Steven B. Roberts

Single‐nucleotide polymorphism (SNP) discovery and applications of SNP genotyping in nonmodel organisms

A supplemental issue on the topic of single-nucleotidepolymorphism-enabled (SNP) research in nonmodel organisms is especially timely. In this issue, organisms with reference genomes are considered to be ‘model’; ‘nonmodel’ organisms are those whose genomes are yet to be sequenced. Advances in DNA sequencing and SNP genotyping have provided profound insights into the genetics of model organisms, but until recently, studies of nonmodel species lagged behind because of the scarcity of sequence and markers (see Fig. 1). In the past year, Tautz et al. (2010) and associated papers in supplemental issue of Molecular Ecology described a revolutionary transition from studies of ‘molecular ecology’ to studies of ‘ecological genomics’. Concurrently, Allendorf et al. (2010) grappled with placing the new-found wealth of sequence and SNP information into a ‘conservation genomics’ context. This revolution in molecular genetics studies would have been difficult to forecast a few years ago. Molecular genetic studies provide exceptional insight into relationships, migration and evolution of natural populations (Morin et al. 2004). During the origins of molecular ecology, in the 1960s and 1970s, it became clear that techniques such as allozyme electrophoresis would provide a basic framework for understanding species interactions and adaptation and for conserving natural genetic variability (Utter et al. 1966, 1974; Avise et al. 1975). Technical limitations at the time restricted both the ability to explore the dynamics of genetic diversity in species exhibiting low levels of variation as well as the direct analysis of adaptive variation in the wild. During the following years, innovators began to dream of potential applications for conservation and management of economically exploited species that included using molecular markers to determine the population-of-origin of migrating animals (see papers in Ryman & Utter 1987; Waples & Aebersold 1990), an important focus of many papers in this issue. Recent decades were punctuated by improvements in molecular and statistical techniques that produced an array of tools relevant to ecological and evolutionary studies such as assignment tests, estimates of effective population size, fine-scale population structure, kinship analyses (e.g. Helyar et al. this issue; Waples & Waples this issue) and genome-wide surveys based upon an ever increasing resolution of individuals and populations. The advantages of genotyping polymorphic SNPs with high-throughput assays have created much interest (Vignal et al. 2002; Brumfield et al. 2003; Morin et al. 2004; Schlötterer 2004). Until recently, however, the scarcity of available DNA sequence data for nonmodel species limited marker development. Further, because of comparatively low mutation rates, cross-species amplification of primers for SNP analyses did not yield the same results as for microsatellites. For example, Miller et al. (2010) tested the OvineSNP50 BeadChip, developed for domestic sheep, in two related ungulates and found only about 1% of the nearly 50 000 SNP loci to be polymorphic. Therefore, the SNP assays or probes developed for one species were not likely to be useful in others, even though primers may cross-amplify. The current supplemental issue contains 22 papers that underline the advantages of SNPs, advocate the need for SNP research in nonmodel organisms, and chart advances in discovery and applications. Although progress is apparent across a broad array of taxa, most papers presented here focus upon species of fish. This outcome, beyond the bias of the workshop organizers, may be in part because of the well-developed multinational collaborations that coordinate the sharing of DNA Correspondence: J. E. Seeb, Fax: (206) 543 5728; E-mail: jseeb@uw.edu

Differential skeletal muscle expression of myostatin across teleost species, and the isolation of multiple myostatin isoforms

Two myostatin (MSTN) isoforms were isolated from brook trout with 92% identity in corresponding regions at the nucleotide level. One isoform was isolated from muscle and brain and the second from ovarian tissue. To our knowledge this is the first time two MSTN isoforms have been isolated from a given vertebrate species. Within the brain, MSTN transcripts were localized to the optic lobes, hindbrain, and hypothalamus. In the trout ovary, MSTN transcripts were upregulated at ovulation in several females. MSTN cDNA fragments were also isolated from several other fish species and differential expression of MSTN among muscle fiber types was observed.

DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas)

BackgroundDNA methylation is an epigenetic mechanism with important regulatory functions in animals. While the mechanism itself is evolutionarily ancient, the distribution and function of DNA methylation is diverse both within and among phylogenetic groups. Although DNA methylation has been well studied in mammals, there are limited data on invertebrates, particularly molluscs. Here we characterize the distribution and investigate potential functions of DNA methylation in the Pacific oyster (Crassostrea gigas).ResultsMethylation sensitive PCR and bisulfite sequencing PCR approaches were used to identify CpG methylation in C. gigas genes and demonstrated that this species possesses intragenic methylation. In silico analysis of CpGo/e ratios in publicly available sequence data suggests that DNA methylation is a common feature of the C. gigas genome, and that specific functional categories of genes have significantly different levels of methylation.ConclusionsThe Pacific oyster genome displays intragenic DNA methylation and contains genes necessary for DNA methylation in animals. Results of this investigation suggest that DNA methylation has regulatory functions in Crassostrea gigas, particularly in gene families that have inducible expression, including those involved in stress and environmental responses.

A genetic basis for the phenotypic differentiation between siscowet and lean lake trout (Salvelinus namaycush)

In Lake Superior there are three principal forms of lake trout (Salvelinus namaycush): lean, siscowet and humper. Wild lean and siscowet differ in the shape and relative size of the head, size of the fins, location and size of the eyes, caudal peduncle shape and lipid content of the musculature. To investigate the basis for these phenotypic differences, lean and siscowet lake trout, derived from gametes of wild populations in Lake Superior, were reared communally under identical environmental conditions for 2.5 years. Fish were analysed for growth, morphometry and lipid content, and differences in liver transcriptomics were investigated using Roche 454 GS‐FLX pyrosequencing. The results demonstrate that key phenotypic differences between wild lean and siscowet lake trout such as condition factor, morphometry and lipid levels, persist in these two forms when reared in the laboratory under identical environmental conditions. This strongly suggests that these differences are genetic and not a result of environmental plasticity. Transcriptomic analysis involving the comparison of hepatic gene frequencies (RNA‐seq) and expression (quantitative reverse transcription–polymerase chain reaction (qPCR)) between the two lake trout forms, indicated two primary gene groups that were differentially expressed; those involving lipid synthesis, metabolism and transport (acyl‐CoA desaturase, acyl‐CoA binding protein, peroxisome proliferator‐activated receptor gamma, and apolipoproteins), and those involved with immunity (complement component C3, proteasome, FK506 binding protein 5 and C1q proteins). The results demonstrate that RNA‐seq can be used to identify differentially expressed genes; however, some discrepancies between RNA‐seq analysis and qPCR indicate that methods for deep sequencing may need to be refined and/or different RNA‐seq platforms utilized.

Rapid accumulation of an interleukin 17 homolog transcript in Crassostrea gigas hemocytes following bacterial exposure.

Interleukin-17 (IL-17) gene models have been found in the sequenced genomes of Strongylocentrotus purpuratus and Caenorhabditis elegans. However, there have been no published reports on the empirical cloning and characterization of any interleukin cDNAs in invertebrates. From a Pacific oyster (Crassostrea gigas) hemocyte cDNA library, two clones were obtained that encoded a protein similar to vertebrate IL-17s. The putative oyster IL-17 homolog (CgIL-17) was 27% identical to rainbow trout IL-17D, 21% to human IL-17D and 24% to an IL-17D-like gene model obtained from the annotation of the sea urchin genome. IL-17s from the oyster, sea urchin, trout and human, contained conserved cysteine residues found in all forms of IL-17 in mammals. Injection of bacteria into C. gigas oysters produced a large and rapid elevation in CgIL-17 transcript abundance in hemocytes, suggesting that this is a very early response gene to pathogens that may be responsible for the stimulation of other immune genes in the oyster.

RNA-Seq Reveals an Integrated Immune Response in Nucleated Erythrocytes

Background Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. Methodology/Principal Findings Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I∶C), polyinosinic∶polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. Conclusions/Significance We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.

Predominant intragenic methylation is associated with gene expression characteristics in a bivalve mollusc

Characterization of DNA methylation patterns in the Pacific oyster, Crassostrea gigas, indicates that this epigenetic mechanism plays an important functional role in gene regulation and may be involved in the regulation of developmental processes and environmental responses. However, previous studies have been limited to in silico analyses or characterization of DNA methylation at the single gene level. Here, we have employed a genome-wide approach to gain insight into how DNA methylation supports the regulation of the genome in C. gigas. Using a combination of methylation enrichment and high-throughput bisulfite sequencing, we have been able to map methylation at over 2.5 million individual CpG loci. This is the first high-resolution methylome generated for a molluscan species. Results indicate that methylation varies spatially across the genome with a majority of the methylated sites mapping to intra genic regions. The bisulfite sequencing data was combined with RNA-seq data to examine genome-wide relationships between gene body methylation and gene expression, where it was shown that methylated genes are associated with high transcript abundance and low variation in expression between tissue types. The combined data suggest DNA methylation plays a complex role in regulating genome activity in bivalves.

Is There a Relationship between DNA Methylation and Phenotypic Plasticity in Invertebrates

There is a significant amount of variation in DNA methylation characteristics across organisms. Likewise, the biological role of DNA methylation varies across taxonomic lineages. The complexity of DNA methylation patterns in invertebrates has only recently begun to be characterized in-depth. In some invertebrate species that have been examined to date, methylated DNA is found primarily within coding regions and patterning is closely associated with gene function. Here we provide a perspective on the potential role of DNA methylation in these invertebrates with a focus on how limited methylation may contribute to increased phenotypic plasticity in highly fluctuating environments. Specifically, limited methylation could facilitate a variety of transcriptional opportunities including access to alternative transcription start sites, increasing sequence mutations, exon skipping, and transient methylation.

Transcriptome sequencing and high‐resolution melt analysis advance single nucleotide polymorphism discovery in duplicated salmonids

Until recently, single nucleotide polymorphism (SNP) discovery in nonmodel organisms faced many challenges, often depending upon a targeted‐gene approach and Sanger sequencing of many individuals. The advent of next‐generation sequencing technologies has dramatically improved discovery, but validating and testing SNPs for use in population studies remain labour intensive. Here, we detail a SNP discovery and validation pipeline that incorporates 454 pyrosequencing, high‐resolution melt analysis (HRMA) and 5′ nuclease genotyping. We generated 4.59 × 108 bp of redundant sequence from transcriptomes of two individual chum salmon, a highly valued species across the Pacific Rim. Nearly 26 000 putative SNPs were identified—some as heterozygotes and some as homozygous for different nucleotides in the two individuals. For validation, we selected 202 templates containing single putative SNPs and conducted HRMA on 10 individuals from each of 19 populations from across the species range. Finally, 5′ nuclease genotyping validated 37 SNPs that conformed to Hardy–Weinberg equilibrium expectations. Putative SNPs expressed as heterozygotes in an ascertainment individual had more than twice the validation rate of those homozygous for different alleles in the two fish, suggesting that many of the latter may have been paralogous sequence variants. Overall, this validation rate of 37/202 suggests that we have found more than 4500 templates containing SNPs for use in this population set. We anticipate using this pipeline to significantly expand the number of SNPs available for the studies of population structure and mixture analyses as well as for the studies of adaptive genetic variation in nonmodel organisms.

Cloning and characterization of prostaglandin endoperoxide synthase-1 and -2 from the brook trout ovary.

Using a combination of reverse transcription-PCR and library screening, the cDNAs for prostaglandin endoperoxide synthase-1 (PGS-1) and 2 (PGS-2) were isolated from the brook trout ovary. The brook trout PGS-1 cDNA encodes for a 598 amino acid protein that is 69% identical to mammalian PGS-1. PGS-1 transcripts were observed in the ovary, spleen, gills, head kidney, trunk kidney, intestine, stomach, skin and heart. To our knowledge, this is the first characterization of a non-mammalian PGS-1 cDNA. The brook trout PGS-2 encodes for a 607 amino acid protein that is 69% identical to mammalian PGS-2 and was observed in the skin, gills, stomach and heart. PGS-2 transcripts were highly upregulated in the ovaries by the phorbol ester, phorbol-12-myristate-13-acetate, in combination with the calcium ionophore, A23187. However, PGS-2 was not observed in the ovary of brook trout undergoing natural oocyte maturation and ovulation.