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Characterization of CENH3 proteins and centromere-associated DNA sequences in diploid and allotetraploid Brassica species

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Abstract

CENH3 is a centromere-specific histone H3 variant and has been used as a marker to identify active centromeres and DNA sequences associated with functional centromere/kinetochore complexes. In this study, up to four distinct CENH3 (BrCENH3) cDNAs were identified in individuals of each of three diploid species of Brassica. Comparison of the BrCENH3 cDNAs implied three related gene families: BrCENH3-A in Brassica rapa (AA), BrCENH3-B in B. nigra (BB), and BrCENH3-C in B. oleracea (CC). Each family encoded a histone fold domain and N-terminal histone tails that vary in length in all three families. The BrCENH3-B cDNAs have a deletion of two exons relative to BrCENH3-A and BrCENH3-C, consistent with the more ancient divergence of the BB genome. Chromatin immunoprecipitation and immunolabeling tests with anti-BrCENH3 antibodies indicated that both centromeric tandem repeats and the centromere-specific retrotransposons of Brassica are directly associated with BrCENH3 proteins. In three allotetraploid species, we find either co-transcription of the BrCENH3 genes of the ancestral diploid species or gene suppression of the BrCENH3 from one ancestor. Although B genome centromeres are occupied by BrCENH3-B in the ancestral species B. nigra, in allotetraploids both BrCENH3-A and BrCENH3-C proteins appear to assemble at these centromeres.

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Acknowledgments

This research was supported by the Natural Science Foundation of China (31025018), by the Ministry of Science and Technology (2011CB944600), and by the Program for National Transgenic Research Program of China (2008ZX08009-001; 2009ZX08010-003B) to W.W. Jin.

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Authors and Affiliations

  1. National Maize Improvement Center of China, Key Laboratory of Crop Genetic Improvement and Genome of Ministry of Agriculture, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100193, China

    Guixiang Wang, Qunyan He & Weiwei Jin

  2. Beijing Vegetable Research Center, National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China

    Guixiang Wang & Fan Liu

  3. State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

    Zhukuan Cheng

  4. Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA, 98109–1024, USA

    Paul B. Talbert

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  1. Guixiang Wang
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  2. Qunyan He
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  3. Fan Liu
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  4. Zhukuan Cheng
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  5. Paul B. Talbert
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  6. Weiwei Jin
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Correspondence to Paul B. Talbert or Weiwei Jin.

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Communicated by L. Comai

Guixiang Wang and Qunyan He contributed equally to this work.

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Fig. S1

RACE sequences from B. rapa cultivar Beijingxin2 (AA). 5-RACE generated 5 clones representing two sequences, A2 and A3. 3-RACE generated 3 clones with a single sequence. The positions of 5′ and 3′ RACE primers are underlined. The start and stop codons of the open reading frame are in bold font (DOC 26 kb)

Fig. S2

Alignment of Brassica cDNAs. Brassica cDNAs comprise three large groups (BrCENH3-A, BrCENH3-B, and BrCENH3-C) differentiated by common nucleotide substitutions and length variations. Nucleotides that differ from the consensus are marked: BrCENH3-A—pink, BrCENH3-B—blue, BrCENH3-C—yellow. The primers used to amplify the cDNAs are shown in gray. The regions of the cDNA clones corresponding to the primers may not reflect exactly the sequences of the underlying genes except for A2 and A3, which represent complete coding sequences of BrCENH3-1 as determined by RACE. Inferred intron positions are marked with |. The predicted coding sequences from four genomic sequences from B. juncea (AB_AB299179 and AB_AB299179) and B. oleracea (C_AB299276 and C_AB299276) are included for reference (DOC 64 kb)

Fig. S3

Genomic sequences from Brassica rapa. Genomic sequences from the BrCENH3-A coding regions of B. rapa accessions Beijingxin2, Jingchunbai98-7, or from both accessions. The genomic sequence of the BrCENH3-A gene of B. juncea (AB299179) is shown for comparison. Overlines indicate the location of PCR primers. Exons are in blue type, and introns are in black type. Alternative donor and acceptor splice sites bordering the third intron are in bold italics (DOC 42.5 kb)

Fig. S4

The Prokaryotic Expression of pGEX-4T-2+BrCENH3. M, protein marker; 1, uninduced bacteria transfected with pGEX-4T-2; 2, bacteria (transfected with pGEX-4T-2) after induction with IPTG; 3 and 5, uninduced bacteria Rosetta transfected with pGEX-4T+BrCENH3; 4 and 6, Bacteria Rosetta (transfected with pGEX-4T+BrCENH3) after induction with IPTG (DOC 206 kb)

Supplementary Table S1

Primers for amplying CENH3 in this study (DOC 44 kb)

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Wang, G., He, Q., Liu, F. et al. Characterization of CENH3 proteins and centromere-associated DNA sequences in diploid and allotetraploid Brassica species. Chromosoma 120, 353–365 (2011). https://doi.org/10.1007/s00412-011-0315-z

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