Search

Single nucleotide polymorphisms (SNPs) in homologous regions play a critical role in the field of genetics. However, genotyping these SNPs is challenging due to the presence of repetitive sequences within genome, which demand specific method. We introduce a new, mid-throughput method that simplifies SNP genotyping in homologous DNA sequences by utilizing a combination of multiplex kb level PCR (PCR size 2.5k-3.5 kb) for capturing targeted regions and multiplex nested PCR library construction for next-generation sequencing (Multi-kb level capture-seq). First of all, we randomly selected 7 SNPs in homologous regions and successfully captured 6-plex kb level amplicons (one of segments contains 2 SNPs, while the remaining segments each have only one SNP) in a single tube. And then, the amplification products were subjected to multiplex nested PCR for library construction and sequenced on Illumina platform. We tested this strategy using 600 amplicons from 100 samples and accurately genotyped 96.8% of target SNPs with a coverage depth of ≥ 15×. For the uniformity within the samples, over 66.7% (4/6) of the amplicons had a coverage depth above 0.2-fold of average sequencing depth. To validate the accuracy of this approach, we performed Ligase detection reaction PCR for genotyping the 100 samples, and found that the genotyping data was 97.71% consistent with our NGS results. In conclusion, we have developed a highly efficient and accurate method for SNP genotyping in homologous regions, which offers researchers a new strategy to explore the complex regions of genome. [ABSTRACT FROM AUTHOR]

Summary: It is generally accepted that kelp populations have a metapopulation structure. Most zoospores settle near to their parent individual, and infrequent but long‐distance dispersal of zoospores contributes to gene flow between local populations. Population genetic analysis of single nucleotide polymorphism genotyping by MIG‐seq (i.e. multiplexed inter‐simple sequence repeats genotyping by sequencing) was performed on the dominant kelp, Eisenia bicyclis, in two areas: Kitsunezaki (a sheltered area) and Shimoda (an exposed site). Regional scale analysis of genetic structure was conducted at six sites in Kitsunezaki and three sites in Shimoda. When viewed on a regional scale based on the inbreeding coefficient within the local population, the inbreeding ratio was higher in Shimoda than in Kitsunezaki, probably as a result of the limited vertical zonal distribution in the exposed environment. Vertically wide distribution in the sheltered environment apparently enabled frequent crossing among individuals in Kitsunezaki. By contrast, when viewed on a local scale based on the pairwise kinship coefficient (FST) between populations, gene flow among local populations in Shimoda occurred over a wide area, but was limited in Kitsunezaki. In Shimoda, genetic exchange between local populations, even if inbreeding is locally active, is likely to support metapopulation maintenance and rapid recovery of local populations. In Kitsunezaki, however, genetic exchange among local populations is limited within few 100 m, and the metapopulation structure will decline in the long term because of inbreeding depression. Local dispersal distance of zoospores was investigated based on parent–offspring analysis in the Kitsunezaki population, which revealed that zoospores mainly settled within 5 m of their parent. However, some zoospores traveled over 27 m within the 4 × 30 m study area. The present study shows the importance of examination at different spatial scales when investigating zoospore dispersal of laminarialean kelps. [ABSTRACT FROM AUTHOR]

Background: Laboratory rats, as model animals, have been extensively used in the fields of life science and medicine. It is crucial to routinely monitor the genetic background of laboratory rats. The conventional approach relies on gel electrophoresis and capillary electrophoresis (CE) technologies. However, the experimental and data analysis procedures for both of these methods are time consuming and costly. Results: We established a single-nucleotide polymorphism (SNP) typing scheme using multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to address the genetic background ambiguity in laboratory rats. This methodology involved three rounds of PCR and two rounds of magnetic bead selection to improve the quality of the sequencing data. We simultaneously analysed 100 laboratory rats (including rats of 5 inbred strains and 2 in-house closed colonies), and the sequencing depth varied from an average of 108.25 to 5189.89, with sample uniformity ranging from 82.5 to 97.5%. A total of 98.9% of the amplicons were successfully genotyped (≥ 30 reads). Genetic background analysis revealed that all 38 experimental rats from the 5 inbred strains were successfully identified (without a heterozygous allele). For the 2 in-house closed colonies, the average heterozygosity (0.162 and 0.169) deviated from the typical range of 0.5–0.7, indicating a departure from the ideal heterozygosity level. Additionally, we employed multiplex PCR-CE to validate the NGS-based method, which yielded consistent results for all the rat strains. These results demonstrated that this approach significantly improves efficiency, saves time, reduces costs and ensures accuracy. Conclusion: By utilizing NGS technology, our developed method leverages SNP genotyping for genetic background identification in laboratory rats, demonstrating advantages in terms of labour efficiency and cost-effectiveness, thereby rendering it well suited for projects involving extensive sample cohorts. [ABSTRACT FROM AUTHOR]

Background: Sheep breeds native to the United Kingdom exhibit a striking diversity of different traits. Some of these traits are highly sustainable, such as seasonal wool shedding in the Wiltshire Horn, and are likely to become more important as pressures on sheep production increase in coming decades. Despite their clear importance to the future of sheep farming, the genetic diversity of native UK sheep breeds is poorly characterised. This increases the risk of losing the ability to select for breed-specific traits from native breeds that might be important to the UK sheep sector in the future. Here, we use 50 K genotyping to perform preliminary analysis of breed relationships and genetic diversity within native UK sheep breeds, as a first step towards a comprehensive characterisation. This study generates novel data for thirteen native UK breeds, including six on the UK Breeds at Risk (BAR) list, and utilises existing data from the publicly available Sheep HapMap dataset to investigate population structure, heterozygosity and admixture. Results: In this study the commercial breeds exhibited high levels of admixture, weaker population structure and had higher heterozygosity compared to the other native breeds, which generally tend to be more distinct, less admixed, and have lower genetic diversity and higher kinship coefficients. Some breeds including the Wiltshire Horn, Lincoln Longwool and Ryeland showed very little admixture at all, indicating a high level of breed integrity but potentially low genetic diversity. Population structure and admixture were strongly influenced by sample size and sample provenance – highlighting the need for equal sample sizes, sufficient numbers of individuals per breed, and sampling across multiple flocks. The genetic profiles both within and between breeds were highly complex for UK sheep, reflecting the complexity in the demographic history of these breeds. Conclusion: Our results highlight the utility of genotyping data for investigating breed diversity and genetic structure. They also suggest that routine generation of genotyping data would be very useful in informing conservation strategies for rare and declining breeds with small population sizes. We conclude that generating genetic resources for the sheep breeds that are native to the UK will help preserve the considerable genetic diversity represented by these breeds, and safe-guard this diversity as a valuable resource for the UK sheep sector to utilise in the face of future challenges. [ABSTRACT FROM AUTHOR]

Background: Anti-calcitonin gene-related peptide (CGRP) monoclonal antibodies have emerged as promising therapeutic options for the treatment of chronic migraine. However, treatment response varies considerably among individuals, suggesting a potential role for genetic factors. This study aimed to identify genetic variants affecting the efficacy of anti-CGRP monoclonal antibody therapy in chronic migraine among the Han Chinese population in Taiwan to enhance treatment precision and to understand the genetic architecture of migraine. Methods: We conducted a quantitative trait locus (QTL) association study in patients with chronic migraines from a tertiary medical center in Taiwan using the Taiwan Precision Medicine Array Chip. The patients received fremanezumab or galcanezumab for at least 12 weeks. Treatment efficacy was assessed based on the improvement rate in monthly migraine days. Genetic variants were identified, and their associations with treatment efficacy were examined through quantitative trait loci analysis, linkage disequilibrium studies, and functional annotations using the Gene Ontology database. Results: Six single nucleotide polymorphisms (SNPs) relative variants were significantly associated with anti-CGRP therapy response (p < 1 × 10− 7): rs116870564, rs75244870, rs56216870, rs12938101, rs74655790, and rs149540851. These variants are located in or near genes, including LRRC4C, ATAD2B, and OXR1, which are involved in neuronal development, DNA-dependent ATPase activity, and oxidation-reduction processes, respectively. The rs116870564 variant in LRRC4C showed the strongest association (β = -0.551, p = 6.65 × 10− 9). The functional impact of these variants is attributed to their regulatory effects on gene expression, which are influenced by intron splicing regulation, transcription factors, and changes in chromatin structure. Conclusion: The identification of key genetic markers associated with response to anti-CGRP therapy emphasizes the importance of genetic variability in treatment efficacy. This could lead to more personalized chronic migraine management strategies and tailored therapeutic approaches based on individual genetic profiles. Further research in larger, diverse populations is warranted to validate these findings and refine our understanding of the role of CGRP in chronic migraine pathophysiology. Trial registration: Not applicable. [ABSTRACT FROM AUTHOR]

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole‐genome single‐nucleotide polymorphism (SNP) quality and quantity using next‐generation sequencing (NGS). We present whole‐genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double‐stranded DNA libraries then enriched for whole‐genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed‐effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole‐genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification. [ABSTRACT FROM AUTHOR]

Abstract Background Laboratory rats, as model animals, have been extensively used in the fields of life science and medicine. It is crucial to routinely monitor the genetic background of laboratory rats. The conventional approach relies on gel electrophoresis and capillary electrophoresis (CE) technologies. However, the experimental and data analysis procedures for both of these methods are time consuming and costly. Results We established a single-nucleotide polymorphism (SNP) typing scheme using multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to address the genetic background ambiguity in laboratory rats. This methodology involved three rounds of PCR and two rounds of magnetic bead selection to improve the quality of the sequencing data. We simultaneously analysed 100 laboratory rats (including rats of 5 inbred strains and 2 in-house closed colonies), and the sequencing depth varied from an average of 108.25 to 5189.89, with sample uniformity ranging from 82.5 to 97.5%. A total of 98.9% of the amplicons were successfully genotyped (≥ 30 reads). Genetic background analysis revealed that all 38 experimental rats from the 5 inbred strains were successfully identified (without a heterozygous allele). For the 2 in-house closed colonies, the average heterozygosity (0.162 and 0.169) deviated from the typical range of 0.5–0.7, indicating a departure from the ideal heterozygosity level. Additionally, we employed multiplex PCR-CE to validate the NGS-based method, which yielded consistent results for all the rat strains. These results demonstrated that this approach significantly improves efficiency, saves time, reduces costs and ensures accuracy. Conclusion By utilizing NGS technology, our developed method leverages SNP genotyping for genetic background identification in laboratory rats, demonstrating advantages in terms of labour efficiency and cost-effectiveness, thereby rendering it well suited for projects involving extensive sample cohorts.

Abstract Background Sheep breeds native to the United Kingdom exhibit a striking diversity of different traits. Some of these traits are highly sustainable, such as seasonal wool shedding in the Wiltshire Horn, and are likely to become more important as pressures on sheep production increase in coming decades. Despite their clear importance to the future of sheep farming, the genetic diversity of native UK sheep breeds is poorly characterised. This increases the risk of losing the ability to select for breed-specific traits from native breeds that might be important to the UK sheep sector in the future. Here, we use 50 K genotyping to perform preliminary analysis of breed relationships and genetic diversity within native UK sheep breeds, as a first step towards a comprehensive characterisation. This study generates novel data for thirteen native UK breeds, including six on the UK Breeds at Risk (BAR) list, and utilises existing data from the publicly available Sheep HapMap dataset to investigate population structure, heterozygosity and admixture. Results In this study the commercial breeds exhibited high levels of admixture, weaker population structure and had higher heterozygosity compared to the other native breeds, which generally tend to be more distinct, less admixed, and have lower genetic diversity and higher kinship coefficients. Some breeds including the Wiltshire Horn, Lincoln Longwool and Ryeland showed very little admixture at all, indicating a high level of breed integrity but potentially low genetic diversity. Population structure and admixture were strongly influenced by sample size and sample provenance – highlighting the need for equal sample sizes, sufficient numbers of individuals per breed, and sampling across multiple flocks. The genetic profiles both within and between breeds were highly complex for UK sheep, reflecting the complexity in the demographic history of these breeds. Conclusion Our results highlight the utility of genotyping data for investigating breed diversity and genetic structure. They also suggest that routine generation of genotyping data would be very useful in informing conservation strategies for rare and declining breeds with small population sizes. We conclude that generating genetic resources for the sheep breeds that are native to the UK will help preserve the considerable genetic diversity represented by these breeds, and safe-guard this diversity as a valuable resource for the UK sheep sector to utilise in the face of future challenges.

Abstract Background Anti-calcitonin gene-related peptide (CGRP) monoclonal antibodies have emerged as promising therapeutic options for the treatment of chronic migraine. However, treatment response varies considerably among individuals, suggesting a potential role for genetic factors. This study aimed to identify genetic variants affecting the efficacy of anti-CGRP monoclonal antibody therapy in chronic migraine among the Han Chinese population in Taiwan to enhance treatment precision and to understand the genetic architecture of migraine. Methods We conducted a quantitative trait locus (QTL) association study in patients with chronic migraines from a tertiary medical center in Taiwan using the Taiwan Precision Medicine Array Chip. The patients received fremanezumab or galcanezumab for at least 12 weeks. Treatment efficacy was assessed based on the improvement rate in monthly migraine days. Genetic variants were identified, and their associations with treatment efficacy were examined through quantitative trait loci analysis, linkage disequilibrium studies, and functional annotations using the Gene Ontology database. Results Six single nucleotide polymorphisms (SNPs) relative variants were significantly associated with anti-CGRP therapy response (p

Abstract 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.

Objective To establish a non-invasive single nucleotide polymorphism genotyping technology based on direct TaqMan-PCR for high-throughput genotyping of site rs688 and rs964184 associated with lipid metabolism to meet the need for early screening of at-risk populations. Methods Extracted DNA was used to optimize the PCR annealing extension temperature and amplification procedure for the designed TaqMan probe; the oral swabs were placed into the sample treatment solution and then briefly centrifuged, and a small amount of the supernatant was taken for the direct qPCR amplification analysis; the freeze-thawing stability of probe and the effects of sample storage time on genotyping results were explored. Results The technology requires only simple treatment of oral swab for PCR analysis and can be stored at room temperature for 4 d. The optimized method was able to distinguish homozygous wild type, homozygous mutant type and heterozygote at rs688 and rs964184 loci. Conclusions A fast, convenient, high-throughput, and low-cost SNP genotyping method has been established, providing an efficient and accurate new approach for large-scale screening of high-risk populations carrying susceptibility genes.

Simple Summary: Pig farming is one of the most profitable components of the livestock sector in agriculture, significantly contributing to economic development, food security, and improved livelihoods for local communities in Africa and globally. However, with the increasing concern for global environmental changes, pig production is considered one of the vulnerable livestock sectors likely to be affected the most. Africa, being a tropical continent with extraordinary geographical and biological diversity, is believed to have varieties of local pigs exhibiting valuable genetic traits that can be used to promote livestock productivity through breeding for climate-resilient breeds. Unfortunately, many of these valuable traits have not been fully identified and exploited. This study provides an overview of the current state of African pig genetic resources by highlighting their diversity and adaptability potential from both phenotypic and genetic evidence. Our results indicate that African local pigs hold potential genetic traits critical for climate change adaptation. However, these traits are threatened due to crossbreeding activities with commercial breeds that are now prevalent across the continent. Thus, to keep up with the rapid speed of climate change, efforts to realize and utilize these considerable potential traits must increase before they are permanently depleted. Africa is home to a wide diversity of locally adapted pig breeds whose genetic architecture offers important insights into livestock adaptation to climate change. However, the majority of these inherent traits have not been fully highlighted. This review presents an overview of the current state of African pig genetic resources, providing highlights on their population and production statistics, production system, population diversity indices, and genomic evidence underlying their evolutionary potential. The study results reveal an incomplete characterization of local pig genotypes across the continent. The characterized population, however, demonstrates moderate to high levels of genetic diversity, enough to support breeding and conservation programs. Owing to low genetic differentiation and limited evidence of distinct population structures, it appears that most local pig populations are strains within larger breeds. Genomic evidence has shown a higher number of selection signatures associated with various economically important traits, thus making them potential candidates for climate change adaptation. The reportedly early evidence of hybridization with wild suid groups further suggests untapped insights into disease resistance and resilience traits that need to be illuminated using higher-density markers. Nevertheless, gene introgression from commercial breeds is prevalent across Africa; thus, efforts to realize and utilize these traits must increase before they are permanently depleted. [ABSTRACT FROM AUTHOR]

The Sahiwal breed of dairy cattle holds significant importance in Pakistan, mostly attributed to its ability to withstand high temperatures, resilience to diseases, and satisfactory performance when fed low-quality roughages. Domestication and breeding of mammals have exerted a consistent selection pressure on a wide range of characteristics in many domesticated species, resulting in discernible genetic modifications at the individual genome level. The study aimed to discover and analyze potential indicators of recent selection in Sahiwal cattle, specifically identifying the genes and quantitative trait loci associated with these selection indicators. The study utilized a sample size of 98 Sahiwal bulls. The genotyping of all animals was conducted using the BovineHD140k BeadChip. After undergoing quality control measures, 87 samples as well as 74,070 SNPs located throughout 29 autosomes were selected as well as included in the study. The selection signatures were examined using the iHS and the Tajima D approach. The result reveals the current positive selections on BTA 1, 2, 6, 11, 12, 15, 17, 21, and 27 with the iHS test, while for the Tajima D test, the current positive selections were detected on BTA 1, 2, 3, 4, 5, 6, 7, 9, 10, 14, 18, 20, 23, and 24. A total of 47 genes were detected within selection regions associated with vital economic traits. The QTL enrichment analysis has shown eight substantial QTLs in BTA19 and BTA20 linked with milk, production, as well as reproduction traits. Therefore, understanding the selection signatures and candidate genes that influence important economic traits can provide foundational knowledge that can be used effectively to gain insight into the underlying mechanisms controlling these traits in Sahiwal cattle. [ABSTRACT FROM AUTHOR]

The curation and preservation of Dutch apple germplasm depends on reliable accession level information. However, many accessions of Dutch heirloom apple cultivars maintained publicly by the "Centre for Genetic Resources, the Netherlands" (CGN) and privately by Dutch pomological societies lack information regarding true-to-typeness and pedigree ancestry. The aim of this study was to address this knowledge gap by genotyping 652 apple accessions maintained in the CGN collection and Dutch private collections, compare their genotypic information to each other and to a large database of apple cultivars from around the world to identify genotypic duplicates and pedigree relationships for the Dutch apple cultivars. Towards this aim, accessions were genotyped on the 20 K Illumina Infinium(R) apple SNP array and with 15 SSR markers. Each accession was assigned to a genotypic profile code (MUNQ codes, as used in previous studies) facilitating communication regarding genotypic duplicates. There were 211 (51.1%) genotypic profiles in the Dutch germplasm which were not identified in other germplasm collections. Private collections maintained many of these unique accessions, including important pedigree ancestors. The study identified a number of common pedigree ancestors of Dutch cultivars, such as 'Herfst Bloem Soete', 'Huismanszoet' (2), and 'Reinette Rouge Étoilée'. The duplicate and pedigree reconstruction results and relevant literature descriptions were used to pomologically verify the identity of relevant accessions. The results of this study resolved identity disputes, helped to decide which accessions should be retained or included in the CGN collection, and benefited ongoing pomological studies in ancestry and provenance of traditional Dutch cultivars. [ABSTRACT FROM AUTHOR]

Current methodologies of genome‐wide single‐nucleotide polymorphism (SNP) genotyping produce large amounts of missing data that may affect statistical inference and bias the outcome of experiments. Genotype imputation is routinely used in well‐studied species to buffer the impact in downstream analysis, and several algorithms are available to fill in missing genotypes. The lack of reference haplotype panels precludes the use of these methods in genomic studies on non‐model organisms. As an alternative, machine learning algorithms are employed to explore the genotype data and to estimate the missing genotypes. Here, we propose an imputation method based on self‐organizing maps (SOM), a widely used neural networks formed by spatially distributed neurons that cluster similar inputs into close neurons. The method explores genotype datasets to select SNP loci to build binary vectors from the genotypes, and initializes and trains neural networks for each query missing SNP genotype. The SOM‐derived clustering is then used to impute the best genotype. To automate the imputation process, we have implemented gtImputation, an open‐source application programmed in Python3 and with a user‐friendly GUI to facilitate the whole process. The method performance was validated by comparing its accuracy, precision and sensitivity on several benchmark genotype datasets with other available imputation algorithms. Our approach produced highly accurate and precise genotype imputations even for SNPs with alleles at low frequency and outperformed other algorithms, especially for datasets from mixed populations with unrelated individuals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many mutant genes causing rare forms of deafness remain to be discovered. A consanguineous Pakistani family segregating nonsyndromic deafness in two sibships were studied using microarrays and exome sequencing. A 1.2 Mb locus (DFNB128) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. There are two previously reported Map3k1-kinase-deficient mouse models that are associated with recessively inherited syndromic deafness. MAP3K1 phosphorylates serine and threonine and functions in a signaling pathway where pathogenic variants of HGF, MET, and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97, and DFNB26, respectively. Our single-cell transcriptome data of mouse cochlea mRNA show expression of Map3k1 and its signaling partners in several inner ear cell types suggesting a requirement of wild-type MAP3K1 for normal hearing. In contrast to dominant variants of MAP3K1 associated with Disorders of Sex Development 46,XY sex-reversal, our computational modeling of the recessive substitution p.(Arg1487His) predicts a subtle structural alteration in MAP3K1, consistent with the limited phenotype of nonsyndromic deafness. [ABSTRACT FROM AUTHOR]

Objective To establish a non-invasive single nucleotide polymorphism genotyping technology based on direct TaqMan-PCR for high-throughput genotyping of site rs688 and rs964184 associated with lipid metabolism to meet the need for early screening of at-risk populations. Methods Extracted DNA was used to optimize the PCR annealing extension temperature and amplification procedure for the designed TaqMan probe; the oral swabs were placed into the sample treatment solution and then briefly centrifuged, and a small amount of the supernatant was taken for the direct qPCR amplification analysis; the freeze-thawing stability of probe and the effects of sample storage time on genotyping results were explored. Results The technology requires only simple treatment of oral swab for PCR analysis and can be stored at room temperature for 4 d. The optimized method was able to distinguish homozygous wild type, homozygous mutant type and heterozygote at rs688 and rs964184 loci. Conclusions A fast, convenient, high-throughput, and low-cost SNP genotyping method has been established, providing an efficient and accurate new approach for large-scale screening of high-risk populations carrying susceptibility genes. [ABSTRACT FROM AUTHOR]

Abstract Objective Data from DNA genotyping via a 96-SNP panel in a study of 25,015 clinical samples were utilized for quality control and tracking of sample identity in a clinical sequencing network. The study aimed to demonstrate the value of both the precise SNP tracking and the utility of the panel for predicting the sex-by-genotype of the participants, to identify possible sample mix-ups. Results Precise SNP tracking showed no sample swap errors within the clinical testing laboratories. In contrast, when comparing predicted sex-by-genotype to the provided sex on the test requisition, we identified 110 inconsistencies from 25,015 clinical samples (0.44%), that had occurred during sample collection or accessioning. The genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density genotyping arrays. It was determined that discrepancies resulted from clerical errors (49.09%), samples from transgender participants (3.64%) and stem cell or bone marrow transplant patients (7.27%) along with undetermined sample mix-ups (40%) for which sample swaps occurred prior to arrival at genome centers, however the exact cause of the events at the sampling sites resulting in the mix-ups were not able to be determined.

Wolves are assumed to be ungulate obligates, however, a recently described pack on Pleasant Island, Alaska USA, is persisting on sea otters and other marine resources without ungulate prey, violating this long‐held assumption. We address questions about these wolves regarding their origin and fate, degree of isolation, risk of inbreeding depression, and diet specialization by individual and sex. We applied DNA metabarcoding and genotyping by amplicon sequencing using 957 scats collected from 2016 to 2022, and reduced representation sequencing of tissue samples to establish a detailed understanding of Pleasant Island wolf ecology and compare them with adjacent mainland wolves. Dietary overlap was higher among individual wolves on Pleasant Island (Pianka's index mean 0.95 ± 0.03) compared to mainland wolves (0.70 ± 0.21). The individual diets of island wolves were dominated by sea otter, ranging from 40.6% to 63.2% weighted percent of occurrence (wPOO) (mean 55.5 ± 8.7). In contrast, individual mainland wolves primarily fed on ungulates (42.2 ± 21.3) or voles during a population outbreak (31.2 ± 23.2). We traced the origin of the Pleasant Island pack to a mainland pair that colonized around 2013 and produced several litters. After this breeding pair was killed, their female offspring and an immigrant male became the new breeders in 2019. We detected 20 individuals of which 8 (40%) were trapped and killed while two died of natural causes during the 6‐year study. Except for the new breeding male, the pedigree analysis and genotype results showed no additional movement to or from the island, indicating limited dispersal but no evidence of inbreeding. Our findings suggest wolves exhibit more flexible foraging behavior than previously believed, and hunting strategies can substantially differ between individuals within or between packs. Nevertheless, anthropogenic and natural mortality combined with limited connectivity to the mainland may inhibit the continued persistence of Pleasant Island wolves. [ABSTRACT FROM AUTHOR]

Improving the seed protein concentration (SPC) of pea (Pisum sativum L.) has turned into an important breeding objective because of the consumer demand for plant-based protein and demand from protein fractionation industries. To support the marker-assisted selection (MAS) of SPC towards accelerated breeding of improved cultivars, we have explored two diverse recombinant inbred line (RIL) populations to identify the quantitative trait loci (QTLs) associated with SPC. The two RIL populations, MP 1918 × P0540-91 (PR-30) and Ballet × Cameor (PR-31), were derived from crosses between moderate SPC × high SPC accessions. A total of 166 and 159 RILs of PR-30 and PR-31, respectively, were genotyped using an Axiom® 90K SNP array and 13.2K SNP arrays, respectively. The RILs were phenotyped in replicated trials in two and three locations of Saskatchewan, Canada in 2020 and 2021, respectively, for agronomic assessment and SPC. Using composite interval mapping, we identified three QTLs associated with SPC in PR-30 and five QTLs in PR-31, with the LOD value ranging from 3.0 to 11.0. A majority of these QTLs were unique to these populations compared to the previously known QTLs for SPC. The QTL SPC-Ps-5.1 overlapped with the earlier reported SPC associated QTL PC-QTL-3. Three QTLs, SPC-Ps-4.2, SPC-Ps-5.1, and SPC-Ps-7.2 with LOD scores of 7.2, 7.9, and 11.3, and which explained 14.5%, 11.6%, and 11.3% of the phenotypic variance, respectively, can be used for marker-assisted breeding to increase SPC in peas. Eight QTLs associated with the grain yield were identified with LOD scores ranging from 3.1 to 8.2. Two sets of QTLs, SPC-Ps-2.1 and GYPs-2.1, and SPC-Ps-5.1 and GY-Ps-5.3, shared the QTL/peak regions. Each set of QTLs contributed to either SPC or grain yield depending on which parent the QTL region is derived from, thus confirming that breeding for SPC should take into consideration the effects on grain yield. [ABSTRACT FROM AUTHOR]

Schizophrenia is a complex condition with entwined genetic and epigenetic risk factors, posing a challenge to disentangle the intermixed pathological and therapeutic epigenetic signatures. To resolve this, we performed 850K methylome-wide and 700K genome-wide studies on the same set of schizophrenia patients by stratifying them into responders, non-responders, and drug-naïve patients. The key genes that signified the response were followed up using real-time gene expression studies to understand the effect of antipsychotics at the gene transcription level. The study primarily implicates hypermethylation in therapeutic response and hypomethylation in the drug-non-responsive state. Several differentially methylated sites and regions colocalized with the schizophrenia genome-wide association study (GWAS) risk genes and variants, supporting the convoluted gene-environment association. Gene ontology and protein-protein interaction (PPI) network analyses revealed distinct patterns that differentiated the treatment response from drug resistance. The study highlights the strong involvement of several processes related to nervous systemdevelopment, cell adhesion, and signaling in the antipsychotic response. The ability of antipsychotic medications to alter the pathology by modulating gene expression or methylation patterns is evident from the general increase in the gene expression of response markers and histone modifiers and the decrease in class II human leukocyte antigen (HLA) genes following treatment with varying concentrations of medications like clozapine, olanzapine, risperidone, and haloperidol. The study indicates a directional overlap of methylation markers between pathogenesis and therapeutic response, thereby suggesting a careful distinction of methylation markers of pathogenesis from treatment response. In addition, there is a need to understand the trade-off between genetic and epigenetic observations. It is suggested that methylomic changes brought about by drugs need careful evaluation for their positive effects on pathogenesis, course of disease progression, symptom severity, side effects, and refractoriness. [ABSTRACT FROM AUTHOR]

Objective: Data from DNA genotyping via a 96-SNP panel in a study of 25,015 clinical samples were utilized for quality control and tracking of sample identity in a clinical sequencing network. The study aimed to demonstrate the value of both the precise SNP tracking and the utility of the panel for predicting the sex-by-genotype of the participants, to identify possible sample mix-ups. Results: Precise SNP tracking showed no sample swap errors within the clinical testing laboratories. In contrast, when comparing predicted sex-by-genotype to the provided sex on the test requisition, we identified 110 inconsistencies from 25,015 clinical samples (0.44%), that had occurred during sample collection or accessioning. The genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density genotyping arrays. It was determined that discrepancies resulted from clerical errors (49.09%), samples from transgender participants (3.64%) and stem cell or bone marrow transplant patients (7.27%) along with undetermined sample mix-ups (40%) for which sample swaps occurred prior to arrival at genome centers, however the exact cause of the events at the sampling sites resulting in the mix-ups were not able to be determined. [ABSTRACT FROM AUTHOR]

Variable Fragment Length Allele-Specific Polymerase Chain Reaction (VFLASP) and Amplification Refractory Mutation System (ARMS) are reliable methods for detecting allelic variations resulting from single base changes within the genome. Due to their widespread application, allele variations caused by Single Nucleotide Polymorphisms (SNPs) can be readily detected using allele-specific primers. In the context of the current study, VFLASP was combined with ARMS method as a novel strategy to enhance the efficacy of both techniques. Clinically important base variations within SNP regions used in the study were detected by a fragment analysis method. To validate the accuracy of the developed VFLASP-ARMS method, specifically designed synthetic sequences were tested using a capillary electrophoresis system. Allele-specific primers exhibit differences solely at the 3′ end based on the sequence of the SNP. Additionally, to increase the specificity of the primers, a base was intentionally added for incompatibility. Therefore, allele discrimination on fragment analysis has been made possible through the 3–6 bp differences in the amplicons.With the optimization of the system, designed synthetic sequences provided reliable and reproducible results in wild-type, heterozygous, and homozygous genotypes using the VFLASP-ARMS method. Hence, our results demonstrated that VFLASP-ARMS method, offers a novel design methodology that can be included in the content of SNP genotyping assays.

Abstract Objectives The collection of genotype data was conducted as an essential part of a pivotal research project with the goal of examining the genetic variability of skin, hair, and iris color among the Kazakh population. The data has practical application in the field of forensic DNA phenotyping (FDA). Due to the limited size of forensic databases from Central Asia (Kazakhstan), it is practically impossible to obtain an individual identification result based on forensic profiling of short tandem repeats (STRs). However, the pervasive use of the FDA necessitates validation of the currently employed set of genetic markers in a variety of global populations. No such data existed for the Kazakhs. The Phenotype Expert kit (DNA Research Center, LLC, Russia) was used for the first time in this study to collect data. Data description The present study provides genotype data for a total of 60 SNP genetic markers, which were analyzed in a sample of 515 ethnic Kazakhs. The dataset comprises a total of 41 single nucleotide polymorphisms (SNPs) obtained from the HIrisPlex-S panel. Additionally, there are 4 SNPs specifically related to the AB0 gene, 1 marker associated with the AMELX/Y genes, and 14 SNPs corresponding to the primary haplogroups of the Y chromosome. The aforementioned data could prove valuable to researchers with an interest in investigating genetic variability and making predictions about phenotype based on eye color, hair color, skin color, AB0 blood group, gender, and biogeographic origin within the male lineage.

Objectives: The collection of genotype data was conducted as an essential part of a pivotal research project with the goal of examining the genetic variability of skin, hair, and iris color among the Kazakh population. The data has practical application in the field of forensic DNA phenotyping (FDA). Due to the limited size of forensic databases from Central Asia (Kazakhstan), it is practically impossible to obtain an individual identification result based on forensic profiling of short tandem repeats (STRs). However, the pervasive use of the FDA necessitates validation of the currently employed set of genetic markers in a variety of global populations. No such data existed for the Kazakhs. The Phenotype Expert kit (DNA Research Center, LLC, Russia) was used for the first time in this study to collect data. Data description: The present study provides genotype data for a total of 60 SNP genetic markers, which were analyzed in a sample of 515 ethnic Kazakhs. The dataset comprises a total of 41 single nucleotide polymorphisms (SNPs) obtained from the HIrisPlex-S panel. Additionally, there are 4 SNPs specifically related to the AB0 gene, 1 marker associated with the AMELX/Y genes, and 14 SNPs corresponding to the primary haplogroups of the Y chromosome. The aforementioned data could prove valuable to researchers with an interest in investigating genetic variability and making predictions about phenotype based on eye color, hair color, skin color, AB0 blood group, gender, and biogeographic origin within the male lineage. [ABSTRACT FROM AUTHOR]

Introduction: Chickpea is a legume crop that thrives in regions with semi-arid or temperate climates. Its seeds are an excellent source of proteins, carbohydrates, and minerals, especially high-quality proteins. Chickpea cultivation faces several challenges including Fusarium wilt (FW), a major fungal disease that significantly reduces productivity. Methods: In this study, a Genome-wide Association Analysis (GWAS) was conducted to identify multiple genomic loci associated with FW resistance in chickpea. We conducted a comprehensive evaluation of 180 chickpea genotypes for FW resistance across three distinct locations (Ethiopia, Tunisia, and Lebanon) during the 2-year span from 2015 to 2016. Disease infection measurements were recorded, and the wilt incidence of each genotype was calculated. We employed a set of 11,979 single nucleotide polymorphisms (SNPs) markers distributed across the entire chickpea genome for SNP genotyping. Population structure analysis was conducted to determine the genetic structure of the genotypes. Results and Discussion: The population structure unveiled that the analyzed chickpea germplasm could be categorized into four sub-populations. Notably, these sub-populations displayed diverse geographic origins. The GWAS identified 11 SNPs associated with FW resistance, dispersed across the genome. Certain SNPs were consistent across trials, while others were specific to particular environments. ChromosomeCA2 harbored five SNPmarkers, CA5 featured two, and CA4, CA6, CA7, andCA8 each had one representative marker. Four SNPs demonstrated an association with FW resistance, consistently observed across a minimum of three distinct environments. These SNPs included SNP5826041, SNP5825086, SNP11063413, SNP5825195, which located in CaFeSOD, CaS13like, CaNTAQ1, and CaAARS genes, respectively. Further investigations were conducted to gain insights into the functions of these genes and their role in FW resistance. This progress holds promise for reducing the negative impact of the disease on chickpea production. [ABSTRACT FROM AUTHOR]

Seed coat color is an important consumer-related trait of the cowpea (Vigna unguiculata [L.] Walp.) and has been a subject of study for over a century. Utilizing newly available resources, including mapping populations, a high-density genotyping platform, and several genome assemblies, the red seed coat color has been mapped to two loci, Red-1 (R-1) and Red-2 (R-2), on Vu03 and Vu07, respectively. A gene model (Vigun03g118700) encoding a dihydroflavonol 4-reductase, a homolog of anthocyanidin reductase 1, which catalyzes the biosynthesis of epicatechin from cyanidin, has been identified as a candidate gene for R-1. Possible causative variants have also been identified for Vigun03g118700. A gene model on Vu07 (Vigun07g118500), with predicted nucleolar function and high relative expression in the developing seed, has been identified as a candidate for R-2. The observed red color is believed to be the result of a buildup of cyanidins in the seed coat. [ABSTRACT FROM AUTHOR]

To establish a genotyping technique for single nucleotide polymorphism(SNP) in highly homologous segments, the homology of 200 and 400 bp segments of SNP was evaluated by running local Blast, and the highly homologous segments SNP were screened out after evaluation. The first round of multiple long-range polymerase chain reaction (PCR) was used to capture long fragments of 9 highly homologous segments of SNP in 329 samples. The purified products of the first round of PCR were used as templates for amplicon library construction and sequencing. The results show that 2 928 SNP loci are detected in 329 samples, and the success rate of sequencing is as high as 98.885 6% . According to the Hardy-Weinberg(HWE) principle, the gene frequencies of SNP loci in 9 high homologous segments were calculated(p values were greater than 0.05). Compared with the gene frequencies obtained from thousands of genomes database in national center for biotechnology information (NCBI), it was found that the single base gene frequencies of the two segments were consistent(deviance<0.15). It is shown that the use of multi-long PCR targeted capture technology combined with second-generation sequencing, provided an accurate, rapid and large sample detection solution for highly homologous segments [ABSTRACT FROM AUTHOR]

Indian cattle breeds are diverse and well adapted to 15 different agro-climatic regions. Assessment of genetic diversity and population structure is highly essential for conservation and utilization of indigenous cattle genetic resources. Genome-wide Single Nucleotide Polymorphism (SNP) information is widely used for genetic characterization among the cattle population. Average heterozygosity was estimated as 0.35 among 11 diverse dairy, dual and draft type Indian cattle breeds, which provided scope for their genetic improvement through selection. Indian indigenous breeds are genetically distinct from exotic dairy cattle breeds, whereas a substantially shorter genetic distance was observed among Indian breeds. This supports the evolutionary theory of Indicine zebu cattle and their divergence from Taurine cattle. Principal component analysis revealed that the draft/dual breeds i.e. Vechur, Kangayam and Ongole are clustered separately from dairy type breeds. Population structure analysis identified the presence of highest level of admixture in Vechur cattle, due to which its population is sizably declining. Genetic characterization is essential to preserve the genomic variability which can further be used for genetic improvement of performance traits in a population. Therefore, this review suggests the genetic characterization of indigenous cattle population for formulating conservation policies in order to prevent our native population from extinction. [ABSTRACT FROM AUTHOR]

Background: Sugarcane (Saccharum spp.) is an economically significant crop for both the sugar and biofuel industries. Breeding sugarcane cultivars with high-performance agronomic traits is the most effective approach for meeting the rising demand for sugar and biofuels. Molecular markers associated with relevant agronomic traits could drastically reduce the time and resources required to develop new sugarcane varieties. Previous sugarcane candidate gene association analyses have found single nucleotide polymorphism (SNP) markers associated with sugar-related traits. This study aims to validate these associated SNP markers of six genes, including Lesion simulating disease 1 (LSD), Calreticulin (CALR), Sucrose synthase 1 (SUS1), DEAD-box ATP-dependent RNA helicase (RH), KANADI1 (KAN1), and Sodium/hydrogen exchanger 7 (NHX7), in a diverse population in 2-year and two-location evaluations. Methods: After genotyping of seven targeted SNP markers was performed by PCR Allelic Competitive Extension (PACE) SNP genotyping, the association with sugar-related traits and important cane yield component traits was determined on a set of 159 sugarcane genotypes. The marker-trait relationships were validated and identified by both t-test analysis and an association analysis based on the general linear model. Results: The mSoSUS1_SNPCh10.T/C and mSoKAN1_SNPCh7.T/C markers that were designed from the SUS1 and KAN1 genes, respectively, showed significant associations with different amounts of sugar-related traits and yield components. The mSoSUS1_SNPCh10.T/C marker was found to have more significant association with sugar-related traits, including pol, CCS, brix, fiber and sugar yield, with p values of 6.08 x 10−6 to 4.35 x 10−2, as well as some cane yield component traits with p values of 1.61 x 10−4 to 3.35 x 10−2. The significant association is consistent across four environments. Conclusion: Sucrose synthase (SUS) is considered a crucial enzyme involved in sucrose metabolism. This marker is a high potential functional marker that may be used in sugarcane breeding programs to select superior sugarcane with good fiber and high sugar contents. [ABSTRACT FROM AUTHOR]

Abstract Wolves are assumed to be ungulate obligates, however, a recently described pack on Pleasant Island, Alaska USA, is persisting on sea otters and other marine resources without ungulate prey, violating this long‐held assumption. We address questions about these wolves regarding their origin and fate, degree of isolation, risk of inbreeding depression, and diet specialization by individual and sex. We applied DNA metabarcoding and genotyping by amplicon sequencing using 957 scats collected from 2016 to 2022, and reduced representation sequencing of tissue samples to establish a detailed understanding of Pleasant Island wolf ecology and compare them with adjacent mainland wolves. Dietary overlap was higher among individual wolves on Pleasant Island (Pianka's index mean 0.95 ± 0.03) compared to mainland wolves (0.70 ± 0.21). The individual diets of island wolves were dominated by sea otter, ranging from 40.6% to 63.2% weighted percent of occurrence (wPOO) (mean 55.5 ± 8.7). In contrast, individual mainland wolves primarily fed on ungulates (42.2 ± 21.3) or voles during a population outbreak (31.2 ± 23.2). We traced the origin of the Pleasant Island pack to a mainland pair that colonized around 2013 and produced several litters. After this breeding pair was killed, their female offspring and an immigrant male became the new breeders in 2019. We detected 20 individuals of which 8 (40%) were trapped and killed while two died of natural causes during the 6‐year study. Except for the new breeding male, the pedigree analysis and genotype results showed no additional movement to or from the island, indicating limited dispersal but no evidence of inbreeding. Our findings suggest wolves exhibit more flexible foraging behavior than previously believed, and hunting strategies can substantially differ between individuals within or between packs. Nevertheless, anthropogenic and natural mortality combined with limited connectivity to the mainland may inhibit the continued persistence of Pleasant Island wolves.

Improving the seed protein concentration (SPC) of pea (Pisum sativum L.) has turned into an important breeding objective because of the consumer demand for plant-based protein and demand from protein fractionation industries. To support the marker-assisted selection (MAS) of SPC towards accelerated breeding of improved cultivars, we have explored two diverse recombinant inbred line (RIL) populations to identify the quantitative trait loci (QTLs) associated with SPC. The two RIL populations, MP 1918 × P0540-91 (PR-30) and Ballet × Cameor (PR-31), were derived from crosses between moderate SPC × high SPC accessions. A total of 166 and 159 RILs of PR-30 and PR-31, respectively, were genotyped using an Axiom® 90K SNP array and 13.2K SNP arrays, respectively. The RILs were phenotyped in replicated trials in two and three locations of Saskatchewan, Canada in 2020 and 2021, respectively, for agronomic assessment and SPC. Using composite interval mapping, we identified three QTLs associated with SPC in PR-30 and five QTLs in PR-31, with the LOD value ranging from 3.0 to 11.0. A majority of these QTLs were unique to these populations compared to the previously known QTLs for SPC. The QTL SPC-Ps-5.1 overlapped with the earlier reported SPC associated QTL PC-QTL-3. Three QTLs, SPC-Ps-4.2, SPC-Ps-5.1, and SPC-Ps-7.2 with LOD scores of 7.2, 7.9, and 11.3, and which explained 14.5%, 11.6%, and 11.3% of the phenotypic variance, respectively, can be used for marker-assisted breeding to increase SPC in peas. Eight QTLs associated with the grain yield were identified with LOD scores ranging from 3.1 to 8.2. Two sets of QTLs, SPC-Ps-2.1 and GY-Ps-2.1, and SPC-Ps-5.1 and GY-Ps-5.3, shared the QTL/peak regions. Each set of QTLs contributed to either SPC or grain yield depending on which parent the QTL region is derived from, thus confirming that breeding for SPC should take into consideration the effects on grain yield.

Schizophrenia is a complex condition with entwined genetic and epigenetic risk factors, posing a challenge to disentangle the intermixed pathological and therapeutic epigenetic signatures. To resolve this, we performed 850K methylome-wide and 700K genome-wide studies on the same set of schizophrenia patients by stratifying them into responders, non-responders, and drug-naïve patients. The key genes that signified the response were followed up using real-time gene expression studies to understand the effect of antipsychotics at the gene transcription level. The study primarily implicates hypermethylation in therapeutic response and hypomethylation in the drug-non-responsive state. Several differentially methylated sites and regions colocalized with the schizophrenia genome-wide association study (GWAS) risk genes and variants, supporting the convoluted gene–environment association. Gene ontology and protein–protein interaction (PPI) network analyses revealed distinct patterns that differentiated the treatment response from drug resistance. The study highlights the strong involvement of several processes related to nervous system development, cell adhesion, and signaling in the antipsychotic response. The ability of antipsychotic medications to alter the pathology by modulating gene expression or methylation patterns is evident from the general increase in the gene expression of response markers and histone modifiers and the decrease in class II human leukocyte antigen (HLA) genes following treatment with varying concentrations of medications like clozapine, olanzapine, risperidone, and haloperidol. The study indicates a directional overlap of methylation markers between pathogenesis and therapeutic response, thereby suggesting a careful distinction of methylation markers of pathogenesis from treatment response. In addition, there is a need to understand the trade-off between genetic and epigenetic observations. It is suggested that methylomic changes brought about by drugs need careful evaluation for their positive effects on pathogenesis, course of disease progression, symptom severity, side effects, and refractoriness.

Background Sugarcane (Saccharum spp.) is an economically significant crop for both the sugar and biofuel industries. Breeding sugarcane cultivars with high-performance agronomic traits is the most effective approach for meeting the rising demand for sugar and biofuels. Molecular markers associated with relevant agronomic traits could drastically reduce the time and resources required to develop new sugarcane varieties. Previous sugarcane candidate gene association analyses have found single nucleotide polymorphism (SNP) markers associated with sugar-related traits. This study aims to validate these associated SNP markers of six genes, including Lesion simulating disease 1 (LSD), Calreticulin (CALR), Sucrose synthase 1 (SUS1), DEAD-box ATP-dependent RNA helicase (RH), KANADI1 (KAN1), and Sodium/hydrogen exchanger 7 (NHX7), in a diverse population in 2-year and two-location evaluations. Methods After genotyping of seven targeted SNP markers was performed by PCR Allelic Competitive Extension (PACE) SNP genotyping, the association with sugar-related traits and important cane yield component traits was determined on a set of 159 sugarcane genotypes. The marker-trait relationships were validated and identified by both t-test analysis and an association analysis based on the general linear model. Results The mSoSUS1_SNPCh10.T/C and mSoKAN1_SNPCh7.T/C markers that were designed from the SUS1 and KAN1 genes, respectively, showed significant associations with different amounts of sugar-related traits and yield components. The mSoSUS1_SNPCh10.T/C marker was found to have more significant association with sugar-related traits, including pol, CCS, brix, fiber and sugar yield, with p values of 6.08 × 10−6 to 4.35 × 10−2, as well as some cane yield component traits with p values of 1.61 × 10−4 to 3.35 × 10−2. The significant association is consistent across four environments. Conclusion Sucrose synthase (SUS) is considered a crucial enzyme involved in sucrose metabolism. This marker is a high potential functional marker that may be used in sugarcane breeding programs to select superior sugarcane with good fiber and high sugar contents.

Simple Summary: Rapid diagnostic tools are critical for the management and eradication of boll weevil, Anthonomus grandis grandis. Here, we present the development and validation of a novel qPCR assay that enables same-day identification of Anthonomus grandis subspecies. Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7–9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts. [ABSTRACT FROM AUTHOR]

Single‐nucleotide polymorphism (SNP) analysis is a powerful tool for population genetics, pedigree reconstruction and phenotypic trait mapping. However, the untapped potential of SNP markers to discriminate the sex of individuals in species with reduced sexual dimorphism or of individuals during immature stages remains a largely unexplored avenue. Here, we developed a novel protocol for molecular sexing of birds based on the detection of unique Z‐ and W‐linked SNP markers. Our method is based on the identification of two unique loci, one in each sexual chromosome. Individuals are considered males when they show no calls for the W‐linked SNP and are heterozygous or homozygous for the Z‐linked SNP, while females exhibit both Z‐ and W‐linked SNP calls. We validated the method in the Jackdaw (Corvus monedula). The reduced sexual dimorphism in this species makes it difficult to identify the sex of individuals in the wild. We assessed the reliability of the method using 36 individuals of known sex and found that their sex was correctly assigned in 100% of cases. The sex‐linked markers also proved to be widely applicable for discriminating males and females from a sample of 927 genotyped individuals at different maturity stages, with an accuracy of 99.5%. Since SNP markers are increasingly used in quantitative genetic analyses of wild populations, the approach we propose has great potential to be integrated into broader genetic research programmes without the need for additional sexing techniques. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus, AACC) was formed by hybridization between progenitor species Brassica rapa (AA) and Brassica oleracea (CC). As a result of a limited number of hybridization events between specific progenitor genotypes and strong breeding selection for oil quality traits, rapeseed has limited genetic diversity. The production of resynthesized B. napus lines via interspecific hybridization of the diploid progenitor species B. rapa and B. oleracea is one possible way to increase genetic variation in rapeseed. However, most resynthesized lines produced so far have been reported to be meiotically unstable and infertile, in contrast to established B. napus cultivars. This hinders both maintenance and use of this germplasm in breeding programs. We characterized a large set of 140 resynthesized lines produced by crosses between B. rapa and B. oleracea, as well as between B. rapa and wild C genome species (B. incana, B. hilarionis, B. montana, B. Bourgeaui, B. villosa and B. cretica) for purity (homozygosity), fertility, and genome stability. Self-pollinated seed set, seeds per ten pods as well as percentage pollen viability were used to estimate fertility. SNP genotyping was performed using the Illumina Infinium Brassica 60K array for 116 genotypes, with at least three individuals per line. Most of the material which had been advanced through multiple generations was no longer pure, with heterozygosity detected corresponding to unknown parental contributions via outcrossing. Fertility and genome stability were both genotype-dependent. Most lines had high numbers of copy number variants (CNVs), indicative of meiotic instability, and high numbers of CNVs were significantly associated with reduced fertility. Eight putatively stable resynthesized B. napus lines were observed. Further investigation of these lines may reveal the mechanisms underlying this effect. Our results suggest that selection of stable resynthesized lines for breeding purposes is possible.

Mitochondrial (mtDNA) and chloroplast (cpDNA) polymorphisms are valuable resources to study past demographic changes, phylogenetics and evolution, especially in forest tree species, where these genomes are haploid and uniparentally transferred. The organellar markers were usually scored separately using direct sequencing or PCR-based approaches, which can be time-consuming and expensive, especially in large-scale population genetics research. In this study, we developed an efficient and cost-effective SNaPshot assay for genotyping preselected mtDNA and cpDNA polymorphism in four closely related pine species including Scots pine (Pinus sylvestris L.) and three taxa from the Pinus mugo complex. We validated the method by genotyping the samples derived from 12 populations of the species from their wide geographical distribution range in Europe. The results proved high accuracy of the method with a genotyping success rate of 99.7%. The set of assayed markers shows significant genetic variation. By using multiplex SNaPshot assay, we provided an efficient and sensitive molecular tool for intra- and interspecific genetic analyses. The presented protocol is useful for fast and relatively cheap SNP genotyping of organelle genome of closely related pine species. The assayed SNPs allow studying the species discrimination and detailed investigations of their population history and structure. Given its numerous benefits and efficient genotyping rate, the SNaPshot method appears to be a valuable and practical resource for studying the genetic makeup of forest tree species. Particularly, it proves to be advantageous for population genetics. [ABSTRACT FROM AUTHOR]

Consumer preferences for cooking-related traits are a deciding factor in the success of new cowpea [Vigna unguiculata (L.) Walp] cultivars. Pigment leaching is an undesirable trait for both consumers and producers alike that occurs during the cooking or canning process and has been a goal for improvement efforts through breeding. This study leverages the power of bulked segregant analysis to identify a locus segregating for the pigment-leaching trait in an F2 population of blackeye seed-type cowpea. A single major locus was identified on Vu06 spanning 1.27 Mb, and SNP haplotypes were identified for low and high pigment-leaching bulks. However, further evaluation of accessions that are unrelated to the F2 population or its progenitors suggests that the trait is polygenic, such that low or high leakage is not determined solely by this locus. Parallels were drawn between cowpea and a close relative, black bean (Phaseolus vulgaris L.), to suggest that additional seed coat or cooking-related traits may also be involved in the pigment-leaching trait. [ABSTRACT FROM AUTHOR]

The potential therapeutic benefits of cannabidiol (CBD) require further study. Here, we report a triple-blind (participant, investigator, and outcome assessor) placebo-controlled crossover study in which 62 hypertensive volunteers were randomly assigned to receive the recently developed DehydraTECH2.0 CBD formulation or a placebo. This is the first study to have been conducted using the DehydraTECH2.0 CBD formulation over a 12-week study duration. The new formulation's long-term effects on CBD concentrations in plasma and urine, as well as its metabolites 7-hydroxy-CBD and 7-carboxy-CBD, were analyzed. The results of the plasma concentration ratio for CBD/7-OH-CBD in the third timepoint (after 5 weeks of use) were significantly higher than in the second timepoint (after 2.5 weeks of use; p = 0.043). In the same timepoints in the urine, a significantly higher concentration of 7-COOH-CBD was observed p < 0.001. Differences in CBD concentration were found between men and women. Plasma levels of CBD were still detectable 50 days after the last consumption of the CBD preparations. Significantly higher plasma CBD concentrations occurred in females compared to males, which was potentially related to greater adipose tissue. More research is needed to optimize CBD doses to consider the differential therapeutic benefits in men and women. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) have been widely used for gene identification. Allelic discrimination for an individual SNP with high reliability and flexibility is critical for the accurate detection of beneficial genes linked to specific SNP sites. Several SNP genotyping platforms have been developed but most exclusively rely on fluorescence signals for allelic differentiation. Genotyping via a fluorescence signal can have a lower accuracy if strong background signal noise is present, a common challenge associated with crop genetics. The semi-thermal asymmetric reverse PCR (STARP) marker system introduces extra SNPs in its forward primers to ensure specificity of the PCR reaction and adds a 4-nucleotide insertion into one universal primer to create fragment length polymorphism among STARP markers, which makes SNP allelic discrimination possible through either fluorescence signals or traditional gel electrophoresis. The STARP marker system is preferable for SNP genotyping in crops such as sugarbeet (Beta vulgaris ssp. Vulgaris L.) that exhibit strong background signal noise during PCR reactions due to an abundant repetitive sequence and high levels of heterozygosity in the genome. In this study, SNPs among sugarbeet lines were detected through genotype-by-sequencing (GBS) and confirmed by sequencing PCR products containing SNP sites. STARP primers were designed, and they generated STARP markers clearly discriminated by SNP alleles among sugarbeet plants either through a fluorescence signal or fragment length polymorphism. In addition, by prolonging 5-nucleotide in an allele-specific forward primer F2 that increased fragment length polymorphism of STARP markers from 4-bp to 9-bp, genotyping individual SNPs can be performed using user-friendly agarose gels. This research resulted in the development of a STARP marker platform for the flexible genotyping of individual SNPs of sugarbeet as well as an improved STARP technique for easy SNP allelic discrimination that also has utilities in other plant species. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD) is a non-psychoactive cannabinoid, and available evidence suggests potential efficacy in the treatment of many disorders. DehydraTECH™2.0 CBD is a patented capsule formulation that improves the bioabsorption of CBD. We sought to compare the effects of CBD and DehydraTECH™2.0 CBD based on polymorphisms in CYP P450 genes and investigate the effects of a single CBD dose on blood pressure. In a randomized and double-blinded order, 12 females and 12 males with reported hypertension were given either placebo capsules or DehydraTECH™2.0 CBD (300 mg of CBD, each). Blood pressure and heart rate were measured during 3 h, and blood and urine samples were collected. In the first 20 min following the dose, there was a greater reduction in diastolic blood pressure (p = 0.025) and mean arterial pressure MAP (p = 0.056) with DehydraTECH™2.0 CBD, which was probably due to its greater CBD bioavailability. In the CYP2C9*2*3 enzyme, subjects with the poor metabolizer (PM) phenotype had higher plasma CBD concentrations. Both CYP2C19*2 (p = 0.037) and CYP2C19*17 (p = 0.022) were negatively associated with urinary CBD levels (beta = −0.489 for CYP2C19*2 and beta = −0.494 for CYP2C19*17). Further research is required to establish the impact of CYP P450 enzymes and the identification of metabolizer phenotype for the optimization of CBD formulations. [ABSTRACT FROM AUTHOR]

Here, we present the R package, minSNPs. This is a re-development of a previously described Java application named Minimum SNPs. MinSNPs assembles resolution- optimised sets of single nucleotide polymorphisms (SNPs) from sequence alignments such as genome-wide orthologous SNP matrices. MinSNPs can derive sets of SNPs optimised for discriminating any user-defined combination of sequences from all others. Alternatively, SNP sets may be optimised to determine all sequences from all other sequences, i.e., to maximise diversity. MinSNPs encompasses functions that facilitate rapid and flexible SNP mining, and clear and comprehensive presentation of the results. The minSNPs' running time scales in a linear fashion with input data volume and the numbers of SNPs and SNPs sets specified in the output. MinSNPs was tested using a previously reported orthologous SNP matrix of Staphylococcus aureus and an orthologous SNP matrix of 3,279 genomes with 164,335 SNPs assembled from four S. aureus short read genomic data sets. MinSNPs was shown to be effective for deriving discriminatory SNP sets for potential surveillance targets and in identifying SNP sets optimised to discriminate isolates from different clonal complexes. MinSNPs was also tested with a large Plasmodium vivax orthologous SNP matrix. A set of five SNPs was derived that reliably indicated the country of origin within three south-east Asian countries. In summary, we report the capacity to assemble comprehensive SNP matrices that effectively capture microbial genomic diversity, and to rapidly and flexibly mine these entities for optimised marker sets. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus, AACC) was formed by hybridization between progenitor species Brassica rapa (AA) and Brassica oleracea (CC). As a result of a limited number of hybridization events between specific progenitor genotypes and strong breeding selection for oil quality traits, rapeseed has limited genetic diversity. The production of resynthesized B. napus lines via interspecific hybridization of the diploid progenitor species B. rapa and B. oleracea is one possible way to increase genetic variation in rapeseed. However, most resynthesized lines produced so far have been reported to be meiotically unstable and infertile, in contrast to established B. napus cultivars. This hinders both maintenance and use of this germplasm in breeding programs. We characterized a large set of 140 resynthesized lines produced by crosses between B. rapa and B. oleracea, as well as between B. rapa and wild C genome species (B. incana, B. hilarionis, B. montana, B. Bourgeaui, B. villosa and B. cretica) for purity (homozygosity), fertility, and genome stability. Self-pollinated seed set, seeds per ten pods as well as percentage pollen viability were used to estimate fertility. SNP genotyping was performed using the Illumina Infinium Brassica 60K array for 116 genotypes, with at least three individuals per line. Most of the material which had been advanced through multiple generations was no longer pure, with heterozygosity detected corresponding to unknown parental contributions via outcrossing. Fertility and genome stability were both genotypedependent. Most lines had high numbers of copy number variants (CNVs), indicative of meiotic instability, and high numbers of CNVs were significantly associated with reduced fertility. Eight putatively stable resynthesized B. napus lines were observed. Further investigation of these lines may reveal the mechanisms underlying this effect. Our results suggest that selection of stable resynthesized lines for breeding purposes is possible. [ABSTRACT FROM AUTHOR]

Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific position in the genome. Until now, 585 million SNPs have been identified in the human genome, and therefore, a widely applicable method is desirable to detect a specific SNP. Herein we report a simple and reliable genotyping assay, which seems to be suitable for medium and small size laboratories, as well, to easily genotype most of the SNPs. In our study, all of the possible base variations (A-T, A-G, A-C, T-G, T-C, G-C) were tested to prove the general feasibility of our technique. The basis of the assay is a fluorescent PCR, in which both allele-specific primers, differing only at the 3′ end according to the sequence of the SNP, were present, and the length of one of them was modified with 3 bp by adding an adapter sequence to the 5’ end of that primer. The competitive presence of both allele-specific primers excludes the false amplification of the absent allele (which can happen in simple allele-specific PCR (AS-PCR)) and ensures the amplification of the proper allele(s). Unlike other complicated genotyping methods that use of manipulation of fluorescent dyes for genotyping, we apply an approach based on the length of amplicons from different alleles to differentiate between them. In our experiment (named variable fragment length allele-specific polymerase chain reaction (VFLASP)), the investigated six SNPs, containing the six available base variations, gave clear and reliable results after detecting the amplicons by capillary electrophoresis.

Seed coat color is an important consumer-related trait of the cowpea (Vigna unguiculata [L.] Walp.) and has been a subject of study for over a century. Utilizing newly available resources, including mapping populations, a high-density genotyping platform, and several genome assemblies, the red seed coat color has been mapped to two loci, Red-1 (R-1) and Red-2 (R-2), on Vu03 and Vu07, respectively. A gene model (Vigun03g118700) encoding a dihydroflavonol 4-reductase, a homolog of anthocyanidin reductase 1, which catalyzes the biosynthesis of epicatechin from cyanidin, has been identified as a candidate gene for R-1. Possible causative variants have also been identified for Vigun03g118700. A gene model on Vu07 (Vigun07g118500), with predicted nucleolar function and high relative expression in the developing seed, has been identified as a candidate for R-2. The observed red color is believed to be the result of a buildup of cyanidins in the seed coat.

The whitefy Bemisia tabaci, a species complex consisting of many morphologically indistinguishable species divided into distinct clades, is one of the most globally important agricultural pests and plant virus vectors. Cassava-colonizing B. tabaci transmits viruses that cause cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Half of all cassava plants in Africa are affected by these viral diseases, resulting in annual production losses of more than US$ 1 billion. Here we report the draft genome of the cassava whitefly B. tabaci Sub-Saharan Africa - East and Central Africa (SSA-ECA), the super-abundant population that has been associated with the rapid spread of viruses causing the pandemics of CMD and CBSD. The SSA-ECA genome assembled from Illumina short reads has a total size of 513.7 Mb and a scaffold N50 length of 497 kb, and contains 15,084 predicted protein-coding genes. Phylogenetic analysis suggests that SSA-ECA diverged from MEAM1 around 5.26 million years ago. A comprehensive genetic analysis of cassava-colonizing B. tabaci in Africa was also conducted, in which a total of 243 whitefly specimens were collected from 18 countries representing all major cassava-growing regions in the continent and genotyped using NextRAD sequencing. Population genomic analyses confirmed the existence of six major populations linked by gene flow and inferred the distribution patterns of these populations across the African continent. The genome of SSA-ECA and the genetic findings provide valuable resources and guidance to facilitate whitefly research and the development of strategies to control cassava viral diseases spread by whiteflies.

Oilseed rape (Brassica napus) is an important oil crop distributed worldwide with a broad adaptation to different climate zones. The cultivation of rapeseed is one of the most commercially viable areas in crop production. Altogether 269,093 ha of rapeseed are cultivated in Kazakhstan. However, all rapeseed cultivars and lines cultivated in Kazakhstan on an industrial scale predominantly belong to the foreign breeding system. Therefore, the formation of a diverse genetic pool for breeding new, highly productive cultivars adopted to the environmental conditions of Kazakhstan is the most important goal in country selection programs. In this work, we have developed ethyl methanesulfonate (EMS) doubled haploid mutant lines from plant material of cultivars ‘Galant’ and ‘Kris’ to broad diversity of rapeseed in Kazakhstan. The development of mutant lines was performed via embryo callusogenesis or embryo secondary callusogenesis. Mutants were investigated by Brassica90k SNP array, and we were able to locate 24,657 SNPs from 26,256 SNPs filtered by quality control on the genome assembly (Bra_napus_v2.0). Only 18,831 SNPs were assigned to the available annotated genomic features. The most frequent combination of mutations according to reference controls was adenine with guanine (70%), followed by adenine with cytosine (28.8%), and only minor fractions were cytosine with guanine (0.54%) and adenine with thymine (0.59%). We revealed 5606.27 markers for ‘Kris’ and 4893.01 markers for ‘Galant’ by mutation occurrence. Most mutation occurrences were occupied by double mutations where progenitors and offspring were homozygous by different alleles, enabling the selection of appropriate genotypes in a short period of time. Regarding the biological impact of mutations, 861 variants were reported as having a low predicted impact, with 1042 as moderate and 121 as high; all others were reported as belonging to non-coding sequences, intergenic regions, and other features with the effect of modifiers. Protein encoding genes, such as wall-associated receptor kinase-like protein 5, TAO1-like disease resistance protein, receptor-like protein 12, and At5g42460-like F-box protein, contained more than two variable positions, with an impact on their biological activities. Nevertheless, the obtained mutant lines were able to survive and reproduce. Mutant lines, which include moderate and high impact mutations in encoding genes, are a perfect pool not only for MAS but also for the investigation of the fundamental basis of protein functions. For the first time, a collection of mutant lines was developed in our country to improve the selection of local rapeseed cultivars.