RESUMO
BACKGROUND: Copy number variation is an important component of genetic variation in higher eukaryotes. The extent of natural copy number variation in C. elegans is unknown outside of 2 highly divergent wild isolates and the canonical N2 Bristol strain. RESULTS: We have used array comparative genomic hybridization (aCGH) to detect copy number variation in the genomes of 12 natural isolates of Caenorhabditis elegans. Deletions relative to the canonical N2 strain are more common in these isolates than duplications, and indels are enriched in multigene families on the autosome arms. Among the strains in our study, the Hawaiian and Madeiran strains (CB4856 and JU258) carry the largest number of deletions, followed by the Vancouver strain (KR314). Overall we detected 510 different deletions affecting 1136 genes, or over 5% of the genes in the canonical N2 genome. The indels we identified had a median length of 2.7 kb. Since many deletions are found in multiple isolates, deletion loci were used as markers to derive an unrooted tree to estimate genetic relatedness among the strains. CONCLUSION: Copy number variation is extensive in C. elegans, affecting over 5% of the genes in the genome. The deletions we have detected in natural isolates of C. elegans contribute significantly to the number of deletion alleles available to researchers. The relationships between strains are complex and different regions of the genome possess different genealogies due to recombination throughout the natural history of the species, which may not be apparent in studies utilizing smaller numbers of genetic markers.
Assuntos
Caenorhabditis elegans/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Genoma Helmíntico , Animais , DNA de Helmintos/genética , Mutação INDEL , Desequilíbrio de Ligação , Recombinação Genética , Deleção de SequênciaRESUMO
Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome.
Assuntos
Caenorhabditis elegans/genética , Hibridização Genômica Comparativa/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Algoritmos , Animais , Bases de Dados Genéticas , Genoma Helmíntico , Genômica/métodos , Mutação Puntual , SoftwareRESUMO
We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of approximately 200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.
Assuntos
Caenorhabditis elegans/genética , Mapeamento Cromossômico/métodos , Hibridização Genômica Comparativa/métodos , Polimorfismo de Nucleotídeo Único/genética , Animais , Cromossomos/genética , Cruzamentos Genéticos , Feminino , Genoma Helmíntico/genética , Masculino , Mutação/genética , Sondas de Oligonucleotídeos/genéticaRESUMO
BACKGROUND: A collection of genetic deficiencies covering over 70% of the Caenorhabditis elegans genome exists, however the application of these valuable biological tools has been limited due to the incomplete correlation between their genetic and physical characterization. RESULTS: We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) to the high resolution, molecular characterization of several genetic deficiency and duplication strains in a 5 Mb region of Chromosome III. We incorporate this data into a physical deficiency map which is subsequently used to direct the positional cloning of essential genes within the region. From this analysis we are able to quickly determine the molecular identity of several previously unidentified mutations. CONCLUSION: We have applied accurate, high resolution molecular analysis to the characterization of genetic mapping tools in Caenorhabditis elegans. Consequently we have generated a valuable physical mapping resource, which we have demonstrated can aid in the rapid molecular identification of mutations of interest.
Assuntos
Caenorhabditis elegans/genética , Mapeamento Cromossômico/métodos , Deleção de Genes , Genoma Helmíntico/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Cromossomos , Dosagem de Genes , Duplicação Gênica , Mapeamento Físico do Cromossomo , Translocação GenéticaRESUMO
The current Caenorhabditis elegans genomic annotation has many genes organized in operons. Using directionally stitched promoterGFP methodology, we have conducted the largest survey to date on the regulatory regions of annotated C. elegans operons and identified 65, over 25% of those studied, with internal promoters. We have termed these operons "hybrid operons." GFP expression patterns driven from internal promoters differ in tissue specificity from expression of operon promoters, and serial analysis of gene expression data reveals that there is a lack of expression correlation between genes in many hybrid operons. The average length of intergenic regions with putative promoter activity in hybrid operons is larger than previous estimates for operons as a whole. Genes with internal promoters are more commonly involved in gene duplications and have a significantly lower incidence of alternative splicing than genes without internal promoters, although we have observed almost all trans-splicing patterns in these two distinct groups. Finally, internal promoter constructs are able to rescue lethal knockout phenotypes, demonstrating their necessity in gene regulation and survival. Our work suggests that hybrid operons are common in the C. elegans genome and that internal promoters influence not only gene organization and expression but also operon evolution.
Assuntos
Caenorhabditis elegans/genética , Óperon , Regiões Promotoras Genéticas , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Evolução Molecular , Duplicação Gênica , Perfilação da Expressão Gênica , Genes de Helmintos , Genes Reporter , Genoma Helmíntico , Genômica , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/genéticaRESUMO
We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50-mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50 kb) multigene deletion and a small (1 kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50 bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8-kb deletion targeting the ast-1 gene on chromosome II and another is a 141-bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to >750 kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira.