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Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-Rye Hybrids
Accepted Manuscript Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-rye Hybrids Shulan Fu, Zhenling Lv, Xiang Guo, Xiangqi Zhang, Fangpu Han PII:
S1673-8527(13)00103-3
DOI:
10.1016/j.jgg.2013.05.005
Reference:
JGG 217
To appear in:
Journal of Genetics and Genomics
Received Date: 14 March 2013 Revised Date:
28 April 2013
Accepted Date: 3 May 2013
Please cite this article as: Fu, S., Lv, Z., Guo, X., Zhang, X., Han, F., Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-rye Hybrids, Journal of Genetics and Genomics (2013), doi: 10.1016/j.jgg.2013.05.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Alteration of Terminal Heterochromatin and Chromosome Rearrangements in
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Derivatives of Wheat-rye Hybrids
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Shulan Fu a, b, Zhenling Lv a, Xiang Guoa, Xiangqi Zhang a, Fangpu Han a
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a
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and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
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b
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611130, China.
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics
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*Corresponding Author
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Phone: +86-10-6480 7926; Fax: +86-10-7485 4467
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E-mail: [email protected]
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Key Laboratory of Plant Breeding and Genetics, Sichuan Agriculture University, Chengdu
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The main text is composed of 16 pages, 4788 words and 6 figures. The number of words in the
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abstract is 153.
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Abstract: Wheat-rye addition and substitution lines and their self progenies revealed variations
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in telomeric heterochromatin and centromeres. Furthermore, a mitotically unstable dicentric
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chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese
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Spring-Imperial rye 3R addition line. An unstable multicentric chromosome was found in the
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progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including
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movement and disappearance of terminal heterochromatin occurred in the progeny of wheat-rye
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addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were
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observed in the progeny of a monosomic 4R addition line, a ditelosomic 5RS addition line and
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a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric
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DNA (TTTAGGG)n, derived from a monosomic 4R addition line are stable and transmissible to
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the next generation. The results indicated that centromeres and terminal heterochromatin can be
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profoundly altered in wheat-rye hybrid derivatives.
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Keywords: Wheat-rye addition lines; Chromosome rearrangements, Multiple Centromeres,
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Minichromosomes; Heterochromatin
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1. INTRODUCTION
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Heterochromatin plays an important role in maintaining the structure and function of
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centromeres and telomeres. Both terminal and centromere regions contain satellite repeats and
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retrotransposons that are usually packaged into heterochromatin (Pearce et al., 1996; Miller et
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al., 1998; Henikoff et al., 2001; Houben and Schubert, 2003; Bühler and Gasser, 2009). The
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dynamics of centromere and telomere evolution in plants has been reviewed (Nagaki et al.,
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2003; Fajkus et al., 2005; Ma et al., 2007). It has been reported that the activation of
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heterochromatic transcription can be affected by environmental and genetic stress conditions
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(Tittel-Elmer et al., 2010). Interspecific hybridization is among the stresses that trigger
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reorganization of the parental genomes (McClintock, 1978). Alteration of rye (Secale cereale
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L.) terminal heterochromatin in triticale (×Triticosecale Wittmack) and in progeny derived
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from triticale × wheat (Triticum ssp.) has been investigated (Appels et al., 1982; Gustafson et
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al., 1983; Lukaszewski and Gustafson 1983; Lapitan et al., 1984). Subsequently, fluorescence
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in situ hybridization (FISH) was used to detect the variation of terminal/subterminal
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heterochromatin of rye chromosomes in wheat-rye addition and substitution lines (Alkhimova
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et al., 1999). These previous studies focused on the variations of rye terminal/subterminal
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heterochromatin and the main variation patterns were deletion and amplification. Several
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studies on centromeres have focused on centromeric DNA sequences and proteins, epigenetic
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regulation and centromeric activity (Henikoff et al., 2001; Heit et al., 2006; Dalal et al., 2007).
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Although centromeres are usually maintained as unique loci on chromosomes, alteration in
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centromeres such as neocentromeres, holocentromeres, dicentric chromosomes, multiple
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centromeres and stable chromosomes without centromeric repeats have been reported (Kynast
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et al., 2000; Hiatt et al., 2002; Nasuda et al., 2005; Guerra et al., 2010; Gao et al., 2011; Fu et
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al., 2012; Neumann et al., 2012; Masonbrink et al., 2013; Fu et al., 2013). Holocentric
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chromosomes contain a kinetochore that spans the whole length of a chromosome.
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Holocentromeres have been reported in organisms such as insects (Heteroptera), worms
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(Parascaris univalens, Caenorhabditis elegans) and plants (Guerra et al., 2010). Dicentric
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chromosomes were formed through asymmetric chromosome translocation. In maize, dicentric
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chromosomes that involved A-B chromosomes and A-A chromosomes were described (Han et
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al., 2006, 2009; Gao et al., 2011). A transmissible dicentric chromosome was mentioned in
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wheat by Sears (1946). Sears and Camara (1952) further observed that this chromosome was
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derived from an isochromosome involving the short arm of chromosome 7, which was later
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specified to be chromosome 7B. This chromosome has three functional centromeres (Zhang et
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al., 2010). Multicentric chromosomes were reported for animals (Paweletz et al., 1989;
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Hadlaczky et al., 1991), wheat (where gametocidal genes could lead to the formation of
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multicentric chromosomes) (Kynast et al., 2000) and pea (Pisum sativum) (Neumann et al.,
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2012).
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In this study, we investigated the alterations of terminal and centromeric heterochromatin in
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wheat-rye alien addition, substitution and translocation lines. Some interesting phenomena
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including change of terminal heterochromatin position and formation of multi-centromeres,
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dicentric chromosomes and minichromosomes were uncovered.
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2. RESULTS
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2.1 Centromere variation in the progeny of the 3RCI disomic addition line
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In the self progeny of a 3RCI disomic addition line, we obtained a dicentric chromosome, which
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was a wheat-rye translocation chromosome (Fig. 1A). This chromosome is unstable because its
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two kinetochores may bind to spindle fibers from opposite poles, resulting in chromosome
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breakage during mitotic divisions, as evidenced by FISH screening in the root tip cells two
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months after planting (Fig. 1B). Simultaneously, a wheat chromosome with multiple
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centromeres was observed in the progeny of the 3RCI disomic addition line. This chromosome
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probably descended from chromosome 1B based on the arm ration and presence of a satellite
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(Fig. 1C). Immunolocalization of H2AThr133ph can detect multiple active centromeres (Dong
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and Han, 2012) that may in some cases behave as a single centromere during mitosis (Fig. 1D).
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In the next generation, this multi-centric chromosome was lost, suggesting its meiotic
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instability.
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2.2 Variations of rye terminal heterochromatin
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In the 3RS ditelosomic line, the rye terminal heterochromatic blocks can be distinguished by
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strong GISH signals at one end of the 3RS telosome (Fig. 2A). Subsequent FISH with the rye-
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specific subtelomeric tandem repeat pSc200 and the highly repetitive DNA sequence pSc119.2
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labeled the terminal region of 3RS (Fig. 2B) which coincided with the rye terminal
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heterochromatin. In another 3RS ditelosomic addition line 3RS-1, the terminal heterochromatin
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of the 3RS has moved to a subterminal position of 3RS (Fig. 2C) as did the signals of repetitive
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sequences, pSc119.2 and pSc200 (Fig. 2D). Among the selfed progeny of 3RS-1, in some
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plants the rye terminal heterochromatin and the highly repetitive DNA sequences pSc119.2 and
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pSc200 were lost (Fig. 2 E and F). 4
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In a line of the selfed progeny of the 5RS ditelosomic addition line (MK5RSdite), all mitotic root
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cells possessed a chromosome fragment containing the terminal heterochromatin of rye
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chromosome arm 5RS (Fig. 3). However, the terminal heterochromatin was moved into a
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subterminal position (Fig. 3A and C). Subsequent FISH using pSc119.2 as probe confirmed this
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presumption (Fig. 3 B and D). Another kind of minichromosomes derived from 5RS, which lost
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the terminal heterochromatin, but retained the CRW signal, were also observed in some mitotic
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cells (Fig. 3C and D)
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2.3 Minichromosomes in progeny of 4R addition lines
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One particular wheat line (MK4Rmon-mini) was detected among the self progeny of the 4R
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monosomic addition line MK4Rmon. GISH and FISH using rye genomic DNA (green) and
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CRW (red) as probes indicated that the two minichromosomes were rye chromosomes (Fig.
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4A). However, only one minichromosome contained telomere signals. The other
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minichromosome is either a ring or a telomere-depleted linear chromosome (Fig. 4B). Some
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self progeny of MK4Rmon-mini still contained minichromosomes, and four types of
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minichromosomes were observed. The first one was the same as that in MK4Rmon-mini. The
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second one contained two minichromosomes with telomere signals indicating a linear structure
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(Fig. 4C). The last two types contained only one minichromosome with or without telomere
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signals (Fig. 4D and E). Immunolocalization revealed CENH3 on minichromosomes with and
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without the telomere signals (Fig. 4F). The minichromosomes with telomere signals could be
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transmitted to the next generation at a frequency of about 55%, while the transmission
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frequency of minichromosomes without telomere oligonucleotide signals was only about 10%.
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2.4 Variation of centromere in 6R/6D substitution line
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In one of the self progeny of the 6R/6D substitution line, several kinds of special chromosomes
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were discovered. These special chromosomes included a minichromosome that was almost
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totally composed of rye centromere sequence (Fig. 5A), a small ring chromosome having two
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rye centromeres (Fig. 5B), a large ring chromosome possessing four rye centromeres (Fig. 5C)
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and a multicentromeric chromosome containing four rye centromeres (Fig. 5D). In this
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multicentromeric chromosome, the four centromeres were similar in size and appeared as
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individual units interspersed by segments of noncentromeric chromatin (Fig. 5D). All
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minichromosomes, small ring chromosomes and multicentromeric chromosomes originated
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from rye chromosome 6R (Fig. 5 E and F).
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3. DISCUSSION
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3.1 Variation in rye terminal heterochromatin
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The mechanism for the observed positional change of terminal rye heterochromatin variation
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(Figs. 2 and 3) remains to be elucidated. It has been inferred that amplification of terminal
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heterochromatin may be caused by non-reciprocal translocation (Lapitan et al., 1984), but non-
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reciprocal translocations have never been proved experimentally. Rather, paracentric inversion
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could have been the reason for a shift into subterminal position, and intrachromosomal
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recombination between directed repeats for deletion of the rye terminal heterochromatin.
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Alternatively, unbalanced segregation from reciprocal translocations can yield daughter nuclei
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with duplications and deletions of chromosomes (Schubert and Lysak, 2011).
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3.2 Formation of minichromosomes and multicentromeres
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Minichromosomes and multicentromeres have been discovered in transformed mouse and rat
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cells (Paweletz et al., 1989; Hadlaczky et al., 1991). Minichromosomes in human cells were
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obtained by adding telomeric DNA to break ends of the Y chromosome (Heller et al., 1996).
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Minichromosomes were also reported for maize (Kato et al., 2005; Han et al., 2007). Maize
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minichromosomes were originally generated by the breakage-fusion-bridge (BFB) cycle
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(McClintock, 1941). Engineered minichromosomes were constructed in maize by modifying
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natural A and supernumerary B chromosomes (Yu et al., 2007). In the present study,
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minichromosomes in progenies of the 4R monosomic addition line, the 5RS ditelosomic
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addition line (Figs. 3 and 4) and the 6R/6D substitution line (Fig. 5) were observed. We assume
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that rye chromosome arms in a wheat background facilitate genomic instability.
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Multicentromeres were observed in the progeny of the 3R disomic addition line on a wheat
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chromosome and in the progeny of 6R/6D substitution lines within the rye part of a wheat-rye
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translocation chromosome (Fig. 1C; Fig. 5Dand E). The mechanisms for the formation of
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multicentromeres in 3R disomic addition and 6R/6D substitution lines should be different from
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the ones in the wheat containing 4Mg chromosome (Kynast et al., 2000) because there are no
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gametocidal genes in the materials used in the present study. Paweletz et al. (1989) put forward
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that the multicentromere was formed through formation of a side-arm bridge and rejoining of
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broken ends. The wheat multicentromere in the 3R disomic addition line should have been
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formed by transposition of centromeric retrotransposons. It has been reported that wide
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(interspecific) hybridization can activate retrotransposons (Scheinker et al., 1990; Jiang et al.,
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2011). Centromeric variations including amplifications of centromere-specific satellite repeats
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and transposable elements were also observed in interspecific macropodid hybrids (Metcalfe et
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al., 2007). It seems possible that the 3R chromosomes added into wheat can provoke structural
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variation of chromosomes and expansion of repetitive DNA sequences by retrotransposition.
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The formation of minichromosomes and multicentromeres occurred together in the 6R/6D
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substitution line. A model is presented to explain their formation (Fig. 6). Breaks within both
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arms of chromosome 6R possibly led to a minichromosome containing a rye centromere. One
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of the break ends of the minichromosome fused after DNA replication and a small chromosome
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containing two rye centromeres −again with two break ends− was formed. There were two
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destinies for the small dicentric chromosome. First, the two broken ends of the small dicentric
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chromosome may fuse and form a dicentric ring chromosome. Second, one of the break ends of
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the linear dicentric chromosome fuses again after replication and a chromosome possessing four
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rye centromeres was formed. Alternatively, the tetracentric chromosome could result from a
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sister chromatid exchange within the dicentric ring chromosome, or the linear chromosome
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with four centromeres formed a ring by fusion of its ends. It has already been reported that
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multicentromeric chromosomes are unstable (Paweletz et al., 1989; Hadlaczky et al., 1991). In
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the present study, the multicentromere in the 6R/6D substitution line was also unstable. During
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mitotic divisions breaks could occur between the centromeres of the dicentric or the tetracentric
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chromosome; thus resulting in minichromosomes derived from dicentric or tetracentric
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chromosomes (Fig. 6).
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Remarkably, the multicentromere in the 3R disomic addition line was stable and could be
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perpetuated through mitosis. Page and Shaffer (1998) have investigated the stability of the
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centromere of a dicentric human chromosome resulting from a Robertsonian translocation
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between chromosomes 13 and 14, and found that close proximity of two functional centromeres
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favoured stability of dicentric chromosomes. Close physical proximity of the multicentromeres
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in the 3R disomic addition line made them behaving as a single centromere because instability
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caused by breakage of mitotic bridges occurs only when sister chromatids between centromeres
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take up a twisted instead of a parallel orientation. The probability of twisting increases with the
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distance between the centromeres.
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3.3 Formation of a dicentric chromosome
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The dicentric chromosome in this study was formed by asymmetric translocation of
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chromosome 3R and a wheat chromosome, similar as in cases described by (Lukaszewski 1995;
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Wang et al., 1995). Apparently, the dicentric chromosome in the present study was unstable,
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indicating that the wheat and the rye centromere were active and the distance between them too
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large to prevent mitotic instability. 7
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In conclusion, our results indicated that the variations of terminal heterochromatin and
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centromeres can be mediated by intergeneric hybridization.
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4.. MATERIALS AND METHODS
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4.1 Plant materials
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1R-7R wheat-rye disomic addition lines, were derived from Chinese Spring wheat × Imperial
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rye (1RCI-7RCI) and the disomic addition lines were from Chinese Spring wheat × KingII rye
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(1RCK-7RCK). These seed stocks were kindly provided by the USDA-Sears Wheat Stock
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Collection. A 3RS monotelosomic addition line and a 3RS ditelosomic line, was selected from
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self progeny of the 3RCI disomic addition line. The 4R monosomic addition line contains a
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single 4R chromosome of rye cultivar Kustro and the entire genome of wheat cultivar
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Mianyang11 (MK4Rmon). The 5RS ditelosomic addition line, a self progeny of 5R monosomic
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addition line, contains two short arms of chromosomes 5R of Kustro and the entire genome of
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wheat cultivar Mianyang11 (MK5RSdite). The wheat-rye 6R/6D substitution line is derived
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from Chinese Spring wheat × Imperial rye, These lines were created by our lab.
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4.2 Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH)
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Chromosome spreads of root tips were prepared according to the method described by Han et
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al. (2006). Rye genomic DNA, the cereal centromere-specific repetitive DNA sequence CRW
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(Liu et al., 2008), the rye centromere-specific repetitive DNA sequence pAWRC.1 (Francki,
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2001), the highly repetitive sequences, pSc119.2 and pSc200, cloned from S. cereale (McIntyre
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et al., 1990; Vershinin et al., 1995), the Aegilops tauschii clone pAs1 (Rayburn and Gill, 1986)
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and wheat telomeric DNA (TTTAGGG) were used as probes. Rye genomic DNA, pSc119.2,
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wheat telomeric DNA and pAWRC.1 were labeled with fluorescein-12-dUTP (PerkinElmer,
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USA). CRW, pSc200 and pAs1 were labeled with Texas-red-5-dUTP (PerkinElmer,USA).
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4.3 Immunolocalization in somatic cells
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Immunostaining of mitotic chromosomes was performed as described (Han et al., 2009), using
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antibodies against histone H2A phosphorylated at threonine residue 133 (H2AThr133ph) (Dong
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and Han, 2012) and a self-made antibody against wheat CENH3. Observations were made
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under an epifluorescence Olympus BX61 microscope (Olympus, Japan) equipped with a cooled
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charge-coupled device camera operated with MetaMorph software, and processed with
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Photoshop CS 3.0. 8
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ACKNOWLEDGEMENTS
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This work was supported by the grants of the National High Technology Research and
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Development Program (“863” Program) of China (No. 2011AA100101) and the Special
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Financial Grant from the China Postdoctoral Science Foundation (No. 2012T50157), and 2011
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collaborative innovation plan of the ministry of education of China. We would like to thank Dr.
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J. Perry Gustafson, University of Missouri, Columbia, USA for providing the wheat-rye
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addition lines used in this study, and Dr. James A. Birchler, University of Missouri, Columbia,
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USA for his critical reading of the manuscript.
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Lukaszewski, A.J., Gustafson, J.P., 1983. Translocations and modifications of chromosomes in triticale × wheat hybrids. Theor. Appl. Genet. 64, 239-248.
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Ma, J.X., Wing, R.A., Bennetzen, J.L., Jackson, S.A., 2007. Plant centromere organization: a dynamic structure with conserved functions. Trends Genet. 23, 134-139. Masonbrink, R.E., Fu,S.L., Han, F.P., Birchler, J.A., 2013. Heritable loss of replication control of a minichromosome derived from the B chromosome of maize. Genetics 193, 77-84.
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McIntyre, C.L., Pereira, S., Moran, L.B., Appels, R., 1990. New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat. Genome 33, 635-640. Metcalfe, C.J., Bulazel, K.V., Ferreri, G.C., Schroeder-Reiter, E., Wanner, G., Rens, W.,
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Obergfell, C., Eldridge, M.D., O'Neill, R.J., 2007. Genomic instability within centromeres of
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Nagaki, K., Song, J., Stupar, R.M., Parokonny, A.S., Yuan, Q.P., Ouyang, S., Liu, J., Hsiao, J.,
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Jones, K.M., Dawe, R.K., Buell, C.R., Jiang, J., 2003. Molecular and cytological analyses of
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large tracks of centromeric DNA reveal the structure and evolutionary dynamics of maize
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Nasuda, S., Hudakova, S., Schubert, I., Houben, A., Endo, T.R., 2005. Stable barley
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chromosomes without centromeric repeats. Proc. Natl. Acad. Sci. USA 102, 9842-9847.
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Neumann, P., Navrátilová, A., Schroeder-Reiter, E., Koblížková, A., Steinbauerová, V.,
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Chocholová, E., Novák, P., Wanner, G., Macas, J., 2012 Stretching the rules: monocentric
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chromosomes with multiple centromere domains. PLoS Genet. 8, e1002777.
Novello, A., Villar, S., 2006. Chromosome plasticity in Ctenomys (Rodentia Octodontidae):
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chromosome 1 evolution and heterochromatin variation. Genetica 127, 303-309. Page, S.L., Shaffer, L.G., 1998. Chromosome stability is maintained by short intercentromeric
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Paweletz, N., Vig, B.K., Finze, E.M., 1989. Evolution of compound centromeres: a new phenomenon. Cancer Genet. Cytogenet. 42, 75-86.
Pearce, S.R., Pich, U., Harrison, G., Flavell, A.J., Heslop-Harrison, J.S., Schubert, I., Kumar,
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the chromosomes but are enriched in the terminal heterochromatin. Chromosome Res. 4,
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Rayburn, A.L., Gill, B.S., 1986. Molecular identification of the D-genome chromosomes of wheat. J. Hered. 77, 253-255.
Scheinker, V.S., Lozovskaya, E.R., Bishop, J.G., Corces, V.G., Evgen'ev, M.B., 1990. A long
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terminal repeat-containing retrotransposon is mobilized during hybrid dysgenesis in
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Drosophila virilis. Proc. Natl. Acad. Sci. USA 87, 9615-9619.
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Schubert, I., Lysak, M.A., 2011. Interpretation of karyotype evolution should consider chromosome structural constraints. Trends Genet. 27, 207-216.
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Sears, E. R., 1946. Isochromosomes and telocentrics in Triticum vulgare. Genetics 31, 229-230.
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Sears, E. R., Camara, A., 1952. A transmissible dicentric chromosome. Genetics 37, 125-135.
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Tittel-Elmer, M., Bucher, E., Broger, L., Mathieu, O., Paszkowski, J., Vaillant I., 2010. Stress-
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induced activation of heterochromatic transcription. PLoS Genet. 6, e1001175. Vershinin, A.V., Schwarzacher, T., Heslop-Harrison, J.S., 1995. The large-scale genomic
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organization of repetitive DNA families at the telomeres of rye chromosomes. Plant Cell 7,
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1823-1833. 12
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Wang, Y.B., Hu, H., Snape, J.W., 1995. Spontaneous wheat/rye translocations from female
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meiotic products of hybrids between octoploid triticale and wheat. Euphytica 81, 265-270.
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Yu, W.C., Han, F.P., Gao, Z., Vega, J.M., Birchler, J.A., 2007. Construction and behavior of
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engineered minichromosomes in maize. Proc. Natl. Acad. Sci. USA 104, 8924-8929. Zhang, W., Friebe, B., Gill, B.S., Jiang, J., 2010. Centromere inactivation and epigenetic
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modifications of a plant chromosome with three functional centromeres. Chromosoma 119,
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553 563.
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FIGURE LEGENDS
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Fig. 1. Formation of dicentric chromosome and multicentromere in the self progeny of the 3RCI
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disomic addition line.
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A: GISH and FISH using rye genomic DNA (green) and CRW (red) as probes to identify the
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dicentric chromosome (arrow). B: GISH and FISH using rye genomic DNA (green) and CRW
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(red) as probes to detect the dicentric chromosome two months after planting. Arrows indicate
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the broken chromosomal arms from the dicentric chromosome. C: GISH and FISH using rye
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genomic DNA (green) and CRW (red) as probes to revealed a multicentromeric chromosome
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(arrow). D: immunolocalization of H2AThr133ph. Arrow indicates active centromeres. Bars =
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10 µm.
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Fig. 2. Variation of terminal heterochromatin in the self progeny of the 3RCI disomic addition
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line.
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A, C and E: GISH and FISH using rye genomic DNA (green) and CRW (red) as probes to
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detect the terminal heterochromatin on 3RS chromosome. B, D and F: FISH using pSc119.2
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(green) and pSc200 (red) to probe the same cell as in (A), (C) and (E). Arrows in (A) and (B)
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indicate the 3RS with normal telomeric heterochromatin at the end of the chromosome 3RS.
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Arrows in (C) and (D) indicate the 3RS arm with re-positioned telomeric heterochromatin and
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the repetitive sequences pSc119.2 and pSc200. Arrows in (E) and (F) indicate the deletion of
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terminal heterochromatin from the chromosome 3RS accompanied by loss of pSc119.2 and
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pSc200 signals. Bars = 10 µm.
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Fig. 3. Variation of terminal heterochromatin in the selfed progeny of 5RS ditelosomic addition
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line (MK5RSdite).
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A: chromosome fragment with terminal heterochromatin in subterminal position (arrow) as
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detected by GISH and FISH with rye genomic DNA (green) and CRW (red). B: FISH using
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pSc119.2 and pAs1 to probe the same cell as in (A). Arrow indicates the pSc119.2 signal at the
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subterminal region. C: a chromosome fragment with terminal heterochromatin at subterminal
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region (arrow) and a minichromosome without terminal heterochromatin (indicated by pointer)
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were detected by GISH and FISH using rye genomic DNA (green) and CRW (red) as probes.
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D: FISH with pSc119.2 and pAs1 to probe the same cell as in (C). Arrow indicates the
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pSc119.2 signal at the subterminal region. Pointer indicates the minichromosome without
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pSc119.2 signal. Bars = 10 µm.
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Fig. 4. Minichromosomes in the self progeny of the 4R monosomic addition line (MK4Rmon).
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A: GISH and FISH using rye genomic DNA (green) and CRW (red) as probes to detect the
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minichromosomes in wheat line MK4Rmon-mini (arrows). B−E: FISH using CRW (red) and
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wheat telomeric DNA (green) as probes to characterise the minichromosomes. Arrows indicate
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the minichromosomes with or without telomere signal. F: immunostaining of centromere-
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specific CENH3 (arrow) on the minichromosome. Bars = 10 µm.
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Fig. 5. Minichromosome and multicentromere in the self progeny of the 6R/6D substitution
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line.
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A: minichromosome (arrow) was detected by FISH using CRW (red) and pAWRC.1 (green) as
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probes. B: small ring chromosome with two rye centromeres (arrow) as detected by FISH using
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CRW (red) and pAWRC.1 (green) as probes. C: small ring chromosome with four rye
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centromeres (arrow) as detected by FISH using CRW (red) and pAWRC.1 (green) as probes.
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D: multicentromeric chromosome (arrow) detected by FISH using CRW (red) and pAWRC.1
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(green) as probes. E: GISH with rye genomic DNA applied to the same cell as in (D). Arrow
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indicates the multicentromeric chromosome. F: GISH with rye genomic DNA applied to the
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same cell as in (C). Arrow indicates the small ring chromosome with four rye centromeres.
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Bars = 10 µm.
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Fig. 6. Model for the formation of ring dicentric and tetracentric chromosomes, as well as of
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minichromosomes within the 6R/6D substitution line.
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S1673-8527(13)00103-3
DOI:
10.1016/j.jgg.2013.05.005
Reference:
JGG 217
To appear in:
Journal of Genetics and Genomics
Received Date: 14 March 2013 Revised Date:
28 April 2013
Accepted Date: 3 May 2013
Please cite this article as: Fu, S., Lv, Z., Guo, X., Zhang, X., Han, F., Alteration of Terminal Heterochromatin and Chromosome Rearrangements in Derivatives of Wheat-rye Hybrids, Journal of Genetics and Genomics (2013), doi: 10.1016/j.jgg.2013.05.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Alteration of Terminal Heterochromatin and Chromosome Rearrangements in
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Derivatives of Wheat-rye Hybrids
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Shulan Fu a, b, Zhenling Lv a, Xiang Guoa, Xiangqi Zhang a, Fangpu Han a
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a
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and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
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b
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611130, China.
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics
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*Corresponding Author
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Phone: +86-10-6480 7926; Fax: +86-10-7485 4467
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E-mail: [email protected]
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Key Laboratory of Plant Breeding and Genetics, Sichuan Agriculture University, Chengdu
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The main text is composed of 16 pages, 4788 words and 6 figures. The number of words in the
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abstract is 153.
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Abstract: Wheat-rye addition and substitution lines and their self progenies revealed variations
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in telomeric heterochromatin and centromeres. Furthermore, a mitotically unstable dicentric
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chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese
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Spring-Imperial rye 3R addition line. An unstable multicentric chromosome was found in the
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progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including
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movement and disappearance of terminal heterochromatin occurred in the progeny of wheat-rye
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addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were
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observed in the progeny of a monosomic 4R addition line, a ditelosomic 5RS addition line and
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a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric
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DNA (TTTAGGG)n, derived from a monosomic 4R addition line are stable and transmissible to
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the next generation. The results indicated that centromeres and terminal heterochromatin can be
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profoundly altered in wheat-rye hybrid derivatives.
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Keywords: Wheat-rye addition lines; Chromosome rearrangements, Multiple Centromeres,
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Minichromosomes; Heterochromatin
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1. INTRODUCTION
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Heterochromatin plays an important role in maintaining the structure and function of
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centromeres and telomeres. Both terminal and centromere regions contain satellite repeats and
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retrotransposons that are usually packaged into heterochromatin (Pearce et al., 1996; Miller et
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al., 1998; Henikoff et al., 2001; Houben and Schubert, 2003; Bühler and Gasser, 2009). The
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dynamics of centromere and telomere evolution in plants has been reviewed (Nagaki et al.,
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2003; Fajkus et al., 2005; Ma et al., 2007). It has been reported that the activation of
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heterochromatic transcription can be affected by environmental and genetic stress conditions
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(Tittel-Elmer et al., 2010). Interspecific hybridization is among the stresses that trigger
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reorganization of the parental genomes (McClintock, 1978). Alteration of rye (Secale cereale
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L.) terminal heterochromatin in triticale (×Triticosecale Wittmack) and in progeny derived
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from triticale × wheat (Triticum ssp.) has been investigated (Appels et al., 1982; Gustafson et
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al., 1983; Lukaszewski and Gustafson 1983; Lapitan et al., 1984). Subsequently, fluorescence
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in situ hybridization (FISH) was used to detect the variation of terminal/subterminal
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heterochromatin of rye chromosomes in wheat-rye addition and substitution lines (Alkhimova
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et al., 1999). These previous studies focused on the variations of rye terminal/subterminal
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heterochromatin and the main variation patterns were deletion and amplification. Several
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studies on centromeres have focused on centromeric DNA sequences and proteins, epigenetic
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regulation and centromeric activity (Henikoff et al., 2001; Heit et al., 2006; Dalal et al., 2007).
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Although centromeres are usually maintained as unique loci on chromosomes, alteration in
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centromeres such as neocentromeres, holocentromeres, dicentric chromosomes, multiple
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centromeres and stable chromosomes without centromeric repeats have been reported (Kynast
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et al., 2000; Hiatt et al., 2002; Nasuda et al., 2005; Guerra et al., 2010; Gao et al., 2011; Fu et
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al., 2012; Neumann et al., 2012; Masonbrink et al., 2013; Fu et al., 2013). Holocentric
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chromosomes contain a kinetochore that spans the whole length of a chromosome.
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Holocentromeres have been reported in organisms such as insects (Heteroptera), worms
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(Parascaris univalens, Caenorhabditis elegans) and plants (Guerra et al., 2010). Dicentric
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chromosomes were formed through asymmetric chromosome translocation. In maize, dicentric
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chromosomes that involved A-B chromosomes and A-A chromosomes were described (Han et
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al., 2006, 2009; Gao et al., 2011). A transmissible dicentric chromosome was mentioned in
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wheat by Sears (1946). Sears and Camara (1952) further observed that this chromosome was
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derived from an isochromosome involving the short arm of chromosome 7, which was later
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specified to be chromosome 7B. This chromosome has three functional centromeres (Zhang et
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al., 2010). Multicentric chromosomes were reported for animals (Paweletz et al., 1989;
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Hadlaczky et al., 1991), wheat (where gametocidal genes could lead to the formation of
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multicentric chromosomes) (Kynast et al., 2000) and pea (Pisum sativum) (Neumann et al.,
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2012).
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In this study, we investigated the alterations of terminal and centromeric heterochromatin in
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wheat-rye alien addition, substitution and translocation lines. Some interesting phenomena
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including change of terminal heterochromatin position and formation of multi-centromeres,
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dicentric chromosomes and minichromosomes were uncovered.
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2. RESULTS
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2.1 Centromere variation in the progeny of the 3RCI disomic addition line
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In the self progeny of a 3RCI disomic addition line, we obtained a dicentric chromosome, which
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was a wheat-rye translocation chromosome (Fig. 1A). This chromosome is unstable because its
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two kinetochores may bind to spindle fibers from opposite poles, resulting in chromosome
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breakage during mitotic divisions, as evidenced by FISH screening in the root tip cells two
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months after planting (Fig. 1B). Simultaneously, a wheat chromosome with multiple
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centromeres was observed in the progeny of the 3RCI disomic addition line. This chromosome
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probably descended from chromosome 1B based on the arm ration and presence of a satellite
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(Fig. 1C). Immunolocalization of H2AThr133ph can detect multiple active centromeres (Dong
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and Han, 2012) that may in some cases behave as a single centromere during mitosis (Fig. 1D).
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In the next generation, this multi-centric chromosome was lost, suggesting its meiotic
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instability.
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2.2 Variations of rye terminal heterochromatin
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In the 3RS ditelosomic line, the rye terminal heterochromatic blocks can be distinguished by
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strong GISH signals at one end of the 3RS telosome (Fig. 2A). Subsequent FISH with the rye-
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specific subtelomeric tandem repeat pSc200 and the highly repetitive DNA sequence pSc119.2
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labeled the terminal region of 3RS (Fig. 2B) which coincided with the rye terminal
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heterochromatin. In another 3RS ditelosomic addition line 3RS-1, the terminal heterochromatin
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of the 3RS has moved to a subterminal position of 3RS (Fig. 2C) as did the signals of repetitive
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sequences, pSc119.2 and pSc200 (Fig. 2D). Among the selfed progeny of 3RS-1, in some
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plants the rye terminal heterochromatin and the highly repetitive DNA sequences pSc119.2 and
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pSc200 were lost (Fig. 2 E and F). 4
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In a line of the selfed progeny of the 5RS ditelosomic addition line (MK5RSdite), all mitotic root
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cells possessed a chromosome fragment containing the terminal heterochromatin of rye
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chromosome arm 5RS (Fig. 3). However, the terminal heterochromatin was moved into a
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subterminal position (Fig. 3A and C). Subsequent FISH using pSc119.2 as probe confirmed this
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presumption (Fig. 3 B and D). Another kind of minichromosomes derived from 5RS, which lost
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the terminal heterochromatin, but retained the CRW signal, were also observed in some mitotic
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cells (Fig. 3C and D)
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2.3 Minichromosomes in progeny of 4R addition lines
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One particular wheat line (MK4Rmon-mini) was detected among the self progeny of the 4R
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monosomic addition line MK4Rmon. GISH and FISH using rye genomic DNA (green) and
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CRW (red) as probes indicated that the two minichromosomes were rye chromosomes (Fig.
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4A). However, only one minichromosome contained telomere signals. The other
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minichromosome is either a ring or a telomere-depleted linear chromosome (Fig. 4B). Some
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self progeny of MK4Rmon-mini still contained minichromosomes, and four types of
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minichromosomes were observed. The first one was the same as that in MK4Rmon-mini. The
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second one contained two minichromosomes with telomere signals indicating a linear structure
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(Fig. 4C). The last two types contained only one minichromosome with or without telomere
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signals (Fig. 4D and E). Immunolocalization revealed CENH3 on minichromosomes with and
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without the telomere signals (Fig. 4F). The minichromosomes with telomere signals could be
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transmitted to the next generation at a frequency of about 55%, while the transmission
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frequency of minichromosomes without telomere oligonucleotide signals was only about 10%.
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2.4 Variation of centromere in 6R/6D substitution line
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In one of the self progeny of the 6R/6D substitution line, several kinds of special chromosomes
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were discovered. These special chromosomes included a minichromosome that was almost
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totally composed of rye centromere sequence (Fig. 5A), a small ring chromosome having two
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rye centromeres (Fig. 5B), a large ring chromosome possessing four rye centromeres (Fig. 5C)
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and a multicentromeric chromosome containing four rye centromeres (Fig. 5D). In this
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multicentromeric chromosome, the four centromeres were similar in size and appeared as
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individual units interspersed by segments of noncentromeric chromatin (Fig. 5D). All
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minichromosomes, small ring chromosomes and multicentromeric chromosomes originated
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from rye chromosome 6R (Fig. 5 E and F).
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3. DISCUSSION
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3.1 Variation in rye terminal heterochromatin
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The mechanism for the observed positional change of terminal rye heterochromatin variation
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(Figs. 2 and 3) remains to be elucidated. It has been inferred that amplification of terminal
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heterochromatin may be caused by non-reciprocal translocation (Lapitan et al., 1984), but non-
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reciprocal translocations have never been proved experimentally. Rather, paracentric inversion
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could have been the reason for a shift into subterminal position, and intrachromosomal
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recombination between directed repeats for deletion of the rye terminal heterochromatin.
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Alternatively, unbalanced segregation from reciprocal translocations can yield daughter nuclei
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with duplications and deletions of chromosomes (Schubert and Lysak, 2011).
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3.2 Formation of minichromosomes and multicentromeres
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Minichromosomes and multicentromeres have been discovered in transformed mouse and rat
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cells (Paweletz et al., 1989; Hadlaczky et al., 1991). Minichromosomes in human cells were
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obtained by adding telomeric DNA to break ends of the Y chromosome (Heller et al., 1996).
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Minichromosomes were also reported for maize (Kato et al., 2005; Han et al., 2007). Maize
153
minichromosomes were originally generated by the breakage-fusion-bridge (BFB) cycle
154
(McClintock, 1941). Engineered minichromosomes were constructed in maize by modifying
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natural A and supernumerary B chromosomes (Yu et al., 2007). In the present study,
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minichromosomes in progenies of the 4R monosomic addition line, the 5RS ditelosomic
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addition line (Figs. 3 and 4) and the 6R/6D substitution line (Fig. 5) were observed. We assume
158
that rye chromosome arms in a wheat background facilitate genomic instability.
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Multicentromeres were observed in the progeny of the 3R disomic addition line on a wheat
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chromosome and in the progeny of 6R/6D substitution lines within the rye part of a wheat-rye
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translocation chromosome (Fig. 1C; Fig. 5Dand E). The mechanisms for the formation of
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multicentromeres in 3R disomic addition and 6R/6D substitution lines should be different from
163
the ones in the wheat containing 4Mg chromosome (Kynast et al., 2000) because there are no
164
gametocidal genes in the materials used in the present study. Paweletz et al. (1989) put forward
165
that the multicentromere was formed through formation of a side-arm bridge and rejoining of
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broken ends. The wheat multicentromere in the 3R disomic addition line should have been
167
formed by transposition of centromeric retrotransposons. It has been reported that wide
168
(interspecific) hybridization can activate retrotransposons (Scheinker et al., 1990; Jiang et al.,
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2011). Centromeric variations including amplifications of centromere-specific satellite repeats
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and transposable elements were also observed in interspecific macropodid hybrids (Metcalfe et
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al., 2007). It seems possible that the 3R chromosomes added into wheat can provoke structural
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variation of chromosomes and expansion of repetitive DNA sequences by retrotransposition.
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The formation of minichromosomes and multicentromeres occurred together in the 6R/6D
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substitution line. A model is presented to explain their formation (Fig. 6). Breaks within both
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arms of chromosome 6R possibly led to a minichromosome containing a rye centromere. One
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of the break ends of the minichromosome fused after DNA replication and a small chromosome
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containing two rye centromeres −again with two break ends− was formed. There were two
178
destinies for the small dicentric chromosome. First, the two broken ends of the small dicentric
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chromosome may fuse and form a dicentric ring chromosome. Second, one of the break ends of
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the linear dicentric chromosome fuses again after replication and a chromosome possessing four
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rye centromeres was formed. Alternatively, the tetracentric chromosome could result from a
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sister chromatid exchange within the dicentric ring chromosome, or the linear chromosome
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with four centromeres formed a ring by fusion of its ends. It has already been reported that
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multicentromeric chromosomes are unstable (Paweletz et al., 1989; Hadlaczky et al., 1991). In
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the present study, the multicentromere in the 6R/6D substitution line was also unstable. During
186
mitotic divisions breaks could occur between the centromeres of the dicentric or the tetracentric
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chromosome; thus resulting in minichromosomes derived from dicentric or tetracentric
188
chromosomes (Fig. 6).
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Remarkably, the multicentromere in the 3R disomic addition line was stable and could be
190
perpetuated through mitosis. Page and Shaffer (1998) have investigated the stability of the
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centromere of a dicentric human chromosome resulting from a Robertsonian translocation
192
between chromosomes 13 and 14, and found that close proximity of two functional centromeres
193
favoured stability of dicentric chromosomes. Close physical proximity of the multicentromeres
194
in the 3R disomic addition line made them behaving as a single centromere because instability
195
caused by breakage of mitotic bridges occurs only when sister chromatids between centromeres
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take up a twisted instead of a parallel orientation. The probability of twisting increases with the
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distance between the centromeres.
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3.3 Formation of a dicentric chromosome
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The dicentric chromosome in this study was formed by asymmetric translocation of
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chromosome 3R and a wheat chromosome, similar as in cases described by (Lukaszewski 1995;
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Wang et al., 1995). Apparently, the dicentric chromosome in the present study was unstable,
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indicating that the wheat and the rye centromere were active and the distance between them too
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large to prevent mitotic instability. 7
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In conclusion, our results indicated that the variations of terminal heterochromatin and
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centromeres can be mediated by intergeneric hybridization.
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4.. MATERIALS AND METHODS
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4.1 Plant materials
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1R-7R wheat-rye disomic addition lines, were derived from Chinese Spring wheat × Imperial
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rye (1RCI-7RCI) and the disomic addition lines were from Chinese Spring wheat × KingII rye
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(1RCK-7RCK). These seed stocks were kindly provided by the USDA-Sears Wheat Stock
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Collection. A 3RS monotelosomic addition line and a 3RS ditelosomic line, was selected from
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self progeny of the 3RCI disomic addition line. The 4R monosomic addition line contains a
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single 4R chromosome of rye cultivar Kustro and the entire genome of wheat cultivar
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Mianyang11 (MK4Rmon). The 5RS ditelosomic addition line, a self progeny of 5R monosomic
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addition line, contains two short arms of chromosomes 5R of Kustro and the entire genome of
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wheat cultivar Mianyang11 (MK5RSdite). The wheat-rye 6R/6D substitution line is derived
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from Chinese Spring wheat × Imperial rye, These lines were created by our lab.
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4.2 Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH)
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Chromosome spreads of root tips were prepared according to the method described by Han et
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al. (2006). Rye genomic DNA, the cereal centromere-specific repetitive DNA sequence CRW
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(Liu et al., 2008), the rye centromere-specific repetitive DNA sequence pAWRC.1 (Francki,
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2001), the highly repetitive sequences, pSc119.2 and pSc200, cloned from S. cereale (McIntyre
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et al., 1990; Vershinin et al., 1995), the Aegilops tauschii clone pAs1 (Rayburn and Gill, 1986)
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and wheat telomeric DNA (TTTAGGG) were used as probes. Rye genomic DNA, pSc119.2,
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wheat telomeric DNA and pAWRC.1 were labeled with fluorescein-12-dUTP (PerkinElmer,
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USA). CRW, pSc200 and pAs1 were labeled with Texas-red-5-dUTP (PerkinElmer,USA).
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4.3 Immunolocalization in somatic cells
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Immunostaining of mitotic chromosomes was performed as described (Han et al., 2009), using
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antibodies against histone H2A phosphorylated at threonine residue 133 (H2AThr133ph) (Dong
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and Han, 2012) and a self-made antibody against wheat CENH3. Observations were made
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under an epifluorescence Olympus BX61 microscope (Olympus, Japan) equipped with a cooled
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charge-coupled device camera operated with MetaMorph software, and processed with
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Photoshop CS 3.0. 8
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ACKNOWLEDGEMENTS
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This work was supported by the grants of the National High Technology Research and
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Development Program (“863” Program) of China (No. 2011AA100101) and the Special
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Financial Grant from the China Postdoctoral Science Foundation (No. 2012T50157), and 2011
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collaborative innovation plan of the ministry of education of China. We would like to thank Dr.
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J. Perry Gustafson, University of Missouri, Columbia, USA for providing the wheat-rye
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addition lines used in this study, and Dr. James A. Birchler, University of Missouri, Columbia,
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USA for his critical reading of the manuscript.
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FIGURE LEGENDS
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Fig. 1. Formation of dicentric chromosome and multicentromere in the self progeny of the 3RCI
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disomic addition line.
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A: GISH and FISH using rye genomic DNA (green) and CRW (red) as probes to identify the
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dicentric chromosome (arrow). B: GISH and FISH using rye genomic DNA (green) and CRW
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(red) as probes to detect the dicentric chromosome two months after planting. Arrows indicate
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the broken chromosomal arms from the dicentric chromosome. C: GISH and FISH using rye
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genomic DNA (green) and CRW (red) as probes to revealed a multicentromeric chromosome
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(arrow). D: immunolocalization of H2AThr133ph. Arrow indicates active centromeres. Bars =
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10 µm.
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Fig. 2. Variation of terminal heterochromatin in the self progeny of the 3RCI disomic addition
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line.
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A, C and E: GISH and FISH using rye genomic DNA (green) and CRW (red) as probes to
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detect the terminal heterochromatin on 3RS chromosome. B, D and F: FISH using pSc119.2
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(green) and pSc200 (red) to probe the same cell as in (A), (C) and (E). Arrows in (A) and (B)
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indicate the 3RS with normal telomeric heterochromatin at the end of the chromosome 3RS.
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Arrows in (C) and (D) indicate the 3RS arm with re-positioned telomeric heterochromatin and
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the repetitive sequences pSc119.2 and pSc200. Arrows in (E) and (F) indicate the deletion of
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terminal heterochromatin from the chromosome 3RS accompanied by loss of pSc119.2 and
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pSc200 signals. Bars = 10 µm.
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Fig. 3. Variation of terminal heterochromatin in the selfed progeny of 5RS ditelosomic addition
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line (MK5RSdite).
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A: chromosome fragment with terminal heterochromatin in subterminal position (arrow) as
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detected by GISH and FISH with rye genomic DNA (green) and CRW (red). B: FISH using
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pSc119.2 and pAs1 to probe the same cell as in (A). Arrow indicates the pSc119.2 signal at the
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subterminal region. C: a chromosome fragment with terminal heterochromatin at subterminal
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region (arrow) and a minichromosome without terminal heterochromatin (indicated by pointer)
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were detected by GISH and FISH using rye genomic DNA (green) and CRW (red) as probes.
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D: FISH with pSc119.2 and pAs1 to probe the same cell as in (C). Arrow indicates the
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pSc119.2 signal at the subterminal region. Pointer indicates the minichromosome without
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pSc119.2 signal. Bars = 10 µm.
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Fig. 4. Minichromosomes in the self progeny of the 4R monosomic addition line (MK4Rmon).
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A: GISH and FISH using rye genomic DNA (green) and CRW (red) as probes to detect the
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minichromosomes in wheat line MK4Rmon-mini (arrows). B−E: FISH using CRW (red) and
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wheat telomeric DNA (green) as probes to characterise the minichromosomes. Arrows indicate
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the minichromosomes with or without telomere signal. F: immunostaining of centromere-
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specific CENH3 (arrow) on the minichromosome. Bars = 10 µm.
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Fig. 5. Minichromosome and multicentromere in the self progeny of the 6R/6D substitution
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line.
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A: minichromosome (arrow) was detected by FISH using CRW (red) and pAWRC.1 (green) as
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probes. B: small ring chromosome with two rye centromeres (arrow) as detected by FISH using
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CRW (red) and pAWRC.1 (green) as probes. C: small ring chromosome with four rye
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centromeres (arrow) as detected by FISH using CRW (red) and pAWRC.1 (green) as probes.
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D: multicentromeric chromosome (arrow) detected by FISH using CRW (red) and pAWRC.1
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(green) as probes. E: GISH with rye genomic DNA applied to the same cell as in (D). Arrow
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indicates the multicentromeric chromosome. F: GISH with rye genomic DNA applied to the
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same cell as in (C). Arrow indicates the small ring chromosome with four rye centromeres.
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Bars = 10 µm.
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Fig. 6. Model for the formation of ring dicentric and tetracentric chromosomes, as well as of
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minichromosomes within the 6R/6D substitution line.
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