Expanding duplication of the testis PHD Finger Protein 7 (PHF7) gene in the chicken genome.

Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-to-histone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH). We selected nine BAC that highlighted nine chromosomal regions for a total of 10 distinct PHF7-like loci on five Gallus gallus chromosomes: Chr1 (three loci), Chr2 (two loci), Chr12 (one locus), Chr19 (one locus) and ChrZ (three loci). We sequenced the corresponding BAC by using high-performance PacBio technology. After assembly, we performed annotation with the FGENESH program: there were a total of 116 peptides, including 39 PHF7-like proteins identified by BLASTP. These proteins share a common exon-intron core structure of 8-11 exons. Phylogeny revealed that the duplications occurred first between chromosomal regions and then inside each region. There are other duplicated genes in the identified BAC sequences, suggesting that these genomic regions exhibit a high rate of tandem duplication. We showed that the PHF7 gene, which is highly expressed in the rooster testis, is a highly duplicated gene family in the chicken genome, and this phenomenon probably concerns other bird species.

Genome organization of major tandem repeats and their specificity for heterochromatin of macro- and microchromosomes in Japanese quail.

Tandemly repeated DNAs form heterochromatic regions of chromosomes, including the vital centromeric chromatin. Despite the progress in new genomic technologies, tandem repeats remain poorly deciphered and need targeted analysis in the species of interest. The Japanese quail is one of the highest-producing poultry species as well as a model organism. Its genome differs by a noticeable accumulation of heterochromatin, which led to an increase by 1/7 compared to the chicken genome size. Prominent heterochromatin blocks occupy the short arms of acrocentric macrochromosomes and of microchromosomes. We have applied de novo repeat finder approach to unassembled raw reads of the Japanese quail genome. We identified the 20 most common tandem repeats with the abundance >1 Mb, which represent about 4.8% of the genome. We found that tandem repeat CjapSAT primarily contributes to the centromeric regions of the macrochromosomes CJA1-8. Cjap31B together with previously characterized BglII makes up centromere regions of microchromosomes and W chromosome. Other repeats populate heterochromatin of microchromosomal short arms in unequal proportions, as revealed by fluorescence in situ hybridization. The Cjap84A, Cjap408A, and CjapSAT repeat sequences show similarities to retrotransposon motifs. This suggests that retroelements may have played a crucial role in the distribution of repeats throughout the Japanese quail genome.

Structure of the intergenic spacers in chicken ribosomal DNA.

BACKGROUND: Ribosomal DNA (rDNA) repeats are situated in the nucleolus organizer regions (NOR) of chromosomes and transcribed into rRNA for ribosome biogenesis. Thus, they are an essential component of eukaryotic genomes. rDNA repeat units consist of rRNA gene clusters that are transcribed into single pre-rRNA molecules, each separated by intergenic spacers (IGS) that contain regulatory elements for rRNA gene cluster transcription. Because of their high repeat content, rDNA sequences are usually absent from genome assemblies. In this work, we used the long-read sequencing technology to describe the chicken IGS and fill the knowledge gap on rDNA sequences of one of the key domesticated animals. METHODS: We used the long-read PacBio RSII technique to sequence the BAC clone WAG137G04 (Wageningen BAC library) known to contain chicken NOR elements and the HGAP workflow software suit to assemble the PacBio RSII reads. Whole-genome sequence contigs homologous to the chicken rDNA repetitive unit were identified based on the Gallus_gallus-5.0 assembly with BLAST. We used the Geneious 9.0.5 and Mega software, maximum likelihood method and Chickspress project for sequence evolution analysis, phylogenetic tree construction and analysis of the raw transcriptome data. RESULTS: Three complete IGS sequences in the White Leghorn chicken genome and one IGS sequence in the red junglefowl contig AADN04001305.1 (Gallus_gallus-5.0) were detected. They had various lengths and contained three groups of tandem repeats (some of them being very GC rich) that form highly organized arrays. Initiation and termination sites of rDNA transcription were located within small and large unique regions (SUR and LUR), respectively. No functionally significant sites were detected within the tandem repeat sequences. CONCLUSIONS: Due to the highly organized GC-rich repeats, the structure of the chicken IGS differs from that of IGS in human, apes, Xenopus or fish rDNA. However, the chicken IGS shares some molecular organization features with that of the turtles, which are other representatives of the Sauropsida clade that includes birds and reptiles. Our current results on the structure of chicken IGS together with the previously reported ribosomal gene cluster sequence provide sufficient data to consider that the complete chicken rDNA sequence is assembled with confidence in terms of molecular DNA organization.

Two new structural mutations in the 5' region of the ASIP gene cause diluted feather color phenotypes in Japanese quail.

BACKGROUND: In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification). RESULTS: For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5' region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5' region of the ASIP gene. CONCLUSIONS: We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5' region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5' region of ASIP are absent in the fawn-2/beige mutant.

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description.

Lampbrush chromosomes are giant, transcriptionally active, meiotic chromosomes found in oocytes of all vertebrates with the exception of mammals. Lampbrush chromosomes offer a convenient tool for cytogenetic mapping and, in particular, have been instrumental in mapping genes and linkage groups on chicken (GGA) chromosomes. Whereas cytogenetic maps of macrochromosome GGA1-10 and microchromosome GGA11-16 lampbrush bivalents have been established, identification and description of smaller microchromosome bivalents are still missing. In this work, we used specific FISH probes for the identification of 12 chicken lampbrush chromosomes formed by GGA17-28. Our observations on chromomere and lateral loop arrangement and chiasma position allowed us to construct the respective cytogenetic maps for these microchromosomes. For the 10 smallest chicken microchromosomes, GGA29-38, no individual molecular tags are available, yet they can be collectively marked using the PO41 repeat. The reported results contribute to building of working cytogenetic maps of the chicken karyotype.

The Rose-comb mutation in chickens constitutes a structural rearrangement causing both altered comb morphology and defective sperm motility.

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.

A duck RH panel and its potential for assisting NGS genome assembly.

BACKGROUND: Owing to the low cost of the high throughput Next Generation Sequencing (NGS) technology, more and more species have been and will be sequenced. However, de novo assemblies of large eukaryotic genomes thus produced are composed of a large number of contigs and scaffolds of medium to small size, having no chromosomal assignment. Radiation hybrid (RH) mapping is a powerful tool for building whole genome maps and has been used for several animal species, to help assign sequence scaffolds to chromosomes and determining their order. RESULTS: We report here a duck whole genome RH panel obtained by fusing female duck embryonic fibroblasts irradiated at a dose of 6,000 rads, with HPRT-deficient Wg3hCl2 hamster cells. The ninety best hybrids, having an average retention of 23.6% of the duck genome, were selected for the final panel. To allow the genotyping of large numbers of markers, as required for whole genome mapping, without having to cultivate the hybrid clones on a large scale, three different methods involving Whole Genome Amplification (WGA) and/or scaling down PCR volumes by using the Fluidigm BioMark(TM) Integrated Fluidic Circuits (IFC) Dynamic Array(TM) for genotyping were tested. RH maps of APL12 and APL22 were built, allowing the detection of intrachromosomal rearrangements when compared to chicken. Finally, the panel proved useful for checking the assembly of sequence scaffolds and for mapping EST located on one of the smallest microchromosomes. CONCLUSION: The Fluidigm BioMark(TM) Integrated Fluidic Circuits (IFC) Dynamic Array(TM) genotyping by quantitative PCR provides a rapid and cost-effective method for building RH linkage groups. Although the vast majority of genotyped markers exhibited a picture coherent with their associated scaffolds, a few of them were discordant, pinpointing potential assembly errors. Comparative mapping with chicken chromosomes GGA21 and GGA11 allowed the detection of the first chromosome rearrangements on microchromosomes between duck and chicken. As in chicken, the smallest duck microchromosomes appear missing in the assembly and more EST data will be needed for mapping them. Altogether, this underlines the added value of RH mapping to improve genome assemblies.

Integrative mapping analysis of chicken microchromosome 16 organization.

BACKGROUND: The chicken karyotype is composed of 39 chromosome pairs, of which 9 still remain totally absent from the current genome sequence assembly, despite international efforts towards complete coverage. Some others are only very partially sequenced, amongst which microchromosome 16 (GGA16), particularly under-represented, with only 433 kb assembled for a full estimated size of 9 to 11 Mb. Besides the obvious need of full genome coverage with genetic markers for QTL (Quantitative Trait Loci) mapping and major genes identification studies, there is a major interest in the detailed study of this chromosome because it carries the two genetically independent MHC complexes B and Y. In addition, GGA16 carries the ribosomal RNA (rRNA) genes cluster, also known as the NOR (nucleolus organizer region). The purpose of the present study is to construct and present high resolution integrated maps of GGA16 to refine its organization and improve its coverage with genetic markers. RESULTS: We developed 79 STS (Sequence Tagged Site) markers to build a physical RH (radiation hybrid) map and 34 genetic markers to extend the genetic map of GGA16. We screened a BAC (Bacterial Artificial Chromosome) library with markers for the MHC-B, MHC-Y and rRNA complexes. Selected clones were used to perform high resolution FISH (Fluorescent In Situ Hybridization) mapping on giant meiotic lampbrush chromosomes, allowing meiotic mapping in addition to the confirmation of the order of the three clusters along the chromosome. A region with high recombination rates and containing PO41 repeated elements separates the two MHC complexes. CONCLUSIONS: The three complementary mapping strategies used refine greatly our knowledge of chicken microchromosome 16 organisation. The characterisation of the recombination hotspots separating the two MHC complexes demonstrates the presence of PO41 repetitive sequences both in tandem and inverted orientation. However, this region still needs to be studied in more detail.

Addition of the microchromosome GGA25 to the chicken genome sequence assembly through radiation hybrid and genetic mapping.

BACKGROUND: The publication of the first draft chicken sequence assembly became available in 2004 and was updated in 2006. However, this does not constitute a definitive and complete sequence of the chicken genome, since the microchromosomes are notably under-represented. In an effort to develop maps for the microchromosomes absent from the chicken genome assembly, we developed radiation hybrid (RH) and genetic maps with markers isolated from sequence currently assigned to "chromosome Unknown" (chrUn). The chrUn is composed of sequence contigs not assigned to named chromosomes. To identify and map sequence belonging to the microchromosomes we used a comparative mapping strategy, and we focused on the small linkage group E26C13. RESULTS: In total, 139 markers were analysed with the chickRH6 panel, of which 120 were effectively assigned to the E26C13 linkage group, the remainder mapping elsewhere in the genome. The final RH map is composed of 22 framework markers extending over a 245.6 cR distance. A corresponding genetic map was developed, whose length is 103 cM in the East Lansing reference population. The E26C13 group was assigned to GGA25 (Gallus gallus chromosome 25) by FISH (fluorescence in situ hybridisation) mapping. CONCLUSION: The high-resolution RH framework map obtained here covers the entire chicken chromosome 25 and reveals the existence of a high number of intrachromosomal rearrangements when compared to the human genome. The strategy used here for the characterization of GGA25 could be used to improve knowledge on the other uncharacterized small, yet gene-rich microchromosomes.

Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution.

BACKGROUND: Comparative genomics is a powerful means of establishing inter-specific relationships between gene function/location and allows insight into genomic rearrangements, conservation and evolutionary phylogeny. The availability of the complete sequence of the chicken genome has initiated the development of detailed genomic information in other birds including turkey, an agriculturally important species where mapping has hitherto focused on linkage with limited physical information. No molecular study has yet examined conservation of avian microchromosomes, nor differences in copy number variants (CNVs) between birds. RESULTS: We present a detailed comparative cytogenetic map between chicken and turkey based on reciprocal chromosome painting and mapping of 338 chicken BACs to turkey metaphases. Two inter-chromosomal changes (both involving centromeres) and three pericentric inversions have been identified between chicken and turkey; and array CGH identified 16 inter-specific CNVs. CONCLUSION: This is the first study to combine the modalities of zoo-FISH and array CGH between different avian species. The first insight into the conservation of microchromosomes, the first comparative cytogenetic map of any bird and the first appraisal of CNVs between birds is provided. Results suggest that avian genomes have remained relatively stable during evolution compared to mammalian equivalents.

A New Chicken Genome Assembly Provides Insight into Avian Genome Structure.

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts.

Integrated maps in quail (Coturnix japonica) confirm the high degree of synteny conservation with chicken (Gallus gallus) despite 35 million years of divergence.

BACKGROUND: By comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps. RESULTS: The integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied. CONCLUSION: Integrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail.

Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype.

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.

Heterogenic final cell cycle by chicken retinal Lim1 horizontal progenitor cells leads to heteroploid cells with a remaining replicated genome.

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.

FISH on avian lampbrush chromosomes produces higher resolution gene mapping.

Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1-6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.

ChickRH6: a chicken whole-genome radiation hybrid panel.

As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6,000 rads with HPRT-deficient hamster Wg3hCl2 cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs.