Advancements in high-throughput nucleotide sequencing techniques have brought with them state-of-the-art bioinformatics programs and software packages. Given the importance of molecular sequence data in contemporary life science research, these software suites are becoming an essential component of many labs and classrooms, and as such are frequently designed for non-computer specialists and marketed as one-stop bioinformatics toolkits. Although beautifully designed and powerful, user-friendly bioinformatics packages can be expensive and, as more arrive on the market each year, it can be difficult for researchers, teachers and students to choose the right software for their needs, especially if they do not have a bioinformatics background. This review highlights some of the currently available and most popular commercial bioinformatics packages, discussing their prices, usability, features and suitability for teaching. Although several commercial bioinformatics programs are arguably overpriced and overhyped, many are well designed, sophisticated and, in my opinion, worth the investment. If you are just beginning your foray into molecular sequence analysis or an experienced genomicist, I encourage you to explore proprietary software bundles. They have the potential to streamline your research, increase your productivity, energize your classroom and, if anything, add a bit of zest to the often dry detached world of bioinformatics.
A lot is known about the evolution and architecture of plastid, mitochondrial, and nuclear genomes, but surprisingly little is known about their relative rates of mutation. Most available relative-rate data come from seed plants, which, with few exceptions, have a mitochondrial mutation rate that is lower than those of the plastid and nucleus. But new findings from diverse plastid-bearing lineages have shown that for some eukaryotes the mitochondrial mutation rate is an order of magnitude greater than those of the plastid and nucleus. Here, we explore for the first time relative rates of mutation within the Glaucophyta-one of three main lineages that make up the Archaeplastida (or Plantae sensu lato). Nucleotide substitution analyses from distinct isolates of the unicellular glaucophyte Cyanophora paradoxa reveal 4-5-fold lower rates of mutation in the plastid and nucleus than the mitochondrion, which is similar to the mutational pattern observed in red algae and haptophytes, but opposite to that of seed plants. These data, together with data from previous reports, suggest that for much of the known photosynthetic eukaryotic diversity, plastid DNA mutations occur less frequently than those in mitochondrial DNA.
Polytomella spp. are free-living, nonphotosynthetic green algae closely related to the model organism Chlamydomonas reinhardtii. Although colorless, Polytomella spp. have a plastid, but it is still unknown whether they harbor a plastid genome. We took a next generation sequencing approach, along with transcriptome sequencing, to search for a plastid genome and an associated gene expression system in Polytomella spp. Illumina sequencing of total DNA from four Polytomella spp. did not produce any recognizable plastid-derived reads but did generate a large number of mitochondrial DNA sequences. Transcriptomic analysis of Polytomella parva uncovered hundreds of putative nuclear-encoded, plastid-targeted proteins, which support the presence of plastid-based metabolic functions, similar to those observed in the plastids of other nonphotosynthetic algae. Conspicuously absent, however, were any plastid-targeted proteins involved in the expression, replication, or repair of plastid DNA. Based on these findings and earlier findings, we argue that the Polytomella genus represents the first well-supported example, to our knowledge, of a primary plastid-bearing lineage without a plastid genome.
Organelle DNA is no stranger to palindromic repeats. But never has a mitochondrial or plastid genome been described in which every coding region is part of a distinct palindromic unit. While sequencing the mitochondrial DNA of the nonphotosynthetic green alga Polytomella magna, we uncovered precisely this type of genic arrangement. The P. magna mitochondrial genome is linear and made up entirely of palindromes, each containing 1-7 unique coding regions. Consequently, every gene in the genome is duplicated and in an inverted orientation relative to its partner. And when these palindromic genes are folded into putative stem-loops, their predicted translational start sites are often positioned in the apex of the loop. Gel electrophoresis results support the linear, 28-kb monomeric conformation of the P. magna mitochondrial genome. Analyses of other Polytomella taxa suggest that palindromic mitochondrial genes were present in the ancestor of the Polytomella lineage and lost or retained to various degrees in extant species. The possible origins and consequences of this bizarre genomic architecture are discussed.
We are just beginning to understand how mutation rates differ among mitochondrial, plastid, and nuclear genomes. In most seed plants the mitochondrial mutation rate is estimated to be lower than those of the plastid and nucleus, whereas in the red alga Porphyra the opposite is true, and in certain green algae all three genomes appear to have similar rates of mutation. Relative rate statistics of organelle vs nuclear genes, however, are lacking for lineages that acquired their plastids through secondary endosymbiosis, but recent organelle DNA analyses suggest that they may differ drastically from what is observed in lineages with primary plastids, such as green plants and red algae. Here, by measuring synonymous nucleotide substitutions, we approximate the relative mutation rates within the haptophyte genus Phaeocystis, which has a red-algal-derived, secondary plastid. Synonymous-site divergence data indicate that for Phaeocystis antarctica and P. globosa the mitochondrial mutation rate is 10 and 3 times that of the plastid and nucleus, respectively. This differs drastically from relative rate estimates for primary-plastid-bearing lineages and presents a much more dynamic view of organelle vs nuclear mutation rates across the eukaryotic domain.
Recently, it was shown that gene conversion between the ends of linear mitochondrial chromosomes can cause telomere expansion and the duplication of subtelomeric loci. However, it is not yet known how widespread this phenomenon is and how significantly it has impacted organelle genome architecture. Using linear mitochondrial DNAs and mitochondrial plasmids from diverse eukaryotes, we argue that telomeric recombination has played a major role in fashioning linear organelle chromosomes. We find that mitochondrial telomeres frequently expand into subtelomeric regions, resulting in gene duplications, homogenizations, and/or fragmentations. We suggest that these features are a product of subtelomeric gene conversion, provide a hypothetical model for this process, and employ genetic diversity data to support the idea that the greater the effective population size the greater the potential for gene conversion between subtelomeric loci.
GenBank is bursting with eukaryotic RNA sequencing (RNA-Seq) results. These data are transforming our view of nuclear transcriptional architecture, but many scientists are ignoring a major component of the data: mitochondrial- and chloroplast-derived sequences. Indeed, organelle transcripts typically represent a significant proportion of the reads generated from eukaryotic RNA-Seq experiments. Here, I argue that these data are an excellent and untapped resource for investigating many aspects of organelle function and evolution.
It has been argued that for certain lineages, noncoding DNA expansion is a consequence of the increased random genetic drift associated with long-term escalations in organism size. But a lack of data has prevented the investigation of this hypothesis in most plastid-bearing protists. Here, using newly sequenced mitochondrial and plastid genomes, we explore the relationship between organelle DNA noncoding content and organism size within volvocine green algae. By looking at unicellular, colonial, and differentiated multicellular algae, we show that organelle DNA complexity scales positively with species size and cell number across the volvocine lineage. Moreover, silent-site genetic diversity data suggest that the volvocine species with the largest cell numbers and most bloated organelle genomes have the smallest effective population sizes. Together, these findings support the view that nonadaptive processes, like random genetic drift, promote the expansion of noncoding regions in organelle genomes.
Animal mitochondrial DNAs (mtDNAs) are typically single circular chromosomes, with the exception of those from medusozoan cnidarians (jellyfish and hydroids), which are linear and sometimes fragmented. Most medusozoans have linear monomeric or linear bipartite mitochondrial genomes, but preliminary data have suggested that box jellyfish (cubozoans) have mtDNAs that consist of many linear chromosomes. Here, we present the complete mtDNA sequence from the winged box jellyfish Alatina moseri (the first from a cubozoan). This genome contains unprecedented levels of fragmentation: 18 unique genes distributed over eight 2.9- to 4.6-kb linear chromosomes. The telomeres are identical within and between chromosomes, and recombination between subtelomeric sequences has led to many genes initiating or terminating with sequences from other genes (the most extreme case being 150 nt of a ribosomal RNA containing the 5 end of nad2), providing evidence for a gene conversion-based model of telomere evolution. The silent-site nucleotide variation within the A. moseri mtDNA is among the highest observed from a eukaryotic genome and may be associated with elevated rates of recombination.
Mitochondrial genomes often contain large amounts of plastid DNA (ptDNA)-derived sequences (MTPTs). It has been suggested that the intercompartmental transfer of ptDNA is greatly reduced in species with only a single plastid per cell (monoplastidic) as compared with those with many plastids per cell (polyplastidic). This hypothesis has not been applied to the movement of DNA from plastids to mitochondria. By analyzing the organelle genomes from diverse mono- and polyplastidic taxa, I show that MTPTs are restricted to the mitochondrial genomes of species with many plastids per cell and are absent from those with one plastid per cell or with monoplastidic meristematic systems. Moreover, the most bloated mitochondrial genomes that were explored had the largest MTPT contents. These data, like previous results on ptDNA-derived sequences in nuclear genomes, support the hypothesis that plastid number and the forces governing the expansion and contraction of noncoding mitochondrial DNA (mtDNA) influence MTPT abundance. I also show that plastid genomes are depauperate in mtDNA-derived sequences (PTMTs), irrespective of the number of mitochondria per cell and plastid genome size, which may reflect the lack of a DNA uptake system in plastids.
Silent-site nucleotide diversity data (?(silent)) can provide insights into the forces driving genome evolution. Here we present ?(silent) statistics for the mitochondrial and nuclear DNAs of Polytomella parva, a nonphotosynthetic green alga with a highly reduced, linear fragmented mitochondrial genome. We show that this species harbors very little genetic diversity, with the exception of the mitochondrial telomeres, which have an excess of polymorphic sites. These data are compared with previously published ?(silent) values from the mitochondrial and nuclear genomes of the model species Chlamydomonas reinhardtii and Volvox carteri, which are close relatives of P. parva, and are used to understand the modes and tempos of genome evolution within green algae.
Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.
Levels of nucleotide substitution at silent sites in organelle versus nuclear DNAs have been used to estimate relative mutation rates among these compartments and explain lineage-specific features of genome evolution. Synonymous substitution divergence values in animals suggest that the rate of mutation in the mitochondrial DNA is 10-50 times higher than that of the nuclear DNA, whereas overall data for most seed plants support relative mutation rates in mitochondrial, plastid, and nuclear DNAs of 1:3:10. Little is known about relative mutation rates in green algae, as substitution rate data is limited to only the mitochondrial and nuclear genomes of the chlorophyte Chlamydomonas. Here, we measure silent-site substitution rates in the plastid DNA of Chlamydomonas and the three genetic compartments of the streptophyte green alga Mesostigma. In contrast to the situation in animals and land plants, our results support similar relative mutation rates among the three genetic compartments of both Chlamydomonas and Mesostigma. These data are discussed in relation to published intra-species genetic diversity data for the three genetic compartments of Chlamydomonas and are ultimately used to address contemporary hypotheses on the organelle genome evolution. To guide future work, we describe evolutionary divergence data of all publically available Mesostigma viride strains and identify, for the first time, three distinct lineages of Mesostigma.
The abundance of nuclear plastid DNA-like sequences (NUPTs) in nuclear genomes can vary immensely; however, the forces responsible for this variation are poorly understood. "The limited transfer window hypothesis" predicts that species with only one plastid per cell will have fewer NUPTs than those with many plastids per cell, but a lack of genome sequence data from monoplastidic species has made this hypothesis difficult to test. Here, by analyzing newly available genome sequences from diverse mono- and polyplastidic taxa, we show that the hypothesis holds. On average, the polyplastidic species we studied had 80 times more NUPTs than those that were monoplastidic. Moreover, NUPT content was positively related to nuclear genome size, indicating that in addition to plastid number, NUPTs are influenced by the forces controlling the expansion and contraction of noncoding nuclear DNA. These findings are consistent with data on nuclear DNAs of mitochondrial origin (NUMTs), suggesting that similar processes govern the abundance of both NUPTs and NUMTs.
Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of beta-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.
The noncoding-DNA content of organelle and nuclear genomes can vary immensely. Both adaptive and nonadaptive explanations for this variation have been proposed. This study addresses a nonadaptive explanation called the mutational-hazard hypothesis and applies it to the mitochondrial, plastid, and nuclear genomes of the multicellular green alga Volvox carteri. Given the expanded architecture of the V. carteri organelle and nuclear genomes (60-85% noncoding DNA), the mutational-hazard hypothesis would predict them to have less silent-site nucleotide diversity (?(silent)) than their more compact counterparts from other eukaryotes-ultimately reflecting differences in 2N(g)? (twice the effective number of genes per locus in the population times the mutation rate). The data presented here support this prediction: Analyses of mitochondrial, plastid, and nuclear DNAs from seven V. carteri forma nagariensis geographical isolates reveal low values of ?(silent) (0.00038, 0.00065, and 0.00528, respectively), much lower values than those previously observed for the more compact organelle and nuclear DNAs of Chlamydomonas reinhardtii (a close relative of V. carteri). We conclude that the large noncoding-DNA content of the V. carteri genomes is best explained by the mutational-hazard hypothesis and speculate that the shift from unicellular to multicellular life in the ancestor that gave rise to V. carteri contributed to a low V. carteri population size and thus a reduced 2N(g)?. Complete mitochondrial and plastid genome maps for V. carteri are also presented and compared with those of C. reinhardtii.
Although DNA sequences of linear mitochondrial genomes are available for a wide variety of species, sequence and conformational data from the extreme ends of these molecules (i.e., the telomeres) are limited. Data on the telomeres is important because it can provide insights into how linear genomes overcome the end-replication problem. This study explores the evolution of linear mitochondrial DNAs (mtDNAs) in the green-algal genus Polytomella (Chlorophyceae, Chlorophyta), the members of which are non-photosynthetic. Earlier works analyzed the linear and linear-fragmented mitochondrial genomes of Polytomella capuana and Polytomella parva. Here we present the mtDNA sequence for Polytomella strain SAG 63-10 [also known as Polytomella piriformis (Pringsheim 1963)], which is the only known representative of a mostly unexplored Polytomella lineage. We show that the P. piriformis mtDNA is made up of two linear fragments of 13 and 3 kb. The telomeric sequences of the large and small fragments are terminally inverted, and appear to end in vitro with either closed (hairpin-loop) or open (nicked-loop) structures as also shown here for P. parva and shown earlier for P. capuana. The structure of the P. piriformis mtDNA is more similar to that of P. parva, which is also fragmented, than to that of P. capuana, which is contained in a single chromosome. Phylogenetic analyses reveal high substitution rates in the mtDNA of all three Polytomella species relative to other chlamydomonadalean algae. These elevated rates could be the result of a greater number of vegetative cell divisions and/or small population sizes in Polytomella species as compared with other chlamydomonadalean algae.
One of the more conspicuous features of plastid DNA (ptDNA) is its low guanine and cytosine (GC) content. As of February 2009, all completely-sequenced plastid genomes have a GC content below 43% except for the ptDNA of the lycophyte Selaginella uncinata, which is 55% GC. The forces driving the S. uncinata ptDNA towards G and C are undetermined, and it is unknown if other Selaginella species have GC-biased plastid genomes. This study presents the complete ptDNA sequence of Selaginella moellendorffii and compares it with the previously reported S. uncinata plastid genome. Partial ptDNA sequences from 103 different Selaginella species are also described as well as a significant proportion of the S. moellendorffii mitochondrial genome. Moreover, S. moellendorffii express sequence tags are data-mined to estimate levels of plastid and mitochondrial RNA editing. Overall, these data are used to show that: (1) there is a genus-wide GC bias in Selaginella ptDNA, which is most pronounced in South American articulate species; (2) within the Lycopsida class (and among plants in general), GC-biased ptDNA is restricted to the Selaginella genus; (3) the cause of this GC bias is arguably a combination of reduced AT-mutation pressure relative to other plastid genomes and a large number of C-to-U RNA editing sites; and (4) the mitochondrial DNA (mtDNA) of S. moellendorffii is also GC biased (even more so than the ptDNA) and is arguably the most GC-rich organelle genome observed to date-the high GC content of the mtDNA also appears to be influenced by RNA editing. Ultimately, these findings provide convincing support for the earlier proposed theory that the GC content of land-plant organelle DNA is positively correlated and directly connected to levels of organelle RNA editing.
The magnitude of noncoding DNA in organelle genomes can vary significantly; it is argued that much of this variation is attributable to the dissemination of selfish DNA. The results of a previous study indicate that the mitochondrial DNA (mtDNA) of the green alga Volvox carteri abounds with palindromic repeats, which appear to be selfish elements. We became interested in the evolution and distribution of these repeats when, during a cursory exploration of the V. carteri nuclear DNA (nucDNA) and plastid DNA (ptDNA) sequences, we found palindromic repeats with similar structural features to those of the mtDNA. Upon this discovery, we decided to investigate the diversity and evolutionary implications of these palindromic elements by sequencing and characterizing large portions of mtDNA and ptDNA and then comparing these data to the V. carteri draft nuclear genome sequence.
The mutational-hazard hypothesis argues that the noncoding-DNA content of a genome is a consequence of the mutation rate (mu) and the effective number of genes per locus in the population (N(g)). The hypothesis predicts that genomes with a high N(g)mu will be more compact than those with a small N(g)mu. Approximations of N(g)mu can be gained by measuring the nucleotide diversity at silent sites (pi(silent)). We addressed the mutation-hazard hypothesis apropos plastid-genome evolution by measuring pi(silent) of the Chlamydomonas reinhardtii plastid DNA (ptDNA), the most noncoding-DNA-dense plastid genome observed to date. The data presented here in conjunction with previously published values of pi(silent) for the C. reinhardtii mitochondrial and nuclear genomes, which are respectively compact and bloated, allow for a complete analysis of nucleotide diversity and genome compactness in all three genetic compartments of this model organism.
Plastid genomes show an impressive array of sizes and compactnesses, but the forces responsible for this variation are unknown. It has been argued that species with small effective genetic population sizes are less efficient at purging excess DNA from their genomes than those with large effective population sizes. If true, one may expect the primary mode of plastid inheritance to influence plastid DNA (ptDNA) architecture. All else being equal, biparentally inherited ptDNAs should have a two-fold greater effective population size than those that are uniparentally inherited, and thus should also be more compact. Here, we explore the relationship between plastid inheritance pattern and ptDNA architecture, and consider the role of phylogeny in shaping our observations. Contrary to our expectations, we found no significant difference in plastid genome size or compactness between ptDNAs that are biparentally inherited relative to those that are uniparentally inherited. However, we also found that there was significant phylogenetic signal for the trait of mode of plastid inheritance. We also found that paternally inherited ptDNAs are significantly smaller (n?=?19, p?=?0.000001) than those that are maternally, uniparentally (when isogamous), or biparentally inherited. Potential explanations for this observation are discussed.
Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits.
This Short Communication highlights the diversity of secondary genome data (like mitochondrial and plastid genomes) that can be gleaned from next-generation sequencing projects, and encourages researchers to be mindful that these data are often as informative and useful as the primary genome data.
Within plastid-bearing species, the relative rates of evolution between mitochondrial and plastid genomes are poorly studied, but for the few lineages in which they have been explored, including land plants and green algae, the mitochondrial DNA mutation rate is nearly always estimated to be lower than or equal to that of the plastid DNA. Here, we show that in protists from three distinct lineages with secondary, red algal-derived plastids, the opposite is true: their mitochondrial genomes are evolving 5-30 times faster than their plastid genomes, even when the plastid is nonphotosynthetic. These findings have implications for understanding the origins and evolution of organelle genome architecture and the genes they encode.
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