RESUMO
KEY MESSAGE: Analysis of a wheat multi-founder population identified 14 yellow rust resistance QTL. For three of the four most significant QTL, haplotype analysis indicated resistance alleles were rare in European wheat. Stripe rust, or yellow rust (YR), is a major fungal disease of wheat (Triticum aestivum) caused by Puccinia striiformis Westend f. sp. tritici (Pst). Since 2011, the historically clonal European Pst races have been superseded by the rapid incursion of genetically diverse lineages, reducing the resistance of varieties previously showing durable resistance. Identification of sources of genetic resistance to such races is a high priority for wheat breeding. Here we use a wheat eight-founder multi-parent population genotyped with a 90,000 feature single nucleotide polymorphism array to genetically map YR resistance to such new Pst races. Genetic analysis of five field trials at three UK sites identified 14 quantitative trait loci (QTL) conferring resistance. Of these, four highly significant loci were consistently identified across all test environments, located on chromosomes 1A (QYr.niab-1A.1), 2A (QYr.niab-2A.1), 2B (QYr.niab-2B.1) and 2D (QYr.niab-2D.1), together explaining ~ 50% of the phenotypic variation. Analysis of these four QTL in two-way and three-way combinations showed combinations conferred greater resistance than single QTL, and genetic markers were developed that distinguished resistant and susceptible alleles. Haplotype analysis in a collection of wheat varieties found that the haplotypes associated with YR resistance at three of these four major loci were rare (≤ 7%) in European wheat, highlighting their potential utility for future targeted improvement of disease resistance. Notably, the physical interval for QTL QYr.niab-2B.1 contained five nucleotide-binding leucine-rich repeat candidate genes with integrated BED domains, of which two corresponded to the cloned resistance genes Yr7 and Yr5/YrSp.
Assuntos
Resistência à Doença/genética , Doenças das Plantas/microbiologia , Puccinia/fisiologia , Triticum/genética , Genótipo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Polimorfismo de Nucleotídeo Único , Puccinia/imunologia , Locos de Características Quantitativas , Triticum/imunologia , Triticum/microbiologiaRESUMO
The pink pigeon (Nesoenas mayeri) is an endemic species of Mauritius that has made a remarkable recovery after a severe population bottleneck in the 1970s to early 1990s. Prior to this bottleneck, an ex situ population was established from which captive-bred individuals were released into free-living subpopulations to increase population size and genetic variation. This conservation rescue led to rapid population recovery to 400-480 individuals, and the species was twice downlisted on the International Union for the Conservation of Nature (IUCN) Red List. We analyzed the impacts of the bottleneck and genetic rescue on neutral genetic variation during and after population recovery (1993-2008) with restriction site-associated sequencing, microsatellite analyses, and quantitative genetic analysis of studbook data of 1112 birds from zoos in Europe and the United States. We used computer simulations to study the predicted changes in genetic variation and population viability from the past into the future. Genetic variation declined rapidly, despite the population rebound, and the effective population size was approximately an order of magnitude smaller than census size. The species carried a high genetic load of circa 15 lethal equivalents for longevity. Our computer simulations predicted continued inbreeding will likely result in increased expression of deleterious mutations (i.e., a high realized load) and severe inbreeding depression. Without continued conservation actions, it is likely that the pink pigeon will go extinct in the wild within 100 years. Conservation rescue of the pink pigeon has been instrumental in the recovery of the free-living population. However, further genetic rescue with captive-bred birds from zoos is required to recover lost variation, reduce expression of harmful deleterious variation, and prevent extinction. The use of genomics and modeling data can inform IUCN assessments of the viability and extinction risk of species, and it helps in assessments of the conservation dependency of populations.
La paloma rosada (Nesoenas mayeri) es una especie endémica de Mauricio que se ha recuperado impresionantemente después de un grave cuello de botella poblacional a principios de la década de 1970 que duró hasta inicios de la década de 1990. Antes de este cuello de botella se había establecido una población ex situ de la cual se liberaban individuos reproducidos en cautiverio a las subpoblaciones en libertad para incrementar la variación genética y el tamaño poblacional. Este rescate de conservación derivó en una recuperación rápida de la población (400-480 individuos) y la especie cambió positivamente de categoría dos veces en la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN). Analizamos los impactos del cuello de botella y el rescate genético sobre la variación genética neutral durante y después de la recuperación poblacional (de 1993 a 2008) mediante secuenciación RAD, análisis de microsatélites y análisis genéticos cuantitativos de los datos del libro genealógico de 1112 aves ubicadas en zoológicos de Europa y los Estados Unidos. Usamos simulaciones por computadora para estudiar los cambios pronosticados en la variación genética y en la viabilidad poblacional del pasado hacia el futuro. La variación genética declinó rápidamente, a pesar de la recuperación poblacional, y el tamaño efectivo de la población fue aproximadamente un orden de magnitud más pequeño que el tamaño del censo. La especie contó con una carga genética elevada de casi 15 equivalentes letales para la longevidad. Nuestras simulaciones pronostican que la endogamia continua probablemente resultará en un incremento en la expresión de mutaciones deletéreas (es decir, una carga realizada elevada) y en una depresión endogámica severa. Sin acciones continuas para la conservación, es probable que la paloma rosada esté extinta en vida libre dentro de cien años. El rescate de conservación de la paloma rosada ha sido fundamental en la recuperación de la población silvestre; sin embargo, se requiere de un rescate genético adicional con las aves de reproducción en cautiverio de los zoológicos para recuperar la variación perdida, reducir la expresión de la variación deletérea dañina y prevenir la extinción. El uso de la genómica y los datos modelados puede orientar las valoraciones de la UICN sobre la viabilidad y el riesgo de extinción de las especies, además de que ayuda en la evaluación de la dependencia que tienen las poblaciones de la conservación.
Assuntos
Aves , Conservação dos Recursos Naturais , Animais , Aves/genética , Espécies em Perigo de Extinção , Europa (Continente) , Variação Genética , Genômica , Densidade DemográficaRESUMO
KEY MESSAGE: Quantitative trait locus (QTL) mapping of 15 yield component traits in a German multi-founder population identified eight QTL each controlling ≥2 phenotypes, including the genetic loci Rht24, WAPO-A1 and WAPO-B1. Grain yield in wheat (Triticum aestivum L.) is a polygenic trait representing the culmination of many developmental processes and their interactions with the environment. Toward maintaining genetic gains in yield potential, 'reductionist approaches' are commonly undertaken by which the genetic control of yield components, that collectively determine yield, are established. Here we use an eight-founder German multi-parental wheat population to investigate the genetic control and phenotypic trade-offs between 15 yield components. Increased grains per ear was significantly positively correlated with the number of fertile spikelets per ear and negatively correlated with the number of infertile spikelets. However, as increased grain number and fertile spikelet number per ear were significantly negatively correlated with thousand grain weight, sink strength limitations were evident. Genetic mapping identified 34 replicated quantitative trait loci (QTL) at two or more test environments, of which 24 resolved into eight loci each controlling two or more traits-termed here 'multi-trait QTL' (MT-QTL). These included MT-QTL associated with previously cloned genes controlling semi-dwarf plant stature, and with the genetic locus Reduced height 24 (Rht24) that further modulates plant height. Additionally, MT-QTL controlling spikelet number traits were located to chromosome 7A encompassing the gene WHEAT ORTHOLOG OF APO1 (WAPO-A1), and to its homoeologous location on chromosome 7B containing WAPO-B1. The genetic loci identified in this study, particularly those that potentially control multiple yield components, provide future opportunities for the targeted investigation of their underlying genes, gene networks and phenotypic trade-offs, in order to underpin further genetic gains in yield.
Assuntos
Cromossomos de Plantas/genética , Genética Populacional , Genoma de Planta , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Triticum/crescimento & desenvolvimento , Mapeamento Cromossômico/métodos , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Triticum/classificação , Triticum/genética , Triticum/metabolismoRESUMO
Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.
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Mapeamento de Sequências Contíguas/métodos , Genoma de Planta , Anotação de Sequência Molecular/métodos , Proteínas de Plantas/genética , Translocação Genética , Triticum/genética , Algoritmos , Mapeamento de Sequências Contíguas/normas , Anotação de Sequência Molecular/normas , Polimorfismo Genético , PoliploidiaRESUMO
KEY MESSAGE: Genetic mapping of sensitivity to the Pyrenophora tritici-repentis effector ToxB allowed development of a diagnostic genetic marker, and investigation of wheat pedigrees allowed transmission of sensitive alleles to be tracked. Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, is a major disease of wheat (Triticum aestivum). Secretion of the P. tritici-repentis effector ToxB is thought to play a part in mediating infection, causing chlorosis of plant tissue. Here, genetic analysis using an association mapping panel (n = 480) and a multiparent advanced generation intercross (MAGIC) population (n founders = 8, n progeny = 643) genotyped with a 90,000 feature single nucleotide polymorphism (SNP) array found ToxB sensitivity to be highly heritable (h2 ≥ 0.9), controlled predominantly by the Tsc2 locus on chromosome 2B. Genetic mapping of Tsc2 delineated a 1921-kb interval containing 104 genes in the reference genome of ToxB-insensitive variety 'Chinese Spring'. This allowed development of a co-dominant genetic marker for Tsc2 allelic state, diagnostic for ToxB sensitivity in the association mapping panel. Phenotypic and genotypic analysis in a panel of wheat varieties post-dated the association mapping panel further supported the diagnostic nature of the marker. Combining ToxB phenotype and genotypic data with wheat pedigree datasets allowed historic sources of ToxB sensitivity to be tracked, finding the variety 'Maris Dove' to likely be the historic source of sensitive Tsc2 alleles in the wheat germplasm surveyed. Exploration of the Tsc2 region gene space in the ToxB-sensitive line 'Synthetic W7984' identified candidate genes for future investigation. Additionally, a minor ToxB sensitivity QTL was identified on chromosome 2A. The resources presented here will be of immediate use for marker-assisted selection for ToxB insensitivity and the development of germplasm with additional genetic recombination within the Tsc2 region.
Assuntos
Ascomicetos , Resistência à Doença/genética , Interações Hospedeiro-Patógeno/genética , Micotoxinas/toxicidade , Doenças das Plantas/genética , Triticum/genética , Mapeamento Cromossômico , Ligação Genética , Marcadores Genéticos , Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Fenótipo , Doenças das Plantas/microbiologia , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Crop populations derived from experimental crosses enable the genetic dissection of complex traits and support modern plant breeding. Among these, multi-parent populations now play a central role. By mixing and recombining the genomes of multiple founders, multi-parent populations combine many commonly sought beneficial properties of genetic mapping populations. For example, they have high power and resolution for mapping quantitative trait loci, high genetic diversity and minimal population structure. Many multi-parent populations have been constructed in crop species, and their inbred germplasm and associated phenotypic and genotypic data serve as enduring resources. Their utility has grown from being a tool for mapping quantitative trait loci to a means of providing germplasm for breeding programmes. Genomics approaches, including de novo genome assemblies and gene annotations for the population founders, have allowed the imputation of rich sequence information into the descendent population, expanding the breadth of research and breeding applications of multi-parent populations. Here, we report recent successes from crop multi-parent populations in crops. We also propose an ideal genotypic, phenotypic and germplasm 'package' that multi-parent populations should feature to optimise their use as powerful community resources for crop research, development and breeding.
Assuntos
Produtos Agrícolas , Genômica , Melhoramento Vegetal , Mapeamento Cromossômico , Produtos Agrícolas/genética , Genoma de Planta , Locos de Características QuantitativasRESUMO
Aphids present an ideal system to study epigenetics as they can produce diverse, but genetically identical, morphs in response to environmental stimuli. Here, using whole genome bisulphite sequencing and transcriptome sequencing of the green peach aphid (Myzus persicae), we present the first detailed analysis of cytosine methylation in an aphid and investigate differences in the methylation and transcriptional landscapes of male and asexual female morphs. We found that methylation primarily occurs in a CG dinucleotide (CpG) context and that exons are highly enriched for methylated CpGs, particularly at the 3' end of genes. Methylation is positively associated with gene expression, and methylated genes are more stably expressed than unmethylated genes. Male and asexual female morphs have distinct methylation profiles. Strikingly, these profiles are divergent between the sex chromosome and the autosomes; autosomal genes are hypomethylated in males compared to asexual females, whereas genes belonging to the sex chromosome, which is haploid in males, are hypermethylated. Overall, we found correlated changes in methylation and gene expression between males and asexual females, and this correlation was particularly strong for genes located on the sex chromosome. Our results suggest that differential methylation of sex-biased genes plays a role in aphid sexual differentiation.
Assuntos
Afídeos/genética , Metilação de DNA/genética , Caracteres Sexuais , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma de Inseto , Masculino , Cromossomo X/genéticaRESUMO
Multi-parent populations (MPPs) are attractive for genetic and breeding studies because they combine genetic diversity with an easy-to-control population structure. Most methods for mapping QTLs in MPPs focus on the detection of QTLs in single environments. Little attention has been given to mapping QTLs in multienvironment trials (METs) and to detecting and modeling QTL-by-environment interactions (QEIs). We present mixed model approaches for the detection and modeling of consistent versus environment-dependent QTLs, i.e., QTL-by-environment interaction (QEI). QTL effects are assumed to be normally distributed with variances expressing consistency or dependence on environments and families. The entries of the corresponding design matrices are functions of identity-by-descent (IBD) probabilities between parents and offspring and follow from the parental origin of offspring DNA. A polygenic effect is added to the models to account for background genetic variation. We illustrate the wide applicability of our method by analyzing several public MPP datasets with observations from METs. The examples include diallel, nested association mapping (NAM), and multi-parent advanced inter-cross (MAGIC) populations. The results of our approach compare favorably with those of previous studies that used tailored methods.
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Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus, recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases (S-RNases) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S7- and S11-Rnases in the S-genotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible 'Comune' clementine and its self-compatible natural mutant 'Monreal'. The reference genome of 'Monreal' clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S7-RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in 'Monreal' compared to the reference genome of C. clementina. Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S7-RNase in 'Monreal' suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous-like MADS-box proteins, to MYB111 and to MLO-like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S7- and S11-RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S7-RNase and consequently of the SI mechanism.
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Zoo populations of threatened species are a valuable resource for the restoration of wild populations. However, their small effective population size poses a risk to long-term viability, especially in species with high genetic load. Recent bioinformatic developments can identify harmful genetic variants in genome data. Here, we advance this approach, analysing the genetic load in the threatened pink pigeon (Nesoenas mayeri). We lifted the mutation-impact scores that had been calculated for the chicken (Gallus gallus) to estimate the genetic load in six pink pigeons. Additionally, we perform in silico crossings to predict the genetic load and realized load of potential offspring. We thus identify the optimal mate pairs that are theoretically expected to produce offspring with the least inbreeding depression. We use computer simulations to show how genomics-informed conservation can reduce the genetic load whilst reducing the loss of genome-wide diversity. Genomics-informed management is likely to become instrumental in maintaining the long-term viability of zoo populations.
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Undomesticated wild species, crop wild relatives, and landraces represent sources of variation for wheat improvement to address challenges from climate change and the growing human population. Here, we study 56,342 domesticated hexaploid, 18,946 domesticated tetraploid and 3,903 crop wild relatives in a massive-scale genotyping and diversity analysis. Using DArTseqTM technology, we identify more than 300,000 high-quality SNPs and SilicoDArT markers and align them to three reference maps: the IWGSC RefSeq v1.0 genome assembly, the durum wheat genome assembly (cv. Svevo), and the DArT genetic map. On average, 72% of the markers are uniquely placed on these maps and 50% are linked to genes. The analysis reveals landraces with unexplored diversity and genetic footprints defined by regions under selection. This provides fertile ground to develop wheat varieties of the future by exploring specific gene or chromosome regions and identifying germplasm conserving allelic diversity missing in current breeding programs.
Assuntos
Variação Genética , Genoma de Planta , Triticum/genética , Alelos , Domesticação , Genótipo , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , TetraploidiaRESUMO
Island species provide excellent models for investigating how selection and drift operate in wild populations, and for determining how these processes act to influence local adaptation and speciation. Here, we examine the role of selection and drift in shaping genomic and phenotypic variation across recently separated populations of Berthelot's pipit (Anthus berthelotii), a passerine bird endemic to three archipelagos in the Atlantic. We first characterized genetic diversity and population structuring that supported previous inferences of a history of recent colonizations and bottlenecks. We then tested for regions of the genome associated with the ecologically important traits of bill length and malaria infection, both of which vary substantially across populations in this species. We identified a SNP associated with variation in bill length among individuals, islands, and archipelagos; patterns of variation at this SNP suggest that both phenotypic and genotypic variation in bill length is largely shaped by founder effects. Malaria was associated with SNPs near/within genes involved in the immune response, but this relationship was not consistent among archipelagos, supporting the view that disease resistance is complex and rapidly evolving. Although we found little evidence for divergent selection at candidate loci for bill length and malaria resistance, genome scan analyses pointed to several genes related to immunity and metabolism as having important roles in divergence and adaptation. Our findings highlight the utility and challenges involved with combining association mapping and population genetic analysis in nonequilibrium populations, to disentangle the effects of drift and selection on shaping genotypes and phenotypes.
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Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.
Assuntos
Ascomicetos/genética , Fraxinus/microbiologia , Genoma Fúngico , Doenças das Plantas/microbiologia , Europa (Continente) , Haplótipos/genéticaRESUMO
Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq was used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.
Assuntos
Diferenciação Celular/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fator de Transcrição GATA3/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Formaldeído/química , Fator de Transcrição GATA3/antagonistas & inibidores , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , PPAR gama/genética , PPAR gama/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Elementos Reguladores de Transcrição , Análise de Sequência de RNA , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Urotélio/citologia , Urotélio/metabolismoRESUMO
Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.