Sequencing and characterization of the guppy (Poecilia reticulata) transcriptome.
ABSTRACT: BACKGROUND:Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata) transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history. RESULTS:We generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs), and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in a congeneric species, the sailfin molly (Poecilia latipinna). Over 40% of reads from the sailfin molly sample aligned to the guppy transcriptome. CONCLUSIONS:We show that next-generation sequencing provided a reliable and broad reference transcriptome. This resource allowed us to identify candidate gene variants, SNPs in coding regions, and sex-specific gene expression, and permitted quantitative analysis of differential gene expression.
Project description:BACKGROUND:The Amazon molly, Poecilia formosa (Teleostei: Poeciliinae) is an unisexual, all-female species. It evolved through the hybridisation of two closely related sexual species and exhibits clonal reproduction by sperm dependent parthenogenesis (or gynogenesis) where the sperm of a parental species is only used to activate embryogenesis of the apomictic, diploid eggs but does not contribute genetic material to the offspring.Here we provide and describe the first de novo assembled transcriptome of the Amazon molly in comparison with its maternal ancestor, the Atlantic molly Poecilia mexicana. The transcriptome data were produced through sequencing of single end libraries (100 bp) with the Illumina sequencing technique. RESULTS:83,504,382 reads for the Amazon molly and 81,625,840 for the Atlantic molly were assembled into 127,283 and 78,961 contigs for the Amazon molly and the Atlantic molly, respectively. 63% resp. 57% of the contigs could be annotated with gene ontology terms after sequence similarity comparisons. Furthermore, we were able to identify genes normally involved in reproduction and especially in meiosis also in the transcriptome dataset of the apomictic reproducing Amazon molly. CONCLUSIONS:We assembled and annotated the transcriptome of a non-model organism, the Amazon molly, without a reference genome (de novo). The obtained dataset is a fundamental resource for future research in functional and expression analysis. Also, the presence of 30 meiosis-specific genes within a species where no meiosis is known to take place is remarkable and raises new questions for future research.
Project description:The unisexual Amazon molly (Poecilia formosa) originated from a hybridization between two sexual species, the sailfin molly (Poecilia latipinna) and the Atlantic molly (Poecilia mexicana). The Amazon molly reproduces clonally via sperm-dependent parthenogenesis (gynogenesis), in which the sperm of closely related species triggers embryogenesis of the apomictic oocytes, but typically does not contribute genetic material to the next generation. We compare for the first time the gonadal transcriptome of the Amazon molly to those of both ancestral species, P. mexicana and P. latipinna.We sequenced the gonadal transcriptomes of the P. formosa and its parental species P. mexicana and P. latipinna using Illumina RNA-sequencing techniques (paired-end, 100 bp). De novo assembly of about 50 million raw read pairs for each species was performed using Trinity, yielding 106,922 transcripts for P. formosa, 115,175 for P. latipinna, and 133,025 for P. mexicana after eliminating contaminations. On the basis of sequence similarity comparisons to other teleost species and the UniProt databases, functional annotation, and differential expression analysis, we demonstrate the similarity of the transcriptomes among the three species. More than 40% of the transcripts for each species were functionally annotated and about 70% were assigned to orthologous genes of a closely related species. Differential expression analysis between the sexual and unisexual species uncovered 2035 up-regulated and 564 down-regulated genes in P. formosa. This was exemplary validated for six genes by qRT-PCR.We identified more than 130 genes related to meiosis and reproduction within the apomictically reproducing P. formosa. Overall expression of these genes seems to be down-regulated in the P. formosa transcriptome compared to both ancestral species (i.e., 106 genes down-regulated, 29 up-regulated). A further 35 meiosis and reproduction related genes were not found in the P. formosa transcriptome, but were only expressed in the sexual species. Our data support the hypothesis of general down-regulation of meiosis-related genes in the apomictic Amazon molly. Furthermore, the obtained dataset and identified gene catalog will serve as a resource for future research on the molecular mechanisms behind the reproductive mode of this unisexual species.
Project description:BACKGROUND:Sexually dimorphic phenotypes are generally associated with differential gene expression between the sexes. The study of molecular evolution and genomic location of these differentially expressed, or sex-biased, genes is important for understanding inter-sexual divergence under sex-specific selection pressures. Teleost fish provide a unique opportunity to examine this divergence in the presence of variable sex-determination mechanisms of recent origin. The guppy, Poecilia reticulata, displays sexual dimorphism in size, ornaments, and behavior, traits shaped by natural and sexual selection in the wild. RESULTS:To gain insight into molecular mechanisms underlying the guppy's sexual dimorphism, we assembled a reference transcriptome combining genome-independent as well as genome-guided assemblies and analyzed sex-biased gene expression between different tissues of adult male and female guppies. We found tissue-associated sex-biased expression of genes related to pigmentation, signal transduction, and spermatogenesis in males; and growth, cell-division, extra-cellular matrix organization, nutrient transport, and folliculogenesis in females. While most sex-biased genes were randomly distributed across linkage groups, we observed accumulation of ovary-biased genes on the sex linkage group, LG12. Both testis-biased and ovary-biased genes showed a significantly higher rate of non-synonymous to synonymous substitutions (dN/dS) compared to unbiased genes. However, in somatic tissues only female-biased genes, including those co-expressed in multiple tissues, showed elevated ratios of non-synonymous substitutions. CONCLUSIONS:Our work identifies a set of annotated gene products that are candidate factors affecting sexual dimorphism in guppies. The differential genomic distribution of gonad-biased genes provides evidence for sex-specific selection pressures acting on the nascent sex chromosomes of the guppy. The elevated rates of evolution of testis-biased and female-biased genes indicate differing evolution under distinct selection pressures on the reproductive versus non-reproductive tissues.
Project description:BACKGROUND:Comparisons of functionally important changes at the molecular level in model systems have identified key adaptations driving isolation and speciation. In cichlids, for example, long wavelength-sensitive (LWS) opsins appear to play a role in mate choice and male color variation within and among species. To test the hypothesis that the evolution of elaborate coloration in male guppies (Poecilia reticulata) is also associated with opsin gene diversity, we sequenced long wavelength-sensitive (LWS) opsin genes in six species of the family Poeciliidae. RESULTS:Sequences of four LWS opsin genes were amplified from the guppy genome and from mRNA isolated from adult guppy eyes. Variation in expression was quantified using qPCR. Three of the four genes encode opsins predicted to be most sensitive to different wavelengths of light because they vary at key amino acid positions. This family of LWS opsin genes was produced by a diversity of duplication events. One, an intronless gene, was produced prior to the divergence of families Fundulidae and Poeciliidae. Between-gene PCR and DNA sequencing show that two of the guppy LWS opsins are linked in an inverted orientation. This inverted tandem duplication event occurred near the base of the poeciliid tree in the common ancestor of Poecilia and Xiphophorus. The fourth sequence has been uncovered only in the genus Poecilia. In the guppies surveyed here, this sequence is a hybrid, with the 5' end most similar to one of the tandem duplicates and the 3' end identical to the other. CONCLUSION:Enhanced wavelength discrimination, a possible consequence of opsin gene duplication and divergence, might have been an evolutionary prerequisite for color-based sexual selection and have led to the extraordinary coloration now observed in male guppies and in many other poeciliids.
Project description:BACKGROUND:The guppy (Poecilia reticulata) is an important model organism for studying sexual selection; male guppies have complex and conspicuous pigmentation, and female guppies exhibit preferences for males with specific color spots. Understanding the genetic basis underlying pigmentation variation in the guppy is important for exploring the factors causing the maintenance of color polymorphism in wild populations. FINDINGS:We focused on the melanic black pigmentation of guppies, and examined genetic variations in the melanocortin 1 receptor (MC1R) gene because variation in this gene is known to contribute to polymorphism of melanin pigmentation in several animal species. The complete coding sequence of the guppy MC1R gene was determined, and two different MC1R alleles (963 and 969 bp) were found in wild populations. Ornamental strain guppies with a 963-bp MC1R tended to show less black pigmentation than those with a 969-bp MC1R, although the association between MC1R genotype and black pigmentation disappeared in the F2 offspring. CONCLUSIONS:The guppy MC1R gene showed variation in the five wild Trinidadian populations we examined, and these populations also differed in terms of allele frequencies. We identified a significant association between black pigmentation and MC1R genotype in fish obtained from aquarium shops. However, the results from F2 families suggest that there are other genes that modify the effects of the MC1R gene.
Project description:Theory predicts that the sexes can achieve greater fitness if loci with sexually antagonistic polymorphisms become linked to the sex determining loci, and this can favor the spread of reduced recombination around sex determining regions. Given that sex-linked regions are frequently repetitive and highly heterozygous, few complete Y chromosome assemblies are available to test these ideas. The guppy system (Poecilia reticulata) has long been invoked as an example of sex chromosome formation resulting from sexual conflict. Early genetics studies revealed that male color patterning genes are mostly but not entirely Y-linked, and that X-linkage may be most common in low-predation populations. More recent population genomic studies of guppies have reached varying conclusions about the size and placement of the Y-linked region. However, this previous work used a reference genome assembled from short-read sequences from a female guppy. Here, we present a new guppy reference genome assembly from a male, using long-read PacBio single-molecule real-time sequencing and chromosome contact information. Our new assembly sequences across repeat- and GC-rich regions and thus closes gaps and corrects mis-assemblies found in the short-read female-derived guppy genome. Using this improved reference genome, we then employed broad population sampling to detect sex differences across the genome. We identified two small regions that showed consistent male-specific signals. Moreover, our results help reconcile the contradictory conclusions put forth by past population genomic studies of the guppy sex chromosome. Our results are consistent with a small Y-specific region and rare recombination in male guppies.
Project description:The all-female Amazon molly (Poecilia formosa) originated from a single hybridization of two bisexual ancestors, Atlantic molly (Poecilia mexicana) and sailfin molly (Poecilia latipinna). As a gynogenetic species, the Amazon molly needs to copulate with a heterospecific male, but the genetic information of the sperm-donor does not contribute to the next generation, as the sperm only acts as the trigger for the diploid eggs' embryogenesis. Here, we study the sequence evolution and gene expression of the duplicated genes coding for androgen receptors (ars) and other pathway-related genes, i.e., the estrogen receptors (ers) and cytochrome P450, family19, subfamily A, aromatase genes (cyp19as), in the Amazon molly, in comparison to its bisexual ancestors. Mollies possess-as most other teleost fish-two copies of the ar, er, and cyp19a genes, i.e., ar?/ar?, er?/er?1, and cyp19a1 (also referred as cyp19a1a)/cyp19a2 (also referred to as cyp19a1b), respectively. Non-synonymous single nucleotide polymorphisms (SNPs) among the ancestral bisexual species were generally predicted not to alter protein function. Some derived substitutions in the P. mexicana and one in P. formosa are predicted to impact protein function. We also describe the gene expression pattern of the ars and pathway-related genes in various tissues (i.e., brain, gill, and ovary) and provide SNP markers for allele specific expression research. As a general tendency, the levels of gene expression were lowest in gill and highest in ovarian tissues, while expression levels in the brain were intermediate in most cases. Expression levels in P. formosa were conserved where expression did not differ between the two bisexual ancestors. In those cases where gene expression levels significantly differed between the bisexual species, P. formosa expression was always comparable to the higher expression level among the two ancestors. Interestingly, er?1 was expressed neither in brain nor in gill in the analyzed three molly species, which implies a more important role of er? in the estradiol synthesis pathway in these tissues. Furthermore, our data suggest that interactions of steroid-signaling pathway genes differ across tissues, in particular the interactions of ars and cyp19as.
Project description:BACKGROUND:Elucidating the genomic basis of adaptation and speciation is a major challenge in natural systems with large quantities of environmental and phenotypic data, mostly because of the scarcity of genomic resources for non-model organisms. The Atlantic molly (Poecilia mexicana, Poeciliidae) is a small livebearing fish that has been extensively studied for evolutionary ecology research, particularly because this species has repeatedly colonized extreme environments in the form of caves and toxic hydrogen sulfide containing springs. In such extreme environments, populations show strong patterns of adaptive trait divergence and the emergence of reproductive isolation. Here, we used RNA-sequencing to assemble and annotate the first transcriptome of P. mexicana to facilitate ecological genomics studies in the future and aid the identification of genes underlying adaptation and speciation in the system. DESCRIPTION:We provide the first annotated reference transcriptome of P. mexicana. Our transcriptome shows high congruence with other published fish transcriptomes, including that of the guppy, medaka, zebrafish, and stickleback. Transcriptome annotation uncovered the presence of candidate genes relevant in the study of adaptation to extreme environments. We describe general and oxidative stress response genes as well as genes involved in pathways induced by hypoxia or involved in sulfide metabolism. To facilitate future comparative analyses, we also conducted quantitative comparisons between P. mexicana from different river drainages. 106,524 single nucleotide polymorphisms were detected in our dataset, including potential markers that are putatively fixed across drainages. Furthermore, specimens from different drainages exhibited some consistent differences in gene regulation. CONCLUSIONS:Our study provides a valuable genomic resource to study the molecular underpinnings of adaptation to extreme environments in replicated sulfide spring and cave environments. In addition, this study adds to the increasing number of genomic resources in the family Poeciliidae, which are widely used in comparative analyses of behavior, ecology, evolution, and medical genetics.
Project description:Guppies (<i>Poecilia reticulata</i>) are frequently introduced to both natural and artificial water bodies as a mosquito control. Laboratory studies have demonstrated that guppies can consume large numbers of larval mosquitoes. Our study investigates how intraspecific variability in guppy phenotype affects their importance as a mosquito biocontrol and how habitat conditions (natural ponds vs. water storage containers) may influence insect biomass and guppy feeding. Using a blocked experimental design, we established stream-side mesocosm ponds with half receiving gravel substrate to simulate pond-bottom habitat. To provide realistic diet choices and insect abundances, we allowed the mesocosms to colonize naturally with aquatic insect larvae for 1 month before introducing guppies. We tested two distinct guppy phenotypes (from high- and low-predation streams) alongside fish-free controls. After 1 month, we measured insect biomass in the mesocosms and examined guppy gut contents to document direct predation. While overall insect biomass was not significantly different across the three fish treatments, we observed a significant reduction in mosquito biomass in fish treatments compared to fish-free controls, as well as intraspecific differences in feeding. Overall insect biomass was significantly higher in mesocosms without gravel, while habitat condition had no effect on mosquito biomass. As guppy phenotype responds to changes in their environments, it is an important consideration for biocontrol policy to anticipate potential ecosystem effects. We close by relating our findings to other studies and by discussing the implications and potential risks of using guppies to control mosquitoes.
Project description:Dwarfism is a condition defined by low harvest weight in fish, but also results in strange body figures which may have potential for the selective breeding of new ornamental fish strains. The objectives of this study are to reveal the physiological causes of dwarfism and identify the genetic loci controlling this trait in the white sailfin molly. Skeletons of dwarf and normal sailfin mollies were observed by X-ray radioscopy and skeletal staining. Genome-wide association studies based on genotyping-by-sequencing (n = 184) were used to map candidate genomic regions associated with the dwarfism trait. Quantitative real-time PCR was performed to determine the expression level of candidate genes in normal (n = 8) and dwarf (n = 8) sailfin mollies. We found that the dwarf sailfin molly has a short and dysplastic spine in comparison to the normal fish. Two regions, located at NW_015112742.1 and NW_015113621.1, were significantly associated with the dwarfism trait. The expression level of three candidate genes, ADAMTS like 1, Larp7 and PPP3CA, were significantly different between the dwarf and normal sailfin mollies in the hepatopancreas, with PPP3CA also showing significant differences in the vertebrae and Larp7 showing significant differences in the muscle. This study identified genomic regions and candidate genes associated with the dwarfism trait in the white sailfin molly and would provide a reference to determine dwarf-causing variations.