ESTs and EST-linked polymorphisms for genetic mapping and phylogenetic reconstruction in the guppy, Poecilia reticulata.
ABSTRACT: BACKGROUND:The guppy, Poecilia reticulata, is a well-known model organism for studying inheritance and variation of male ornamental traits as well as adaptation to different river habitats. However, genomic resources for studying this important model were not previously widely available. RESULTS:With the aim of generating molecular markers for genetic mapping of the guppy, cDNA libraries were constructed from embryos and different adult organs to generate expressed sequence tags (ESTs). About 18,000 ESTs were annotated according to BLASTN and BLASTX results and the sequence information from the 3' UTRs was exploited to generate PCR primers for re-sequencing of genomic DNA from different wild type strains. By comparison of EST-linked genomic sequences from at least four different ecotypes, about 1,700 polymorphisms were identified, representing about 400 distinct genes. Two interconnected MySQL databases were built to organize the ESTs and markers, respectively. A robust phylogeny of the guppy was reconstructed, based on 10 different nuclear genes. CONCLUSION:Our EST and marker databases provide useful tools for genetic mapping and phylogenetic studies of the guppy.
Project description: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 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:Guppies (Poecilia reticulata) 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:Many fishes are able to jump out of the water and launch themselves into the air. Such behavior has been connected with prey capture, migration and predator avoidance. We found that jumping behavior of the guppy Poecilia reticulata is not associated with any of the above. The fish jump spontaneously, without being triggered by overt sensory cues, is not migratory and does not attempt to capture aerial food items. Here, we use high speed video imaging to analyze the kinematics of the jumping behavior P. reticulata. Fish jump from a still position by slowly backing up while using its pectoral fins, followed by strong body trusts which lead to launching into the air several body lengths. The liftoff phase of the jump is fast and fish will continue with whole body thrusts and tail beats, even when out of the water. This behavior occurs when fish are in a group or in isolation. Geography has had substantial effects on guppy evolution, with waterfalls reducing gene flow and constraining dispersal. We suggest that jumping has evolved in guppies as a behavioral phenotype for dispersal.
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:The diversity of visual systems in fish has long been of interest for evolutionary biologists and neurophysiologists, and has recently begun to attract the attention of molecular evolutionary geneticists. Several recent studies on the copy number and genomic organization of visual pigment proteins, the opsins, have revealed an increased opsin diversity in fish relative to most vertebrates, brought about through recent instances of opsin duplication and divergence. However, for the subfamily of opsin genes that mediate vision at the long-wavelength end of the spectrum, the LWS opsins, it appears that most fishes possess only one or two loci, a value comparable to most other vertebrates. Here, we characterize the LWS opsins from cDNA of an individual guppy, Poecilia reticulata, a fish that is known exhibit variation in its long-wavelength sensitive visual system, mate preferences and colour patterns. RESULTS:We identified six LWS opsins expressed within a single individual. Phylogenetic analysis revealed that these opsins descend from duplication events both pre-dating and following the divergence of the guppy lineage from that of the bluefin killifish, Lucania goodei, the closest species for which comparable data exists. Numerous amino acid substitutions exist among these different LWS opsins, many at sites known to be important for visual pigment function, including spectral sensitivity and G-protein activation. Likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy LWS opsin lineages. CONCLUSION:The guppy displays an unusually high number of LWS opsins compared to other fish, and to vertebrates in general. Observing both substitutions at functionally important sites and the persistence of lineages across species boundaries suggests that these opsins might have functionally different roles, especially with regard to G-protein activation. The reasons why are currently unknown, but may relate to aspects of the guppy's behavioural ecology, in which both male colour patterns and the female mate preferences for these colour patterns experience strong, highly variable selection pressures.
Project description:Poecilia reticulata is one of the most popular ornamental fish species with a higher demand for males due to their large colorful fins. The objectives of this study were mapping of the sex-determining (SD) region on linkage group 12 of guppy, and identification of a sex specific marker. We generated eight polymorphic microsatellite markers distributed along the distal 5.4 Mbp sequence of the previously identified SD region on linkage group (LG) 12. The markers were tested for association with sex in 156 individuals of the Red Blonde and Flame strains, and 126 progeny of four full-sibs Red Blonde families. A male-specific allele was found for microsatellite gu1066 at position of 25.3 Mbp on LG12 for both strains, and gu832 at position 24.4 Mbp for the Flame strain. Thus, a region of 0.9 Mbp between these markers, harboring 27 annotated genes, was selected for analysis. Based on association of copy number variation and sex determination we mapped a duplicated region between LGs 9 and 12, of 1.26 Mbp, containing 59 genes on LG12. The common interval between the segment bounded by gu1066 and gu832, and the duplicated region of 0.43 Mbp on LG12 harbors 11 genes of which 6 have multiple copies (54%). Growth arrest and DNA damage inducible gamma-like (GADD45G-like) is a plausible positional and functional candidate gene for its role in male fertility. We characterized the genomic structure of the gene in guppy, and found two isoforms; but no sex-biased differences were evident in genomic sequence and gene expression.
Project description:The guppy (Poecilia reticulata) is a model species in ecology and evolution. Many studies have examined effects of predators on guppy behaviour, reproduction, survival strategies, feeding and other life-history traits, but few have studied variation in their parasite diversity. We surveyed parasites of 18 Trinidadian populations of guppy, to provide insight on the geographical mosaic of parasite variability, which may act as a source of natural selection acting on guppies. We found 21 parasite species, including five new records for Trinidad. Spatial variation in parasite diversity was significantly higher than that of piscine predators, and significant variation in parasite richness among individuals and populations was correlated with: (i) host size, (ii) snail species richness, and (iii) the distance between populations. Differences in parasite species richness are likely to play an important, yet underestimated role in the biology of this model species of vertebrate ecology and evolution.
Project description:Guppy (Poecilia reticulata) is an ideal model for studying environmental estrogens, and its large caudal fin has a high capacity to regenerate. This study analyzed the feasibility of caudal fin for detecting vitellogenin (Vtg), the most commonly used biomarker of environmental estrogens. Firstly, a sandwich ELISA for guppy Vtg was developed using purified lipovitellin and its antibody and it had a working range of 7.8-1000?ng/mL and detection limit of 3.1?ng/mL. The ELISA was used to detect tissue distribution of Vtg. In male guppy exposed to 50 and 100?ng/L 17?-estradiol (E2), Vtg concentration in caudal fin was higher than that in whole fish, brain, eyes, gonad, and skin, and was close to that in the liver. Furthermore, male guppies were exposed to environmental concentrations of 17a-ethinylestradiol (EE2) and bisphenol S (BPS) to validate the utility of caudal fin Vtg for detecting estrogenic activities. The lowest observed effect concentration of EE2 and BPS were lower than 2?ng/L and 1??g/L, which were below or equal to the values reported for other species, demonstrating that caudal fin Vtg was highly sensitive to estrogenic chemicals. Therefore, caudal fins of guppies are suggested as alternative samples for Vtg biomarker detection.