RESUMO
Pyrosequencing is a unique sequencing method that was developed as an alternative to classical DNA sequencing for short- to medium-read applications. Compared to other methods, it is highly quantitative, fast and inexpensive. Additional advantages include high accuracy, flexibility and ability to automate sample preparation. This article presents a historical overview of pyrosequencing, improvements that have been made through the years and its evolution into a platform that vastly expanded the scope of genetic analysis that could be performed outside of a big sequencing center. In addition, we describe numerous applications in microbiology that have benefited from the pyrosequencing method.
Assuntos
Análise de Sequência de DNA/métodos , Genes , Genoma , Microbiologia/instrumentação , Análise de Sequência de DNA/instrumentaçãoRESUMO
In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.
Assuntos
Cacau/genética , Etiquetas de Sequências Expressas , Doenças das Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Biblioteca Gênica , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genéticaRESUMO
Aluminum (Al) tolerance mechanisms in crop plants have been extensively researched, but our understanding of the physiological mechanisms underlying Al tolerance in trees is still limited. To investigate Al tolerance in eucalypts, seedlings of six species (Eucalyptus globulus Labill., Eucalyptus urophylla S.T. Blake, Eucalyptus dunnii Maiden, Eucalyptus saligna Sm., Eucalyptus cloeziana F. J. Muell. and Eucalyptus grandis w. Hill ex Maiden) and seedlings of six clones of Eucalyptus species were grown for 10 days in nutrient solutions containing Al concentrations varying from 0 to 2.5 microM (0 to 648 microM Al3+ activities). Root elongation of most species was inhibited only by high Al3+ activities. Low to intermediate Al3+ activities were beneficial to root elongation of all species and clones. Among the species tested, E. globulus and E. urophylla were more tolerant to Al toxicity, whereas E. grandis and E. cloeziana were more susceptible to Al-induced damage. Although E. globulus seedlings were tolerant to Al toxicity, they were highly sensitive to lanthanum (La), indicating that the tolerance mechanism is specific for Al. Fine roots accumulated more Al and their elongation was inhibited more than that of thick roots. In E. globulus, accumulation of Al in root tips increased linearly with increasing Al concentration in the nutrient solution. The majority of Al taken up was retained in the root system, and the small amounts of Al translocated to the shoot system were found mainly in older leaves. No more than 60% of the Al in the thick root tip was in an exchangeable form in the apoplast that could be removed by sequential citrate rinses. Gas chromatography/mass spectrometry and ion chromatography analyses indicated that root exposure to Al led to a greater than 200% increase in malic acid concentration in the root tips of all eucalypt species. The increase in malate concentration in response to Al treatment correlated with the degree of Al tolerance of the species. A small increase in citric acid concentration was also observed in all species, but there were no consistent changes in the concentrations of other organic acids in response to Al treatment. In all eucalypt species, Al treatment induced the secretion of citric and malic acid in root exudates, but no trend with respect to Al tolerance was observed. Thus, although malate and citrate exudation by roots may partially account for the overall high Al tolerance of these eucalypt species, it appears that tolerance is mainly derived from the internal detoxification of Al by complexation with malic acid.
Assuntos
Alumínio/farmacologia , Eucalyptus/efeitos dos fármacos , Árvores/efeitos dos fármacos , Alumínio/análise , Ácido Cítrico/metabolismo , Eucalyptus/fisiologia , Coifa/química , Raízes de Plantas/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Brotos de Planta/química , Árvores/fisiologiaRESUMO
Polymerase chain reaction has been used to detect the presence of the virulence associated gene, tcpA and part of the promoter distal region of the toxin-co-regulated pilus cluster in non-O1, non-toxigenic, Vibrio cholerae. The amplified regions were characterised by restriction fragment length polymorphism and heteroduplex motility assay. We describe the nucleotide sequence of the tcpA gene fragment from non-toxigenic vibrios from clinical and environmental sources. The present study shows that there are at least three types of the tcpA gene among V. cholerae and the primers specific for the classical tcpA gene, amplify all biotypes. A sequence similarity in other regions of the toxin-co-regulated pilus cluster is suggested. The evidences for the presence of this cluster among non-toxigenic vibrios is, to our knowledge, reported for the first time. The use of restriction fragment length polymorphism for typing the tcpA and studying the alleles distribution is proposed.