Cross-species overgo hybridization and comparative physical mapping within avian genomes

The chicken genome sequence facilitates comparative genomics within other avian species. We performed cross-species hybridizations using overgo probes designed from chicken genomic and zebra finch expressed sequence tags (ESTs) to turkey and zebra finch BAC libraries. As a result, 3772 turkey BACs were assigned to 336 markers or genes, and 1662 zebra finch BACs were assigned to 164 genes. As expected, cross-hybridization was more successful with overgos within coding sequences than within untranslated region, intron or flanking sequences and between chicken and turkey, when compared with chicken-zebra finch or zebra finch-turkey cross-hybridization. These data contribute to the comparative alignment of avian genome maps using a 'one sequence, multiple genomes' strategy.     

A genome-wide set of 106 microsatellite markers for the blue tit (Cyanistes caeruleus)

We have characterized a set of 106 microsatellite markers in 26-127 individual blue tits (Cyanistes caeruleus), and assigned their location on the zebra finch (Taeniopygia guttata) and on the chicken (Gallus gallus) genome on the basis of sequence homology. Thirty-one markers are newly designed from zebra finch EST (expressed sequence tags) sequences, 22 markers were developed by others from EST sequences using different methods and the remaining 53 loci were previously designed or modified passerine markers. The 106 microsatellite markers are distributed over 26 and 24 chromosomes in the zebra finch and in the chicken genome respectively and the number of alleles varies between 2 and 49. Eight loci deviate significantly from Hardy-Weinberg equilibrium and show a high frequency of null alleles, and three pairs of markers located in the same chromosome appear to be in linkage disequilibrium. With the exception of these few loci, the polymorphic microsatellite markers presented here provide a useful genome-wide resource for population and evolutionary genetic studies of the blue tit, in addition to their potential utility in other passerine birds.     

Beyond an AFLP genome scan towards the identification of immune genes involved in plague resistance in Rattus rattus from Madagascar

Genome scans using amplified fragment length polymorphism (AFLP) markers became popular in nonmodel species within the last 10 years, but few studies have tried to characterize the anonymous outliers identified. This study follows on from an AFLP genome scan in the black rat (Rattus rattus), the reservoir of plague (Yersinia pestis infection) in Madagascar. We successfully sequenced 17 of the 22 markers previously shown to be potentially affected by plague‐mediated selection and associated with a plague resistance phenotype. Searching these sequences in the genome of the closely related species Rattus norvegicus assigned them to 14 genomic regions, revealing a random distribution of outliers in the genome (no clustering). We compared these results with those of an in silico AFLP study of the R. norvegicus genome, which showed that outlier sequences could not have been inferred by this method in R. rattus (only four of the 15 sequences were predicted). However, in silico analysis allowed the prediction of AFLP markers distribution and the estimation of homoplasy rates, confirming its potential utility for designing AFLP studies in nonmodel species. The 14 genomic regions surrounding AFLP outliers (less than 300 kb from the marker) contained 75 genes encoding proteins of known function, including nine involved in immune function and pathogen defence. We identified the two interleukin 1 genes (Il1a and Il1b) that share homology with an antigen of Y. pestis, as the best candidates for genes subject to plague‐mediated natural selection. At least six other genes known to be involved in proinflammatory pathways may also be affected by plague‐mediated selection.     

Genetic mapping of the major histocompatibility complex in the zebra finch (<Emphasis Type="Italic">Taeniopygia guttata</Emphasis>)

Genes of the major histocompatibility complex (MHC) have received much attention in immunology, genetics, and ecology because they are highly polymorphic and play important roles in parasite resistance and mate choice. Until recently, the MHC of passerine birds was not well-described. However, the genome sequencing of the zebra finch (Taeniopygia guttata) has partially redressed this gap in our knowledge of avian MHC genes. Here, we contribute further to the understanding of the zebra finch MHC organization by mapping SNPs within or close to known MHC genes in the zebra finch genome. MHC class I and IIB genes were both mapped to zebra finch chromosome 16, and there was no evidence that MHC class I genes are located on chromosome 22 (as suggested by the genome assembly). We confirm the location in the MHC region on chromosome 16 for several other genes (BRD2, FLOT1, TRIM7.2, GNB2L1, and CSNK2B). Two of these (CSNK2B and FLOT1) have not previously been mapped in any other bird species. In line with previous results, we also find that orthologs to the immune-related genes B-NK and CLEC2D, which are part of the MHC region in chicken, are situated on zebra finch chromosome Z and not among other MHC genes in the zebra finch.     

Mass production of SNP markers in a nonmodel passerine bird through RAD sequencing and contig mapping to the zebra finch genome

Here, we present an adaptation of restriction‐site‐associated DNA sequencing (RAD‐seq) to the Illumina HiSeq2000 technology that we used to produce SNP markers in very large quantities at low cost per unit in the Réunion grey white‐eye (Zosterops borbonicus), a nonmodel passerine bird species with no reference genome. We sequenced a set of six pools of 18–25 individuals using a single sequencing lane. This allowed us to build around 600 000 contigs, among which at least 386 000 could be mapped to the zebra finch (Taeniopygia guttata) genome. This yielded more than 80 000 SNPs that could be mapped unambiguously and are evenly distributed across the genome. Thus, our approach provides a good illustration of the high potential of paired‐end RAD sequencing of pooled DNA samples combined with comparative assembly to the zebra finch genome to build large contigs and characterize vast numbers of informative SNPs in nonmodel passerine bird species in a very efficient and cost‐effective way.     

Microsatellite resources for Passeridae species: a predicted microsatellite map of the house sparrow Passer domesticus

We identified microsatellite sequences of potential utility in the house sparrow (Passer domesticus) and assigned their predicted genome locations. These sequences included newly isolated house sparrow loci, which we fully characterized. Many of the newly isolated loci were polymorphic in two other species of Passeridae: Berthelot's pipit Anthus berthelotii and zebra finch Taeniopygia guttata. In total, we identified 179 microsatellite markers that were either isolated directly from, or are of known utility in, the house sparrow. Sixty-seven of these markers were designed from unique sequences that we isolated from a house sparrow genomic library. These new markers were combined with 36 house sparrow markers isolated by other studies and 76 markers isolated from other passerine species but known to be polymorphic in the house sparrow. We utilized sequence homology to assign chromosomal locations for these loci in the assembled zebra finch genome. One hundred and thirty-four loci were assigned to 25 different autosomes and eight loci to the Z chromosome. Examination of the genotypes of known-sex house sparrows for 37 of the new loci revealed a W-linked locus and an additional Z-linked locus. Locus Pdoμ2, previously reported as autosomal, was found to be Z-linked. These loci enable the creation of powerful and cost-effective house sparrow multiplex primer sets for population and parentage studies. They can be used to create a house sparrow linkage map and will aid the identification of quantitative trait loci in passerine species.     

Karyotypic polymorphism of the zebra finch Z chromosome

We describe a karyotypic polymorphism on the zebra finch Z chromosome. This polymorphism was discovered because of a difference in the position of the centromere and because it occurs at varying frequencies in domesticated colonies in the USA and Germany and among two zebra finch subspecies. Using DNA fluorescent in situ hybridization to map specific Z genes and measurements of DNA replication, we show that this polymorphism is the result of a large pericentric inversion involving the majority of the chromosome. We sequenced a likely breakpoint for the inversion and found many repetitive sequences. Around the breakpoint, there are numerous repetitive sequences and several copies of PAK3 (p21-activated kinase 3)-related sequences (PAK3Z) which showed testes-specific expression by RT-PCR. Our findings further suggest that the sequenced genome of the zebra finch may be derived from a male heterozygote for the Z chromosome polymorphism. This finding, in combination with regional differences in the frequency of the polymorphism, has important consequences for future studies using zebra finches.     

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

Background

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.

Results

The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.

Conclusion

The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.     

Nucleotide Variation, Linkage Disequilibrium and Founder-Facilitated Speciation in Wild Populations of the Zebra Finch (Taeniopygia guttata)

The zebra finch has long been an important model system for the study of vocal learning, vocal production, and behavior. With the imminent sequencing of its genome, the zebra finch is now poised to become a model system for population genetics. Using a panel of 30 noncoding loci, we characterized patterns of polymorphism and divergence among wild zebra finch populations. Continental Australian populations displayed little population structure, exceptionally high levels of nucleotide diversity (π = 0.010), a rapid decay of linkage disequilibrium (LD), and a high population recombination rate (ρ ≈ 0.05), all of which suggest an open and fluid genomic background that could facilitate adaptive variation. By contrast, substantial divergence between the Australian and Lesser Sunda Island populations (K_ST = 0.193), reduced genetic diversity (π = 0.002), and higher levels of LD in the island population suggest a strong but relatively recent founder event, which may have contributed to speciation between these populations as envisioned under founder-effect speciation models. Consistent with this hypothesis, we find that under a simple quantitative genetic model both drift and selection could have contributed to the observed divergence in six quantitative traits. In both Australian and Lesser Sundas populations, diversity in Z-linked loci was significantly lower than in autosomal loci. Our analysis provides a quantitative framework for studying the role of selection and drift in shaping patterns of molecular evolution in the zebra finch genome.     

珍珠鸟(Taeniopygia guttata)小染色体基因表达的密码子偏性

从NCBI数据库（http：／／www．ncbi．nlm．nih．gov／projects／mapview／map）下载珍珠鸟全部小染色体基因的cDNA序列,最终共有1586个基因的CDS序列纳入统计分析。密码子的偏性分析使用CodonW（1．4．2）完成,初步确定了UUC、UCC、UCG等27个密码子为珍珠鸟小染色体基因表达的“最优”密码子。对应分析表明,影响珍珠鸟小染色体基因密码子使用的主要因素分别为GC3s、CDS的GC含量基以及因的表达丰度。珍珠鸟小染色体基因的密码子用法受到了基因碱基组成的显著影响,其密码子的偏性是碱基组成及选择等因素综合作用的结果。本研究的目的是系统探究珍珠乌小染色体基因的密码子用法,探究鸟类基因表达的分子调控机制。     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

Molecular evolution of genes in avian genomes

Background

Obtaining a draft genome sequence of the zebra finch (Taeniopygia guttata), the second bird genome to be sequenced, provides the necessary resource for whole-genome comparative analysis of gene sequence evolution in a non-mammalian vertebrate lineage. To analyze basic molecular evolutionary processes during avian evolution, and to contrast these with the situation in mammals, we aligned the protein-coding sequences of 8,384 1:1 orthologs of chicken, zebra finch, a lizard and three mammalian species.

Results

We found clear differences in the substitution rate at fourfold degenerate sites, being lowest in the ancestral bird lineage, intermediate in the chicken lineage and highest in the zebra finch lineage, possibly reflecting differences in generation time. We identified positively selected and/or rapidly evolving genes in avian lineages and found an over-representation of several functional classes, including anion transporter activity, calcium ion binding, cell adhesion and microtubule cytoskeleton.

Conclusions

Focusing specifically on genes of neurological interest and genes differentially expressed in the unique vocal control nuclei of the songbird brain, we find a number of positively selected genes, including synaptic receptors. We found no evidence that selection for beneficial alleles is more efficient in regions of high recombination; in fact, there was a weak yet significant negative correlation between ω and recombination rate, which is in the direction predicted by the Hill-Robertson effect if slightly deleterious mutations contribute to protein evolution. These findings set the stage for studies of functional genetics of avian genes.     

Molecular Phylogenetic Analysis of Zebra Finch Basic Helix-Loop-Helix Transcription Factors

The zebra finch (Taeniopygia guttata) is an established organism for developmental, behavioral, and neurological research. In this study, we conducted a genomewide survey using the zebra finch genome project databases and identified 86 bHLH sequences in silico in the zebra finch genome. Phylogenetic analysis revealed that 85 proteins belong to 38 families with 29, 18, 18, 3, 11, and 6 bHLH members in supergroups A, B, C, D, E, and F, respectively. One orphan member belonged to none of these groups. Comparisons of zebra finch with chicken and human bHLH repertoires suggested that both humans and birds have a number of lineage-specific bHLH members. Chromosome distribution patterns and phylogenetic analysis suggest that the zebra finch bHLH members should have arisen through gene duplication. This study provides useful information for further research using zebra finch as a model system.     

Explorative genome scan to detect candidate loci for adaptation along a gradient of altitude in the common frog (Rana temporaria)

Today, with the rapid development of population genomics, the genetic basis of adaptation can be unraveled directly at the genome level, without any prerequisites about the selectively advantageous genes or traits. For nonmodel species, it is now possible to screen many markers randomly scattered across the genome and to distinguish between the neutral genetic background and outlier loci displaying an atypical behavior (e.g., a higher differentiation between populations). This study investigated the genetic frame of adaptation to a gradient of altitude in the common frog (Rana temporaria) by means of a genome scan based on 392 amplified fragment length polymorphism markers. Using two outlier detection methods never applied to dominant data so far, we sought for loci with a genetic differentiation diverging from neutral expectations when comparing populations from different altitudes. All the detected loci were sorted out according to their most probable cause for outlier behavior and classified as false positives, outliers due to local effects, or outliers associated with altitude. Altogether, eight good candidate loci were identified as potentially involved in adaptation to altitude because they were picked out in several independent interaltitude comparisons. This result illustrated the potential of genome-wide surveys to reveal selection signatures along selection gradients, where the association between environmental variables and fitness-related traits may be complex and/or cryptic. In this article, we also underlined the need for confirmation of the selection footprints for the outlier loci. Finally, we provided some preliminary insights into the genetic basis of adaptation along an altitudinal cline in the common frog.     

Molecular cloning and characterization of the germline-restricted chromosome sequence in the zebra finch

The zebra finch (Taeniopygia guttata) germline-restricted chromosome (GRC) is the largest chromosome and has a unique system of transmission in germ cells. In the male, the GRC exists as a single heterochromatic chromosome in the germline and is eliminated from nuclei in late spermatogenesis. In the female, the GRC is bivalent and euchromatic and experiences recombination. These characteristics suggest a female-specific or female-beneficial function of the GRC. To shed light on the function of GRC, we cloned a portion of the GRC using random amplified polymorphic DNA–polymerase chain reaction and analyzed it using molecular genetic and cytogenetic methods. The GRC clone hybridized strongly to testis but not blood DNA in genomic Southern blots. In fluorescent in situ hybridization analysis on meiotic chromosomes from synaptonemal complex spreads, the probe showed hybridization across a large area of the GRC, suggesting that it contains repetitive sequences. We isolated a sequence homologous to the GRC from zebra finch chromosome 3 and a region of chicken chromosome 1 that is homologous to zebra finch chromosome 3; the phylogenetic analysis of these three sequences suggested that the GRC sequence and the zebra finch chromosome 3 sequence are most closely related. Thus, the GRC sequences likely originated from autosomal DNA and have evolved after the galliform–passeriform split. The present study provides a foundation for further study of the intriguing GRC.     

The Turkey Ig-Like Receptor Family: Identification,Expression and Function

The chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes.