Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres

Abstract

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

“…From a library constructed using the affinity-purified DNA, 276 clones were randomly picked up, and their DNA sequences were determined. Among these sequences, seven and five were similar to the previously reported centromeric retrotransposons NtCR and NtoCR (Nagaki et al 2011), respectively. Furthermore, six different tandem repetitive DNA sequences were identified (Table 1), and the sizes of the repeat units ranged from 48 to 180 bp ( Table 1).…”

“…2). In this qPCR analysis, 25SrDNA and 5SrDNA were used as non-centromeric controls, and NtCR and NtoCR were used as centromeric controls (Nagaki et al 2011). As a result, NtCR and NtoCR were enriched significantly (P \ 0.0001 for both) in the NtCENH3-bound DNA pool compared with 25SrDNA and 5SrDNA, which were not enriched significantly (P \ 0.8) in the pool (Fig.…”

“…Fluorescence in situ hybridization FISH analysis of mitotic metaphase chromosomes was performed as previously described (Nagaki et al 2011). Chromosomes were prepared from the root tips of tobacco SR1 plants.…”

“…Recently, two centromeric retrotransposons, NtCR and NtoCR, have been identified by ChIP using antiNtCENH3 antibodies (Nagaki et al 2009a(Nagaki et al , 2011. NtoCR is specifically located on the centromeres of T-genome chromosomes derived from N. tomentosiformis, whereas NtCR is located on the centromeres of both ancestral species (Nagaki et al 2011). NtCR shows a sequence similarity with a tomato centromeric retrotransposon, TGRIV (Chang et al 2008;Nagaki et al 2011).…”

“…NtoCR is specifically located on the centromeres of T-genome chromosomes derived from N. tomentosiformis, whereas NtCR is located on the centromeres of both ancestral species (Nagaki et al 2011). NtCR shows a sequence similarity with a tomato centromeric retrotransposon, TGRIV (Chang et al 2008;Nagaki et al 2011). However, tandem repeats or micro-satellites, which are found in most centromeres of higher eukaryotes, have not been identified in the Nicotiana species.…”