Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

The population biology of transposable elements

A transposable element can be defined as a DNA sequence capable of moving to new sites in the genome. Such DNA sequences have been described in a wide range of organisms. The evolutionary processes affecting transposable elements can thus be divided into two categories: changes in sequence and changes in genomic location. As with other types of evolutionary change, the nature of the evolutionary process will be reflected in the extent and type of genetic variation existing in wild populations. Quantitative models of the evolution of transposable element sequences and positions will be outlined, and related to relevant data. The extent to which models designed to describe obvious transposable elements such as the mobile sequences of Drosophila are also applicable to interspersed repetitive DNAs from other species will be discussed.     

Genome sequences and evolutionary biology, a two-way interaction

Complete genome sequences are accumulating rapidly, culminating with the announcement of the human genome sequence in February 2001. In addition to cataloguing the diversity of genes and other sequences, genome sequences will provide the first detailed and complete data on gene families and genome organization, including data on evolutionary changes. Reciprocally, evolutionary biology will make important contributions to the efforts to understand functions of genes and other sequences in genomes. Large-scale, detailed and unbiased comparisons between species will illuminate the evolution of genes and genomes, and population genetics methods will enable detection of functionally important genes or sequences, including sequences that have been involved in adaptive changes.     

Alignment-free comparison of genome sequences by a new numerical characterization

In order to compare different genome sequences, an alignment-free method has proposed. First, we presented a new graphical representation of DNA sequences without degeneracy, which is conducive to intuitive comparison of sequences. Then, a new numerical characterization based on the representation was introduced to quantitatively depict the intrinsic nature of genome sequences, and considered as a 10-dimensional vector in the mathematical space. Alignment-free comparison of sequences was performed by computing the distances between vectors of the corresponding numerical characterizations, which define the evolutionary relationship. Two data sets of DNA sequences were constructed to assess the performance on sequence comparison. The results illustrate well validity of the method. The new numerical characterization provides a powerful tool for genome comparison.     

Simple sequence repeats for genetic studies of alpaca

Trace sequences from the 2X alpaca genome sequencing effort were examined to identify simple sequence repeats (microsatellites) for genetic studies. A total of 6,685 repeat-containing sequences were downloaded from GenBank, processed, and assembled into contigs representing an estimated 4,278 distinct sequences. This sequence set contained 2,290 sequences of length > 100 nucleotides that contained microsatellites of length > or = 14 dinucleotide or 10 trinucleotide repeats with purity equal to 100%. An additional 13 sequences contained a GC microsatellite of length > or = 12 repeats (purity = 100%) were also obtained. Primer pairs for amplification of 1,516 putative loci are presented. Amplification of genomic DNA from alpaca and llama by PCR was demonstrated for 14 primer sets including one from each of the microsatellite repeat types. Comparative chromosomal location for the alpaca markers was predicted in the bovine genome by BLAT searches against assembly 4.0 of the bovine whole genome sequence. A total of 634 markers (41.8%) returned BLAT hits with score > 100 and Identity > 85%, with the majority assignable to unique locations. We show that microsatellites are abundant and easily identified within the alpaca genome sequence. These markers will provide a valuable resource for further genetic studies of the alpaca and related species.     

Phylogenetic inferences from molecular sequences: review and critique

Conflicting results often accompany phylogenetic analyses of RNA, DNA, or protein sequences across diverse species. Causes contributing to these conflicts relate to ambiguities in identifying homologous characters of alignments, sensitivity of tree-making methods to unequal evolutionary rates, biases in species sampling, unrecognized paralogy, functional differentiation, loss of phylogenetic informational content due to long branches or fast evolution, and difficulties with the assumptions and approximations used to infer phylogenetic relationships. Attempts to surmount these conflicts by averaging over many proteins are problematic due to inherent biases of selected families, lack of signal in others, and events of lateral transfer, fusion, and/or chimerism. The process of assessing reliability of the results using the bootstrap method is strewn with obstacles because of lack of independence and inhomogeneity in the molecular data. Problems inherent to the three major procedures for developing phylogenetic trees--parsimony, likelihood, distance--are reviewed. Special attention is given to the problem of inferring evolutionary distances from patterns of similarity among sequences. The difficulties encountered by methods of phylogenetic reconstructions based on the analysis of divergent sequence families make new methods based on the analysis of complete genomes reasonable alternatives. Several of these are considered, including the signature sequences of Gupta and associates, the study of genome profiles, and the genomic signature set forth by Karlin and colleagues.     

Piperprotrusum (Piperaceae), a new species from southern Thailand based on morphological and molecular evidence

Piper protrusum Chaveer.& Tanee,sp.nov.is described and illustrated.It dominantly comprises three branching types with three different types of leaf blades,bases,and apexes.The critical distinguishing character is the protruded receptacle having a bract and nine stamens.Individual plants have been discovered in areas of Southern Thailand since 2004 without reproductive parts.The investigated sites were revisited several times,and an individual with flowers was finally found in July 2009.Phylogenetic analysi...     

Global variation in G+C content along vertebrate genome DNA. Possible correlation with chromosome band structures

The global, rather than local, variation in G+C content along the nuclear DNA sequences of various organisms was studied using GenBank sequence data. When long DNA sequences of the genomes of Escherichia coli and Saccharomyces cerevisiae were examined, the levels of their G+C content (G+C%) were found to be within a narrow range around that of the whole genome. The G+C% levels for sequences of vertebrate genomes, however, were found to cover a wide range, showing that their genome is a mosaic of sequences with different G+C% levels, in each of which the sequence is fairly homogeneous in its G+C% for a very long distance. Through surveying a human genetic map and GenBank DNA sequences, the global variations in G+C% along the human genome DNA were found to be correlated with chromosome band structures.     

16S～23S RDNA间区在链球菌和流感嗜血杆菌分类中的应用

利用16S~23S rDNA间区（intergenic spacer regions,ISR）在不同细菌中拷贝数、碱基排列、序列长度及所含tRNA基因种类和数目的差异,对15株链球菌和流感嗜血杆菌进行属、种、型和株系的分类鉴定。在16S rDNA的3′端和23S rDNA的5′端的保守区中合成引物,PCR扩增16S~23S rDNA ISR序列,对多态片段切胶纯化直接测序。在GenBank上查找对应细菌的ISR序列。用DNAMAN软件进行系统进化分析。链球菌属为单拷贝16S~23Sr RNA ISR、有一个tRNAAla基因编码区、分子大小在269~446bp之间,序列分成4个保守区和4个可变区,可变区碱基排列方式和数目的不同是种分类的依据。7株链球菌的同源率在78%~88%。同种异株的差异反映在碱基的插入和缺失上。流感嗜血杆菌各生物型均为2个拷贝的ISR,小片段为514~519bp,编码1个tRNAGlu基因,有3个狭窄可变区。大片段富含A T碱基,在I、II和IV型中分别是868、848和856bp,编码一个tRNAIle基因和一个tRNAAla基因。不同生物型小分子ISR与标准菌株比较,同源性在97.3%~99.6 %之间。 ISR作为细菌分类的目的基因具有属、种、型和株特异性与灵敏性。简单的基因分离分析技术为认识病原微生物提供了更多的机会。 Abstract:To facilitate species level identification of bacteria without the requirement of presumptive identification,the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S~23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus.Three pairs of primer targeting conserved sequences flanking the 3′ end of 16S and the 5′end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains.The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon).The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kit——Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer.Then those sequences were compared with known seqnences on the GenBank.The alignment of nucleotide sequence,evolutionary distances and phylogenetic tress were analyzed by software DANMAN version 4.0.The PCR products were showed polymorphism patterns with agarose gel.One band was contained in streptococcus genus.The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446bp.All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera.It was found that some spacer sequence blocks were highly conserved between operons of a genome,whereas the presence of others was variable,three regions showed significant spatial variation.Most of the differences between the sequences came from several bases insertions/deletions and substitutions.There are two major bands in the Haemophilus biotypes(515 and 884bp),the small ISR amplicon contained one tDNA coding for tRNAGlu.In contrast to the large one contained two tRNA genes coding for tRANAla and tRNAIle.Two regions of repeating motifs with only A or T were present in higher copy numbers between tRANAla and tRNAIle.The phylogenetic trees varied from 97.5 to 98.8%.The PCR and direct sequencing of 16S~23S rRAN ISR were successful in the pathogen species identification.     

Phylogenetic relationships amongTetrahymena species determined using the polymerase chain reaction

Summary The species of theTetrahymena pyriformis complex present a conundrum with regard to their highly conservative morphology and widely divergent molecular characteristics. We have investigated the phylogenetic relationships among these species using the nucleotide sequences from the histone H3II/H4II region of the genome. This region includes portions of the two histone coding sequences, as well as the intergenic region. The DNA sequences of these regions were amplified by the polymerase chain reaction (PCR) and the sequence of each was determined. Nucleotide substitutions and insertions/deletions within this set of sequences were compared to determine the phylogenetic relationships among the species of the complex. These data yield phylogenetic trees with identical topologies when different tree-building routines are used, indicating that the data are very robust.Glaucoma chattoni was used as an outgroup to root the trees for this analysis. The genome organization ofG. chattoni and the divergence of its histone H3II/H4II region sequence relative to those of the complex clearly indicate that this species has diverged considerably from the complex. These results show that PCR amplification analysis is feasible over considerable evolutionary distances. However, DNA-DNA hybridization may be more useful than sequence analysis in resolving the relationships among the closely related species in the complex.     

On the use of DNA sequences for determining the species limits of a polymorphic new species in the stink bug genus Halys (Heteroptera: Pentatomidae) from Pakistan

Abstract.  We describe a new species of Halys Fabricius (Pentatomidae: Pentatominae: Halyini) based on morphological and DNA sequence data, and demonstrate the value of DNA sequences for taxonomic problems that are difficult to resolve on the basis of morphology alone. Halys sindillus Memon, Meier & Manan, sp.n. varies with regard to characters that are usually constant within the genus (spermathecal bulb of females; blade of male clasper; ratio between the second and third antennomeres; length of labium). The surprising levels of variation raised the question as to how many species were represented in three series of specimens from Pakistan. Because the morphological variability was largely continuous, we hypothesized the presence of one new species, and confirm this result here using sequence data from two mitochondrial markers. The data reveal very little molecular variation within the newly described species (COI: 730 bp: 0–0.16%; COI/tRNA_Leu/COII: 563 bp: 0–0.36%), that is, morphology and DNA sequences show very different patterns of variability. The new species is compared with the closely related Halys sulcatus (Thunberg) whose sequences are distinctly different and whose spermathecal bulbs are largely invariable (I: 2.87–3.28%; II: 2.13–2.49%). We discuss the shortcomings of mitochondrial data in taxonomy and compare the genetic distances in Halys with frequency distributions of intra- and interspecific distances obtained for all 878 Hemiptera COI sequences in GenBank. We conclude that the observed distances for Halys are consistent with our taxonomic conclusions, thus demonstrating the usefulness of DNA sequences for Halys taxonomy. However, the observed overlap between intra- and interspecific sequence variability in Hemiptera is so wide that it questions the feasibility of approaches to taxonomy based predominantly on DNA sequences (e.g. DNA taxonomy, DNA barcoding).     

Characterization of a Trypanosoma cruzi genomic fragment complementary to several species-specific mRNAs but different from the spliced leader sequence

We have isolated a clone of Trypanosoma cruzi genomic DNA, lambda 3b2-5, which contains sequences that are reiterated in the genome. Northern blot analysis showed that clone 3b2-5 hybridizes to 1,200-5,000 bases different mRNA species. The number of mRNAs species hybridized to clone 3b2-5 exceeds its coding capacity showing that this clone carries sequences that are common to several mRNAs species and conserved in the poly A(+) RNA. These sequences are not homologous to the T. cruzi spliced leader sequence, since clone 3b2-5 does not hybridize to a synthetic 20 nucleotide complementary to the spliced leader sequence. Clone 3b2-5 does not hybridize to DNA and RNA from several genera of Trypanosomatidae and other Trypanosoma species indicating that it carries T. cruzi species-specific sequences.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

The PhyLoTA Browser: processing GenBank for molecular phylogenetics research

As an archive of sequence data for over 165,000 species, GenBank is an indispensable resource for phylogenetic inference. Here we describe an informatics processing pipeline and online database, the PhyLoTA Browser (http://loco.biosci.arizona.edu/pb), which offers a view of GenBank tailored for molecular phylogenetics. The first release of the Browser is computed from 2.6 million sequences representing the taxonomically enriched subset of GenBank sequences for eukaryotes (excluding most genome survey sequences, ESTs, and other high-throughput data). In addition to summarizing sequence diversity and species diversity across nodes in the NCBI taxonomy, it reports 87,000 potentially phylogenetically informative clusters of homologous sequences, which can be viewed or downloaded, along with provisional alignments and coarse phylogenetic trees. At each node in the NCBI hierarchy, the user can display a "data availability matrix" of all available sequences for entries in a subtaxa-by-clusters matrix. This matrix provides a guidepost for subsequent assembly of multigene data sets or supertrees. The database allows for comparison of results from previous GenBank releases, highlighting recent additions of either sequences or taxa to GenBank and letting investigators track progress on data availability worldwide. Although the reported alignments and trees are extremely approximate, the database reports several statistics correlated with alignment quality to help users choose from alternative data sources.     

Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA''s of B77 Avian Sarcoma Virus

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.     

五种啮齿类动物mtDNA蛋白编码基因序列变化的比较

目的利用本实验室测定的中国地鼠、金黄地鼠和GenBank中田鼠、小鼠、大鼠的线粒体全基因组序列,比较分析五种啮齿类动物的mtDNA蛋白编码基因序列的变异,探讨其分子进化关系。方法将五种动物各自的13个蛋白编码基因分别连接成一个序列,用DNAstar-EditSeq分析软件计算每个序列的碱基长度和组成,计算蛋白编码基因的碱基和氨基酸的差异。以人为外群,基于连接在一起的13个蛋白编码基因的氨基酸序列,用MEGA4.0软件通过最大简约性法(MP)和非加权成对平均数法(UPGMA)构建进化树。结果在五种啮齿动物的13个蛋白基因序列中,A、T、C、G碱基的平均含量为32.4%、29.6%、26.2%和11.9%,中国地鼠mtDNA各蛋白编码序列以及其编码的氨基酸序列与其他物种相比,与金黄地鼠的相应序列差异最小,与大鼠mtDNA各蛋白编码序列以及其编码的氨基酸序列差异较大。分子进化树也显示中国地鼠和金黄地鼠的亲缘关系最近,与小鼠、大鼠存在的差异相对大。结论五种动物的碱基组成的百分比中显示G的相对缺乏,相互之间的进化关系与传统的分类地位基本吻合。     

Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.     

Simulation of Genomes: A Review

There is an increasing role of population genetics in human genetic research linking empirical observations with hypotheses about sequence variation due to historical and evolutionary causes. In addition, the data sets are increasing in size, with genome-wide data becoming a common place in many empirical studies. As far as more information is available, it becomes clear that simplest hypotheses are not consistent with data. Simulations will provide the key tool to contrast complex hypotheses on real data by generating simulated data under the hypothetical historical and evolutionary conditions that we want to contrast. Undoubtedly, developing tools for simulating large sequences that at the same time allow simulate natural selection, recombination and complex demography patterns will be of great interest in order to better understanding the trace left on the DNA by different interacting evolutionary forces. Simulation tools will be also essential to evaluate the sampling properties of any statistics used on genome-wide association studies and to compare performance of methods applied at genome-wide scales. Several recent simulation tools have been developed. Here, we review some of the currently existing simulators which allow for efficient simulation of large sequences on complex evolutionary scenarios. In addition, we will point out future directions in this field which are already a key part of the current research in evolutionary biology and it seems that it will be a primary tool in the future research of genome and post-genomic biology.     

DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae).

DNA barcoding has been touted as a program that will efficiently and relatively cheaply inform on biological diversity; yet many exemplars purporting to demonstrate the efficacy of the method have been undertaken by its principal proponents. Critics of DNA barcoding identify insufficient within-taxon sampling coupled with the knowledge that levels of haplotypic paraphyly are rather high as key reasons to be sceptical of the value of an exclusively DNA-based taxonomic. Here I applied a DNA barcoding approach using mtDNA sequences from the cytochrome oxidase I gene to examine diversity in a group of endemic New Zealand grasshoppers belonging to the genus Sigaus . The mtDNA data revealed high genetic distances among individuals of a single morpho-species, but this diversity was geographically partitioned. Phylogenetic analysis supported at least four haplogroups within one species ( Sigaus australis ) but paraphyly of this species with respect to several others. In some instances two morphologically and ecologically distinct species shared identical mtDNA haplotypes. The mismatch of genealogy and taxonomy revealed in the Sigaus australis complex indicates that, if used in isolation, DNA barcoding data can be highly misleading about biodiversity. Furthermore, failure to take into account evidence from natural history and morphology when utilizing DNA barcoding will tend to conceal the underlying evolutionary processes associated with speciation.
© The Willi Hennig Society 2007.