Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat

Abstract

The kinetochore is a protein complex including kinetochore-specific proteins that plays a role in chromatid segregation during mitosis and meiosis. The complex associates with centromeric DNA sequences that are usually species-specific. In plant species, tandem repeats including satellite DNA sequences and retrotransposons have been reported as centromeric DNA sequences. In this study on sunflowers, a cDNA-encoding centromere-specific histone H3 (CENH3) was isolated from a cDNA pool from a seedling, and an antibody was raised against a peptide synthesized from the deduced cDNA. The antibody specifically recognized the sunflower CENH3 (HaCENH3) and showed centromeric signals by immunostaining and immunohistochemical staining analysis. The antibody was also applied in chromatin immunoprecipitation (ChIP)-Seq to isolate centromeric DNA sequences and two different types of repetitive DNA sequences were identified. One was a long interspersed nuclear element (LINE)-like sequence, which showed centromere-specific signals on almost all chromosomes in sunflowers. This is the first report of a centromeric LINE sequence, suggesting possible centromere targeting ability. Another type of identified repetitive DNA was a tandem repeat sequence with a 187-bp unit that was found only on a pair of chromosomes. The HaCENH3 content of the tandem repeats was estimated to be much higher than that of the LINE, which implies centromere evolution from LINE-based centromeres to more stable tandem-repeat-based centromeres. In addition, the epigenetic status of the sunflower centromeres was investigated by immunohistochemical staining and ChIP, and it was found that centromeres were heterochromatic.

Keywords: ChIP-seq; centromere; centromeric DNA; centromeric histone H3; sunflower (Helianthus annuus).

Figure 1

Phylogenetic tree based on the amino acid sequences of plant CENH3. The species name and GenBank accession number are indicated in parentheses. Rice canonical histone H3 was used as an outgroup. Bootstrap values greater than 800 in 1000 tests are indicated on the branches.

Figure 2

Immunostaining of sunflower metaphase chromosomes using an anti-HaCENH3 antibody. (A) DAPI-stained chromosomes. (B) Immunosignals of an anti-HaCENH3 antibody. (C) Merged image of (A,B). Scale bar, 10 μm.

Figure 3

ChIP-qPCR analysis of centromeric DNA sequences of sunflowers. The columns and error bars represent the average relative enrichments (REs) and the standard errors from four independent ChIP reactions, respectively. The coding region of the sunflower ubiquitin gene (X14333) was used as a non-centromeric (negative) control in the anti-HaCENH3 ChIP and as a negative control in the anti-H3K9me2 ChIP. Since HaCENH3CL124 showed the lowest REs among the sequences in the anti-H3K4me2 and H4Ac ChIP, HaCENH3CL124 was used as a negative control. The statistical significance of differences between the negative controls and other sequences was determined using Student's t-test (^*P < 0.01).

Figure 4

FISH using the enriched sequence from ChIP-Seq. (A,E,I,N,R) DAPI-stained sunflower chromosomes. (B,F,J) FISH signals of pHaCENH3CL124-1. (C) FISH signals of rDNA. (G,K,P,T) FISH signals of HAG004N15. (O) FISH signals of pHaCENH3CL20-1. (S) FISH signals of the PCR products of HaCENH3CL20. (D) A merged image of (A–C). (H) A merged image of (E–G). (L) A merged image of (I–K). (Q) A merged image of (N–P). (U) A merged image of (R–T). (I–L) Enlarged images of (E–H). (M) Karyograms from Ceccarelli's report (2007). White arrowheads in (E–H) and black arrowheads in (M) indicate centromeres on the HaCENH3CL124-positive chromosomes and the reported chromosomes, respectively. Scale bar, 10 μm.

Figure 5

Immunostaining using the anti-HaCENH3 and anti-H3K4me2 antibodies. (A–E) An interphase nucleus. (F–J) Prophase chromosomes. (K–O) Metaphase chromosomes. Scale bar, 10 μm.

Figure 6

Immunostaining and immunohistochemical staining using the anti-HaCENH3 and anti-H3K9me2 antibodies. (A–O) Immunostaining images. (A–E) An interphase nucleus. (F–J) Prophase chromosomes. (K–O) Metaphase chromosomes. (P–T) Immunohistochemical staining images of metaphase chromosomes. Scale bar, 10 μm. (U) Scanning of metaphase chromosomes. Scanned positions are indicated as red lines. (V) A vertical scan of the chromosome. (W) A horizontal scan of the chromosome.