An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and cryptophyte plastids are sisters

Background  

Horizontal gene transfer (HGT) to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid.     

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus <Emphasis Type="Italic">Cuscuta</Emphasis>

Background  

Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle.     

Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

Background  

Parthenium argentatum (guayule) is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of P. argentatum was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines.     

Effective Extraction and Assembly Methods for Simultaneously Obtaining Plastid and Mitochondrial Genomes

Background

In conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously.

Results

First, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately.

Conclusion

A simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.     

<Emphasis Type="Italic">Tracembler</Emphasis> – software for <Emphasis Type="Italic">in-silico</Emphasis> chromosome walking in unassembled genomes

Background  

Whole genome shotgun sequencing produces increasingly higher coverage of a genome with random sequence reads. Progressive whole genome assembly and eventual finishing sequencing is a process that typically takes several years for large eukaryotic genomes. In the interim, all sequence reads of public sequencing projects are made available in repositories such as the NCBI Trace Archive. For a particular locus, sequencing coverage may be high enough early on to produce a reliable local genome assembly. We have developed software, Tracembler, that facilitates in silico chromosome walking by recursively assembling reads of a selected species from the NCBI Trace Archive starting with reads that significantly match sequence seeds supplied by the user.     

An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection

Background

Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses.

Methodology/Principal Findings

We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge.

Conclusions/Significance

The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.     

The complete plastid genome sequence of the parasitic green alga Helicosporidium sp. is highly reduced and structured

Background  

Loss of photosynthesis has occurred independently in several plant and algal lineages, and represents a major metabolic shift with potential consequences for the content and structure of plastid genomes. To investigate such changes, we sequenced the complete plastid genome of the parasitic, non-photosynthetic green alga, Helicosporidium.     

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales)

Premise

Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome.

Methods

We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω).

Results

Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK.

Conclusions

We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.     

Tracking <Emphasis Type="Italic">Alu</Emphasis> evolution in New World primates

Background  

Alu elements are Short INterspersed Elements (SINEs) in primate genomes that have proven useful as markers for studying genome evolution, population biology and phylogenetics. Most of these applications, however, have been limited to humans and their nearest relatives, chimpanzees. In an effort to expand our understanding of Alu sequence evolution and to increase the applicability of these markers to non-human primate biology, we have analyzed available Alu sequences for loci specific to platyrrhine (New World) primates.     

A SNP transferability survey within the genus <Emphasis Type="Italic">Vitis</Emphasis>

Background  

Efforts to sequence the genomes of different organisms continue to increase. The DNA sequence is usually decoded for one individual and its application is for the whole species. The recent sequencing of the highly heterozygous Vitis vinifera L. cultivar Pinot Noir (clone ENTAV 115) genome gave rise to several thousand polymorphisms and offers a good model to study the transferability of its degree of polymorphism to other individuals of the same species and within the genus.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies

Background

The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.

Results

We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.

Conclusions

In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.     

VIGOR,an annotation program for small viral genomes

Background  

The decrease in cost for sequencing and improvement in technologies has made it easier and more common for the re-sequencing of large genomes as well as parallel sequencing of small genomes. It is possible to completely sequence a small genome within days and this increases the number of publicly available genomes. Among the types of genomes being rapidly sequenced are those of microbial and viral genomes responsible for infectious diseases. However, accurate gene prediction is a challenge that persists for decoding a newly sequenced genome. Therefore, accurate and efficient gene prediction programs are highly desired for rapid and cost effective surveillance of RNA viruses through full genome sequencing.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Ori-Finder: A web-based system for finding <Emphasis Type="Italic">oriC</Emphasis> s in unannotated bacterial genomes

Background  

Chromosomal replication is the central event in the bacterial cell cycle. Identification of replication origins (oriCs) is necessary for almost all newly sequenced bacterial genomes. Given the increasing pace of genome sequencing, the current available software for predicting oriCs, however, still leaves much to be desired. Therefore, the increasing availability of genome sequences calls for improved software to identify oriCs in newly sequenced and unannotated bacterial genomes.     

A high density of ancient spliceosomal introns in oxymonad excavates

Background  

Certain eukaryotic genomes, such as those of the amitochondriate parasites Giardia and Trichomonas, have very low intron densities, so low that canonical spliceosomal introns have only recently been discovered through genome sequencing. These organisms were formerly thought to be ancient eukaryotes that diverged before introns originated, or at least became common. Now however, they are thought to be members of a supergroup known as excavates, whose members generally appear to have low densities of canonical introns. Here we have used environmental expressed sequence tag (EST) sequencing to identify 17 genes from the uncultivable oxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group.     

Phylogenetic detection of horizontal gene transfer during the step-wise genesis of Mycobacterium tuberculosis

Background  

In the past decade, the availability of complete genome sequence data has greatly facilitated comparative genomic research aimed at addressing genetic variability within species. More recently, analysis across species has become feasible, especially in genera where genome sequencing projects of multiple species have been initiated. To understand the genesis of the pathogen Mycobacterium tuberculosis within a genus where the majority of species are harmless environmental organisms, we have used genome sequence data from 16 mycobacteria to look for evidence of horizontal gene transfer (HGT) associated with the emergence of pathogenesis. First, using multi-locus sequence analysis (MLSA) of 20 housekeeping genes across these species, we derived a phylogeny that serves as the basis for HGT assignments. Next, we performed alignment searches for the 3989 proteins of M. tuberculosis H37Rv against 15 other mycobacterial genomes, generating a matrix of 59835 comparisons, to look for genetic elements that were uniquely found in M. tuberculosis and closely-related pathogenic mycobacteria. To assign when foreign genes were likely acquired, we designed a bioinformatic program called mycoHIT (mycobacterial homologue investigation tool) to analyze these data in conjunction with the MLSA-based phylogeny.     

Differentiation of regions with atypical oligonucleotide composition in bacterial genomes

Background  

Complete sequencing of bacterial genomes has become a common technique of present day microbiology. Thereafter, data mining in the complete sequence is an essential step. New in silico methods are needed that rapidly identify the major features of genome organization and facilitate the prediction of the functional class of ORFs. We tested the usefulness of local oligonucleotide usage (OU) patterns to recognize and differentiate types of atypical oligonucleotide composition in DNA sequences of bacterial genomes.     

The frontier between cell and organelle: genome analysis of <Emphasis Type="Italic">Candidatus</Emphasis> Carsonella ruddii

Background  

Bacterial symbioses are widespread among insects. The early establishment of such symbiotic associations has probably been one of the key factors for the evolutionary success of insects, since it may have allowed access to novel ecological niches and to new imbalanced food resources, such as plant sap or blood. Several genomes of bacterial endosymbionts of different insect species have been recently sequenced, and their biology has been extensively studied. Recently, the complete genome sequence of Candidatus Carsonella ruddii, considered the primary endosymbiont of the psyllid Pachpsylla venusta, has been published. This genome consists of a circular chromosome of 159,662 bp and has been proposed as the smallest bacterial endosymbiont genome known to date.