-Tx. Broad inter-host diversity contrasts with intra-host genotype sequence stability upon transmission, during follow-up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long-term in subsequent episodes of infection, indicating replication of both sources despite pre-existing immunity.

Genetic Diversity and Population Structure of a Large USDA Sesame Collection.

Sesame, Sesamum indicum L., is one of the oldest domesticated crops used for its oil and protein in many parts of the world. To build genomic resources for sesame that could be used to improve sesame productivity and responses to stresses, a USDA sesame germplasm collection of 501 accessions originating from 36 countries was used in this study. The panel was genotyped using genotyping-by-sequencing (GBS) technology to explore its genetic diversity and population structure and the relatedness among its accessions. A total of 24,735 high-quality single-nucleotide polymorphism (SNP) markers were identified over the 13 chromosomes. The marker density was 1900 SNP per chromosome, with an average polymorphism information content (PIC) value of 0.267. The marker polymorphisms and heterozygosity estimators indicated the usefulness of the identified SNPs to be used in future genetic studies and breeding activities. The population structure, principal components analysis (PCA), and unrooted neighbor-joining phylogenetic tree analyses classified two distinct subpopulations, indicating a wide genetic diversity within the USDA sesame collection. Analysis of molecular variance (AMOVA) revealed that 29.5% of the variation in this population was due to subpopulations, while 57.5% of the variation was due to variation among the accessions within the subpopulations. These results showed the degree of differentiation between the two subpopulations as well as within each subpopulation. The high fixation index (FST) between the distinguished subpopulations indicates a wide genetic diversity and high genetic differentiation among and within the identified subpopulations. The linkage disequilibrium (LD) pattern averaged 161 Kbp for the whole sesame genome, while the LD decay ranged from 168 Kbp at chromosome LG09 to 123 Kbp in chromosome LG05. These findings could explain the complications of linkage drag among the traits during selections. The selected accessions and genotyped SNPs provide tools to enhance genetic gain in sesame breeding programs through molecular approaches.