New Types of Wheat Chromosomal Structural Variations in Derivatives of Wheat-Rye Hybrids

Background Chromosomal rearrangements induced by wheat-rye hybridization is a very well investigated research topic. However, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported. Methodology/Principal Findings Octoploid triticale lines were derived from common wheat Triticum. aestivum L. ‘Mianyang11’×rye Secale cereale L. ‘Kustro’. Some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ and common wheat T. aestivum L. ‘Chuannong27’ followed by self-fertilization. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and rye genomic DNA as probes were used to analyze the mitotic chromosomes of these progeny. Alterations of wheat chromosomes including 5A, 6A, 1B, 2B, 6B, 7B, 1D, 3D and 7D were observed. 5AL arm carrying intercalary Oligo-pSc119.2-1, Oligo-pTa535-1 or both Oligo-pSc119.2-1 and Oligo-pTa535-1 signals, 6AS, 1BS and 1DL arms containing terminal Oligo-pSc119.2-1 signal, 6BS and 3DS arms without terminal Oligo-pSc119.2-1 signal, 7BS without subtelomeric Oligo-pSc119.2-1 signal and 7DL with intercalary Oligo-pSc119.2-1 signal have been observed. However, these changed wheat chromosomes have not been detected in ‘Mianyang11’ and Chuannong 27. The altered 5A, 6A, 7B and 7D chromosomes in this study have not been reported and represent several new karyotype structures of common wheat chromosomes. Conclusions/Significance These rearranged wheat chromosomes in the present study afford some new genetic variations for wheat breeding program and are valuable materials for studying the biological function of tandem repetitive DNA sequences.


Introduction
Wide hybridization is very important for wheat (Triticum aestivum L.) cultivar improvement because it can enrich the cultivated gene pools by incorporating favourable alleles, genes or gene complexes from wild relatives [1]. In the program of using wide hybridization methods to improve wheat cultivars, attention was often directed at alien elite gene that has been introgressed into wheat background, however, the structural variations of wheat chromosomes in the derivatives from wheat6wild relatives is notable. Chromosome rearrangements including deletions, translocations, ring chromosomes, dicentric chromosomes and a paracentric inversion were observed during the production of a substitution of chromosome 6B s from Triticum speltoides (Tausch) Gren. ex Richter for chromosome 6B of Chinese Spring wheat (T. aestivum L.) [2]. In newly synthesized amphiploids of Aegilops and Triticum, the reduction or amplification of subtelomeric repeated sequences Spelt1 and Spelt52 have been observed [3]. In synthetic amphiploid derived from T. aestivum (accession 252) 6 Ae. speltoides (accession 15-1), drastic physical elimination of tandem DNA repeat pGc1R-1 has been detected [4]. Localized genomic alterations have been discovered in newly synthesized allotetraploid wheats [5]. These previous studies indicate that the structure of wheat chromosomes could be changed in derivatives, which were derived from wide hybridization. Wide hybridization between wheat (T. aestivum L.) and rye (Secale cereale L.) has been successfully used in wheat breeding programs. Alterations of chromosomal structure of rye chromosomes in triticales, wheat-rye disomic addition lines and wheat-rye substitution lines have been discovered [6][7][8][9][10][11]. Genome rearrangements and chromosome instability in wheat-rye disomic addition lines have also been detected [12][13]. Structural variations of wheat chromosomes induced by wheat-rye monosomic addition lines have been reported [14]. These studies mentioned above indicated that both wheat and rye chromosomes could change in derivatives from wheat6rye. Although abundant genetic diversity was stored in wheat-rye hybrids, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported. In fact, more attentions should be paid on the variations of wheat chromosomes in wheat-rye hybrids. In the present study, some new types of wheat chromosomal structural variations in derivatives from wheat 6 rye were discovered and discussed.
GISH and FISH analysis of line 1-6-7B-2 and its self-fertilized progeny  Figure 2B-C). These two translocation chromosomes were arised from reciprocal translocation between 1B and 2B chromosomes at centromere. No obvious structural variations of wheat chromosomes were observed in the other 29 self-fertilized progeny of line 1-6-7B-2. These results indicate that a single 4R chromosome added into wheat genetic background could induce not only localized structural variation of wheat chromosomes but also reciprocal translocation between wheat chromosomes.
GISH and FISH analysis of lines 12FT1290 and 12FT1378 and their self-fertilized progeny 12FT1290 is a wheat-rye 2R monosomic addition line ( Figure 3A). Line 12FT1378 contained a 2RL/6RL translocation chromosome, a 4RL arm, and a 1R chromosome ( Figure 3B). Compared with their parental wheat 'Mianyang11', the structures of 5A and 7D chromosomes in the two lines have changed. That is, strong intercalary Oligo-pSc119.2-1 signals existed on two 5AL arms and one 7DL arm ( Figure 3A-C).
Twenty-six seeds (13FT279.1-13FT279.26) were randomly selected from the self-fertilized progeny of 12FT1378 for FISH and GISH analyses. All the 26 seeds contained two changed 5A chromosomes ( Figure 5), eight of the 26 seeds contained one changed 7D chromosome ( Figure 5A), ten seeds contained two changed 7D chromosome ( Figure 5B) and eight seeds contained normal 7D chromosomes ( Figure 5C). These results indicate that the changed 5A and 7D chromosomes could be transmitted stably to the offspring. Additionally, in line 13FT279.26, terminal Oligo-pSc119.2-1 signals appeared on a 1DL arm and disappeared from a 3DS arm and a 6BS arm ( Figure 5A, D).
A 1R chromosome, a 1RL arm, a 7B MY chromosome and a 7B CN chromosome existed in line 12FT1997 ( Figure 7A). Oligo-pSc119.2-1 signals could be observed on both of the short arms of 7B chromosomes ( Figure 7A). Twenty-five seeds (13FT236.1-13FT236.25) were randomly selected from the self-fertilized progeny of 12FT1997 for FISH and GISH analyses. Among the 25 progeny, 15 lines contained a 7B MY chromosome and a 7B CN chromosome ( Figure 7B), four lines contained two 7B MY chromosomes ( Figure 7C) and six lines contained two 7B CN chromosomes ( Figure 7D). Among the six lines, two lines carried one changed 7B CN chromosome and two lines had two changed 7B CN chromosomes. That is, the subtelomeric Oligo-pSc119.2-1 signals disappeared from the 7B CN S arm(s) ( Figure 7E-G).
The data about the transmission frequency and structural variations of rye chromosomes were not shown because the focus of this study is on the structural alterations of wheat chromosomes.

Wheat chromosomal alterations induced by rye chromosomes
Wide hybridization is one of the stresses that might trigger reorganization of the parental genomes [19]. Common wheat (T. aestivum L.) is formed through natural wide hybridization and allopoplyploidy. During the evolutionary process of common wheat, chromosomal structural variations such as 4AL-5AL-7BS, 2AS-4BS and 2AL-4BL translocations have been detected [20][21]. Wide hybridization between wheat (T. aestivum) and rye (S. cereale) is an important cytogenetic and breeding tool in wheat. The purpose of crossing wheat with rye is to enrich the cultivated gene pools of wheat. In fact, chromosomal rearrangement of wheat induced by wide hybridization is noteworthy. Chromosomal variations of wheat chromosomes induced by wheat-rye monosomic addition lines have been reported [14]. In this study, the wheat lines with changed wheat chromosomes also contained one or several rye chromosome(s). Both the previous study and this study indicate that rye chromosomes added to wheat background could induce modifications of wheat chromosomes. The question arises as to the possible cause of the structural variations of wheat chromosomes. The reciprocal translocation between 1B and 2B chromosomes might be caused by the 4R chromosome added into wheat genetic background, because wheat-rye monosomic addition lines can easily induce structural variation of chromosome and high frequency of chromosome translocation [22]. The other structural alterations of wheat chromosomes were revealed by FISH analysis using tandem repetitive DNA sequences as probes. It has been suggested that major structural chromosomal rearrangements including deletions, duplications, translocations and inversions are very often associated with cytogenetically detectable regions that are composed of repetitive DNA sequences [18]. Wide hybridization could accelerate repetitive DNA sequence evolution [23]. It has already been reported that retrotransposon-like sequences formed the junctions of tandem repetitive sequences [24]. It was supposed that the deletion and expansion of tandem repetitive sequences in wheat-rye addition and substitution lines might be related to retrotransposon [9]. Therefore, the structural variations of wheat chromosomes in this study might also be related to retrotransposon. Of course, further evidence for this hypothesis is needed. Localized rapid genomic changes involving loss or gain of pSc119.2 repeat in newly synthesized allotetraploid wheats have been reported and these localized systemic genomic changes may have played a role in karyotype stabilization [5]. Therefore, the biological function of repetitive DNA sequences in wide hybrids is worth studying. Undoubtedly, the structural modifications of wheat chromosomes in this study represent a new genetic variation of wheat genome. This kind of genetic variation might have the potential to impact positively on wheat improvement.
In conclusion, structural modifications of wheat chromosomes could occur in derivatives of wheat-rye hybrids. Perhaps, wheat lines with these new modified wheat chromosomes contain the potential value fro wheat breeding program. Additionally, the alterations of wheat chromosomes were displayed by the dynamic changes of tandem repetitive DNA sequences, therefore, these wheat lines are valuable materials for studying the biological function of tandem repetitive DNA sequences.
Cytological techniques and in situ hybridization FISH and GISH were used to analyze the mitotic metaphase cells of materials used in this study. The genomic DNA of rye 'Kustro', Oligo-pSc119.2-1 and Oligo-pTa535-1 [15] were used as probes. The genomic DNA of rye 'Kustro' was labeled with Texas Red-5-dUTP (Invitrogen). Oligo-pSc119.2-1 and Oligo-pTa535-1 were 59 end-labelled with 6-carboxyfluorescein (6-FAM) and 6carboxytetramethylrhodamine (Tamra), respectively [15]. Oligonucleotide probes were synthesized by Shanghai Invitrogen Biotechnology Co. Ltd. (Shanghai, China). The two synthesized probes were diluted by using 16TE solution and the amount applied was operated according to Tang et al. [15]. The chromosome spreads of materials were prepared through the methods described by Han et al. [25]. Probe labeling and in situ hybridization were also operated according to Han et al. [25]. Images were taken using an epifluorescence microscope (BX51, Olympus) equipped with a cooled charge-coupled device camera operated with HCIMAGE Live software (version 2.0.1.5) and processed with photoshop CS 3.0.