Eli Meyer

Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx

BackgroundNew methods are needed for genomic-scale analysis of emerging model organisms that exemplify important biological questions but lack fully sequenced genomes. For example, there is an urgent need to understand the potential for corals to adapt to climate change, but few molecular resources are available for studying these processes in reef-building corals. To facilitate genomics studies in corals and other non-model systems, we describe methods for transcriptome sequencing using 454, as well as strategies for assembling a useful catalog of genes from the output. We have applied these methods to sequence the transcriptome of planulae larvae from the coral Acropora millepora.ResultsMore than 600,000 reads produced in a single 454 sequencing run were assembled into ~40,000 contigs with five-fold average sequencing coverage. Based on sequence similarity with known proteins, these analyses identified ~11,000 different genes expressed in a range of conditions including thermal stress and settlement induction. Assembled sequences were annotated with gene names, conserved domains, and Gene Ontology terms. Targeted searches using these annotations identified the majority of genes associated with essential metabolic pathways and conserved signaling pathways, as well as novel candidate genes for stress-related processes. Comparisons with the genome of the anemone Nematostella vectensis revealed ~8,500 pairs of orthologs and ~100 candidate coral-specific genes. More than 30,000 SNPs were detected in the coral sequences, and a subset of these validated by re-sequencing.ConclusionThe methods described here for deep sequencing of the transcriptome should be widely applicable to generate catalogs of genes and genetic markers in emerging model organisms. Our data provide the most comprehensive sequence resource currently available for reef-building corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widely-studied coral will enable research into the biological processes underlying stress responses in corals and evolutionary adaptation to global climate change.

2b-RAD: a simple and flexible method for genome-wide genotyping

We describe 2b-RAD, a streamlined restriction site–associated DNA (RAD) genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Well-studied accessions of Arabidopsis thaliana were genotyped to validate the methods accuracy and to demonstrate fine-tuning of marker density as needed. The simplicity of the 2b-RAD protocol makes it particularly suitable for high-throughput genotyping as required for linkage mapping and profiling genetic variation in natural populations.

Profiling gene expression responses of coral larvae (Acropora millepora) to elevated temperature and settlement inducers using a novel RNA‐Seq procedure

Elevated temperatures resulting from climate change pose a clear threat to reef‐building corals; however, the traits that might influence corals’ survival and dispersal during climate change remain poorly understood. Global gene expression profiling is a powerful hypothesis‐forming tool that can help elucidate these traits. Here, we applied a novel RNA‐Seq protocol to study molecular responses to heat and settlement inducers in aposymbiotic larvae of the reef‐building coral Acropora millepora. This analysis of a single full‐sibling family revealed contrasting responses between short‐ (4‐h) and long‐term (5‐day) exposures to elevated temperatures. Heat shock proteins were up‐regulated only in the short‐term treatment, while the long‐term treatment induced the down‐regulation of ribosomal proteins and up‐regulation of genes associated with ion transport and metabolism (Ca2+ and CO32−). We also profiled responses to settlement cues using a natural cue (crustose coralline algae, CCA) and a synthetic neuropeptide (GLW‐amide). Both cues resulted in metamorphosis, accompanied by differential expression of genes with known developmental roles. Some genes were regulated only by the natural cue, which may correspond to the recruitment‐associated behaviour and morphology changes that precede metamorphosis under CCA treatment, but are bypassed under GLW‐amide treatment. Validation of these expression profiles using qPCR confirmed the quantitative accuracy of our RNA‐Seq approach. Importantly, qPCR analysis of different larval families revealed extensive variation in these responses depending on genetic background, including qualitative differences (i.e. up‐regulation in one family and down‐regulation in another). Future studies of gene expression in corals will have to address this genetic variation, which could have important adaptive consequences for corals during global climate change.

Bacterial Diversity in Solenopsis invicta and Solenopsis geminata Ant Colonies Characterized by 16S amplicon 454 Pyrosequencing

Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000–10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

Genomic determinants of coral heat tolerance across latitudes

Some like it hot Coral reefs are threatened by increasing temperatures. Acute temperature increases stress and damage corals. However, more gradual temperature changes can result in adaptation and subsequent tolerance for higher temperatures. Dixon et al. show that the heat tolerance that currently exists across coral populations from different latitudes can be inherited. Thus, natural variation in temperature tolerance may facilitate rapid adaptation among corals as our climate warms. Science, this issue p. 1460 In a warming world, existing variation in heat tolerance could help corals beat the heat. As global warming continues, reef-building corals could avoid local population declines through “genetic rescue” involving exchange of heat-tolerant genotypes across latitudes, but only if latitudinal variation in thermal tolerance is heritable. Here, we show an up–to–10-fold increase in odds of survival of coral larvae under heat stress when their parents come from a warmer lower-latitude location. Elevated thermal tolerance was associated with heritable differences in expression of oxidative, extracellular, transport, and mitochondrial functions that indicated a lack of prior stress. Moreover, two genomic regions strongly responded to selection for thermal tolerance in interlatitudinal crosses. These results demonstrate that variation in coral thermal tolerance across latitudes has a strong genetic basis and could serve as raw material for natural selection.

Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas)

Compared with understanding of biological shape and form, knowledge is sparse regarding what regulates growth and body size of a species. For example, the genetic and physiological causes of heterosis (hybrid vigor) have remained elusive for nearly a century. Here, we investigate gene-expression patterns underlying growth heterosis in the Pacific oyster (Crassostrea gigas) in two partially inbred (f = 0.375) and two hybrid larval populations produced by a reciprocal cross between the two inbred families. We cloned cDNA and generated 4.5 M sequence tags with massively parallel signature sequencing. The sequences contain 23,274 distinct signatures that are expressed at statistically nonzero levels and show a highly positively skewed distribution with median and modal counts of 9.25 million and 3 transcripts per million, respectively. For nearly half of these signatures, expression level depends on genotype and is predominantly nonadditive (hybrids deviate from the inbred average). Statistical contrasts suggest ≈350 candidate genes for growth heterosis that exhibit concordant nonadditive expression in reciprocal hybrids; this represents only ≈1.5% of the >20,000 transcripts. Patterns of gene expression, which include dominance for low expression and even underdominance of expression, are more complex than predicted from classical dominant or overdominant explanations of heterosis. Preliminary identification of ribosomal proteins among candidate genes supports the suggestion from previous studies that efficiency of protein metabolism plays a role in growth heterosis.

Gene expression under chronic heat stress in populations of the mustard hill coral (Porites astreoides) from different thermal environments

Recent evidence suggests that corals can acclimatize or adapt to local stress factors through differential regulation of their gene expression. Profiling gene expression in corals from diverse environments can elucidate the physiological processes that may be responsible for maximizing coral fitness in their natural habitat and lead to a better understanding of the corals capacity to survive the effects of global climate change. In an accompanying paper, we show that Porites astreoides from thermally different reef habitats exhibit distinct physiological responses when exposed to 6 weeks of chronic temperature stress in a common garden experiment. Here, we describe expression profiles obtained from the same corals for a panel of 9 previously reported and 10 novel candidate stress response genes identified in a pilot RNA‐Seq experiment. The strongest expression change was observed in a novel candidate gene potentially involved in calcification, SLC26, a member of the solute carrier family 26 anion exchangers, which was down‐regulated by 92‐fold in bleached corals relative to controls. The most notable signature of divergence between coral populations was constitutive up‐regulation of metabolic genes in corals from the warmer inshore location, including the gluconeogenesis enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase and the lipid beta‐oxidation enzyme acyl‐CoA dehydrogenase. Our observations highlight several molecular pathways that were not previously implicated in the coral stress response and suggest that host management of energy budgets might play an adaptive role in holobiont thermotolerance.

Comparative genomics explains the evolutionary success of reef-forming corals

Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years. DOI: http://dx.doi.org/10.7554/eLife.13288.001

Microbiomes of ant castes implicate new microbial roles in the fungus-growing ant Trachymyrmex septentrionalis

Fungus-growing ants employ several defenses against diseases, including disease-suppressing microbial biofilms on their integument and in fungal gardens. Here, we compare the phenology of microbiomes in natural nests of the temperate fungus-growing ant Trachymyrmex septentrionalis using culture-dependent isolations and culture-independent 16S-amplicon 454-sequencing. 454-sequencing revealed diverse actinobacteria associated with ants, including most prominently Solirubrobacter (12.2–30.9% of sequence reads), Pseudonocardia (3.5–42.0%), and Microlunatus (0.4–10.8%). Bacterial abundances remained relatively constant in monthly surveys throughout the annual active period (late winter to late summer), except Pseudonocardia abundance declined in females during the reproductive phase. Pseudonocardia species found on ants are phylogenetically different from those in gardens and soil, indicating ecological separation of these Pseudonocardia types. Because the pathogen Escovopsis is not known to infect gardens of T. septentrionalis, the ant-associated microbes do not seem to function in Escovopsis suppression, but could protect against ant diseases, help in nest sanitation, or serve unknown functions.

Construction of a high-resolution genetic linkage map and comparative genome analysis for the reef-building coral Acropora millepora

BackgroundWorldwide, coral reefs are in decline due to a range of anthropogenic disturbances, and are now also under threat from global climate change. Virtually nothing is currently known about the genetic factors that might determine whether corals adapt to the changing climate or continue to decline. Quantitative genetics studies aiming to identify the adaptively important genomic loci will require a high-resolution genetic linkage map. The phylogenetic position of corals also suggests important applications for a coral genetic map in studies of ancestral metazoan genome architecture.ResultsWe constructed a high-resolution genetic linkage map for the reef-building coral Acropora millepora, the first genetic map reported for any coral, or any non-Bilaterian animal. More than 500 single nucleotide polymorphism (SNP) markers were developed, most of which are transferable in populations from Orpheus Island and Great Keppel Island. The map contains 429 markers (393 gene-based SNPs and 36 microsatellites) distributed in 14 linkage groups, and spans 1,493 cM with an average marker interval of 3.4 cM. Sex differences in recombination were observed in a few linkage groups, which may be caused by haploid selection. Comparison of the coral map with other metazoan genomes (human, nematode, fly, anemone and placozoan) revealed synteny regions.ConclusionsOur study develops a framework that will be essential for future studies of adaptation in coral and it also provides an important resource for future genome sequence assembly and for comparative genomics studies on the evolution of metazoan genome structure.