Improved Reference Genome Uncovers Novel Sex-Linked Regions in the Guppy (Poecilia reticulata).

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.

The Genome of the Trinidadian Guppy, Poecilia reticulata, and Variation in the Guanapo Population.

For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-predation site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individuals. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adaptation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish.

Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

Improved white spruce (Picea glauca) genome assemblies and annotation of large gene families of conifer terpenoid and phenolic defense metabolism.

White spruce (Picea glauca), a gymnosperm tree, has been established as one of the models for conifer genomics. We describe the draft genome assemblies of two white spruce genotypes, PG29 and WS77111, innovative tools for the assembly of very large genomes, and the conifer genomics resources developed in this process. The two white spruce genotypes originate from distant geographic regions of western (PG29) and eastern (WS77111) North America, and represent elite trees in two Canadian tree-breeding programs. We present an update (V3 and V4) for a previously reported PG29 V2 draft genome assembly and introduce a second white spruce genome assembly for genotype WS77111. Assemblies of the PG29 and WS77111 genomes confirm the reconstructed white spruce genome size in the 20 Gbp range, and show broad synteny. Using the PG29 V3 assembly and additional white spruce genomics and transcriptomics resources, we performed MAKER-P annotation and meticulous expert annotation of very large gene families of conifer defense metabolism, the terpene synthases and cytochrome P450s. We also comprehensively annotated the white spruce mevalonate, methylerythritol phosphate and phenylpropanoid pathways. These analyses highlighted the large extent of gene and pseudogene duplications in a conifer genome, in particular for genes of secondary (i.e. specialized) metabolism, and the potential for gain and loss of function for defense and adaptation.

Reasons for the invasive success of a guppy (Poecilia reticulata) population in Trinidad.

The introduction of non-native species into new habitats poses a major threat to native populations. Of particular interest, though often overlooked, are introductions of populations that are not fully reproductively isolated from native individuals and can hybridize with them. To address this important topic we used different approaches in a multi-pronged study, combining the effects of mate choice, shoaling behaviour and genetics. Here we present evidence that behavioural traits such as shoaling and mate choice can promote population mixing if individuals do not distinguish between native and foreign conspecifics. We examined this in the context of two guppy (Poecilia reticulata) populations that have been subject to an introduction and subsequent population mixing event in Trinidad. The introduction of Guanapo River guppies into the Turure River more than 50 years ago led to a marked reduction of the original genotype. In our experiments, female guppies did not distinguish between shoaling partners when given the choice between native and foreign individuals. Introduced fish are therefore likely to benefit from the protection of a shoal and will improve their survival chances as a result. The additional finding that male guppies do not discriminate between females on the basis of origin will further increase the process of population mixing, especially if males encounter mixed shoals. In a mesocosm experiment, in which the native and foreign populations were allowed to mate freely, we found, as expected on the basis of these behavioural interactions, that the distribution of offspring genotypes could be predicted from the proportions of the two types of founding fish. This result suggests that stochastic and environmental processes have reinforced the biological ones to bring about the genetic dominance of the invading population in the Turure River. Re-sampling the Turure for genetic analysis using SNP markers confirmed the population mixing process and showed that it is an on-going process in this river and has led to the nearly complete disappearance of the original genotype.

Paired-end RAD-seq for de novo assembly and marker design without available reference.

MOTIVATION: Next-generation sequencing technologies have facilitated the study of organisms on a genome-wide scale. A recent method called restriction site associated DNA sequencing (RAD-seq) allows to sample sequence information at reduced complexity across a target genome using the Illumina platform. Single-end RAD-seq has proven to provide a large number of informative genetic markers in reference as well as non-reference organisms. RESULTS: Here, we present a method for de novo assembly of paired-end RAD-seq data in order to produce extended contigs flanking a restriction site. We were able to reconstruct one-tenth of the guppy genome represented by 200-500 bp contigs associated to EcoRI recognition sites. In addition, these contigs were used as reference allowing the detection of thousands of new polymorphic markers that are informative for mapping and population genetic studies in the guppy. AVAILABILITY: A perl and C++ implementation of the method demonstrated in this article is available under http://guppy.weigelworld.org/weigeldatabases/radMarkers/ as package RApiD. CONTACT: christine.dreyer@tuebingen.mpg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Gene duplication and divergence of long wavelength-sensitive opsin genes in the guppy, Poecilia reticulata.

Female preference for male orange coloration in the genus Poecilia suggests a role for duplicated long wavelength-sensitive (LWS) opsin genes in facilitating behaviors related to mate choice in these species. Previous work has shown that LWS gene duplication in this genus has resulted in expansion of long wavelength visual capacity as determined by microspectrophotometry (MSP). However, the relationship between LWS genomic repertoires and expression of LWS retinal cone classes within a given species is unclear. Our previous study in the related species, Xiphophorus helleri, was the first characterization of the complete LWS opsin genomic repertoire in conjunction with MSP expression data in the family Poeciliidae, and revealed the presence of four LWS loci and two distinct LWS cone classes. In this study we characterized the genomic organization of LWS opsin genes by BAC clone sequencing, and described the full range of cone cell types in the retina of the colorful Cumaná guppy, Poecilia reticulata. In contrast to X. helleri, MSP data from the Cumaná guppy revealed three LWS cone classes. Comparisons of LWS genomic organization described here for Cumaná to that of X. helleri indicate that gene divergence and not duplication was responsible for the evolution of a novel LWS haplotype in the Cumaná guppy. This lineage-specific divergence is likely responsible for a third additional retinal cone class not present in X. helleri, and may have facilitated the strong sexual selection driven by female preference for orange color patterns associated with the genus Poecilia.

Genome-wide single nucleotide polymorphisms reveal population history and adaptive divergence in wild guppies.

Adaptation of guppies (Poecilia reticulata) to contrasting upland and lowland habitats has been extensively studied with respect to behaviour, morphology and life history traits. Yet population history has not been studied at the whole-genome level. Although single nucleotide polymorphisms (SNPs) are the most abundant form of variation in many genomes and consequently very informative for a genome-wide picture of standing natural variation in populations, genome-wide SNP data are rarely available for wild vertebrates. Here we use genetically mapped SNP markers to comprehensively survey genetic variation within and among naturally occurring guppy populations from a wide geographic range in Trinidad and Venezuela. Results from three different clustering methods, Neighbor-net, principal component analysis (PCA) and Bayesian analysis show that the population substructure agrees with geographic separation and largely with previously hypothesized patterns of historical colonization. Within major drainages (Caroni, Oropouche and Northern), populations are genetically similar, but those in different geographic regions are highly divergent from one another, with some indications of ancient shared polymorphisms. Clear genomic signatures of a previous introduction experiment were seen, and we detected additional potential admixture events. Headwater populations were significantly less heterozygous than downstream populations. Pairwise F(ST) values revealed marked differences in allele frequencies among populations from different regions, and also among populations within the same region. F(ST) outlier methods indicated some regions of the genome as being under directional selection. Overall, this study demonstrates the power of a genome-wide SNP data set to inform for studies on natural variation, adaptation and evolution of wild populations.

Genetic linkage map of the guppy, Poecilia reticulata, and quantitative trait loci analysis of male size and colour variation.

We report construction of a genetic linkage map of the guppy genome using 790 single nucleotide polymorphism markers, integrated from six mapping crosses. The markers define 23 linkage groups (LGs), corresponding to the known haploid number of guppy chromosomes. The map, which spans a genetic length of 899 cM, includes 276 markers linked to expressed genes (expressed sequence tag), which have been used to derive broad syntenic relationships of guppy LGs with medaka chromosomes. This combined linkage map should facilitate the advancement of genetic studies for a wide variety of complex adaptive phenotypes relevant to natural and sexual selection in this species. We have used the linkage data to predict quantitative trait loci for a set of variable male traits including size and colour pattern. Contributing loci map to the sex LG for many of these traits.

Linkage analysis reveals the independent origin of Poeciliid sex chromosomes and a case of atypical sex inheritance in the guppy (Poecilia reticulata).

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.

Natural variation of male ornamental traits of the guppy, Poecilia reticulata.

Male ornamental traits of the guppy, Poecilia reticulata, provide an outstanding example of natural variation in sex-linked male-advantageous traits that are shaped by both sexual and environmental selection. A substantial fraction of the underlying genes is known to be genetically linked to the sex-determining region on the differentiating Y-chromosome. Intercrosses between parental populations originating from geographically distant locations in East Trinidad and Cumaná (Venezuela) were used to study segregation of ornamental traits in male progeny. In addition, we performed backcrosses to compare segregation of ornaments in presence or absence of prominent traits linked to the Y-chromosome. Another backcross strategy involving XY females from the laboratory strain zebrinus maculatus allowed studying additive and dominant effects of alleles on two different Y-chromosomes on pattern formation. For genetic mapping, we have previously developed nuclear SNP markers linked to expressed genes, including several genes known to be important for pattern formation in other species. Of these candidate genes 15 were placed on 11 different linkage groups. Our phenotypic and genotypic analysis of progeny from mapping crosses and backcrosses suggests several genetic mechanisms that enhance natural variation, namely, additive effects of codominant alleles, suppressive actions of dominant alleles, and a complex interplay between sex-linked and autosomal cofactors.

ESTs and EST-linked polymorphisms for genetic mapping and phylogenetic reconstruction in the guppy, Poecilia reticulata.

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.

Opsin gene duplication and diversification in the guppy, a model for sexual selection.

Identification of genes that control variation in adaptive characters is a prerequisite for understanding the processes that drive sexual and natural selection. Male coloration and female colour perception play important roles in mate choice in the guppy (Poecilia reticulata), a model organism for studies of natural and sexual selection. We examined a potential source for the known variation in colour perception, by analysing genomic and complementary DNA sequences of genes that code for visual pigment proteins. We find high sequence variability, both within and between populations, and expanded copy number for long-wave sensitive (LWS) opsin genes. Alleles with non-synonymous changes that suggest dissimilar spectral tuning properties occur in the same population and even in the same individual, and the high frequency of non-synonymous substitutions argues for diversifying selection acting on these proteins. Therefore, variability in tuning amino acids is partitioned within individuals and populations of the guppy, in contrast to variability for LWS at higher taxonomic levels in cichlids, a second model system for differentiation owing to sexual selection. Since opsin variability parallels the extreme male colour polymorphism within guppy populations, we suggest that mate choice has been a major factor driving the coevolution of opsins and male ornaments in this species.