Chromatin immunoprecipitation cloning reveals rapid evolutionary patterns of centromeric DNA in Oryza species

Abstract

The functional centromeres of rice (Oryza sativa, AA genome) chromosomes contain two key DNA components: the CRR centromeric retrotransposons and a 155-bp satellite repeat, CentO. However, several wild Oryza species lack the CentO repeat. We developed a chromatin immunoprecipitation-based technique to clone DNA fragments derived from chromatin containing the centromeric histone H3 variant CenH3. Chromatin immunoprecipitation cloning was carried out in the CentO-less species Oryza rhizomatis (CC genome) and Oryza brachyantha (FF genome). Three previously uncharacterized genome-specific satellite repeats, CentO-C1, CentO-C2, and CentO-F, were discovered in the centromeres of these two species. An 80-bp DNA region was found to be conserved in CentO-C1, CentO, and centromeric satellite repeats from maize and pearl millet, species which diverged from rice many millions of years ago. In contrast, the CentO-F repeat shows no sequence similarity to other centromeric repeats but has almost completely replaced other centromeric sequences in O. brachyantha, including the CRR-related sequences that normally constitute a significant fraction of the centromeric DNA in grass species.

Fig. 1.

Presence of the CentO repeat and CenH3 protein in different Oryza species. (A) Southern blot hybridization of the CentO repeat to TaqI-digested genomic DNA from lanes: 1, O. sativa (spp. japonica); 2, O. sativa (spp. indica); 3, O. glaberrima;4, O. rufipogon;5, O. nivara;6, O. meridinalis;7, O. minuta;8, O. officinalis;9, O. eichingeri; 10, O. rhizomatis; 11, O. alta; 12, O. grandiglumis; 13, O. latifolia; 14, O. australiensis; 15, O. brachyantha; 16, O. granulata; 17, O. meyeriana. The genome assignment of each species is indicated at the top of each lane. Lane M is a 100-bp DNA ladder. Note that no hybridization signals were detected in CC-, FF- and GG-genome species. Weak hybridization was observed in lane 13 after a longer exposure of the film. (B) Immunoassaying on somatic metaphase chromosomes of O. rhizomatis by using a rice anti-CenH3 antibody. (C) Immunoassaying on somatic metaphase chromosomes of O. brachyantha by using a rice anti-CenH3 antibody. (Scale bars: 5 μm.)

Fig. 2.

The proportions of centromeric DNA elements isolated by ChIP cloning in O. rhizomatis and O. brachyantha. Note that the ChIP cloning libraries may contain various type of DNA sequences, and only the preselected high-copy elements are included in the diagrams.

Fig. 3.

Distribution of the centromeric satellite repeats CentO-C1, CentO-C2, and CentO-F in different Oryza species. (A) HaeIII-digested genomic DNAs were probed with the CentO-C1 repeat. (B) TaqI-digested genomic DNAs were probed with the CentO-C2 repeat. (C) Tru9-digested genomic DNAs were probed with the CentO-F repeat. Lanes: 1, O. sativa (spp. japonica); 2, O. sativa (spp. indica); 3, O. glaberrima;4, O. rufipogon;5, O. nivara;6, O. meridinalis; 7, O. minuta;8, O. officinalis;9, O. eichingeri; 10, O. rhizomatis; 11, O. alta; 12, O. grandiglumis; 13, O. latifolia; 14, O. australiensis; 15, O. brachyantha; 16, O. granulata; 17, O. meyeriana. The genome assignment of each species is indicated at the top of each lane.

Fig. 4.

FISH mapping of centromeric repeats. (A) FISH signals derived from satellite repeat CentO-C1. Arrows point to nine unambiguous hybridization sites. (B) FISH signals derived from the rice CRR probe (23). (C) The FISH signals from CentO-C1 and CRR are merged with the pachytene chromosomes of O. rhizomatis.(D) FISH signals derived from satellite repeat CentO-C2. Arrowheads point to hybridization sites at the centromeres. (E) The FISH signals from CentO-C2 are merged with the somatic metaphase chromosomes of O. rhizomatis. (F) FISH mapping of CentO-C2 to the pachytene chromosomes of O. rhizomatis. Arrows point to unambiguous centromeric signals. (G) FISH mapping of CentO-C1 (red signals) and CentO (green signals) to the somatic metaphase chromosomes of O. punctata (BBCC). (H) FISH mapping of CentO-C1 (red signals) and CentO (green signals) to the somatic metaphase chromosomes of O. latifolia (CCDD). (I) FISH mapping of the CentO-F satellite repeat to the pachytene chromosomes of O. brachyantha. Chromosomes were counterstained with DAPI in all images and pseudocolored in either blue or red. (Scale bars: 5 μm in G and H; 10 μm in C, E, F, and I.)

Fig. 5.

Evolution of the CentO-C1 and its related centromeric satellite repeats. (A) Consensus sequences of CentO-C1 repeat of O. rhizomatis displayed as a sequence logo. The line indicates the location of the 80-bp region that is conserved among the CentO-C1, CentO, and CentC repeats. (B) Alignment of the 80-bp region from the consensus sequences of the CentO-C1, CentO (from O. sativa with AA genomes and O. alta with CCDD genomes), CentC (from Z. mays and T. Tripsacum genomes), and the centromeric repeat from P. glaucum. (C) Neighbor-joining tree of the 80-bp region from representative monomers of CentO-C1, CentO, CentC, and the Pennisetum subfamilies. The tree was constructed by using mega3 (Jukes–Cantor model, γ parameter of 2 for rate variation between sites, gaps included in the calculation). Numbers at branch points represent bootstrap values for 500 replications, and the scale bar represents estimated substitutions per site.