Zebra Finch Genome Research Articles

Improved zebra finch brain transcriptome identifies novel proteins with sex differences

The zebra finch (Taeniopygia guttata), a representative oscine songbird species, has been widely studied to investigate behavioral neuroscience, most notably the neurobiological basis of vocal learning, a rare trait shared in only a few animal groups including humans. In 2019, an updated zebra finch genome annotation (bTaeGut1_v1.p) was released from the Ensembl database and is substantially more comprehensive than the first version published in 2010. In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the bird transcriptome. To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines. From our analysis, we added 220 novel genes and 8,134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation. We further validated 18 different novel proteins by using mass-spectrometry data generated from zebra finch caudal telencephalon tissue. Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.

Transcript- and annotation-guided genome assembly of the European starling.

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second, North American, short-read genome assembly (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterization. S. vulgaris vAU combined 10× genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 6222 scaffolds (7.6Mb scaffold N50, 94.6% busco completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Species-specific transcript mapping and gene annotation revealed good gene-level assembly and high functional completeness. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, saaga) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counterintuitive behaviour in traditional busco metrics, and present buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. This work expands our knowledge of avian genomes and the available toolkit for assessing and improving genome quality. The new genomic resources presented will facilitate further global genomic and transcriptomic analysis on this ecologically important species.

Identification of Centromere-Specific Repeats in the Zebra Finch Genome

Tandem repetitive sequences represent a significant part of many genomes but remain poorly characterized due to various methodological difficulties. Here, we describe the tandem repeat composition in the genome of zebra finch, Taeniopygia guttata, a species that has long served as an animal model, primarily in neurobiology and comparative genomics. Using available genome sequencing raw read datasets, we bioinformatically reconstructed consensus sequences of several tandem repeats and proved that the most abundant ones, Tgut191A and Tgut716A, are centromere-associated in chromosomes. Each centromeric region can have a different number of copies of each repeat, with Tgut716A enrichment in almost all microchromosomes and sex chromosomes. Sequences similar to Tgut191A and Tgut716A found in other Estrildidae and Viduidae species can be considered as candidate centromeric sequences, but this requires further cytogenetic verification.

Genome Stability Is in the Eye of the Beholder: CR1 Retrotransposon Activity Varies Significantly across Avian Diversity.

Since the sequencing of the zebra finch genome it has become clear that avian genomes, while largely stable in terms of chromosome number and gene synteny, are more dynamic at an intrachromosomal level. A multitude of intrachromosomal rearrangements and significant variation in transposable element (TE) content have been noted across the avian tree. TEs are a source of genome plasticity, because their high similarity enables chromosomal rearrangements through nonallelic homologous recombination, and they have potential for exaptation as regulatory and coding sequences. Previous studies have investigated the activity of the dominant TE in birds, chicken repeat 1 (CR1) retrotransposons, either focusing on their expansion within single orders, or comparing passerines with nonpasserines. Here, we comprehensively investigate and compare the activity of CR1 expansion across orders of birds, finding levels of CR1 activity vary significantly both between and within orders. We describe high levels of TE expansion in genera which have speciated in the last 10 Myr including kiwis, geese, and Amazon parrots; low levels of TE expansion in songbirds across their diversification, and near inactivity of TEs in the cassowary and emu for millions of years. CR1s have remained active over long periods of time across most orders of neognaths, with activity at any one time dominated by one or two families of CR1s. Our findings of higher TE activity in species-rich clades and dominant families of TEs within lineages mirror past findings in mammals and indicate that genome evolution in amniotes relies on universal TE-driven processes.

Remnant of Unrelated Amniote Sex Chromosomal Linkage Sharing on the Same Chromosome in House Gecko Lizards, Providing a Better Understanding of the Ancestral Super-Sex Chromosome.

Comparative chromosome maps investigating sex chromosomal linkage groups in amniotes and microsatellite repeat motifs of a male house gecko lizard (Hemidactylus frenatus, HFR) and a flat-tailed house gecko lizard (H. platyurus, HPL) of unknown sex were examined using 75 bacterial artificial chromosomes (BACs) from chicken and zebra finch genomes. No massive accumulations of microsatellite repeat motifs were found in either of the gecko lizards, but 10 out of 13 BACs mapped on HPL chromosomes were associated with other amniote sex chromosomes. Hybridization of the same BACs onto multiple different chromosome pairs suggested transitions to sex chromosomes across amniotes. No BAC hybridization signals were found on HFR chromosomes. However, HFR diverged from HPL about 30 million years ago, possibly due to intrachromosomal rearrangements occurring in the HFR lineage. By contrast, heterochromatin likely reshuffled patterns between HPL and HFR, as observed from C-positive heterochromatin distribution. Six out of ten BACs showed partial homology with squamate reptile chromosome 2 (SR2) and snake Z and/or W sex chromosomes. The gecko lizard showed shared unrelated sex chromosomal linkages—the remnants of a super-sex chromosome. A large ancestral super-sex chromosome showed a correlation between SR2 and snake W sex chromosomes.

The design and application of a 50 K SNP chip for a threatened Aotearoa New Zealand passerine, the hihi.

Next-generation sequencing has transformed the fields of ecological and evolutionary genetics by allowing for cost-effective identification of genome-wide variation. Single nucleotide polymorphism (SNP) arrays, or "SNP chips", enable very large numbers of individuals to be consistently genotyped at a selected set of these identified markers, and also offer the advantage of being able to analyse samples of variable DNA quality. We used reduced representation restriction-aided digest sequencing (RAD-seq) of 31 birds of the threatened hihi (Notiomystis cincta; stitchbird) and low-coverage whole genome sequencing (WGS) of 10 of these birds to develop an Affymetrix 50K SNP chip. We overcame the limitations of having no hihi reference genome and a low quantity of sequence data by separate and pooled de novo assembly of each of the 10WGS birds. Reads from all individuals were mapped back to these de novo assemblies to identify SNPs. A subset of RAD-seq and WGS SNPs were selected for inclusion on the chip, prioritising SNPs with the highest quality scores whose flanking sequence uniquely aligned to the zebra finch (Taeniopygia guttata) genome. Of the 58,466 SNPs manufactured on the chip, 72% passed filtering metrics and were polymorphic. By genotyping 1,536hihi on the array, we found that SNPs detected in multiple assemblies were more likely to successfully genotype, representing a cost-effective approach to identify SNPs for genotyping. Here, we demonstrate the utility of the SNP chip by describing the high rates of linkage disequilibrium in the hihi genome, reflecting the history of population bottlenecks in the species.

As above, so below: Whole transcriptome profiling demonstrates strong molecular similarities between avian dorsal and ventral pallial subdivisions.

Over the last two decades, beginning with the Avian Brain Nomenclature Forum in 2000, major revisions have been made to our understanding of the organization and nomenclature of the avian brain. However, there are still unresolved questions on avian pallial organization, particularly whether the cells above the vestigial ventricle represent distinct populations to those below it or similar populations. To test these two hypotheses, we profiled the transcriptomes of the major avian pallial subdivisions dorsal and ventral to the vestigial ventricle boundary using RNA sequencing and a new zebra finch genome assembly containing about 22,000 annotated, complete genes. We found that the transcriptomes of neural populations above and below the ventricle were remarkably similar. Each subdivision in dorsal pallium (Wulst) had a corresponding molecular counterpart in the ventral pallium (dorsal ventricular ridge). In turn, each corresponding subdivision exhibited shared gene co‐expression modules that contained gene sets enriched in functional specializations, such as anatomical structure development, synaptic transmission, signaling, and neurogenesis. These findings are more in line with the continuum hypothesis of avian brain subdivision organization above and below the vestigial ventricle space, with the pallium as a whole consisting of four major cell populations (intercalated pallium, mesopallium, hyper‐nidopallium, and arcopallium) instead of seven (hyperpallium apicale, interstitial hyperpallium apicale, intercalated hyperpallium, hyperpallium densocellare, mesopallium, nidopallium, and arcopallium). We suggest adopting a more streamlined hierarchical naming system that reflects the robust similarities in gene expression, neural connectivity motifs, and function. These findings have important implications for our understanding of overall vertebrate brain evolution.

Genomic evidence of neo-sex chromosomes in the eastern yellow robin

BACKGROUND: Understanding sex-biased natural selection can be enhanced by access to well-annotated chromosomes including ones inherited in sex-specific fashion. The eastern yellow robin (EYR) is an endemic Australian songbird inferred to have experienced climate-driven sex-biased selection and is a prominent model for studying mitochondrial-nuclear interactions in the wild. However, the lack of an EYR reference genome containing both sex chromosomes (in birds, a female bearing Z and W chromosomes) limits efforts to understand the mechanisms of these processes. Here, we assemble the genome for a female EYR and use low-depth (10×) genome resequencing data from 19 individuals of known sex to identify chromosome fragments with sex-specific inheritance. FINDINGS: MaSuRCA hybrid assembly using Nanopore and Illumina reads generated a 1.22-Gb EYR genome in 20,702 scaffolds (94.2% BUSCO completeness). Scaffolds were tested for W-linked (female-only) inheritance using a k-mer approach, and for Z-linked inheritance using median read-depth test in male and female reads (read-depths must indicate haploid female and diploid male representation). This resulted in 2,372 W-linked scaffolds (total length: 97,872,282 bp, N50: 81,931 bp) and 586 Z-linked scaffolds (total length: 121,817,358 bp, N50: 551,641 bp). Anchoring of the sex-linked EYR scaffolds to the reference genome of a female zebra finch revealed 2 categories of sex-linked genomic regions. First, 653 W-linked scaffolds (25.7 Mb) were anchored to the W sex chromosome and 215 Z-linked scaffolds (74.4 Mb) to the Z. Second, 1,138 W-linked scaffolds (70.9 Mb) and 179 Z-linked scaffolds (51.0 Mb) were anchored to a large section (coordinates ∼5 to ∼60 Mb) of zebra finch chromosome 1A. The first ∼5 Mb and last ∼14 Mb of the reference chromosome 1A had only autosomally behaving EYR scaffolds mapping to them. CONCLUSIONS: We report a female (W chromosome-containing) EYR genome and provide genomic evidence for a neo-sex (neo-W and neo-Z) chromosome system in the EYR, involving most of a large chromosome (1A) previously only reported to be autosomal in passerines.

Genomic evidence of neo-sex chromosomes in the eastern yellow robin.

BackgroundUnderstanding sex-biased natural selection can be enhanced by access to well-annotated chromosomes including ones inherited in sex-specific fashion. The eastern yellow robin (EYR) is an endemic Australian songbird inferred to have experienced climate-driven sex-biased selection and is a prominent model for studying mitochondrial-nuclear interactions in the wild. However, the lack of an EYR reference genome containing both sex chromosomes (in birds, a female bearing Z and W chromosomes) limits efforts to understand the mechanisms of these processes. Here, we assemble the genome for a female EYR and use low-depth (10×) genome resequencing data from 19 individuals of known sex to identify chromosome fragments with sex-specific inheritance.FindingsMaSuRCA hybrid assembly using Nanopore and Illumina reads generated a 1.22-Gb EYR genome in 20,702 scaffolds (94.2% BUSCO completeness). Scaffolds were tested for W-linked (female-only) inheritance using a k-mer approach, and for Z-linked inheritance using median read-depth test in male and female reads (read-depths must indicate haploid female and diploid male representation). This resulted in 2,372 W-linked scaffolds (total length: 97,872,282 bp, N50: 81,931 bp) and 586 Z-linked scaffolds (total length: 121,817,358 bp, N50: 551,641 bp). Anchoring of the sex-linked EYR scaffolds to the reference genome of a female zebra finch revealed 2 categories of sex-linked genomic regions. First, 653 W-linked scaffolds (25.7 Mb) were anchored to the W sex chromosome and 215 Z-linked scaffolds (74.4 Mb) to the Z. Second, 1,138 W-linked scaffolds (70.9 Mb) and 179 Z-linked scaffolds (51.0 Mb) were anchored to a large section (coordinates ∼5 to ∼60 Mb) of zebra finch chromosome 1A. The first ∼5 Mb and last ∼14 Mb of the reference chromosome 1A had only autosomally behaving EYR scaffolds mapping to them.ConclusionsWe report a female (W chromosome–containing) EYR genome and provide genomic evidence for a neo-sex (neo-W and neo-Z) chromosome system in the EYR, involving most of a large chromosome (1A) previously only reported to be autosomal in passerines.

Transcriptomic analysis of immune response to bacterial lipopolysaccharide in zebra finch (Taeniopygia guttata)

BackgroundDespite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (LPS) or vehicle to stimulate the innate immune system. 2 hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells.ResultsIn hypothalamus, there were 707 significantly upregulated transcripts, as well as 564 and 144 in the spleen and RBCs, respectively, relative to controls. Also, 155 transcripts in the hypothalamus, 606 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated.ConclusionsThese results suggest a potential trade-off in expression of genes that regulate immunity and metabolism in birds challenged with LPS. This finding is consistent with a hypothermic response to LPS treatment in small birds. Unlike mammals, birds have nucleated RBCs, and these results support a novel transcriptomic response of avian RBCs to immune challenge.

Exploring the molecular basis of neuronal excitability in a vocal learner

BackgroundVocal learning, the ability to learn to produce vocalizations through imitation, relies on specialized brain circuitry known in songbirds as the song system. While the connectivity and various physiological properties of this system have been characterized, the molecular genetic basis of neuronal excitability in song nuclei remains understudied. We have focused our efforts on examining voltage-gated ion channels to gain insight into electrophysiological and functional features of vocal nuclei. A previous investigation of potassium channel genes in zebra finches (Taeniopygia guttata) revealed evolutionary modifications unique to songbirds, as well as transcriptional specializations in the song system [Lovell PV, Carleton JB, Mello CV. BMC Genomics 14:470 2013]. Here, we expand this approach to sodium, calcium, and chloride channels along with their modulatory subunits using comparative genomics and gene expression analysis encompassing microarrays and in situ hybridization.ResultsWe found 23 sodium, 38 calcium, and 33 chloride channel genes (HGNC-based classification) in the zebra finch genome, several of which were previously unannotated. We determined 15 genes are missing relative to mammals, including several genes (CLCAs, BEST2) linked to olfactory transduction. The majority of sodium and calcium but few chloride channels showed differential expression in the song system, among them SCN8A and CACNA1E in the direct motor pathway, and CACNG4 and RYR2 in the anterior forebrain pathway. In several cases, we noted a seemingly coordinated pattern across multiple nuclei (SCN1B, SCN3B, SCN4B, CACNB4) or sparse expression (SCN1A, CACNG5, CACNA1B).ConclusionThe gene families examined are highly conserved between avian and mammalian lineages. Several cases of differential expression likely support high-frequency and burst firing in specific song nuclei, whereas cases of sparse patterns of expression may contribute to the unique electrophysiological signatures of distinct cell populations. These observations lay the groundwork for manipulations to determine how ion channels contribute to the neuronal excitability properties of vocal learning systems.

The Genome of Blue-Capped Cordon-Bleu Uncovers Hidden Diversity of LTR Retrotransposons in Zebra Finch.

Avian genomes have perplexed researchers by being conservative in both size and rearrangements, while simultaneously holding the blueprints for a massive species radiation during the last 65 million years (My). Transposable elements (TEs) in bird genomes are relatively scarce but have been implicated as important hotspots for chromosomal inversions. In zebra finch (Taeniopygia guttata), long terminal repeat (LTR) retrotransposons have proliferated and are positively associated with chromosomal breakpoint regions. Here, we present the genome, karyotype and transposons of blue-capped cordon-bleu (Uraeginthus cyanocephalus), an African songbird that diverged from zebra finch at the root of estrildid finches 10 million years ago (Mya). This constitutes the third linked-read sequenced genome assembly and fourth in-depth curated TE library of any bird. Exploration of TE diversity on this brief evolutionary timescale constitutes a considerable increase in resolution for avian TE biology and allowed us to uncover 4.5 Mb more LTR retrotransposons in the zebra finch genome. In blue-capped cordon-bleu, we likewise observed a recent LTR accumulation indicating that this is a shared feature of Estrildidae. Curiously, we discovered 25 new endogenous retrovirus-like LTR retrotransposon families of which at least 21 are present in zebra finch but were previously undiscovered. This highlights the importance of studying close relatives of model organisms.

De novo annotation of the transcriptome of the Northern Wheatear (Oenanthe oenanthe).

We have sequenced a partial transcriptome of the Northern Wheatear (Oenanthe oenanthe), a species with one of the longest migrations on Earth. The transcriptome was constructed de novo using RNA-Seq sequence data from the pooled mRNA of six different tissues: brain, muscle, intestine, liver, adipose tissue and skin. The samples came from nine captive-bred wheatears collected at three different stages of the endogenous autumn migratory period: (1) lean birds prior the onset of migration, (2) during the fattening stage and (3) individuals at their migratory body mass plateau, when they have almost doubled their lean body mass. The sample structure used to build up the transcriptome of the Northern Wheatears concerning tissue composition and time guarantees the future survey of the regulatory genes involved in the development of the migratory phenotype. Through the pre-migratory period, birds accomplish outstanding physical and behavioural changes that involve all organ systems. Nevertheless, the molecular mechanisms through which birds synchronize and control hyperphagia, fattening, restlessness increase, immunity boosting and tuning the muscles for such endurance flight are still largely unknown. The use of RNA-Seq has emerged as a powerful tool to analyse complex traits on a broad scale, and we believe it can help to characterize the migratory phenotype of wheatears at an unprecedented level. The primary challenge to conduct quantitative transcriptomic studies in non-model species is the availability of a reference transcriptome, which we have constructed and described in this paper. The cDNA was sequenced by pyrosequencing using the Genome Sequencer Roche GS FLX System; with single paired-end reads of about 400 bp. We estimate the total number of genes at 15,640, of which 67% could be annotated using Turkey and Zebra Finch genomes, or protein sequence information from SwissProt and NCBI databases. With our study, we have made a first step towards understanding the migratory phenotype regarding gene expression of a species that has become a model to study birds long-distance migrations.

The first set of universal nuclear protein-coding loci markers for avian phylogenetic and population genetic studies

Multiple nuclear markers provide genetic polymorphism data for molecular systematics and population genetic studies. They are especially required for the coalescent-based analyses that can be used to accurately estimate species trees and infer population demographic histories. However, in avian evolutionary studies, these powerful coalescent-based methods are hindered by the lack of a sufficient number of markers. In this study, we designed PCR primers to amplify 136 nuclear protein-coding loci (NPCLs) by scanning the published Red Junglefowl (Gallus gallus) and Zebra Finch (Taeniopygia guttata) genomes. To test their utility, we amplified these loci in 41 bird species representing 23 Aves orders. The sixty-three best-performing NPCLs, based on high PCR success rates, were selected which had various mutation rates and were evenly distributed across 17 avian autosomal chromosomes and the Z chromosome. To test phylogenetic resolving power of these markers, we conducted a Neoavian phylogenies analysis using 63 concatenated NPCL markers derived from 48 whole genomes of birds. The resulting phylogenetic topology, to a large extent, is congruence with results resolved by previous whole genome data. To test the level of intraspecific polymorphism in these makers, we examined the genetic diversity in four populations of the Kentish Plover (Charadrius alexandrinus) at 17 of NPCL markers chosen at random. Our results showed that these NPCL markers exhibited a level of polymorphism comparable with mitochondrial loci. Therefore, this set of pan-avian nuclear protein-coding loci has great potential to facilitate studies in avian phylogenetics and population genetics.

Discovery of the First Germline-Restricted Gene by Subtractive Transcriptomic Analysis in the Zebra Finch, Taeniopygia guttata

Developmentally programmed genome rearrangements are rare in vertebrates, but have been reported in scattered lineages including the bandicoot, hagfish, lamprey, and zebra finch (Taeniopygia guttata) [1]. Inthe finch, a well-studied animal model for neuroendocrinology and vocal learning [2], one such programmed genome rearrangement involves a germline-restricted chromosome, or GRC, which is found in germlines of both sexes but eliminated from mature sperm [3, 4]. Transmitted only through the oocyte, it displays uniparental female-driven inheritance, and early in embryonic development is apparently eliminated from all somatic tissue in both sexes [3, 4]. The GRC comprises the longest finch chromosome at over 120 million base pairs [3], and previously the only known GRC-derived sequence was repetitive and non-coding [5]. Because the zebra finch genome project was sourced from male muscle (somatic) tissue [6], the remaining genomic sequence and protein-coding content of the GRC remain unknown. Here we report the first protein-coding gene from the GRC: a member of the α-soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein (α-SNAP) family hitherto missing from zebra finch gene annotations. In addition to the GRC-encoded α-SNAP, we find an additional paralogous α-SNAP residing in the somatic genome (a somatolog)-makingthe zebra finch the first example in which α-SNAP is not a single-copy gene. We show divergent, sex-biased expression for the paralogs and also thatpositive selection is detectable across the birdα-SNAP lineage, including the GRC-encoded α-SNAP. This study presents the identification and evolutionary characterization of the first protein-coding GRC gene in any organism.

Fitness consequences of polymorphic inversions in the zebra finch genome

BackgroundInversion polymorphisms constitute an evolutionary puzzle: they should increase embryo mortality in heterokaryotypic individuals but still they are widespread in some taxa. Some insect species have evolved mechanisms to reduce the cost of embryo mortality but humans have not. In birds, a detailed analysis is missing although intraspecific inversion polymorphisms are regarded as common. In Australian zebra finches (Taeniopygia guttata), two polymorphic inversions are known cytogenetically and we set out to detect these two and potentially additional inversions using genomic tools and study their effects on embryo mortality and other fitness-related and morphological traits.ResultsUsing whole-genome SNP data, we screened 948 wild zebra finches for polymorphic inversions and describe four large (12–63 Mb) intraspecific inversion polymorphisms with allele frequencies close to 50 %. Using additional data from 5229 birds and 9764 eggs from wild and three captive zebra finch populations, we show that only the largest inversions increase embryo mortality in heterokaryotypic males, with surprisingly small effect sizes. We test for a heterozygote advantage on other fitness components but find no evidence for heterosis for any of the inversions. Yet, we find strong additive effects on several morphological traits.ConclusionsThe mechanism that has carried the derived inversion haplotypes to such high allele frequencies remains elusive. It appears that selection has effectively minimized the costs associated with inversions in zebra finches. The highly skewed distribution of recombination events towards the chromosome ends in zebra finches and other estrildid species may function to minimize crossovers in the inverted regions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1056-3) contains supplementary material, which is available to authorized users.

Red Carotenoid Coloration in the Zebra Finch Is Controlled by a Cytochrome P450 Gene Cluster

Bright-red colors in vertebrates are commonly involved in sexual, social, and interspecific signaling [1-8] and are largely produced by ketocarotenoid pigments. In land birds, ketocarotenoids such as astaxanthin are usually metabolically derived via ketolation of dietary yellow carotenoids [9, 10]. However, the molecular basis of this gene-environment mechanism has remained obscure. Here we use the yellowbeak mutation in the zebra finch (Taeniopygia guttata) to investigate the genetic basis of red coloration. Wild-type ketocarotenoids were absent in the beak and tarsus of yellowbeak birds. The yellowbeak mutation mapped to chromosome 8, close to a cluster of cytochrome P450 loci (CYP2J2-like) that are candidates for carotenoid ketolases. The wild-type zebra finch genome was found to have three intact genes in thiscluster: CYP2J19A, CYP2J19B, and CYP2J40. In yellowbeak, there are multiple mutations: loss of acomplete CYP2J19 gene, a modified remaining CYP2J19 gene (CYP2J19(yb)), and a non-synonymous SNP in CYP2J40. In wild-type birds, CYP2J19 loci are expressed in ketocarotenoid-containing tissues: CYP2J19A only in the retina and CYP2J19B in the beak and tarsus and to a variable extent in the retina. In contrast, expression of CYP2J19(yb) is barely detectable in the beak of yellowbeak birds. CYP2J40 has broad tissue expression and shows no differences between wild-type and yellowbeak. Our results indicate that CYP2J19 genes are strong candidates for the carotenoid ketolase and imply that ketolation occurs in the integument in zebra finches. Since cytochrome P450 enzymes include key detoxification enzymes, our results raise the intriguing possibility that red coloration may be an honest signal of detoxification ability.

A genome-wide search for epigenetically [corrected] regulated genes in zebra finch using MethylCap-seq and RNA-seq.

Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA- and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2′-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control.

A microsatellite-based linkage map for song sparrows (Melospiza melodia).

Although linkage maps are important tools in evolutionary biology, their availability for wild populations is limited. The population of song sparrows (Melospiza melodia) on Mandarte Island, Canada, is among the more intensively studied wild animal populations. Its long-term pedigree data, together with extensive genetic sampling, have allowed the study of a range of questions in evolutionary biology and ecology. However, the availability of genetic markers has been limited. We here describe 191 new microsatellite loci, including 160 high-quality polymorphic autosomal, 7 Z-linked and 1 W-linked markers. We used these markers to construct a linkage map for song sparrows with a total sex-averaged map length of 1731 cM and covering 35 linkage groups, and hence, these markers cover most of the 38-40 chromosomes. Female and male map lengths did not differ significantly. We then bioinformatically mapped these loci to the zebra finch (Taeniopygia guttata) genome and found that linkage groups were conserved between song sparrows and zebra finches. Compared to the zebra finch, marker order within small linkage groups was well conserved, whereas the larger linkage groups showed some intrachromosomal rearrangements. Finally, we show that as expected, recombination frequency between linked loci explained the majority of variation in gametic phase disequilibrium. Yet, there was substantial overlap in gametic phase disequilibrium between pairs of linked and unlinked loci. Given that the microsatellites described here lie on 35 of the 38-40 chromosomes, these markers will be useful for studies in this species, as well as for comparative genomics studies with other species.

In silico identification and characterisation of 17 polymorphic anonymous non-coding sequence markers (ANMs) for red grouse (Lagopus lagopus scotica)

Anonymous non-coding sequence markers (ANMs) are powerful neutral genetic markers with great utility in phylogeography, population genetics and population genomics. Developing ANMs has previously relied on sequencing random fragments of genomic DNA in the target species and then querying bioinformatics databases to identify unannotated, putatively neutral fragments. Here, we describe an alternative in silico approach that is based on identifying large unannotated genomic regions in model species to provide a priori neutral targets for candidate ANMs that are remote from exonic regions. We illustrate this approach by developing a set of 17 polymorphic ANMs for red grouse (Lagopus lagopus scotica) from c. 1 Mbp non-coding chromosome regions of chicken, turkey and zebrafinch genomes. This pipeline presents a powerful and efficient approach when appropriate model genomes are available for the target species of interest.