RESUMO
Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave is based on highly multiplexed ligation, followed by amplification of up to 20 ligated probes in a single PCR. Depending on the multiplexing level of the ligation reaction, the latter employs selective amplification using the amplified fragment length polymorphism (AFLP) technology. Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. The SNPWave technique is illustrated by a 100-plex genotyping assay for Arabidopsis, a 40-plex assay for tomato and a 10-plex assay for Caenorhabditis elegans, detected on the MegaBACE 1000 capillary sequencer.
Assuntos
Arabidopsis/genética , Caenorhabditis elegans/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Solanum lycopersicum/genética , Alelos , Animais , DNA/análise , DNA/genética , Sondas de DNA/genética , Genótipo , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Bacterial symbioses are widespread among insects, probably being one of the key factors of their evolutionary success. We present the complete genome sequence of Blochmannia floridanus, the primary endosymbiont of carpenter ants. Although these ants feed on a complex diet, this symbiosis very likely has a nutritional basis: Blochmannia is able to supply nitrogen and sulfur compounds to the host while it takes advantage of the host metabolic machinery. Remarkably, these bacteria lack all known genes involved in replication initiation (dnaA, priA, and recA). The phylogenetic analysis of a set of conserved protein-coding genes shows that Bl. floridanus is phylogenetically related to Buchnera aphidicola and Wigglesworthia glossinidia, the other endosymbiotic bacteria whose complete genomes have been sequenced so far. Comparative analysis of the five known genomes from insect endosymbiotic bacteria reveals they share only 313 genes, a number that may be close to the minimum gene set necessary to sustain endosymbiotic life.
Assuntos
Gammaproteobacteria/genética , Genoma Bacteriano , Animais , Insetos , Modelos Biológicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Análise de Sequência de DNARESUMO
We have sequenced the genome of the intracellular symbiont Buchnera aphidicola from the aphid Baizongia pistacea. This strain diverged 80-150 million years ago from the common ancestor of two previously sequenced Buchnera strains. Here, a field-collected, nonclonal sample of insects was used as source material for laboratory procedures. As a consequence, the genome assembly unveiled intrapopulational variation, consisting of approximately 1,200 polymorphic sites. Comparison of the 618-kb (kbp) genome with the two other Buchnera genomes revealed a nearly perfect gene-order conservation, indicating that the onset of genomic stasis coincided closely with establishment of the symbiosis with aphids, approximately 200 million years ago. Extensive genome reduction also predates the synchronous diversification of Buchnera and its host; but, at a slower rate, gene loss continues among the extant lineages. A computational study of protein folding predicts that proteins in Buchnera, as well as proteins of other intracellular bacteria, are generally characterized by smaller folding efficiency compared with proteins of free living bacteria. These and other degenerative genomic features are discussed in light of compensatory processes and theoretical predictions on the long-term evolutionary fate of symbionts like Buchnera.