Dyomin A et al. (2019), Structure of the intergenic spacers in chicken ...

XB-ART-56410

Genet Sel Evol 2019 Oct 26;511:59. doi: 10.1186/s12711-019-0501-7.

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Structure of the intergenic spacers in chicken ribosomal DNA.

Dyomin A , Galkina S , Fillon V , Cauet S , Lopez-Roques C , Rodde N , Klopp C , Vignal A , Sokolovskaya A , Saifitdinova A , Gaginskaya E .

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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.

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	Fig. 1. Chicken rDNA repeat structure. Structure of the WAG137G4_utg0 contig obtained by PacBio sequencing of the WAG137G04 chicken BAC clone. The positions of three complete and one incomplete rRNA gene clusters together with three intercalary IGS are indicated. An enlarged diagram of the rRNA gene cluster is shown separately
	Fig. 2. Chicken IGS length variants. Structure of three IGS from the WAG137G04 BAC clone (IGS_I, IGS_II, IGS_III) and an IGS present in the AADN04001305.1 contig of Gallus_gallus-5.0. a Detailed comparative figures of IGS structural elements distribution. Repeat deficiency regions are designated with fine black lines. b Contracted IGS figures, central block repeats are organized into tetrads
	Fig. 3. Tandem repeats at the chicken IGS 5′ end. a Svetlana (SV) repeat unit; b Alsu (AL) repeat unit. Both repeats are consensus sequences
	Fig. 4. Elena repeats in chicken IGS. Relationships between repeats in the Elena (EL) group. The figure was plotted using the maximum likelihood method. The numbers following the repeat names indicate the repeat ordinal position in the IGS, and the numbers following after a space—the IGS ordinal position in WAG137G4_utg0 contig. An expanded figure is attached in Additional file 4
	Fig. 5. Consensus sequences of Elena (EL) repeat variants. Elena (EL) repeat variants: alignment of the sequences
	Fig. 6. Valerie repeats in chicken IGS. Relationships between repeats in the Valerie group. The figure was plotted using the maximum likelihood method. The numbers following the repeat names indicate the repeat ordinal position in the IGS, and the numbers following a space—the IGS ordinal position in WAG137G4_utg0 contig
	Fig. 7. Consensus sequences of Valerie (VAL) repeat variants. Valerie (VAL) repeat variants: alignment of the sequences
	Fig. 8. GC content in Sauropsida and Mammalia IGS. (C+G) content and putative CpG island distribution in the IGS of chicken Gallus gallus, terrapin Malaclemys terrapin, human Homo sapiens, and mouse Mus musculus. Regions containing repeats are designated with horizontal green blocks (Rep); putative CpG islands—with light-green boxes (CpG Island); GC pairs distribution is shown in the graphs (GC content)
	Fig. 9. IGS transcription. Analysis of the IGS transcription in different chicken organs. The vertical axis represents read counts aligned to each nucleotide position