Haplotype Patterns Research Articles

Nanopore sequencing with unique molecular identifiers enables accurate mutation analysis and haplotyping in the complex lipoprotein(a) KIV-2 VNTR.

Repetitive genome regions, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR), are major constituents of the uncharted dark genome and evade conventional sequencing approaches. The protein-coding LPA kringle IV type-2 (KIV-2) VNTR (5.6kb per unit, 1-40 units per allele) is a medically highly relevant example with a particularly intricate structure, multiple haplotypes, intragenic homologies, and an intra-VNTR STR. It is the primary regulator of plasma lipoprotein(a) [Lp(a)] concentrations, an important cardiovascular risk factor. Lp(a) concentrations vary widely between individuals and ancestries. Multiple variants and functional haplotypes in the LPA gene and especially in the KIV-2 VNTR strongly contribute to this variance. We evaluated the performance of amplicon-based nanopore sequencing with unique molecular identifiers (UMI-ONT-Seq) for SNP detection, haplotype mapping, VNTR unit consensus sequence generation, and copy number estimation via coverage-corrected haplotypes quantification in the KIV-2 VNTR. We used 15 human samples and low-level mixtures (0.5 to 5%) of KIV-2 plasmids as a validation set. We then applied UMI-ONT-Seq to extract KIV-2 VNTR haplotypes in 48 multi-ancestry 1000 Genome samples and analyzed at scale a poorly characterized STR within the KIV-2 VNTR. UMI-ONT-Seq detected KIV-2 SNPs down to 1% variant level with high sensitivity, specificity, and precision (0.977 ± 0.018; 1.000 ± 0.0005; 0.993 ± 0.02) and accurately retrieved the full-length haplotype of each VNTR unit. Human variant levels were highly correlated with next-generation sequencing (R2 = 0.983) without bias across the whole variant level range. Six reads per UMI produced sequences of each KIV-2 unit with Q40 quality. The KIV-2 repeat number determined by coverage-corrected unique haplotype counting was in close agreement with droplet digital PCR (ddPCR), with 70% of the samples falling even within the narrow confidence interval of ddPCR. We then analyzed 62,679 intra-KIV-2 STR sequences and explored KIV-2 SNP haplotype patterns across five ancestries. UMI-ONT-Seq accurately retrieves the SNP haplotype and precisely quantifies the VNTR copy number of each repeat unit of the complex KIV-2 VNTR region across multiple ancestries. This study utilizes the KIV-2 VNTR, presenting a novel and potent tool for comprehensive characterization of medically relevant complex genome regions at scale.

Pharmacogenetic analysis of structural variation in the 1000 genomes project using whole genome sequences

While significant strides have been made in understanding pharmacogenetics (PGx) and gene-drug interactions, there remains limited characterization of population-level PGx variation. This study aims to comprehensively profile global star alleles (haplotype patterns) and phenotype frequencies in 58 pharmacogenes associated with drug absorption, distribution, metabolism, and excretion. PyPGx, a star-allele calling tool, was employed to identify star alleles within high-coverage whole genome sequencing (WGS) data from the 1000 Genomes Project (N = 2504; 26 global populations). This process involved detecting structural variants (SVs), such as gene deletions, duplications, hybrids, as well as single nucleotide variants and insertion-deletion variants. The majority of our PyPGx calls for star alleles and phenotype frequencies aligned with the Pharmacogenomics Knowledge Base, although notable population-specific frequencies differed at least twofold. Validation efforts confirmed known SVs while uncovering several novel SVs currently undefined as star alleles. Additionally, we identified 210 small nucleotide variants associated with severe functional consequences that are not defined as star alleles. The study serves as a valuable resource, providing updated population-level star allele and phenotype frequencies while incorporating SVs. It also highlights the burgeoning potential of cost-effective WGS for PGx genotyping, offering invaluable insights to improve tailored drug therapies across diverse populations.

Ecological connectivity of the Qiongzhou Strait: a case form Orangefin Ponyfish (Photopectoralis bindus) haplotype diversity and genetic structure

Grasping the genetic structure of marine fish populations is vital for comprehending species connectivity patterns and determining the appropriate spatiotemporal scales for conservation management strategies. Here, we analyzed the population genetics of the Orangefin Ponyfish (Photopectoralis bindus Valenciennes, 1835) by examining a portion of the gene coding for the mitochondrial cytochrome c oxidase subunit I. The aim was to evaluate the haplotype pattern, genetic structure, demographic history, as well as the influence of ecological connectivity through the Qiongzhou Strait on the distribution patterns of this species in the northern South China Sea and the Beibu Gulf. In total, 257 specimens yielded only 13 haplotypes, with the predominant haplotype present at all sampling locations. The analysis revealed a “star-like” haplotype pattern, indicating low levels of both haplotype and nucleotide diversity. Additionally, a small but significant genetic structure was observed between the coastal regions flanking the Leizhou Peninsula. These patterns in the haplotype network and genetic structure may be significantly influenced by contemporary currents, particularly through the connectivity of the Qiongzhou Strait. Tajima’s D and Fu’s Fs demonstrated pronouncedly negative values, along with a unimodal mismatch distribution, suggested a recent demographic expansion of Photopectoralis bindus during the late Pleistocene, likely influenced by fluctuations in sea levels.

Whole-genome sequencing of Ganzi horse reveals the genetic diversity and provides unique insights into its plateau adaptation

Ganzi horse is distributed in the Ganzi Tibetan Autonomous Prefecture. As we all know, this region on the southeast of the Tibetan Plateau is characterized by low oxygen levels, low atmospheric pressure, and intense ultraviolet light. The extreme environment causes the genomic variation of plateau animals for environmental adaptation. At present, the genetic diversity and the genetic basis of high-altitude adaptation of Ganzi horses remain unclear. Here, an extensive genetic analysis was conducted utilizing whole-genome sequencing data from 145 individual horses. The analysis of autosomal genetic diversity confirmed that Ganzi horses had experienced low intensity artificial selection and had high genetic diversity. Examination of mitochondrial DNA information from whole-genome sequencing data revealed multiple maternal origins for Ganzi horses. Further, four different selective scanning methods were used to identify positive selected genomic regions and genes associated with high-altitude adaptation. We identified genes associated with altitude adaptation at selection windows in Ganzi horses, such as EPAS1, ABTB2, RHOQ, and TMEM247. Notably, EPAS1 and OR52A1J gene exhibited high select signal values, different nucleotide diversities, and haplotype patterns, and missense mutations in EPAS1 (A > T) and OR52A1J (C > T) were found to be more frequent in high-altitude horses. Moreover, our research illuminated significant gene flow between Ganzi and Chaidamu horses, which may be related to the formation of Ganzi horses. In general, these findings not only enhance our understanding of this unique native breed but also further our understanding of Ganzi horse's high-altitude adaptation.

Signatures of selective sweeps in continuous-space populations.

Selective sweeps describe the process by which an adaptive mutation arises and rapidly fixes in the population, thereby removing genetic variation in its genomic vicinity. The expected signatures of selective sweeps are relatively well understood in panmictic population models, yet natural populations often extend across larger geographic ranges where individuals are more likely to mate with those born nearby. To investigate how such spatial population structure can affect sweep dynamics and signatures, we simulated selective sweeps in populations inhabiting a two-dimensional continuous landscape. The maximum dispersal distance of offspring from their parents can be varied in our simulations from an essentially panmictic population to scenarios with increasingly limited dispersal. We find that in low-dispersal populations, adaptive mutations spread more slowly than in panmictic ones, while recombination becomes less effective at breaking up genetic linkage around the sweep locus. Together, these factors result in a trough of reduced genetic diversity around the sweep locus that looks very similar across dispersal rates. We also find that the site frequency spectrum around hard sweeps in low-dispersal populations becomes enriched for intermediate-frequency variants, making these sweeps appear softer than they are. Furthermore, haplotype heterozygosity at the sweep locus tends to be elevated in low-dispersal scenarios as compared to panmixia, contrary to what we observe in neutral scenarios without sweeps. The haplotype patterns generated by these hard sweeps in low-dispersal populations can resemble soft sweeps from standing genetic variation that arose from substantially older alleles. Our results highlight the need for better accounting for spatial population structure when making inferences about selective sweeps.

Genetic diversity and connectivity of the invasive gastropod, Callinina georgiana (Caenogastropoda: Viviparidae) across a fragmented riverscape: A mitonuclear perspective

Abstract Aquatic invasive species are a significant threat to global freshwater biodiversity. This study focuses on the banded mystery snail, Callinina georgiana, an invasive species in the Adirondack region of northern New York—an important section of the New York Great Lakes Basin. This project aims to explore the genetic connectivity of C. georgiana within its invasive range using a combination of mitochondrial and nuclear markers. Sampling was conducted in the Raquette River and adjacent waterways, with a total of 229 snails collected from 16 distinct populations distributed across eight different waterbodies. Also included were two populations from the species' native range in the southern U.S.A. DNA was extracted, and a 710‐bp fragment of the mitochondrial DNA marker cytochrome c oxidase 1 and a 351‐bp fragment of nuclear marker histone‐3 were amplified. Population genetic analyses including haplotype patterning, AMOVA and genetic diversity estimates, neutrality tests and tests for isolation by distance were performed to assess connectivity patterns. Results showed moderate to high levels of genetic admixture within the snail's invasive range as indicated by the lack of geographic patterning of haplotypes and low to moderate levels of genetic differentiation across multiple sites. Demographic analyses combined with high numbers of private haplotypes indicate historic population expansion. Interestingly, a case of mitonuclear discordance was detected for native and invasive populations as evident by incongruent haplotype patterns for the cytochrome c oxidase 1 and histone‐3 markers. Callinina georgiana exhibits a high level of genetic connectivity in its invasive range. The presence of dams does not significantly affect apparent gene flow, indicating that anthropogenic activities, such as boat traffic might be key in dispersing the snails across this fragmented freshwater system. This study offers new insights into the dispersal and genetic structure of an invasive freshwater snail. It highlights the importance of considering anthropogenic factors when confronting complex patterns of genetic diversity. The findings are significant for biodiversity conservation and provide a basis for developing strategies to manage and contain the spread of aquatic invasive species such as C. georgiana, especially in regions with high human activity.

Genetic association study for three single nucleotide polymorphisms related to type 2 diabetes in Egyptian population

BackgroundDiabetes mellitus is a disease that may result from interaction between environmental factors and a strong genetic component. The current study is aimed at exploring three single nucleotide polymorphisms to identify the associated ones with type 2 diabetes in the Egyptian society. The studied single nucleotide polymorphisms (rs10096097 in GOAT, rs6740584 in CREB1, and rs62521874 in MAFA) were examined via genotyping cases (n = 98) and irrelevant healthy subjects (n = 82).ResultsAssociations were checked using dominant, recessive, genotypic, allelic, and Cochran–Armitage trend models. By comparing diabetic patients with controls, rs6740584 was associated with type 2 diabetes by employing all used models except the recessive model. Rs10096097 was connected with type 2 diabetes using the genotypic association, Cochran–Armitage trend test, and recessive model and not any other model. Rs62521874 was not linked with type 2 diabetes in all models. Moreover, haplotype association for rs10096097 and rs62521874 was conducted as these two single nucleotide polymorphisms were located on the same chromosome. The haplotype pattern rs10096097:G—rs62521874:A was identified as a biomarker for type 2 diabetes susceptibility in the Egyptian community.ConclusionsThe GOAT and CREB1 polymorphisms showed susceptibility to type 2 diabetes. Moreover, MAFA had no role in the disease except through the haplotype with GOAT polymorphism.

Parallel Evolution in Mosquito Vectors-A Duplicated Esterase Locus is Associated With Resistance to Pirimiphos-methyl in Anopheles gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programs. The organophosphate (OP), pirimiphos-methyl, is a relatively new chemical in the vector control armory but is now widely used in indoor-residual spray campaigns. While generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in A. gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to OPs in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in A. gambiae, A. coluzzii and A. arabiensis. As in C. pipiens, copy number variants have arisen at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in A. gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programs.

FBLN2 is associated with Goldenhar syndrome and is essential for cranial neural crest cell development.

Goldenhar syndrome, a rare craniofacial malformation, is characterized by developmental anomalies in the first and second pharyngeal arches. Its etiology is considered to be heterogenous, including both genetic and environmental factors that remain largely unknown. To further elucidate the genetic cause in a five-generation Goldenhar syndrome pedigree and exploit the whole-exome sequencing (WES) data of this pedigree, we generated collapsed haplotype pattern markers based on WES and employed rare variant nonparametric linkage analysis. FBLN2 was identified as a candidate gene via analysis of WES data across the significant linkage region. A fbln2 knockout zebrafish line was established by CRISPR/Cas9 to examine the gene's role in craniofacial cartilage development. fbln2 was expressed specifically in the mandible during the zebrafish early development, while fbln2 knockout zebrafish exhibited craniofacial malformations with abnormal chondrocyte morphologies. Functional studies revealed that fbln2 knockout caused abnormal chondrogenic differentiation, apoptosis, and proliferation of cranial neural crest cells (CNCCs), and downregulated the bone morphogenic protein (BMP) signaling pathway in the zebrafish model. This study demonstrates the role of FBLN2 in CNCC development and BMP pathway regulation, and highlights FBLN2 as a candidate gene for Goldenhar syndrome, which may have implications for the selection of potential screening targets and the development of treatments for conditions like microtia-atresia.

Mapping the Pharmacogenetic Landscape in a Ugandan Population: Implications for Personalized Medicine in an Underrepresented Population.

Africans are extremely underrepresented in global genomic research. African populations face high burdens of communicable and non-communicable diseases and experience widespread polypharmacy. As population-specific genetic studies are crucial to understanding unique genetic profiles and optimizing treatments to reduce medication-related complications in this diverse population, the present study aims to characterize the pharmacogenomics profile of a rural Ugandan population. We analyzed low-pass whole genome sequencing data from 1998 Ugandans to investigate 18 clinically actionable pharmacogenes in this population. We utilized PyPGx to identify star alleles (haplotype patterns) and compared allele frequencies across populations using the Pharmacogenomics Knowledgebase PharmGKB. Clinical interpretations of the identified alleles were conducted following established dosing guidelines. Over 99% of participants displayed actionable phenotypes across the 18 pharmacogenes, averaging 3.5 actionable genotypes per individual. Several variant alleles known to affect drug metabolism (i.e., CYP3A5*1, CYP2B6*9, CYP3A5*6, CYP2D6*17, CYP2D6*29, and TMPT*3C)-which are generally more prevalent in African individuals-were notably enriched in the Ugandan cohort, beyond reported frequencies in other African peoples. More than half of the cohort exhibited a predicted impaired drug response associated with CFTR, IFNL3, CYP2B6, and CYP2C19, and approximately 31% predicted altered CYP2D6 metabolism. Potentially impaired CYP2C9, SLCO1B1, TPMT, and DPYD metabolic phenotypes were also enriched in Ugandans compared with other African populations. Ugandans exhibit distinct allele profiles that could impact drug efficacy and safety. Our findings have important implications for pharmacogenomics in Uganda, particularly with respect to the treatment of prevalent communicable and non-communicable diseases, and they emphasize the potential of pharmacogenomics-guided therapies to optimize healthcare outcomes and precision medicine in Uganda.

Cytological Observation and RNA-Seq Analyses Reveal miR9564 and Its Target Associated with Pollen Sterility in Autotetraploid Rice.

Understanding the regulation of autotetraploid sterility is essential for harnessing the strong advantages in genomic buffer capacity, biodiversity, and heterosis of autotetraploid rice. miRNAs play crucial roles in fertility regulation, yet information about their reproductive roles and target genes in tetraploid rice remains limited. Here, we used three tetraploid lines, H1 (fertile), HF (fertile), and LF (sterile), to investigate cytological features and identify factors associated with autotetraploid sterility. LF showed abnormal meiosis, resulting in low pollen fertility and viability, ultimately leading to scarce fertilization and a low-seed setting compared to H1 and HF. RNA-seq revealed 30 miRNA-candidate target pairs related to autotetraploid pollen sterility. These pairs showed opposite expression patterns, with differential expression between fertile lines (H1 and HF) and the sterile line (LF). qRT-PCR confirmed that miR9564, miR528, and miR27874 were highly expressed in the anthers of H1 and HF but not in LF, while opposite results were obtained in their targets (ARPS, M2T, and OsRPC53). Haplotype and expression pattern analyses revealed that ARPS was specifically expressed in lines with the same haplotype of MIR9564 (the precursor of miR9564) as LF. Furthermore, the Dual-GFP assay verified that miR9564 inhibited the fluorescence signal of ARPS-GFP. The over-expression of ARPS significantly decreased the seed setting rate (59.10%) and pollen fertility (50.44%) of neo-tetraploid rice, suggesting that ARPS plays important roles in autotetraploid pollen sterility. This study provides insights into the cytological characteristic and miRNA expression profiles of tetraploid lines with different fertility, shedding light on the role of miRNAs in polyploid rice.

Polymorphisms of the Leptin gene in Jabres cattle

Many genes, including the leptin gene, control growth performance. Polymorphism or SNPs variant within the gene could change its expression in phenotypes. This study aimed to identify the SNP and haplotype variation of the leptin gene in Jabres cattle (n = 47 head). The SNPs variant was detected using the BioEdit version 7.0 program by sequence alignment. The HaploView program analyzed the haplotype pattern created from the SNP variants. As a result, 20 SNPs were found within the partial sequence of the leptin gene. Only 3 SNPs are located in the coding sequence (CDS) region, SNP g. 12215T>C, g.12237C>T, and g.12238G>A. For the haplotype analysis, we used only SNPs with HW p-value cutoff and minimum minor allele frequency (MAF) higher than 0.05 (Jabres = 12 SNPs). The result showed a distinctive haplotype pattern of SNPs. All the blocks of LD plot in Jabres cattle showed a high linked disequilibrium (LD) (R2 > 0.33, LOD > 2) except for the block containing SNP g.12238G>A (R2 < 0.33, LOD < 2). In conclusion, the polymorphism and haplotype pattern found in this study could be used for further association analysis to the phenotypes, and its utilization could be used as an effective marker selection tool.

Genetic variation in the CYP1A gene caused by laboratory exposure of benzo (a) pyrene to common carp

Polycyclic aromatic hydrocarbons (PAHs) are pervasive environmental contaminants, with benzo(a)pyrene (B(a)P) standing out as a prototypical example. B(a)P is a known mutagen and carcinogen that, upon metabolic activation stimulated by cytochromes P450 (CYP1A) with microsomal epoxide hydrolase, reacts with DNA. In this study, common carp (Cyprinus carpio) were subjected to three different doses of B(a)P (1μg/kg and 10μg/kg) to investigate the mutagenic effects on B(a)P-derived DNA in vivo.Blood samples were collected, followed by thorough DNA extraction and polymerase chain reaction analysis. The findings revealed a pronounced mutational impact of B(a)P on the amplified segment of the CYP1A gene. Specifically, at a concentration of 1μg/kg, B(a)P induced 8 mutations, 3 amino acid alterations, and the emergence of 5 new haplotype patterns. In contrast, at a concentration of 10μg/kg, B(a)P resulted in 21 mutations, 10 changes in amino acids, and the generation of 7 new haplotype patterns. Additionally, alterations in the three-dimensional protein composition were observed in both dosage groups.This study underscores the significant mutagenic potential of B(a)P, shedding light on its capacity to induce genetic changes and protein structure modifications, thereby emphasizing the importance of monitoring and addressing the environmental presence of PAHs for both ecological and human health considerations

Abstract 3493: Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay

Abstract The abnormal changes in DNA methylation are linked to the early stages of carcinogenesis. Identifying these epigenetic changes in circulating tumor DNA (ctDNA) can reveal potential biomarkers for the early diagnosis of various cancers. However, analyzing such data poses bioinformatics challenges due to the lack of sensitivity in detecting the low abundance ctDNA signals in biopsy samples, which are often overwhelmed by the complexity of libraries containing hundreds of targeted regions. Read-level methylation analysis holds the promise of more in-depth DNA methylation detection due to the wide coverage and high sensitivity of rare signals. However, this approach is hindered by the absence of a standardized workflow capable of generating interpretable reports suitable for both bench scientists and professional bioinformaticians. Here, we present a bioinformatics workflow that examines next-generation sequencing (NGS) data and characterizes the read-level methylation patterns of amplicons. Compared to other currently available tools, our method is designed to work with high-multiplex, large-scale targeted assays. It effectively eliminates the undesired noise derived from sequencing byproducts such as false CpG calls, dimers, and off-target alignments. Additionally, to accommodate the substantial volume of data generated by state-of-the-art NGS platforms, the workflow enables parallel processing of samples compatible with both cloud-based and on-premises computing resources. This workflow provides a comprehensive per-sample visualization of DNA methylation patterns and reports read-level methylation results in a “pattern-as-a-feature” table. In this table, the occurrence of an amplicon epiallelic haplotype (pattern) for every sample is represented as a “feature column” and is aggregated with all patterns discovered in the experiment. These read-level patterns, along with other information, can be used to develop machine learning algorithms to reiteratively harvest true predictive features and penalize confounding signals in predicting cancer diagnosis. Citation Format: Mingda Jin, Masatomo Kaneko, Steven Cen, Hongtao Li, Wei Guo, Xinyi Zhou, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, Andre L. Abreu, Jeffrey Bhasin, Xiaojing Yang, Xi-Yu Jia. Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3493.

First molecular description of Neorhadinorhynchus nudus (Acanthocephala: Cavisomidae) from fish in the pacific coast of Vietnam, with notes on biogeography.

Neorhadinorhynchus nudus (Harada, 1938) Yamaguti, 1939 (Cavisomidae) was morphologically described from the frigate tuna Auxis thazard (Lacépède) (Scombridae) in Nha Trang, Pacific south Vietnam. Females of N. nudus were fully described for the first time in the Pacific. Its original inadequate description as Rhadinorhynchus nudus (Harada, 1938) was corrected in material from Fiji Island, the Red Sea and Pacific Vietnam and errors in the text and line drawings of Harada were repeated in subsequent major publications where it underwent considerable nomenclature changes. New descriptive and biogeographical notes are included. We also provided here the molecular characterization of the nuclear gene (18S) and the mitochondrial cytochromecoxidase subunit 1 (cox1) sequence data ofN. nudus. Furthermore, to elucidate the phylogenetic relationship of N. nudus within the family Cavisomidae and with other isolates were performed incorporating nuclear (18S) and mitochondrial (cox1) sequence data using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic results showed thatN. nudus has a relationship withother isolates of the same species and the median-joining network showed the pattern of haplotypes that reflected the structure of the populations.

Interleukin-10 promoter polymorphisms and haplotypes in patients with Guillain-Barré syndrome.

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts both pro- and anti-inflammatory effects on the immune system as well as in the pathogenesis of Guillain-Barré syndrome (GBS). We investigated whether the three common polymorphisms -1082 G/A(rs1800896), -819 C/T(rs1800871), and -592 C/A(rs1800872) in the promoter region of IL-10 have any influence on the susceptibility, severity, and clinical outcome of GBS. IL-10 promoter polymorphisms were investigated in 152 patients with GBS and 152 healthy controls from Bangladesh using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), and allele-specific oligonucleotide-PCR (ASO-PCR). Haplotype patterns and frequencies were analyzed using Heatmaply R-package, chi-square, and Fisher's exact test. The serum level of IL-10 was measured through enzyme-linked immunosorbent assays. p-values < 0.05 were considered statistically significant. IL-10 promoter polymorphisms -1082 G/A, -819 C/T, and -592 C/A were not associated with GBS susceptibility. The homozygous -819 TT genotype showed a tendency with susceptibility (p = 0.029; pc = 0.08) and was prevalent in axonal variants of GBS compared to demyelinating subtypes and controls (p = 0.042, OR = 8.67, 95% CI = 1.03-72.97; pc = 0.123 and p = 0.005, OR = 4.2, 95% CI = 1.55-11.40; pc = 0.015, respectively). Haplotype analysis revealed 19 patterns of genotypes and high IL-10 expression haplotype combinations (GCC/GTA, GCC/ATA, and GCC/GCA) may have influence on disease severity (p = 0.026; pc = 0.078). Serum expression of IL-10 was elevated in GBS patients ([GBS, 12.16 ± 45.71] vs. [HC, 0.65 ± 5.17] pg/mL; p = 0.0027) and varied with disease severity ([severe-GBS, 15.25 ± 51.72] vs. [mild-GBS, 3.59 ± 19.79] pg/mL, p = 0.046). The -819 TT genotypes influence axonal GBS, and high frequency of IL-10 expression haplotype combination with elevated serum IL-10 may play an important role in disease severity.

PSXII-1 Genetic Regions Associated with Cryptorchidism in Related Wagyu Cattle

Abstract Wagyu cattle have gained popularity in recent years due to their increased amounts of intramuscular marbling and their calving ease in crossbred cattle. The small effective population size of Wagyu cattle outside of Japan has led to inbreeding and an increased presentation of autosomal recessive traits. Cryptorchidism, the absence of at least one testicle from the scrotum, is the most common genital defect in males and is seen more commonly in inbred populations suggesting an autosomal recessive inheritance. The objective of this study was to identify genetic regions associated with unilateral cryptorchidism in related Wagyu cattle. All individuals in the study shared a common cow ancestor in their pedigree. Fourteen individuals, three cases and eleven controls, were genotyped using the Illumina BovineHD BeadChip (San Diego, CA). There were 450,083 SNPs that remained for the haplotype analysis after SNPs were removed for quality control parameters of genotyping call rate (&amp;lt; 90%), minor allele frequency (&amp;lt; 1%) and failure to meet Hardy Weinberg equilibrium testing (P &amp;lt; 1 x 10-5). No animals were removed due to low call rate (&amp;lt; 90%). Haplotypes were detected within the SNP and Variation Suite v8.2 (Golden Helix, Bozeman, MT) for an association with cryptorchidism using a chi-square test (P &amp;lt; 0.05) and false discovery rate (FDR &amp;lt; 0.05). There were 107,439 different SNP haplotype combinations among 49,627 haplotypes identified in the Wagyu cattle. The average haplotype size was 21.7 kb in Wagyu cattle. Prior to multiple testing correction, 1,233 haplotypes were associated (P &amp;lt; 0.05), with 6 haplotypes on BTA7 (P = 0.001 to P = 0.002) being of particular interest as the SNPs for those haplotypes were identical and homozygous, whereas none of the controls were homozygous for the SNPs. The SNP haplotype pattern observed on BTA7 was consistent with an autosomal recessive trait. When applying multiple testing corrections (FDR &amp;lt; 0.05), none of the haplotypes were significant. Additional unilateral cryptorchid bulls that share the same common cow ancestor have been identified and will be tested to see if the haplotypes on BTA7 are segregating with the phenotype. Identifying genetic regions associated with cryptorchidism will allow the identification of carriers for this trait that can then be used for choosing mates that are not carrying this trait. Reducing the number of cryptorchid males in a breed with a small effective population size would allow breeders to utilize the genetics of carriers without increasing the frequency of cryptorchidism.

Limited Polymorphism in the Dihydrofolate Reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand

BackgroundThe 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr–pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program. MethodsPkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein–ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates. ResultsFive nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein–ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr–pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates. ConclusionsA new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.

Multiple introductions and human-aided dispersal of the UK’s most widespread non-native amphibian

The alpine newt Ichthyosaura alpestris has achieved a widespread distribution as a non-native (alien) species in Britain since its initial introduction over a century ago, but the patterns of its release and subsequent dispersal have never yet been collectively analysed. We employed a multi-disciplinary combination of methods, using geographic profiling to estimate the likely number and locations of introductions, and mitochondrial DNA polymorphisms to investigate the likely geographic source of primary introductions, including the potential role of the pet trade. In parallel we used population genetic analysis and coalescence-based modelling to infer the demographics and directionality of dispersal from founding populations. Our results show that alpine newts have been released at multiple sites. We found a close resemblance between patterns of mtDNA haplotypes in the pet trade and those of established alpine newt populations, suggesting a relationship between trade, releases, and dispersal. Results from demographic modelling using Approximate Bayesian Computation are also consistent with multiple independent introductions with limited local dispersal, and additionally suggest that releases may occur from intermediate sources, such as captive populations. Our results support the hypothesis that deliberate human activity is largely responsible for both introductions of alpine newts into the UK and their wider dispersal post-introduction. The likely involvement of the international pet trade highlights the risk that ongoing releases of I. alpestris may expose native species to pathogens, whether pre-existing or novel.

Genome-wide association study of cooking-caused grain expansion in rice (Oryza sativa L.).

Cooking-caused rice grain expansion (CCRGE) is a critical trait for evaluating the cooking quality of rice. Previous quantitative trait locus (QTL) mapping studies on CCRGE have been limited to bi-parental populations, which restrict the exploration of natural variation and mapping resolution. To comprehensively and precisely dissect the genetic basis of CCRGE, we performed a genome-wide association study (GWAS) on three related indices: grain breadth expansion index (GBEI), grain length expansion index (GLEI), and grain length-breadth ratio expansion index (GREI), using 345 rice accessions grown in two years (environments) and 193,582 SNP markers. By analyzing each environment separately using seven different methods (3VmrMLM, mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, pKWmEB, ISIS EM-BLASSO), we identified a total of 32, 19 and 27 reliable quantitative trait nucleotides (QTNs) associated with GBEI, GLEI and GREI, respectively. Furthermore, by jointly analyzing the two environments using 3VmrMLM, we discovered 19, 22 and 25 QTNs, as well as 9, 5 and 7 QTN-by-environment interaction (QEIs) associated with GBEI, GLEI and GREI, respectively. Notably, 12, 9 and 15 QTNs for GBEI, GLEI and GREI were found within the intervals of previously reported QTLs. In the vicinity of these QTNs or QEIs, based on analyses of mutation type, gene ontology classification, haplotype, and expression pattern, we identified five candidate genes that are related to starch synthesis and endosperm development. The five candidate genes, namely, LOC_Os04g53310 (OsSSIIIb, near QTN qGREI-4.5s), LOC_Os05g02070 (OsMT2b, near QTN qGLEI-5.1s), LOC_Os06g04200 (wx, near QEI qGBEI-6.1i and QTNs qGREI-6.1s and qGLEI-6.1t), LOC_Os06g12450 (OsSSIIa, near QTN qGLEI-6.2t), and LOC_Os08g09230 (OsSSIIIa, near QTN qGBEI-8.1t), are predicted to be involved in the process of rice grain starch synthesis and to influence grain expansion after cooking. Our findings provide valuable insights and will facilitate genetic research and improvement of CCRGE.