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Identification of the centromeric repeat in the threespine stickleback fish (Gasterosteus aculeatus)

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Abstract

Centromere sequences exist as gaps in many genome assemblies due to their repetitive nature. Here we take an unbiased approach utilizing centromere protein A (CENP-A) chomatin immunoprecipitation followed by high-throughput sequencing to identify the centromeric repeat sequence in the threespine stickleback fish (Gasterosteus aculeatus). A 186-bp, AT-rich repeat was validated as centromeric using both fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on interphase nuclei and metaphase spreads. This repeat hybridizes strongly to the centromere on all chromosomes, with the exception of weak hybridization to the Y chromosome. Together, our work provides the first validated sequence information for the threespine stickleback centromere.

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Abbreviations

BAC:

Bacterial artificial chromosome

BSA:

Bovine serum albumin

CEN:

Centromeric repeat sequence

CenH3:

Centromeric histone H3

CENP-A:

Centromere protein A

CENP-B:

Centromere protein B

ChIP:

Chromatin immunopreciptiation

ChIP-seq:

Chromatin immunopreciptiation sequencing

DAPI:

4’,6’-Diamidino-2-phenlyindole

dpf:

Days post-fertilization

EDTA:

Ethylenediaminetetraacetic acid

FISH:

Fluorescence in situ hybridization

GacCEN:

Threespine stickleback (Gasterosteus aculeatus) centromeric repeat sequence

H3:

Histone H3

HOR:

Higher-order repeat

IF-FISH:

Immunofluorescence combined with FISH

IP:

Immunoprecipitation

KCl:

Potassium chloride

MNase:

Micrococcal nuclease

PBS:

Phosphate-buffered saline

PBST:

Phosphate-buffered saline Tween-20

PCR:

Polymerase chain reaction

PMSF:

Phenylmethanesulfonylfluoride

POF1:

Pacific Ocean female 1

POF2:

Pacific Ocean female 2

RPM:

Reads per million

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SSC:

Saline-sodium citrate

3′ RACE:

Rapid amplification of cDNA ends

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Acknowledgments

We thank Sue Biggins and Steve Henikoff for reading this manuscript. We thank Ryan Basom of the Fred Hutchinson Cancer Research Center Genomics Shared Resource for help with ChIP-seq data analysis, Jaki Braggin for help with the Western blots, and the Henikoff, Malik, and Peichel labs for helpful discussions. This research was supported by a National Science Foundation Graduate Research Fellowship (DGE-1256082), the National Institutes of Health Chromosome Metabolism and Cancer Training Grant (T32 CA009657), and the Fred Hutchinson Cancer Research Center.

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Correspondence to Catherine L. Peichel.

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Responsible Editor: Rachel O'Neill, Ph.D.

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Fig. S1

Threespine stickleback Pacific Ocean CENP-A cDNA, protein, and antibody. a Full-length cDNA sequence for the threespine stickleback CENP-A protein. This sequence has been deposited in GenBank (accession number KT321854). b Alignment between the Danio rerio CENP-A protein (D.re CENP-A), the threespine stickleback CENP-A protein (G.ac CENP-A), and the threespine stickleback H3 protein (G.ac H3) reveals extensive divergence in the N-terminal tail (red). The threespine stickleback H3 protein sequence is a product of the gene ENSGACG00000005779 (Ensembl BROAD S1; Feb 2006). The antibody was raised against the peptide sequence highlighted with a box. c Western blot of the CENP-A antibody on protein lysates from threespine stickleback liver and kidney. (PDF 183 kb)

Fig. S2

Alignment of the enriched repeats found in the most abundant clusters from both ChIP-seq samples. All repeats that were enriched in the IP relative to input from the 500 most abundant clusters in each sample were aligned in Geneious (Biomatters, New Zealand), but only the 44 repeats (Pacific Ocean female 1 (POF1)) and 41 repeats (Pacific Ocean female 2 (POF2)) that were enriched in the IP relative to input from the 50 most abundant clusters are shown here for simplicity. The blue box indicates the 186 bp repeating unit found in all enriched clusters. (PDF 1016 kb)

Fig. S3

Alignment of the consensus repeating unit from each independent ChIP-seq sample. The consensus repeat sequences from the Pacific Ocean female 1 (POF1) and Pacific Ocean female 2 (POF2) were aligned to create the final consensus centromere repeat sequence shown in Fig. 2. Nucleotide ambiguities: R = A or G; W = A or T. (PDF 92 kb)

Fig. S4

Assembled scaffolds containing centromere repeats were found on both edges of the gap in the chromosome 9 assembly corresponding to the position of the centromere, and on a single edge of the gaps corresponding to the centromeres of chromosomes 1, 3, 5, 7, 17, 18, 20, and the X chromosome (Urton et al. 2011). Additional GacCEN like repeats were found on four scaffolds from unassembled regions of the genome. Each arrow shows the size of the repeat variant, and the percent identity to GacCEN is represented by color: blue <90 %; green 90-95 %; red >95 %. The direction of the arrow points towards the center of the genome assembly gap. The dark shaded regions in each chromosome drawing depicts the region where the GacCEN containing scaffold is found; relative position of the centromeres is based on Urton et al. 2011. (PDF 170 kb)

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Cech, J.N., Peichel, C.L. Identification of the centromeric repeat in the threespine stickleback fish (Gasterosteus aculeatus). Chromosome Res 23, 767–779 (2015). https://doi.org/10.1007/s10577-015-9495-3

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  • DOI: https://doi.org/10.1007/s10577-015-9495-3

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