Abstract
Holocentric chromosomes occur in a number of independent eukaryotic lineages, and they form holokinetic kinetochores along the entire poleward chromatid surfaces. Due to this alternative chromosome structure, Luzula elegans sister chromatids segregate already in anaphase I followed by the segregation of the homologues in anaphase II. However, not yet known is the localization and dynamics of cohesin and the structure of the synaptonemal complex (SC) during meiosis. We show here that the α-kleisin subunit of cohesin localizes at the centromeres of both mitotic and meiotic metaphase chromosomes and that it, thus, may contribute to assemble the centromere in L. elegans. This localization and the formation of a tripartite SC structure indicate that the prophase I behaviour of L. elegans is similar as in monocentric species.
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Abbreviations
- dNTP:
-
Deoxynucleoside triphosphates
- PCR:
-
Polymerase chain reaction
- DNA:
-
Deoxyribonucleic acid
- SIM:
-
Structured illumination microscopy
- SMC:
-
Structural maintenance of chromosome
- SC:
-
Synaptonemal complex
- RACE:
-
Rapid amplification of cDNA ends
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Acknowledgments
We are grateful to all members of the Chromosome Structure & Function laboratory (IPK Gatersleben), Ingo Schubert (IPK) and E-Eva Tomaštíková (Centre of the Region Haná for Biotechnological and Agricultural, Research, Institute of Experimental Botany, Olomouc, Czech Republic) for fruitful discussions; to Karla Meier, Katrin Kumke, Oda Weiß, Isolde Tillack and Gresch Ulrike (IPK) for excellent technical assistance; to Anne Fiebig for sequence submission; and to Karin Lipfert (IPK) for help with artwork. This work was supported by the China CSC scholarship, the Deutsche Forschungsgemeinschaft (SPP 1384, HO 1779/17-1) and the IPK Gatersleben.
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Supplemental Figure 1
CENH3 of L. elegans. (A) Gene structure model of LeCENH3, the positions of start/stop codons, and the in silico identified sequence. The obtained 5’ and 3’ RACE sequences and primer sites are indicated. (B) Alignment of CENH3 sequences from two Luzula species. The conserved domains of CENH3 are indicated by rectangle frames. (C) Phylogenetic analysis of CENH3 proteins from different species. (D) Western blot analysis using anti-LeCENH3, anti-histone H3 and anti-α-tubulin (as control) antibodies. The triangle indicates the band observed corresponding to the LeCENH3 protein. The total protein was extracted from L. elegans flower buds. (PDF 466 kb) (PDF 466 kb)
Supplemental Figure 2
The α-kleisins of L. elegans. (A) Phylogenetic analysis of α-kleisin-like proteins from different plant species. Reference IDs for the phylogenetic analysis of the α-kleisin sequences used in this study are available in Supplemental Table 2. (B) Protein structure model of four Luzula α-kleisin-like proteins based on in silico identification. The similarity of N-terminal conserved regions (red) among each other is indicated. (C) Gene structure model of Leα-kleisin-1 transcripts. Positions of start/stop codon and primer sites are indicated. (D) The purified recombinant Leα-kleisin protein was analyzed by Coomassie staining (blue gel on left) and Western blotting (black picture on right) with anti-6X His tag antibodies. The major band observed corresponds to the Leα-kleisin protein (triangle). (E) The purified recombinant Leα-kleisin protein was analyzed by Leα-kleisin recombinant antibody. The major band observed corresponds to the Leα-kleisin protein (triangle). (PDF 589 kb)
Supplemental Figure 3
The distribution of Leα-kleisin (red) and LeCENH3 (green) at pachytene of L. elegans was identified by SIM. The panels below show the regions of interest (rectangle) further magnified. (PDF 94 kb)
Supplemental Figure 4
The distribution of anti-OsSGO1 (red) along metaphase II chromosomes of L. elegans. (PDF 43 kb)
Supplemental Figure 5
The centromere localization of Leα-kleisin (red) in somatic metaphase chromosomes of V. faba was identified by SIM. (PDF 119 kb)
Supplemental Table 1
List of primer sequences for PCR, RT-PCR and FISH. (DOCX 18 kb)
Supplemental Table 2
List of sequence identifiers and description of α-kleisin sequences used for phylogenetic tree construction. (DOCX 17 kb)
Supplemental Table 3
The similarity of different Leα-kleisin protein sequences. (DOCX 17 kb)
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Ma, W., Schubert, V., Martis, M.M. et al. The distribution of α-kleisin during meiosis in the holocentromeric plant Luzula elegans . Chromosome Res 24, 393–405 (2016). https://doi.org/10.1007/s10577-016-9529-5
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DOI: https://doi.org/10.1007/s10577-016-9529-5