RESUMO
Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.
Assuntos
Arabidopsis/genética , Marcadores Genéticos/genética , Genoma de Planta , Ascomicetos/crescimento & desenvolvimento , Mapeamento Cromossômico , DNA de Plantas/genética , Genes de Plantas/genética , Predisposição Genética para Doença , Genótipo , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Polimorfismo GenéticoAssuntos
Arabidopsis/genética , Mapeamento Cromossômico/métodos , DNA de Plantas/isolamento & purificação , Marcadores Genéticos , Polimorfismo Genético , Sequência de Bases , Primers do DNA , DNA de Plantas/química , Eletroforese em Gel de Ágar/métodos , Indicadores e Reagentes , Reação em Cadeia da Polimerase/métodosRESUMO
Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Membrana Transportadoras , Proteínas de Plantas/biossíntese , Transcrição Gênica , Agrobacterium tumefaciens , Arabidopsis/genética , Southern Blotting , Proteínas de Transporte/genética , Mapeamento Cromossômico , DNA Antissenso , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/biossíntese , Sementes/fisiologiaRESUMO
By exploiting the ability of Pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P. aeruginosa pathogenesis. Our studies reveal a remarkable degree of conservation in the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins.
Assuntos
Arabidopsis/microbiologia , Pseudomonas aeruginosa/patogenicidade , Virulência , Animais , Evolução Biológica , Queimaduras/microbiologia , Camundongos , PlantasRESUMO
We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.