Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n=9) wild relatives

Diversity in the chloroplast genome of 171 accessions representing the Brassica 'C' (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23-0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus.

A phylogenetic reappraisal of the Peltophorum group (Caesalpinieae: Leguminosae) based on the chloroplast trnL-F, rbcL and rps16 sequence data

The monophyly of the Peltophorum group, one of nine informal groups recognized by Polhill in the Caesalpinieae, was tested using sequence data from the trnL-F, rbcL, and rps16 regions of the chloroplast genome. Exemplars were included from all 16 genera of the Peltophorum group, and from 15 genera representing seven of the other eight informal groups in the tribe. The data were analyzed separately and in combined analyses using parsimony and Bayesian methods. The analysis method had little effect on the topology of well-supported relationships. The molecular data recovered a generally well-supported phylogeny with many intergeneric relationships resolved. Results show that the Peltophorum group as currently delimited is polyphyletic, but that eight genera plus one undescribed genus form a core Peltophorum group, which is referred to here as the Peltophorum group sensu stricto. These genera are Bussea, Conzattia, Colvillea, Delonix, Heteroflorum (inedit.), Lemuropisum, Parkinsonia, Peltophorum, and Schizolobium. The remaining eight genera of the Peltophorum group s.l. are distributed across the Caesalpinieae. Morphological support for the redelimited Peltophorum group and the other recovered clades was assessed, and no unique synapomorphy was found for the Peltophorum group s.s. A proposal for the reclassification of the Peltophorum group s.l. is presented.

Evolutionary patterns in neotropical Helieae (Gentianaceae): evidence from morphology, chloroplast and nuclear DNA sequences

Parsimony-based phylogenetic analyses of the neotropical tribe Helieae (Gentianaceae) are presented, including 22 of the 23 genera and 60 species. This study is based on data from morphology, palynology, and seed micromorphology (127 structural characters), and DNA sequences (matK, trnL intron, ITS). Phylogenetic reconstructions based on ITS and morphology provided the greatest resolution, morphological data further helping to tentatively place several taxa for which DNA was not available (Celiantha, Lagenanthus, Rogersonanthus, Roraimaea, Senaea, Sipapoantha, Zonanthus). Celiantha, Prepusa and Senaea together appear as the sister clade to the rest of Helieae. The remainder of Helieae is largely divided into two large subclades, the Macrocarpaea subclade and the Symbolanthus subclade. The first subclade includes Macrocarpaea, sister to Chorisepalum, Tochia, and Zonanthus. Irlbachia and Neblinantha are placed as sisters to the Symbolanthus subclade, which includes Aripuana, Calolisianthus, Chelonanthus, Helia, Lagenanthus, Lehmanniella, Purdieanthus, Rogersonanthus, Roraimaea, Sipapoantha, and symbolanthus. Generic-level polyphyly is detected in Chelonanthus and Irlbachia. Evolution of morphological characters is discussed, and new pollen and seed characters are evaluated for the first time in a combined morphological-molecular phylogenetic analysis.

Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

Background: the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).Results: Populations of R. solani AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of R. solani AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of R. solani AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Phi(ST) = 0.257, significant at P < 0.05) but not for locus pP42F (Phi(ST) = 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of R. solani AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of R. solani AG-3.Conclusion: the two sisters populations of the basidiomycete fungus R. solani AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.

Mitochondrial DNA-like sequence in the nuclear genome of Saguinus (Callitrichinae, Primates): transfer estimation

Seqüências tipo mitocondriais têm comumente sido encontradas no genoma nuclear de diversos organismos. Quando acidentalmente incluídas em estudos de seqüências mitocondriais, diversas conclusões errôneas podem ser obtidas. No entanto, estes pseudogenes nucleares tipo mitocondriais podem ser usados para a estimativa da taxa relativa de evolução de genes mitocondriais e também como grupo externo em análises filogenéticas. No presente trabalho, seqüências mitocondriais com características do tipo de pseudogene, tais como deleções e/ou inserções e códons de parada, foram encontradas em tamarins (Saguinus spp., Callitrichinae, Primates). A análise filogenética permitiu a estimativa do tempo da migração da seqüência mitocondrial para o genoma nuclear e algumas inferências filogenéticas. A escolha de um grupo externo não adequado (Aotus infulatus) não permitiu uma reconstrução filogenética confiável da subfamília Callitrichinae. A divergência bastante antiga de Cebidae (Callitrichinae, Aotinae e Cebinae) pode ter favorecido o aparecimento de homoplasias, obscurecendo a análise.

Chromosomal Mapping of Repetitive DNA and Cytochrome C Oxidase I Sequence Analysis Reveal Differentiation among Sympatric Samples of Astyanax fasciatus (Characiformes, Characidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

REPSA: A method for determining the sequence specificity of DNA binding ligands

We developed a novel combinatorial method termed restriction endonuclease protection selection and amplification (REPSA) to identify consensus binding sites of DNA-binding ligands. REPSA uses a unique enzymatic selection based on the inhibition of cleavage by a type IIS restriction endonuclease, an enzyme that cleaves DNA at a site distal from its recognition sequence. Sequences bound by a ligand are protected from cleavage while unprotected sequences are cleaved. This enzymatic selection occurs in solution under mild conditions and is dependant only on the DNA-binding ability of the ligand. Thus, REPSA is useful for a broad range of ligands including all classes of DNA-binding ligands, weakly binding ligands, mixed populations of ligands, and unknown ligands. Here I describe REPSA and the application of this method to select the consensus DNA-binding sequences of three representative DNA-binding ligands; a nucleic acid (triplex-forming single-stranded DNA), a protein (the TATA-binding protein), and a small molecule (Distamycin A). These studies generated new information regarding the specificity of these ligands in addition to establishing their DNA-binding sequences. ^

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions. (C) 2002 American Society for Mass Spectrometry.

CopywriteR: DNA copy number detection from off-target sequence data.

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting 'off-target' sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools.

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5′ side or a G on the 3′ side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

Subtraction hybridization identifies a transformation progression-associated gene PEG-3 with sequence homology to a growth arrest and DNA damage-inducible gene

Cancer is a progressive multigenic disorder characterized by defined changes in the transformed phenotype that culminates in metastatic disease. Determining the molecular basis of progression should lead to new opportunities for improved diagnostic and therapeutic modalities. Through the use of subtraction hybridization, a gene associated with transformation progression in virus- and oncogene-transformed rat embryo cells, progression elevated gene-3 (PEG-3), has been cloned. PEG-3 shares significant nucleotide and amino acid sequence homology with the hamster growth arrest and DNA damage-inducible gene gadd34 and a homologous murine gene, MyD116, that is induced during induction of terminal differentiation by interleukin-6 in murine myeloid leukemia cells. PEG-3 expression is elevated in rodent cells displaying a progressed-transformed phenotype and in rodent cells transformed by various oncogenes, including Ha-ras, v-src, mutant type 5 adenovirus (Ad5), and human papilloma virus type 18. The PEG-3 gene is transcriptionally activated in rodent cells, as is gadd34 and MyD116, after treatment with DNA damaging agents, including methyl methanesulfonate and γ-irradiation. In contrast, only PEG-3 is transcriptionally active in rodent cells displaying a progressed phenotype. Although transfection of PEG-3 into normal and Ad5-transformed cells only marginally suppresses colony formation, stable overexpression of PEG-3 in Ad5-transformed rat embryo cells elicits the progression phenotype. These results indicate that PEG-3 is a new member of the gadd and MyD gene family with similar yet distinct properties and this gene may directly contribute to the transformation progression phenotype. Moreover, these studies support the hypothesis that constitutive expression of a DNA damage response may mediate cancer progression.

Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

Short DNA Fragments without Sequence Similarity Are Initiation Sites for Replication in the Chromosome of the Yeast Yarrowia lipolytica

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (Vernis et al., 1997). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.