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1.
Genet Sel Evol ; 53(1): 63, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301193

RESUMO

BACKGROUND: Linkage disequilibrium (LD) is a key parameter to study the history of populations and to identify and fine map quantitative trait loci (QTL) and it has been studied for many years in animal populations. The advent of new genotyping technologies has allowed whole-genome LD studies in most cattle populations. However, to date, long-range LD (LRLD) between distant variants on the genome has not been investigated in detail in cattle. Here, we present the first comprehensive study of LRLD in French beef cattle by analysing data on 672 Charolais (CHA), 462 Limousine (LIM) and 326 Blonde d'Aquitaine (BLA) individuals that were genotyped on the Illumina BovineHD Beadchip. Furthermore, whole-genome LD and haplotype block structure were analysed in these three breeds. RESULTS: We computed linkage disequilibrium (r2) values for 5.9, 5.6 and 6.0 billion pairs of SNPs on the 29 autosomes of CHA, LIM and BLA, respectively. Mean r2 values drop to less than 0.1 for distances between SNPs greater than 120 kb. However, for the first time, we detected the existence of LRLD in the three main French beef breeds. In total, 598, 266, and 795 LRLD events (r2 ≥ 0.6) were detected in CHA, LIM and BLA, respectively. Each breed had predominantly population-specific LRLD interactions, although shared LRLD events occurred in a number of regions (55 LRLD events were shared between two breeds and nine between the three breeds). Examples of possible functional gene interactions and QTL co-location were observed with some of these LRLD events, which suggests epistatic selection. CONCLUSIONS: We identified long-range linkage disequilibrium for the first time in French beef cattle populations. Epistatic selection may be the main source of the observed LRLD events, but other forces may also be involved. LRLD information should be accounted for in genome-wide association studies.


Assuntos
Bovinos/genética , Desequilíbrio de Ligação , Animais , Estudo de Associação Genômica Ampla/métodos , Técnicas de Genotipagem/métodos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Carne Vermelha/normas
2.
Genet Sel Evol ; 53(1): 64, 2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34325663

RESUMO

BACKGROUND: With the completion of a single nucleotide polymorphism (SNP) chip for honey bees, the technical basis of genomic selection is laid. However, for its application in practice, methods to estimate genomic breeding values need to be adapted to the specificities of the genetics and breeding infrastructure of this species. Drone-producing queens (DPQ) are used for mating control, and usually, they head non-phenotyped colonies that will be placed on mating stations. Breeding queens (BQ) head colonies that are intended to be phenotyped and used to produce new queens. Our aim was to evaluate different breeding program designs for the initiation of genomic selection in honey bees. METHODS: Stochastic simulations were conducted to evaluate the quality of the estimated breeding values. We developed a variation of the genomic relationship matrix to include genotypes of DPQ and tested different sizes of the reference population. The results were used to estimate genetic gain in the initial selection cycle of a genomic breeding program. This program was run over six years, and different numbers of genotyped queens per year were considered. Resources could be allocated to increase the reference population, or to perform genomic preselection of BQ and/or DPQ. RESULTS: Including the genotypes of 5000 phenotyped BQ increased the accuracy of predictions of breeding values by up to 173%, depending on the size of the reference population and the trait considered. To initiate a breeding program, genotyping a minimum number of 1000 queens per year is required. In this case, genetic gain was highest when genomic preselection of DPQ was coupled with the genotyping of 10-20% of the phenotyped BQ. For maximum genetic gain per used genotype, more than 2500 genotyped queens per year and preselection of all BQ and DPQ are required. CONCLUSIONS: This study shows that the first priority in a breeding program is to genotype phenotyped BQ to obtain a sufficiently large reference population, which allows successful genomic preselection of queens. To maximize genetic gain, DPQ should be preselected, and their genotypes included in the genomic relationship matrix. We suggest, that the developed methods for genomic prediction are suitable for implementation in genomic honey bee breeding programs.


Assuntos
Abelhas/genética , Modelos Genéticos , Seleção Artificial , Animais , Genoma de Inseto , Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/normas , Técnicas de Genotipagem/métodos
3.
Genes (Basel) ; 12(5)2021 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-34065649

RESUMO

The aroma of grapes and derived wines has long been one of the major traits considered in the selection of grapevine varieties through the centuries. In particular, Muscat aromatic grapes have been highly appreciated and widespread since ancient times. Monoterpenes are the key compounds responsible for the Muscat flavor. A major QTL affecting monoterpene level has been found to co-localize with the 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) gene, encoding for the 1-deoxy-D-xylulose 5-phosphate synthase enzyme involved in the plastidial pathway of terpene biosynthesis. In more detail, a single nucleotide polymorphism (SNP 1822) in the coding region of the gene causes a "gain of function" mutation, which is involved in Muscat flavor. In this work, we have developed a digital PCR-based assay to target allelic variations in the VvDXS gene, SNP1822, with the aim to propose a fast and sensitive analytical tool for targeting Muscat-flavored grapevine genotypes. The assay accurately predicts the genetic structure at 1822 SNP, critical for the development of the aroma in the great majority of Muscats. In the case of grapes in which the aromatic component is due to mutations other than SNP 1822 (e.g., Chasselas Musqué and Chardonnay Muscat), further specific assays can be developed.


Assuntos
Técnicas de Genotipagem/métodos , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Transferases/genética , Vitis/genética , Mutação com Ganho de Função , Melhoramento Vegetal/métodos , Reação em Cadeia da Polimerase/métodos , Vitis/metabolismo
4.
J Med Microbiol ; 70(6)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34165424

RESUMO

We characterized 515 Mycoplasma pneumoniae specimens in Hokkaido. In 2013 and 2014, the p1 gene type 1 strain, mostly macrolide-resistant, was dominant and the prevalence of macrolide resistance was over 50 %. After 2017, the p1 gene type 2 lineage, mostly macrolide-sensitive, increased and the prevalence of macrolide resistance became 31.0 % in 2017, 5.3 % in 2018 and 16.3 % in 2019.


Assuntos
Macrolídeos/farmacologia , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/epidemiologia , Criança , Farmacorresistência Bacteriana/genética , Técnicas de Genotipagem/métodos , Humanos , Japão/epidemiologia , Mutação , Mycoplasma pneumoniae/classificação , Mycoplasma pneumoniae/efeitos dos fármacos , Nasofaringe/microbiologia , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/microbiologia , RNA Ribossômico 23S/genética
5.
Genes (Basel) ; 12(5)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946496

RESUMO

The article highlighted the problem of meat cattle genetic defects. The aim was the development of DNA tests for some genetic defects diagnostics, the determination of the animal carriers and their frequencies tracking in time. The 1490 DNA samples from the Aberdeen Angus (n = 701), Hereford (n = 385), Simmental (n = 286) and Belgian Blue (n = 118) cattle have been genotyped on the genetic defects by newly created and earlier developed DNA tests based on AS-PCR and PCR-RFLP methods. The Aberdeen Angus cattle genotyping has revealed 2.38 ± 0.31% AMC-cows and 1.67 ± 0.19 % AMC-bulls, 0.65 ± 0.07% DDC-cows and 0.90 ± 0.10% DDC-bulls. The single animals among the Hereford cattle were carriers of MSUD and CWH (on 0.27 ± 0.05%), ICM and HY (on 0.16 ± 0.03%). The Simmental cattle were free from OS. All Belgian Blue livestock were M1- and 0.84%-CMD1-carriers. The different ages Aberdeen Angus cattle genotyping has shown the tendency of the AMC- and DDC frequencies to increase in the later generations. The statistically significant increase of DDC of 1.17% in the cows' population born in 2019 compared to those born in 2015 allows concluding the further development of the DNA analysis-based measures preventing the manifestation of the genetic anomalies in meat cattle herds is necessary.


Assuntos
Doenças dos Bovinos/genética , Bovinos/genética , Triagem de Portadores Genéticos/veterinária , Animais , Doenças dos Bovinos/diagnóstico , Triagem de Portadores Genéticos/métodos , Triagem de Portadores Genéticos/normas , Técnicas de Genotipagem/métodos , Técnicas de Genotipagem/normas , Técnicas de Genotipagem/veterinária , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade
6.
Genes (Basel) ; 12(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946707

RESUMO

Microsatellite DNA analysis is a powerful tool for assessing population genetics. The main aim of this study was to assess the genetic potential of the peregrine falcon population covered by the restitution program. We characterized individuals from breeders that set their birds for release into the wild and birds that have been reintroduced in previous years. This was done using a well-known microsatellite panel designed for the peregrine falcon containing 10 markers. We calculated the genetic distance between individuals and populations using the UPGMA (unweighted pair group method with arithmetic mean) method and then performed a Principal Coordinates Analysis (PCoA) and constructed phylogenetic trees, to visualize the results. In addition, we used the Bayesian clustering method, assuming 1-15 hypothetical populations, to find the model that best fit the data. Units were segregated into groups regardless of the country of origin, and the number of alleles and observed heterozygosity were different in different breeding groups. The wild and captive populations were grouped independent of the original population.


Assuntos
Falconiformes/genética , Genótipo , Repetições de Microssatélites , Animais , Ecossistema , Espécies em Perigo de Extinção , Falconiformes/classificação , Falconiformes/fisiologia , Técnicas de Genotipagem/métodos , Técnicas de Genotipagem/normas , Filogenia , Polônia , Polimorfismo Genético , Padrões de Referência
7.
Mol Biol Rep ; 48(4): 3593-3604, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33973139

RESUMO

Variants in the epidermal growth factor receptor (EGFR) gene are recognized as predictors of therapy response and are correlated with progression-free and overall survival in non-small cell lung cancer (NSCLC) patients. Molecularly guided therapy needs precise and cost-effective molecular tests. This review focused primarily on screening or target methods for the EGFR variants detection with diagnostic and prognostic potential in the clinical research published papers. Concerning the inclusion and exclusion criteria, the search interval comprised available articles published from 2010 until 2020 in three electronic databases, ISI Web of Science, Pub Med, and Scopus. The analysis of eligible studies started with 5647 and obtained the final 987 full-text articles analyzed as clinical research. The regions comprised were Africa, America, Australia, Asia, Euro-Asia, Europe, or a consortium of different countries. All of the tested methods were applied prevalently in Asia. In clinical research, the polymerase chain reaction (PCR), followed by sequencing methods have been involved mostly over the years. The identified high-through output approaches evolved to improve the survival and quality of the NSCLC patient's life becoming more sensitive, specific, and cost-effective.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Testes Genéticos/métodos , Técnicas de Genotipagem/métodos , Neoplasias Pulmonares/genética , Receptores ErbB/genética , Humanos , Polimorfismo Genético
8.
Eur J Protistol ; 79: 125799, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34044353

RESUMO

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.


Assuntos
Cryptosporidium parvum/classificação , Cryptosporidium parvum/genética , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da Espécie
9.
Nat Commun ; 12(1): 2981, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016985

RESUMO

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Recombinação Genética , Espermatócitos/metabolismo , Animais , Evolução Biológica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos , Cromossomos/genética , Europa (Continente) , Fertilidade/genética , Técnicas de Genotipagem/métodos , Masculino , Camundongos , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Análise do Sêmen , Espermatócitos/citologia
10.
Genes (Basel) ; 12(4)2021 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801723

RESUMO

Tan spot, caused by the fungus Pyrenophoratritici-repentis (Ptr), is a severe foliar disease of wheat (Triticumaestivum L.). Improving genetic resistance is a durable strategy to reduce Ptr-related losses. Here, we dissected Ptr-infection's genetic basis in 372 European wheat varieties via single sequence repeats (SSR) in addition to 35k and 90k single nucleotide polymorphism (SNP) marker platforms. In our phenotypic data analyses, Ptr infection showed a significant genotypic variance and a significant negative correlation with plant height. Genome-wide association studies revealed a highly quantitative nature of Ptr infection and identified two quantitative trait loci (QTL), viz., QTs.ipk-7A and QTs.ipk-7B, which imparted 21.23 and 5.84% of the genotypic variance, respectively. Besides, the Rht-D1 gene showed a strong allelic influence on the resistance scores. Due to the complex genetic nature of the Ptr infection, the potential of genome-wide prediction (GP) was assessed via three different genetic models on individual and combined marker platforms. The GP results indicated that the marker density and marker platforms do not considerably impact prediction accuracy (~40-42%) and that higher-order epistatic interactions may not be highly pervasive. Our results provide a further understanding of Ptr-infection's genetic nature, serve as a resource for marker-assisted breeding, and highlight the potential of genome-wide selection for improved Ptr resistance.


Assuntos
Ascomicetos/patogenicidade , Resistência à Doença , Técnicas de Genotipagem/métodos , Locos de Características Quantitativas , Triticum/genética , Estudo de Associação Genômica Ampla , Repetições de Microssatélites , Modelos Genéticos , Fenótipo , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Triticum/microbiologia
11.
Malar J ; 20(1): 194, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879156

RESUMO

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test. METHODS: A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai-Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed. RESULTS: The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87-100 %] with specificity of 100 % (95 % CI: 87.66-100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females. CONCLUSIONS: The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine.


Assuntos
Técnicas de Genotipagem/métodos , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Malária Vivax/epidemiologia , Feminino , Deficiência de Glucosefosfato Desidrogenase/genética , Humanos , Malária Vivax/parasitologia , Masculino , Tailândia/epidemiologia
12.
Int J Legal Med ; 135(4): 1329-1339, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33884487

RESUMO

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.


Assuntos
Cor de Olho/genética , Genótipo , Técnicas de Genotipagem/instrumentação , Técnicas de Genotipagem/métodos , Cor de Cabelo/genética , Fenótipo , Brasil/etnologia , Genética Forense/métodos , Humanos
13.
Genes (Basel) ; 12(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805090

RESUMO

BACKGROUND: Several genes and single nucleotide polymorphisms (SNPs) have been associated with early childhood caries. However, they are highly age- and population-dependent and the majority of existing caries prediction models are based on environmental and behavioral factors only and are scarce in infants. METHODS: We examined 6 novel and previously analyzed 22 SNPs in the cohort of 95 Polish children (48 caries, 47 caries-free) aged 2-3 years. All polymorphisms were genotyped from DNA extracted from oral epithelium samples. We used Fisher's exact test, receiver operator characteristic (ROC) curve and uni-/multi-variable logistic regression to test the association of SNPs with the disease, followed by the neural network (NN) analysis. RESULTS: The logistic regression (LogReg) model showed 90% sensitivity and 96% specificity, overall accuracy of 93% (p < 0.0001), and the area under the curve (AUC) was 0.970 (95% CI: 0.912-0.994; p < 0.0001). We found 90.9-98.4% and 73.6-87.2% prediction accuracy in the test and validation predictions, respectively. The strongest predictors were: AMELX_rs17878486 and TUFT1_rs2337360 (in both LogReg and NN), MMP16_rs1042937 (in NN) and ENAM_rs12640848 (in LogReg). CONCLUSIONS: Neural network prediction model might be a substantial tool for screening/early preventive treatment of patients at high risk of caries development in the early childhood. The knowledge of potential risk status could allow early targeted training in oral hygiene and modifications of eating habits.


Assuntos
Biologia Computacional/métodos , Suscetibilidade à Cárie Dentária/genética , Redes Reguladoras de Genes , Técnicas de Genotipagem/métodos , Polimorfismo de Nucleotídeo Único , Amelogenina/genética , Estudos de Casos e Controles , Pré-Escolar , Proteínas do Esmalte Dentário/genética , Proteínas da Matriz Extracelular/genética , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Metaloproteinase 16 da Matriz/genética , Redes Neurais de Computação , Polônia
14.
Genes (Basel) ; 12(4)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923933

RESUMO

Traits such as plant height (PH), juvenile growth habit (GH), heading date (HD), and tiller number are important for both increasing yield potential and improving crop adaptation to climate change. In the present study, these traits were investigated by using the same bi-parental population at early (F2 and F2-derived F3 families) and late (F6 and F7, recombinant inbred lines, RILs) generations to detect quantitative trait loci (QTLs) and search for candidate genes. A total of 176 and 178 lines were genotyped by the wheat Illumina 25K Infinium SNP array. The two genetic maps spanned 2486.97 cM and 3732.84 cM in length, for the F2 and RILs, respectively. QTLs explaining the highest phenotypic variation were found on chromosomes 2B, 2D, 5A, and 7D for HD and GH, whereas those for PH were found on chromosomes 4B and 4D. Several QTL detected in the early generations (i.e., PH and tiller number) were not detected in the late generations as they were due to dominance effects. Some of the identified QTLs co-mapped to well-known adaptive genes (i.e., Ppd-1, Vrn-1, and Rht-1). Other putative candidate genes were identified for each trait, of which PINE1 and PIF4 may be considered new for GH and TTN in wheat. The use of a large F2 mapping population combined with NGS-based genotyping techniques could improve map resolution and allow closer QTL tagging.


Assuntos
Cromossomos de Plantas/genética , Técnicas de Genotipagem/métodos , Locos de Características Quantitativas , Triticum/genética , Pão , Mapeamento Cromossômico , Endogamia , Fenótipo , Melhoramento Vegetal
15.
Genes (Basel) ; 12(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916492

RESUMO

The genome of the SARS-CoV-2 virus, the causal agent of the COVID-19 pandemic, has diverged due to multiple mutations since its emergence as a human pathogen in December 2019. Some mutations have defined several SARS-CoV-2 clades that seem to behave differently in terms of regional distribution and other biological features. Next-generation sequencing (NGS) approaches are used to classify the sequence variants in viruses from individual human patients. However, the cost and relative scarcity of NGS equipment and expertise in developing countries prevent studies aimed to associate specific clades and variants to clinical features and outcomes in such territories. As of March 2021, the GR clade and its derivatives, including the B.1.1.7 and B.1.1.28 variants, predominate worldwide. We implemented the post-PCR small-amplicon high-resolution melting analysis to genotype SARS-CoV-2 viruses isolated from the saliva of individual patients. This procedure was able to clearly distinguish two groups of samples of SARS-CoV-2-positive samples predicted, according to their melting profiles, to contain GR and non-GR viruses. This grouping of the samples was validated by means of amplification-refractory mutation system (ARMS) assay as well as Sanger sequencing.


Assuntos
COVID-19/virologia , Técnicas de Genotipagem/métodos , SARS-CoV-2/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Desnaturação de Ácido Nucleico , RNA Viral/isolamento & purificação
17.
Clin Microbiol Infect ; 27(7): 1036.e1-1036.e8, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33813118

RESUMO

OBJECTIVES: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping. METHODS: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination. RESULTS: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020. CONCLUSIONS: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples.


Assuntos
COVID-19/diagnóstico , Técnicas de Genotipagem/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , SARS-CoV-2/genética , Sequenciamento Completo do Genoma/normas , Artefatos , COVID-19/virologia , Genoma Viral , Técnicas de Genotipagem/métodos , Guias como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Viral , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma/métodos , Fluxo de Trabalho
18.
Int J Legal Med ; 135(4): 1257-1265, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33754178

RESUMO

Evaluating the short tandem repeat (STR) in the field is important for the timely identification of a suspect. Several lines showed that the RapidHIT® ID system is reliable for DNA genotyping with buccal swabs and naked DNA. However, the application of this approach with blood samples has been poorly investigated. Because blood samples are among the most common forensic samples in our laboratory, further studies should be conducted. Here, we assessed the analytical performance of 19 STR loci with a newly developed RapidINTEL (RI) Sample Cartridge Kit by using the blood samples with known genotypes. Several commonly used substrates were included in the sensitivity study, and FTA cards proved to be the most promising sample carrier for blood storage and later identification. There was superior sensitivity and specificity with a 100% concordance rate for 0.5 µL of blood or 7 ng of genomic DNA. The performance for blood samples was comparable with that for the standard protocol. High success rate (90.57%) and high-concordance (100%) genotyping were automatically achieved over a wide range of operating conditions except for TH01. No contamination was observed throughout the study. Hematin, indigo, and humic acid had limited influence on the instrument system, while urea and melanin dramatically affected the genotyping results. Generally, the newly developed RI sample cartridge provided an alternative method for the STR genotyping of single-source blood samples over a wide range of operating conditions.


Assuntos
DNA/sangue , Técnicas de Genotipagem/instrumentação , Técnicas de Genotipagem/métodos , Repetições de Microssatélites , Feminino , Humanos , Masculino , Sensibilidade e Especificidade
19.
J Vet Sci ; 22(2): e24, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33774940

RESUMO

BACKGROUND: Bovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea. OBJECTIVES: To survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing. METHODS: A total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs. RESULTS: We identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes. CONCLUSIONS: Through SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.


Assuntos
Cervos , Técnicas de Genotipagem/veterinária , Mycobacterium bovis/genética , Polimorfismo de Nucleotídeo Único , Sus scrofa , Tuberculose Bovina/microbiologia , Animais , Bovinos , Técnicas de Genotipagem/métodos , Mycobacterium bovis/isolamento & purificação , República da Coreia
20.
Genetics ; 217(1): 1-10, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33683359

RESUMO

Errors in genotype calling can have perverse effects on genetic analyses, confounding association studies, and obscuring rare variants. Analyses now routinely incorporate error rates to control for spurious findings. However, reliable estimates of the error rate can be difficult to obtain because of their variance between studies. Most studies also report only a single estimate of the error rate even though genotypes can be miscalled in more than one way. Here, we report a method for estimating the rates at which different types of genotyping errors occur at biallelic loci using pedigree information. Our method identifies potential genotyping errors by exploiting instances where the haplotypic phase has not been faithfully transmitted. The expected frequency of inconsistent phase depends on the combination of genotypes in a pedigree and the probability of miscalling each genotype. We develop a model that uses the differences in these frequencies to estimate rates for different types of genotype error. Simulations show that our method accurately estimates these error rates in a variety of scenarios. We apply this method to a dataset from the whole-genome sequencing of owl monkeys (Aotus nancymaae) in three-generation pedigrees. We find significant differences between estimates for different types of genotyping error, with the most common being homozygous reference sites miscalled as heterozygous and vice versa. The approach we describe is applicable to any set of genotypes where haplotypic phase can reliably be called and should prove useful in helping to control for false discoveries.


Assuntos
Genótipo , Técnicas de Genotipagem/métodos , Linhagem , Animais , Aotidae , Feminino , Técnicas de Genotipagem/normas , Masculino , Padrões de Referência , Reprodutibilidade dos Testes
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