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1.
An Acad Bras Cienc ; 91(suppl 3): e20190339, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31460595

RESUMO

Genetic drift is the fortuitous occurrence of genetic events that when they become fixed modify the genome of populations. They can take the form of mutations of single nucleotides (SNPs), the insertion or deletion of short sequences (Indels) or the repetitions of short sequences (CNV i.e. copy number variants) or long insertions or deletion (structural modifications). Their frequency is 10-9 to 10-8 depending on the species, or 50 to 100 per birth in humans. The incidence of these de novo mutations is higher when the father is old at conception. It thus appears that genetic drift, which constitutes the initial element of evolution, has a very strong dynamics. Its intervention in the appearance or disappearance of some major phenotypes is complicated by the uncertainties about the genetic mechanisms in heritability which, paradoxically, are only partially understood.


Assuntos
Evolução Molecular , Deriva Genética , Mutação INDEL/genética , Mamíferos/genética , Animais , Humanos , Camundongos , Ratos
2.
BMC Bioinformatics ; 20(1): 342, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208315

RESUMO

BACKGROUND: Whole exome sequencing (WES) is a cost-effective method that identifies clinical variants but it demands accurate variant caller tools. Currently available tools have variable accuracy in predicting specific clinical variants. But it may be possible to find the best combination of aligner-variant caller tools for detecting accurate single nucleotide variants (SNVs) and small insertion and deletion (InDels) separately. Moreover, many important aspects of InDel detection are overlooked while comparing the performance of tools, particularly its base pair length. RESULTS: We assessed the performance of variant calling pipelines using the combinations of four variant callers and five aligners on human NA12878 and simulated exome data. We used high confidence variant calls from Genome in a Bottle (GiaB) consortium for validation, and GRCh37 and GRCh38 as the human reference genome. Based on the performance metrics, both BWA and Novoalign aligners performed better with DeepVariant and SAMtools callers for detecting SNVs, and with DeepVariant and GATK for InDels. Furthermore, we obtained similar results on human NA24385 and NA24631 exome data from GiaB. CONCLUSION: In this study, DeepVariant with BWA and Novoalign performed best for detecting accurate SNVs and InDels. The accuracy of variant calling was improved by merging the top performing pipelines. The results of our study provide useful recommendations for analysis of WES data in clinical genomics.


Assuntos
Simulação por Computador , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Exoma , Pareamento de Bases/genética , Exoma/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Curva ROC
3.
Mol Genet Genomics ; 294(5): 1343-1357, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31197471

RESUMO

China, inhabited by over 1.3 billion people and known for its genetic, cultural and linguistic diversity, is considered to be indispensable for understanding the association between language families and genetic diversity. In order to get a better understanding of the genetic diversity and forensic characteristics of Tai-Kadai-speaking populations in Southwest China, we genotyped 30 insertion/deletion (InDel) markers and amelogenin in 205 individuals from Tai-Kadai-speaking Bouyei people using the Qiagen Investigator DIPplex amplification kit. We carried out a comprehensive population genetic relationship investigation among 14,303 individuals from 84 worldwide populations based on allele frequency correlation and 4907 genotypes of 30 InDels from 36 populations distributed in all continental or major subregions and seven linguistic phyla in China. Forensic parameters observed show highly polymorphic and informative features for Asians, although the DIPplex kit was developed focusing on Europeans, and indicate that this amplification system is appropriate to forensic personal identification and parentage testing. Patterns of InDel variations revealed by principal components analysis, multidimensional scaling plots, phylogenetic relationship exploration, model-based clustering as well as four pairwise genetic distances (Fst, Nei, Cavalli-Sforza and Reynolds) demonstrate significant genetic differentiation at the continental scale and genetic uniformity in Asia except for Tibeto-Burman and Turkic-speaking populations. Additionally, Tai-Kadai speakers, including Bouyei, Zhuang and Dong, share more genetic ancestry components than with other language speakers, and in general they are genetically very similar to Hmong-Mien-speaking populations. The dataset of Bouyei people generated in the present study is valuable for forensic identification and parentage tests in China.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Grupos Étnicos/genética , Marcadores Genéticos/genética , Mutação INDEL/genética , Mutagênese Insercional/genética , China , Genética Forense/métodos , Deleção de Genes , Frequência do Gene/genética , Genética Populacional/métodos , Genótipo , Humanos , Filogenia , Análise de Componente Principal/métodos
4.
Med Sci Monit ; 25: 3390-3396, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31064975

RESUMO

BACKGROUND This study aimed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion polymorphisms with left ventricular hypertrophy (LVH) in Han and Uighur hypertension-OSAHS (obstructive sleep apnea hypopnea syndrome) patients in China. MATERIAL AND METHODS A total of 162 Han and 72 Uygur patients with hypertension-OSAHS were independently subdivided into an LVH group and a non-LVH (NLVH) group based on the left ventricular mass index. The insertion/deletion polymorphisms of ACE gene were determined by polymerase chain reaction. The association of ACE gene insertion/deletion polymorphisms with LVH was assessed by chi-squared test. Logistic regression analysis was performed to obtain the odds ratios and 95% confidence intervals for the risk of LVH after adjusting for confounding factors. RESULTS In Uighur patients, the distributions of D allele and DD genotype showed significant differences between the LVH group and the NLVH group. The difference of DD genotype remained significant after multivariate adjustment. In contrast, no significant differences were observed in the distributions of D allele and DD genotype between the LVH group and the NLVH group in Han patients. Moreover, moderate-severe OSAHS was an independent risk factor for LVH. CONCLUSIONS D allele and DD genotype of ACE gene are possible genetic markers for the risk of LVH in Uighur but not Han hypertension-OSAHS patients.


Assuntos
Hipertrofia Ventricular Esquerda/genética , Peptidil Dipeptidase A/genética , Apneia Obstrutiva do Sono/genética , Adulto , Alelos , Grupo com Ancestrais do Continente Asiático/genética , China , Grupos Étnicos/genética , Feminino , Frequência do Gene/genética , Genótipo , Humanos , Hipertensão/complicações , Hipertensão/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/fisiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Fatores de Risco , Apneia Obstrutiva do Sono/complicações
5.
J Hum Genet ; 64(6): 535-543, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30944401

RESUMO

Insertion and deletion markers (InDels) have gained considerable attentions in population genetics and forensic research. In this study, we investigated genetic distributions of 30 InDels in Gansu Yugur and Guizhou Miao groups and evaluated their forensic application values. Genetic relationship analyses between Gansu Yugur, Guizhou Miao groups and other published populations were conducted based on these 30 InDels. Power of discrimination and power of exclusion in trio and duo cases of 30 InDels ranged from 0.3528 to 0.6247, 0.0937 to 0.1873, and 0.0219 to 0.1247 in Gansu Yugur group; and they ranged from 0.2579 to 0.6247, 0.0671 to 0.1874, and 0.0105 to 0.1247 in Guizhou Miao group. Obtained cumulative power of discrimination values indicated these InDels could be used for forensic individual identifications in both ethnic groups. Principal component analysis and phylogenetic reconstruction revealed that Gansu Yugur and Guizhou Miao groups had close affinities with their neighboring populations. Genetic structure analyses among these populations also indicated that studied Gansu Yugur and Guizhou Miao groups showed similar genetic structure with their neighboring populations. Further analyses of Y-STR, mtDNA, and ancestry informative markers should be conducted to better understand genetic backgrounds of Gansu Yugur and Guizhou Miao groups in the future.


Assuntos
Cromossomos Humanos Y/genética , DNA Mitocondrial/genética , Genética Populacional , Mutação INDEL/genética , Alelos , Grupo com Ancestrais do Continente Asiático/genética , China , Grupos Étnicos/genética , Feminino , Ciências Forenses , Testes Genéticos , Variação Genética/genética , Genótipo , Haplótipos/genética , Humanos , Masculino
6.
Nat Biotechnol ; 37(5): 561-566, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936564

RESUMO

Benchmark small variant calls are required for developing, optimizing and assessing the performance of sequencing and bioinformatics methods. Here, as part of the Genome in a Bottle (GIAB) Consortium, we apply a reproducible, cloud-based pipeline to integrate multiple short- and linked-read sequencing datasets and provide benchmark calls for human genomes. We generate benchmark calls for one previously analyzed GIAB sample, as well as six genomes from the Personal Genome Project. These new genomes have broad, open consent, making this a 'first of its kind' resource that is available to the community for multiple downstream applications. We produce 17% more benchmark single nucleotide variations, 176% more indels and 12% larger benchmark regions than previously published GIAB benchmarks. We demonstrate that this benchmark reliably identifies errors in existing callsets and highlight challenges in interpreting performance metrics when using benchmarks that are not perfect or comprehensive. Finally, we identify strengths and weaknesses of callsets by stratifying performance according to variant type and genome context.


Assuntos
Benchmarking , Biologia Computacional/tendências , Genoma Humano/genética , Genômica/tendências , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único , Software/tendências
7.
Mol Biol Rep ; 46(2): 2387-2394, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30848448

RESUMO

Chemokine CC receptor type 5 (CCR5) is a cell surface receptor that has high affinity for chemotropic cytokines called chemokines. The CCR5 gene contains a 32 base pairs (bp) deletion (CCR5Δ32). This deletion may result in a malformed and nonfunctional receptor, reported to be responsible for the development and dissemination of different cancers. CCR5Δ32 exists in two allelic forms i.e. deletion (D) and wild type (WT). This study aims to detect the role of CCR5Δ32 in breast cancer development. Blood samples were collected from breast cancer patients (330) and controls of same gender (306). Along with this histopathologically diagnosed malignant tissue samples were also excised from breast lesions of 100 patients. Genetic variations within the blood and tissue samples were examined by PCR then observed through gel electrophoresis and confirmed by direct DNA sequencing. Obtained DNA sequences were aligned and analyzed by MEGA6 software. Genotypic and association analyses were done by SPSS software version 17.0. Deletion of 32 bp in CCR5 gene has been analyzed. Genotypic variations of CCR5Δ32 are; homozygous wild type (WT/WT), heterozygous deletion (WT/D) and homozygous deletion (D/D). Statistical analyses of CCR5Δ32 data revealed that WT/D was significantly higher in blood samples of breast cancer patients (7.27% (24/330)) as compare to controls (1.30% (4/306)). In tumor tissue samples WT/WT being the most frequent genotype (99.00% (99/100)) with 1.00 (1/100) of D/D which suggested that it may be acquired. Hence, association analysis showed that CCR5Δ32 is positively associated with breast cancer in Pakistan (p < 0.001). The risk ratio of CCR5Δ32 was 5.6610 (95% confidence interval: 2.0377 to 15.7267) and odds ratio was calculated to be 6.0335 (95% confidence interval: 2.1288 to 17.0999) which signifies that deletion also increases the risk of breast cancer development. Moreover, association analyses also revealed that clinicopathological features do not have any impact on the CCR5Δ32 genotype of breast cancer. This suggests that deletion of 32 bp in CCR5 gene may be associated with breast cancer. CCR5 signals the activation and migration of immune cells at the site of tumor formation. Because of deletion; deformed CCR5 receptor might be unable to express and function properly which may subdue the immunity against cancer hence, leading to its progression.


Assuntos
Neoplasias da Mama/genética , Receptores CCR5/genética , Adulto , Idoso , Alelos , Sequência de Bases/genética , Estudos de Casos e Controles , Estudos Transversais , Feminino , Frequência do Gene/genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Heterozigoto , Humanos , Mutação INDEL/genética , Pessoa de Meia-Idade , Paquistão , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Receptores CCR5/metabolismo , Deleção de Sequência/genética
8.
Methods Mol Biol ; 1961: 29-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912038

RESUMO

Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com ). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Quebras de DNA de Cadeia Dupla , Mutação INDEL/genética , Mutagênese , Mutação/genética , Mutação Puntual/genética
9.
Methods Mol Biol ; 1961: 45-66, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912039

RESUMO

Recent developments in gene targeting methodologies such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionized approaches for gene modifications in cells, tissues, and whole animals showing great promise for translational applications. With regard to CRISPR/Cas9, a variety of repurposed systems have been developed to achieve gene knock-out, base editing, targeted knock-in, gene activation/repression, epigenetic modulation, and locus-specific labeling. A functional communality of all CRISPR/Cas9 applications is the gRNA-dependent targeting specificity of the Cas9/gRNA complex that, for gene knock-out (KO) purposes, has been shown to dictate the indel formation potential. Therefore, the objective of a CRISPR/Cas9 KO set up is to identify gRNA designs that enable maximum out-of-frame insertion and/or deletion (indel) formation and thus, gRNA design becomes a proxy for optimal functionality of CRISPR/Cas9 KO and repurposed systems. To this end, validation of gRNA functionality depends on efficient, accurate, and sensitive identification of indels induced by a given gRNA design. For in vitro indel profiling the most commonly used methods are based on amplicon size discrimination or sequencing. Indel detection by amplicon analysis (IDAA™) is an alternative sensitive, fast, and cost-efficient approach ideally suited for profiling of indels induced by Cas9/gRNA with similar sensitivity, specificity, and resolution, down to single base discrimination, as the preferred next-generation sequencing-based indel profiling methodologies. Here we provide a protocol that is based on complexed Cas9/gRNA RNPs delivered to primary peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals followed by quantitative IDAA indel profiling. Importantly, the protocol described benefits from a short "sample-to-data" turnaround time of less than 5 h. Thus, this protocol describes a methodology that provides a suitable and effective solution to validate and quantify the extent of ex vivo CRISPR/Cas9 targeting in primary cells.


Assuntos
Sistemas CRISPR-Cas/genética , Células Cultivadas , Edição de Genes , Humanos , Mutação INDEL/genética , Leucócitos Mononucleares/metabolismo , RNA Guia/genética
10.
J Renin Angiotensin Aldosterone Syst ; 20(1): 1470320319836302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30854921

RESUMO

OBJECTIVE:: Meta-analysis was performed in the current study to evaluate the relationship of the angiotensin-converting enzyme insertion/deletion polymorphism with the risk of the incidence of Henoch-Schönlein purpura. METHODS:: The electronic databases, including Embase, PubMed and Google scholar, were systemically retrieved to search for related articles. Meanwhile, statistical analysis was performed using the odds ratio and the corresponding 95% confidence interval. RESULTS:: A total of six articles enrolling 504 patients and 706 healthy controls was enrolled into the current meta-analysis. Results of the meta-analysis suggested that the angiotensin-converting enzyme D allele was markedly correlated with the risk of the incidence of Henoch-Schönlein purpura among the general population (deletion (D) vs. insertion (I): odds ratio (OR) 1.42, 95% confidence interval (CI) 1.05-1.93; DD vs. II: OR 2.23, 95% CI 1.06-4.70; DI vs. II: OR 1.36, 95% CI 1.00-1.85; dominant model: OR 1.56, 95% CI 1.00-2.42; recessive model: OR 1.83, 95% CI 1.06-3.16). Moreover, such a polymorphism was found to correlate with the susceptibility to Henoch-Schönlein purpura when studies were stratified according to the sample size of over 200. In addition, such a polymorphism was recognised to be remarkably associated with the susceptibility to Henoch-Schönlein purpura in the Caucasian population, which was not found in the Asian population. CONCLUSIONS:: The results of the current meta-analysis indicate that the angiotensin-converting enzyme D allele might be a risk factor against the risk of Henoch-Schönlein purpura, especially in Caucasians.


Assuntos
Predisposição Genética para Doença , Mutação INDEL/genética , Peptidil Dipeptidase A/genética , Púrpura de Schoenlein-Henoch/genética , Grupo com Ancestrais do Continente Europeu/genética , Humanos , Interleucina-18/genética , Viés de Publicação
11.
Nat Commun ; 10(1): 1459, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926794

RESUMO

Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.


Assuntos
Linfoma de Burkitt/genética , Genoma Humano , Transcriptoma/genética , Adolescente , Processamento Alternativo/genética , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criança , Pré-Escolar , Pontos de Quebra do Cromossomo , Estudos de Coortes , Metilação de DNA/genética , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação INDEL/genética , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequenciamento Completo do Genoma
12.
Int J Mol Sci ; 20(5)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818833

RESUMO

It is clear that the incompatibility system in Fragaria is gametophytic, however, the genetic mechanism behind this remains elusive. Eleven second-generation lines of Fragaria viridis with different compatibility were obtained by manual self-pollination, which can be displayed directly by the level of fruit-set rate. We sequenced two second-generation selfing lines with large differences in fruit-set rate: Ls-S2-53 as a self-incompatible sequencing sample, and Ls-S2-76 as a strong self-compatible sequencing sample. Fragaria vesca was used as a completely self-compatible reference sample, and the genome-wide variations were identified and subsequently annotated. The distribution of polymorphisms is similar on each chromosome between the two sequencing samples, however, the distribution regions and the number of homozygous variations are inconsistent. Expression pattern analysis showed that six candidate genes were significantly associated with self-incompatibility. Using F. vesca as a reference, we focused our attention on the gene FIP2-like (FH protein interacting protein), associated with actin cytoskeleton formation, as the resulting proteins in Ls-S2-53 and Ls-S2-76 have each lost a number of different amino acids. Suppression of FIP2-like to some extent inhibits germination of pollen grains and growth of pollen tubes by reducing F-actin of the pollen tube tips. Our results suggest that the differential distribution of homozygous variations affects F. viridis fruit-set rate and that the fully encoded FIP2-like can function normally to promote F-actin formation, while the new FIP2-like proteins with shortened amino acid sequences have influenced the (in)compatibility of two selfing lines of F. viridis.


Assuntos
Fragaria/genética , Genes de Plantas , Estudos de Associação Genética , Variação Genética , Autoincompatibilidade em Angiospermas/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Cruzamentos Genéticos , Frutas/genética , Regulação da Expressão Gênica de Plantas , Germinação , Homozigoto , Mutação INDEL/genética , Anotação de Sequência Molecular , Mapeamento Físico do Cromossomo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único/genética
13.
Mar Biotechnol (NY) ; 21(3): 301-309, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30810831

RESUMO

The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few years, the genetic tools for gene functional studies have thus far been limited to RNA interference technology. In this study, we developed a gene editing system for C. gigas based on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 ribonucleoprotein complexes. Two candidate genes, myostatin (MSTN) and Twist, were selected as targets. After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, CRISPR-induced indel mutations were detected in the target genes. The CRISPR/Cas9-induced mutations were predominantly small indel mutations ranging in size from 1 to 24 bp in these two target genes. These results demonstrate that CRISPR/Cas9 can be successfully used as an effective targeted gene editing system in C. gigas. The method reported here provides a powerful tool for gene functional studies in oysters and other marine bivalves, and potentially as a new technology for genetic engineering to improve oyster traits for aquaculture.


Assuntos
Aquicultura/métodos , Sistemas CRISPR-Cas , Crassostrea/genética , Edição de Genes/métodos , Animais , Edição de Genes/normas , Mutação INDEL/genética
14.
Planta ; 249(5): 1599-1615, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30771045

RESUMO

MAIN CONCLUSION: Alternative splicing (AS) events were identified and verified in cabbage by comparative transcriptome analysis. The corresponding markers were developed and the germplasm resources were identified. Alternative splicing (AS) is a central regulatory mechanism that greatly contributes to plant gene expression and transcriptome diversity. A large body of evidence has shown that AS complexity is relevant for plant development, evolution, complexity, and adaptation. Both insertion/deletion (InDel) and single nucleotide polymorphism (SNP) are typically co-dominant inheritance markers and have abundant polymorphisms. These have been widely used for marker-assisted selection, genetic mapping, and germplasm identification in plants. However, little is known about the molecular mechanisms underlying AS events and the development of markers including SNP and InDel from the cabbage transcriptome. In this study, three cabbage transcriptome datasets were collected and aligned to the cabbage reference genome to analyze AS events and marker development. 31,524 AS events were identified from three cabbage genotypes, accounting for 20.8% of the total cabbage genes. Alternative 3' splice site donor (A3SS) was the most frequent type of the four main AS events in cabbage. 70,475 InDels and 706,269 SNPs were identified with average frequencies of 1 InDel/6.9 kb and 1 SNP/0.7 kb, respectively. 71,942 potential SSRs were identified in 53,129 assembled unigenes with a density of 1 SSR/6.8 kb. The ratio of SNPs with synonymous/non-synonymous mutations was 1:0.65. 142 InDels and 36 SNPs were randomly selected and validated via Sanger sequencing and polymorphism was found among 66.2% of the InDels and 78.6% of the SNPs. Furthermore, 35 informative InDel markers were successfully used for genetic diversity analysis on 36 cabbage accessions. These results facilitate understanding of the molecular regulation mechanism underlying AS events in cabbage. They also provide molecular marker resource data for genetic mapping construction and germplasm identification, and facilitate the genetic improvement of cabbage via breeding.


Assuntos
Processamento Alternativo/genética , Brassica/genética , Mapeamento Cromossômico/métodos , Marcadores Genéticos/genética , Genoma de Planta/genética , Genótipo , Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único/genética , Transcriptoma/genética
15.
Gene ; 695: 92-98, 2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-30769141

RESUMO

Zinc finger proteins are a class of transcription factors with finger-like domains and have diverse uses in biological processes, including development, differentiation, and metabolism. In this study, we identified the absence of the 24 bp sequence in the third exon of the zinc finger protein 764-like (ZNF764L) gene that lead to the production of two new transcripts, ZNF764L-SV1 and ZNF764L-SV2, and the sum of the expression levels of the two transcripts is approximately equal the total RNA expression level. Temporal and spatial expression showed that ZNF764L had higher expression during the embryonic stage. Moreover, the research study revealed a 22-bp indel mutation in the first exon region of ZNF764L gene. Statistically significant results (P < 0.05) were encountered for this indel for chicken growth and carcass traits, which include birth weight, chest breadth and body slanting length at 4 weeks of age and subcutaneous fat weight and others. Genetic parameter analysis showed that D is the predominant allele in the commercial chicken population. Gene expression for each genotype showed that birds carrying the II allele had a higher expression level than the other genotypes. These findings enrich the understanding of ZNF764L gene function and enhance reproduction in the chicken industry.


Assuntos
Galinhas/genética , Dedos de Zinco/genética , Alelos , Processamento Alternativo/genética , Animais , Éxons , Estudos de Associação Genética , Genótipo , Mutação INDEL/genética , Carne , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética
16.
Plant Cell Rep ; 38(4): 503-510, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30783736

RESUMO

KEY MESSAGE: Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.


Assuntos
Sistemas CRISPR-Cas/genética , Oryza/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes , Mutação INDEL/genética
17.
PLoS One ; 14(1): e0208356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30641545

RESUMO

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009-10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing >80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention.


Assuntos
Cápsulas Bacterianas/metabolismo , Epidemiologia Molecular , Polissacarídeos Bacterianos/metabolismo , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Animais , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Cápsulas Bacterianas/genética , Vias Biossintéticas/genética , Modelos Animais de Doenças , Feminino , Humanos , Mutação INDEL/genética , Soros Imunes/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Óperon/genética , Proteínas Opsonizantes/metabolismo , Fagocitose , Polimorfismo de Nucleotídeo Único/genética , Polissacarídeos Bacterianos/genética , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Estados Unidos/epidemiologia
18.
Methods Mol Biol ; 1931: 75-84, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30652284

RESUMO

Marker assisted selection (MAS), an advance tool in plant breeding that allows accurate and efficient introgression of important agronomic trait(s) from a germplasm source to desired elite lines, has been applied to sorghum recently. Here, we report the methods for the deployment of MAS for trait introgression using endpoint genotyping technology for single nucleotide polymorphism (SNP)/insertion deletion (InDel) coupled with an application of KASP (Kompetitive Allele Specific Polymerase Chain Reaction [PCR]) chemistry allowing for the selection of parents for generational advancement without going through the laborious and time consuming phenotypic selection and additional generations for selection of desired individuals. This MAS-SNP marker assisted backcrossing scheme can be applied to accurately select heterozygotes for use as an allele donor parent in each backcross generation, thus expediting the backcrossing scheme and resulting in time savings of 3 years compared to conventional methods of introgression practiced in sorghum breeding and improvement.


Assuntos
Marcadores Genéticos/genética , Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único/genética , Sorghum/genética , Alelos , Edição de Genes/métodos , Genótipo , Fenótipo , Melhoramento Vegetal/métodos , Locos de Características Quantitativas/genética
19.
BMC Plant Biol ; 19(1): 15, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621598

RESUMO

BACKGROUND: Leaf mold, one of the major diseases of tomato caused by Cladosporium fulvum (C. fulvum), can dramatically reduce the yield and cause multimillion dollar losses annually worldwide. Mapping the resistance genes (R genes) of C. fulvum and devising MAS based strategies for breeding new cultivars is an effective approach to improve the resistance in tomato. Up to now, many C. fulvum genes or QTLs have been mapped using different genetic materials, but few studies focused on Cf-10 gene positioning. RESULTS: In this study, we investigated the genetic rules for Cf-10 and used a novel combinatorial strategy to rapidly map the Cf-10 gene. Initially, the performance of F1, F2 and BC1F1 individuals after infection, demonstrated that the resistance against C. fulvum was controlled by a single dominant gene. Two pools of resistant and susceptible individuals from F2 population were investigated, using mapping by sequencing approach and Cf-10 was found to be localized to 3.35 Mb and 3.74 Mb on chromosome 1, employing SNP/InDel index methods, respectively. After accounting for overlapping regions, these two algorithms yielded a total length of 3.29 Mb, narrowing down the target region. We further developed five serviceable KASP markers for this region based on sequencing data and conducted local QTL mapping using individuals from the F2 population, except for mapping by sequencing as mentioned above. Finally Cf-10 gene was mapped spanning a region of 790 kb, where only one gene (Solyc01g007130.3) was annotated as probable receptor protein kinase TMK1 with a LRR motif, a common R gene characteristic. The RT-qPCR analysis further confirmed the localization and the relative expression of Solyc01g007130.3 in Ontario 792 and was found to be significantly higher than that in Moneymaker at 9 dpi and 12 dpi, respectively. CONCLUSION: This study proposed a novel combinatorial strategy by combining SNP-index, InDel-index analyses and local QTL mapping using KASP genotyping approach to rapidly map genes responsible for specific traits and provided a robust base for cloning the Cf-10 gene. Furthermore, these analyses suggest that Solyc01g007130.3 is a potential candidate to be regarded as Cf-10 gene.


Assuntos
Ligação Genética/genética , Mutação INDEL/genética , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Cladosporium/patogenicidade , Genótipo
20.
Genet Epidemiol ; 43(1): 112-117, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30565766

RESUMO

It is unclear whether insertions and deletions (indels) are more likely to influence complex traits than abundant single-nucleotide polymorphisms (SNPs). We sought to understand which category of variation is more likely to impact health. Using the SardiNIA study as an exemplar, we characterized 478,876 common indels and 8,246,244 common SNPs in up to 5,949 well-phenotyped individuals from an isolated valley in Sardinia. We assessed association between 120 traits, resulting in 89 nonoverlapping-associated loci.We evaluated whether indels were enriched among credible sets of potential causal variants. These credible sets included 1,319 SNPs and 88 indels. We did not find indels to be significantly enriched. Indels were the most likely causal variant in seven loci, including one locus associated with monocyte count where an indel with causality and mechanism previously demonstrated (rs200748895:TGCTG/T) had a 0.999 posterior probability. Overall, our results show a very modest and nonsignificant enrichment for common indels in associated loci.


Assuntos
Mutação INDEL/genética , Polimorfismo de Nucleotídeo Único/genética , Loci Gênicos , Humanos , Itália , Anotação de Sequência Molecular
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