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Molecular adn biochemical characterization of a non-Robertsonian wheat-rye chromosome translocation line.

Several principles and methodologies of inducing chromosome translocations have been reviewed by Sears (1981), Feldman (1988), and Jiang et al. (1994). These techniques can be classified into two major groups. The first group of methods of inducing alien-wheat translocations is by exploiting homoeologous chromosome pairing. Alien chromosomes can pair with their wheat homoeologues, though at a low frequency in F1 hybrids between alien species and wheat (Triticum aestivum L.) lacking Ph1 gene, the major inhibitor of homoeologous pairing. The second method involves spontaneous chromosome breakage and reunion or by ionizing irradiation, tissue culture (Larkin and Skowcraft, 1981; Lapitan et al., 1986; Hu et al., 1995), or the application of gametocidal genes (Endo, 1988).

In this paper, we report characterization of a novel non-Robertsonian wheat-rye chromosome translocation in line WER-1-1 by biochemical, molecular, and cytogenetic methods.


The plant materials examined in this study included (i) the second (DH2, double haploid), third(DH3), and fourth (DH4) generations of pollen-derived line WER-1-1, which is from the cross between M27 (a disomic substitution line 1R(1D) where 1R chromosome of rye is substituted for chromosome 1D of wheat) and Chinese Spring; (ii) pollen-derived line WER-4, a disomic 1RS isochromosome addition line, derived from the same cross as WER-1-1; (iii) M27, Chinese Spring (CS), and nullisomic-tetrasomic stocks of CS N1B-T1A and CS N1D-T1B; and (iv) Secale cereale L. cv. Austria Rye.

Biochemical Assays

Ten percent polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) was used for the analysis of high molecular weight glutenin subunits (HMW-GS) according to the method of Payne and Corfield (1979). Gliadin components were evaluated by acid polyacrylamide gel electrophoresis (APAGE) following the method described by Tao et al. (1991) with some modifications.

Southern Hybridization

Probes pSc119.2 and pSc 5.3H3 were kindly provided by Phil Larkin (CRISO Division of Plant Industry, Institute of Plant Production and Processing, Australia). DNA of leaves from 2-wk-old seedlings was isolated by the method of Sharp et al. (1988). Approximately 5 [micro]g of DNA was digested to completion with restriction endonucleases, separated on 1.2% (w/v) agarose gel, and transferred to a Zeta-probe blotting membrane (Bio-Rad Laboratories, Richmond, CA; Catalog Number 162-0159) as suggested by the manufacturer. The membrane was prehybridized for 6 h at 65 [degrees] C and hybridized overnight at 65 [degrees] C, according to Sharp et al. (1988). Probes were labeled with [sup.32]p with the "random priming" labeling kit from Life Technologies, Inc. (Gaithersburg, MD; Catalog Number 18187-013). Five 20-min posthybridization washes were carried out as follows: (i) 2 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.5% (w/v) SDS at 65 [degrees] C; (ii) 1 x SSC, 0.5% (w/v) SDS at 65 [degrees] C; (iii) 0.5 x SSC, 0.5% (w/v) SDS at 65 [degrees] C; (iv) 0.1 x SSC, 0.5% (w/v) SDS at 65 [degrees] C, and (v) 0.1x SSC at room temperature. The membrane was exposed to X-ray film (Kodak, Japan; Catalog Number 6351068) at -70 [degrees] C with intensifying screens for a period of 12 to 24 h.


The C-banding technique followed the method of Tao et al. (1991) with minor modifications, and chromosomes were identified based on standard karyotypes of wheat (Gill and Kimber, 1974a; Gill et al., 1991) and rye (Gill and Kimber, 1974b; Mukai et al., 1992).

Genomic In Situ Hybridization (GISH)

The protocol of Le et al. (1989) was followed with some modifications. Genomic DNA of S. cereale was isolated, digested with HindIII, and then labeled with digoxigenin-11-UTP (Boehringer Mannheim, Mannheim, Germany; Catalog Number 1093088) by nick translation. The hybridization signals were detected by peroxidase--diaminobenzidine tetrahydrochloride (POD--DAB). After staining, washing, and airdrying, the preparations were examined and photographed with an Olympus BH-2 microscope.

Fluorescence In Situ Hybridization (FISH)

Hybridization was carried out in a 15-[micro]L mixture per slide, which contained 0.1 [micro]g of Biotin-11-dUTP-labeled S. cereale genomic DNA, 5 [micro]g of sheared salmon sperm DNA, 5 [micro]g of sheared wheat genomic DNA, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 0.4% (w/v) SDS, and 2 x SSC. Sites of probe hybridization were detected with mouse anti-biotin (Boehringer Mannheim, Mannheim, Germany; Catalog Number 1297597) and FITC-conjugated sheep anti-mouse antibodies (Boehringer Mannheim, Germany; Catalog Number 821 462). Slides were mounted with an antifading solution containing 1 [micro]g/mL propidium iodine, and examined under a Nikon fluorescence microscope. Chromosome images were obtained with a computer-aided confocal laser scanning system (MRC-600, Bio-Rad), linked to the Nikon fluorescence microscope.


Production of WER-1-1

M27 was used as the 1R donor. Previous study showed that M27 is a disomic substitution line 1R(1D) where an intact 1R chromosome pair of rye is substituted for chromosome pair 1D of wheat (Tao et al., 1991). Chinese Spring was chosen as one of the parents because of its well-established genetic marker systems, which make it easy to characterize the chromosome constitution of its derivatives. In this study, the chromosomal constitution of M27 was confirmed via cytological methods. C-banding and GISH on root tip preparations revealed the presence of only a pair of intact 1R chromosomes in M27 (Fig. 1 a, b).


In spring 1993, the F1 hybrid was obtained from CS as the female parent and M27 as the male parent. Eight pollen-derived plants, numbered from WER-1 to WER-8, were produced through anther culturing the F1 hybrid. In DH1 generation, pollen-derived plant WER-1 possessed two spikes which set seeds. These two spikes were named WER-1-1 and WER-1-2. Spike WER-1-1 was selfed for three consecutive generations.

Biochemical Assays

Loci encoding Glu-1 and Sec-3, on the long arms of wheat homoeologous group 1 chromosomes and 1R (Payne, 1987; Lawrence and Shepherd, 1981) were analyzed by SDS-PAGE. The SDS-PAGE patterns of M27, WER-1-1, WER-4, Chinese Spring, and nulli-tetrasomic stocks of homoeologous group 1 are shown in Fig. 2. Line WER-1-1 has the two 1RL specific bands, but lacks the bands encoded by 1DL, as does M27. A close observation on the low molecular weight glutenins and gliadins showed that 1RS-encoded band exists in M27, WER-1-1, and WER-4, the 1DS-encoded band occurs in WER-4, Chinese Spring, and CS N1B-T1A, but is absent from M27, WER-1-1, and CS N1D-T1B (Fig. 2).


In wheat, the genes encoding [Gamma] and [Omega] gliadins are located on the short arms of homoeologous group 1 chromosomes of wheat and rye (Payne, 1987; Lawrence and Shepherd, 1981). Figure 3 presents the gliadin APAGE patterns of M27, WER-1-1, WER-4, Chinese Spring, and its nulli-tetrasomic stocks of homoeologous group 1. It was clear that WER-1-1 has the specific band of 1RS and lacks the 1DS bands (Fig. 3). The results from these two biochemical assays revealed that pollen-derived line WER-1-1 is a 1R(1D) substitution line.


Southern Hybridization

To further verify the presence of rye chromatin in WER-1-1, Southern hybridization of dispersed rye repeat sequence pSc 119.2 was applied to Chinese Spring, M27, WER-4, WER-1-1, and a rye cultivar (Fig. 4). The smeared hybridization pattern on rye confirmed that pSc 119.2 was a dispersed rye repeat sequence. However, distinctive banding patterns were observed among Chinese Spring and the other 1R-containing materials. Fragment a, present in WER-4 and M27 but absent from Chinese Spring, must be located on the short arm of 1R. M27 carries, in addition, fragment b, which Chinese Spring and WER-4 lack, which therefore indicates that this fragment is probably located on 1R long arm. A comparison of the hybridization pattern of WER-1-1 with those of Chinese Spring, WER-4, and M27 showed that 1R-specific fragments a and b are present in WER-1-1, thus providing evidence that WER-1-1 contains the elements from both arms of 1R. In addition, the hybridization of dispersed rye specific repeat sequence pSc5.3H3 to HindIII-restricted DNAs from Chinese Spring, M27, WER-4, WER-1-1, and rye also revealed the presence of rye chromatin in WER-1-1 (data not shown).


Cytogenetic Analyses

Ten DH1 seeds (DH2 generation) were obtained from spike WER-1-1. Of the 10 DH1 seeds, four seeds did not germinate, whereas the remaining six seeds germinated normally. It was found through conventional squashing preparation that all the six seeds had a chromosome number of 2n = 42. C-banding patterns of the six seeds showed that, among the 42 chromosomes, there existed a pair of chromosomes with two terminal heterochromatin bands on the short arms and one interstitial band on the long arm, and without the diagnostic terminal band on the long arm (Fig. 5a). So, chromosome 1R pair has lost distal parts of the long arm. The breakpoint is located between the interstitial band and the terminal band on 1RL. GISH patterns of the six seeds unambiguously revealed that in addition to a pair of rye chromosomes, WER-1-1 also possessed a pair of wheat-rye non-Robertsonian translocation chromosomes (Fig. 5b). Furthermore, FISH on randomly selected eight DH2 seeds (DH3 generation) and 22 DH3 seeds (DH4 generation) showed that all the 30 seeds tested had the same chromosome constitution as the DH2 generation (Fig. 6). Combining the results from C-banding and in situ hybridization, it can be deduced that the translocated rye segments present in the wheat-rye translocation chromosome should be the lost distal portions of 1RL since the 1R donor parent, M27, had no rye chromatin but a pair of intact 1R chromosomes. Moreover, the translocated rye fragment with the length less than one third of that of 1RL, can be stably transmitted to the offspring.


The observations on biochemical assays, Southern hybridization, and cytogenetic analyses led to the convincing conclusion that in pollen-derived line WER-1-1, wheat chromosomes 1D were substituted by a pair of abnormal 1R chromosomes, in which distal parts of the long arms were translocated to the long arm ends of a pair of wheat chromosomes. Moreover, translocation line WER-1-1 was cytologically stable over generations.


From the C-banding patterns of the abnormal 1R chromosomes present in WER-1-1 and the fact that the 1R donor parent M27 had no rye chromatin but a pair of intact 1R chromosomes, it was concluded that the rye segments on the wheat-rye translocation chromosomes were the distal parts of 1R long arms. The following observation added further evidence supporting this conclusion. The cytological stability of the chromosome constitution of translocation line WER-1-1 was maintained in subsequent generations (from DH2-DH4), indicating that the abnormal chromosome 1R, plus the rye segment present on the wheat-rye translocation chromosome, should constitute an intact 1R chromosome or at least the main "functioning parts" of 1R. If this were not so, chromosomal duplications and deletions would occur, resulting in the cytological instability of the chromosome constitution of WER-1-1.

The presence of Phi gene and the coexistence of the abnormal 1R chromosomes and the 1R-wheat translocation chromosomes in WER-1-1 largely excludes the possibility that the 1R-wheat translocation was the result of either homoeologous or non-homoeologous recombination events. Common wheat is a typical allohexaploid species with three closely related genomes. However, the Ph system does not permit pairing among homoeologous chromosomes. Even in the haploid level, where only one dosage of each of the genomes A, B, and D exists, homoeologues of wheat pair at a low frequency (Hao et al., 1981; Miao, 1987). For the more distant species such as S. cereale, homoeologous pairing occurs rarely (Zhang et al., 1998), even in the absence of Phi gene (Koebner and Shepherd, 1986). Our data showed that at male meiosis I, the F1 hybrid between Chinese Spring and M27 had a mean chromosome configuration of [2.64.sup.I] (1-7) + [19.47.sup.II] (16-22) + [0.01.sup.III] (0- 2) + 0.06.sup.IV] (01) in 150 pollen mother cells observed, indicating that 1R paired little, if at all, with wheat chromosomes (unpublished data). Furthermore, if the monosomic 1R chromosome did pair with one of the wheat chromosomes at male meiosis of the F1 hybrid, from which WER-1-1 was derived, the recombinant chromosomes, i.e., the abnormal 1R chromosome and the 1R-wheat translocation chromosome, will go into different tetraspores, and after anther culture, these two chromosomes should appear in different pollen-derived plants. Therefore, the translocation arose in the male gametophyte following meiosis as also suggested by Badaeva et al. (1995). Obviously, further molecular analysis of the fine structure of the 1R-wheat translocation chromosome such as the breakpoint position, the size of the rye segment, and the identity of the wheat chromosome is needed.


Erming Wang thanks Dr. C.X. Cheng and Ms. H.M. Mou for their technical assistance in Southern hybridization. Also thanked are Drs. Wenjun Zhang and Xiangqi Zhang for their constructive discussions and their generosity in sharing their experience. We are grateful to Dr. B.S. Gill for his assistance in manuscript preparation.


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State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics, Chinese Academy of Sciences, Beijing 100101. Received 21 Sep. 1997. (*) Corresponding author (rxwei@public.east.
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Author:Wang, Erming; Xing, Hongyan; Wen, Yuxiang; Zhou, Wenjuan; Wei, Rongxuan; Han, Hu
Publication:Crop Science
Date:Jul 1, 1998
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