Production and Identification of Wheat-Agropyron cristatum 2P Translocation Lines

Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), a wild relative of common wheat, possesses many potentially valuable traits that can be transferred to common wheat through breeding programs. The wheat-A. cristatum disomic addition and translocation lines can be used as bridge materials to introduce alien chromosomal segments to wheat. Wheat-A. cristatum 2P disomic addition line II-9-3 was highly resistant to powdery mildew and leaf rust, which was reported in our previous study. However, some translocation lines induced from II-9-3 have not been reported. In this study, some translocation lines were induced from II-9-3 by 60Co-γ irradiation and gametocidal chromosome 2C and then identified by cytological methods. Forty-nine wheat-A. cristatum translocation lines were obtained and various translcoation types were identified by GISH (genomic in situ hybridization), such as whole-arm, segmental and intercalary translocations. Dual-color FISH (fluorescent in situ hybridization) was applied to identify the wheat chromosomes involved in the translocations, and the results showed that A. cristatum 2P chromosome segments were translocated to the different wheat chromosomes, including 1A, 2A, 3A, 4A, 5A, 6A, 7A, 3B, 5B, 7B, 1D, 4D and 6D. Many different types of wheat-A. cristatum alien translocation lines would be valuable for not only identifying and cloning A. cristatum 2P-related genes and understanding the genetics and breeding effects of the translocation between A. cristatum chromosome 2P and wheat chromosomes, but also providing new germplasm resources for the wheat genetic improvement.


Introduction
Wheat (Triticum aestivum L.), widely planted in different parts of the world, is the third most important cereal behind maize and rice. However, modern breeding was challenged by the narrow genetic variation [1,2], which affected further improvements in wheat yield and quality. Wild relatives of wheat possessed many desirable and valuable traits that could be used as gene resources for wheat improvement [3][4][5]. For example, Agropyron cristatum chromosome 6P with the genes controlling large numbers of florets and kernels per spike and multiple fertile tiller numbers per plant [6,7]; Dasypyrum villosum chromosome 1V including the seed storage

Induction techniques and cross combination
Wheat-A. cristatum 2P disomic addition line II-9-3 was overwintered in the field and transplanted into pots before jointing. The plants at the booting stage were irradiated with 60 Co gamma rays at a dose of 20 Gray (Gy) and a dose rate of 0.5 Gy/min at the cobalt source chamber of Beijing University [30]. Fresh pollen collected from the donor parent 'Fukuhokomugi' was used to pollinate the irradiated spikes of addition line II-9-3, which had been artificially emasculated 1-3 days prior. Mature hybrid seeds were harvested and used to produce a M 1 BC 1 population. Pollen collected from 'Fukuhokomugi' was used to pollinate the untreated spikes of wheat-A. cristatum 2P disomic addition line II-9-3 as a control.
Crosses were carried out using the CS-G2C addition line as the female parent and the wheat-A. cristatum 2P disomic addition line II-9-3 as the male parent. Mature hybrid seeds were harvested and selfed to produce a F 2 population.
All the materials surveyed in this study were planted at the Chinese Academy of Agricultural Science Experiment Station in Beijing (39°57'13"N, 116°19'20"E) during the 2013-2014 growing season.

Chromosome preparation
The exposed seeds were dipped into water in Petri dishes with double moistened filter papers at room temperature for one day, and then the water was absorbed by dry filter papers. The seeds were transferred into a refrigerator at 4°C for 48 h and germinated at 23°C in an incubator. Roots were sampled when the length of the roots was approximately 1.5-2.0 cm; these samples were pretreated in ice water for 24-48 h, fixed in a solution of absolute ethanol-acetic acid (3:1, v:v) at 4°C for 48 h, and then kept in 70% ethanol solution at -20°C. The chromosome slides were treated with 45% glacial acetic acid [32]. Cytological observations were made under a BX51 Olympus phase contrast microscope (Olympus Corp., Tokyo, Japan), and the images were captured using a digital camera. The slides were fixed using ultraviolet light in a TL-2000 Ultraviolet Translinker (Japan) for one minute when needed for GISH detection.

GISH analysis
GISH was used to analyse the mitotic metaphase cells of the materials used in this study. A. cristatum and 'Fukuhokomugi' genomic DNA was isolated by a modified CTAB (hexadecyl trimethyl ammonium bromide) method [33]. The purity and concentration of DNA were measured using a spectrophotometer. The P-genomic DNA and 'Fukuhokomugi' genomic DNA were used as probe and block, at a ratio of 1:40, respectively, to identify the A. cristatum chromosomal fragments. The total genomic DNA of A. cristatum was labelled with digoxigenin-11-dUTP and used as a probe for GISH. The GISH procedure followed that described by Liu et al. [31]. The GISH images were observed under a Nikon Eclipse E600 (Japan) fluorescence microscope and captured with a CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).

FISH analysis
FISH was used to characterize the translocated wheat chromosome, and dual-color FISH/GISH was employed by using repetitive DNA clones, i.e., pAs1, pHvG39, pSc119.2 and the total genomic DNA of A. cristatum as probes. The clones pAs1, pHvG39 and pSc119.2 were labelled with digoxigenin-11-dUTP, biotin-16-dUTP and biotin-16-dUTP, respectively, and were used as probes for dual-color FISH. Chromosomes were counterstained with DAPI (4', 6-diamidino-2-phenylindole). The hybridization signals were examined using a Nikon Eclipse E600 (Japan) fluorescence microscope, and the FISH images were captured using a CCD camera. After FISH, the hybridization signals were washed with PBS (phosphate-buffered saline). The total genomic DNA of A. cristatum was labelled with digoxigenin-11-dUTP as a probe for subsequent GISH. Finally, according to the two hybridization results for the same cell, the homoeologous groups of the translocated wheat chromosomes were analysed by reference to the standard idiogram of the chromosomes of Chinese Spring wheat showing the locations of FISH signals [34,35].

GISH detection of M 1 BC 1 and F 2 plants induced by 60 Co-γ irradiation and gametocidal chromosome 2C
The GISH results were shown in Table 1. GISH analysis of 263 M 1 BC 1 progenies from 60 Co-γ irradiation showed that 39 plants had translocations between chromosome 2P and the wheat chromosomes, 204 plants had a whole chromosome 2P and 20 plants had no chromosome 2P. The translocation frequency induced by irradiation was 14.83%.
Among the 148 F 2 hybrid progenies of the CS-G2C and wheat-A. cristatum 2P addition lines, 51 plants had P chromosomal segments, accounting for 34.46% of the examined plants. Among them, there were 10 translocated plants (6.76%), 4 double P fragments addition plants (2.70%) and 37 single P fragment addition plants (25.00%). At the same time, neither translocations nor any other types of chromosomal structural changes were detected in any of the wheat-A. cristatum 2P disomic addition line/'Fukuhokomugi' F 1 plants (control). This indicated that the translocation lines in this experiment totally resulted from the irradiation and gametocidal chromosome effect. Moreover, the frequency of alien translocation induced by irradiation was significantly higher than that induced by gametocidal chromosome 2C.

The alien translocation types and frequencies of the M 1 BC 1 and F 2 progenies
Among the 411 M 1 BC 1 and F 2 progenies, 49 translocation plants were verified and various translocation types were obtained. The translocation types and frequencies between wheat and A. cristatum 2P chromosomes were shown in Table 2. The translocation plants were divided into two categories: the translocation plants with or without an intact chromosome 2P. There were 3 alien translocation plants with one intact 2P, including one plant (2.04%) with both small and large alien segmental translocation, as well as 2 plants (4.08%) with small alien segmental translocation (Fig 1A). There were 46 alien translocation plants without one intact 2P: 2 plants (4.08%) having large alien segmental and whole-arm translocation (Fig 1B), 10 plants (20.41%) having both large and small alien segmental reciprocal translocation (Fig 1C), 10 plants (20.41%) having large alien segmental translocation (Fig 1D), 1 plant (2.04%) with two small alien segmental and two intercalary translocations, 1 plant (2.04%) with two small alien segmental and one intercalary translocation (Fig 1E), 2 plants (4.08%) with two wholearm reciprocal translocation (Fig 1F) In this paper, we reported 74 translocated chromosomes including 24 large alien segmental translocations (LASTs), 25 small alien segmental translocations (SASTs), 8 intercalary translocations (ITs) and 17 whole arm translocations (WATs) (Fig 2). This result showed an ascending order of IT < WAT < LAST % SAST in the occurrence frequency ( Table 3). The ratio of   Table 3; Fig 2), indicating that 60 Co-γ irradiation and gametocidal chromosome 2C had very high efficiency in inducing interstitial breakage. Sixty-six translocations (89.8%, including 17 WATs, 24 LASTs and 25 SASTs) involved one-breakage fusion events, while only 8 ITs involved two-breakage fusion events. This indicated that the major effect of irradiation and gametocidal chromosome 2C was to induce one-breakage fusion events.
We performed dual-color FISH/GISH using P genomic DNA and the clone pAs1 labelled with digoxigenin-11-dUTP, the clones pHvG39 and pSc119.2 labelled with biotin-16-dUTP. Plant 2P-23 has 2 translocated chromosomes; the small alien segmental translocation of 2P-23 showed an obvious green fluorescent signal near the centromeric region of the short arm and a faint green signal in the middle of the long arm; the arm ratio is large. Accordingly, it was a 5BS.L-2P translocation with a small 2P fragment. Further, the large alien segmental translocation of 2P-23 exhibited a faint green signal at the end of the arm; it was a 3A-2P translocation with a large 2P fragment ( Fig 3A 1 and 3A 2 ). Plant 2P-35 contained a 7A-2PL.2PS-7A intercalary translocation ( Fig 3B 1 and 3B 2 ). Plant 2P-40 contained a 4DL.4DS-2PL small-segment translocation containing the whole 2P chromosome short arm. C: Small alien segmental translocation containing the partial 2P chromosome long arm. D: Small alien segmental translocation containing the partial 2P chromosome short arm. E: Whole arm translocation containing the whole 2P chromosome long arm. F: Whole arm translocation containing the whole 2P chromosome short arm. G: Intercalary translocation.

Discussion
Chromosome translocations can be induced by Ph-system, tissue culture, gametocidal chromosomes and ionization irradiation. Allosyndetic associations between wheat and A. cristatum are rare [23,[36][37][38]; consequently, homoeologous pairing between wheat and A. cristatum chromosomes rarely takes place even when induced with a Ph-system [39]. Translocations induced by tissue culture are very technical and troublesome because they require a long period to  obtain regenerative plants [40]. Compared with the first two approaches, the gametocidal chromosome from Aegilops cylindrica Host was successfully used to induce chromosome mutations in common wheat in nature. These chromosome variations could be transmitted to stable offspring [41], which was considered as an effective method to induce chromosomal structural variations [42]. Ionizing irradiation can induce chromosome breakage randomly and yield many translocation types including small segment translocations and intercalary translocations [43], which has been widely used in transferring useful genes from wild relatives to wheat for the improvement of resistance or tolerance to biotic and abiotic stresses [8,29,44,45]. In our study, forty-nine 2P translocated plants were successfully obtained, which was the first study to report such a finding. Abundant types of alien chromosome translocations were induced by both ionizing irradiation and gametocidal chromosome 2C, which indicated that these two methods were useful for producing wheat-A. cristatum 2P translocations. Ionizing irradiation has been proven as an effective method to induce chromosomal translocations [46]. Dry seed, plants at meiosis and spikes at the pollen stage can be used as irradiated materials [29,30,47]. The present study showed that irradiation of plants at meiosis was highly effective. We only pollinated irradiated spikes using fresh pollen from untreated plants. More M 1 BC 1 seeds can be obtained as long as donor parents (untreated plants) have sufficient pollen. Furthermore, the treatment of booting stage plants in the pots was more convenient than that on spikes. Additionally, chromosomal structural variants can be directly identified in the M 1 BC 1 generation by GISH.
To date, many translocation lines have been screened and identified involving three wheat-A. cristatum disomic addition lines [29][30][31]. A new strategy to rapidly produce a large number of translocations between wheat and A. cristatum has been developed [30]. This strategy was used to induce additional four wheat-A. cristatum disomic addition lines and to produce wheat-A. cristatum alien translocation lines with different breakpoints and P segments of different sizes. The translocation lines can be further used to construct a deletion bin map of each P chromosome.
The transfer of desirable genes from the tertiary gene pool could be an efficient way to increase genetic diversity and improve cultivated wheat [28,48]. Translocation lines, especially small alien segment translocations, would be genetically more stable and desirable [49]. The production of small alien segment translocations makes it possible to transfer useful genes from wild relatives to wheat compared with the low fertility and genetic instability of addition and substitution lines [50][51][52]. A. cristatum has many potentially valuable traits that can be used in wheat improvement, so it is important to produce wheat-A. cristatum 2P small-segment translocations to transfer useful genes to wheat for broadening wheat genetic diversity. Huang et al. [53] identified two homozygous wheat-A. cristatum 6P small intercalary translocation lines. These lines did not carry undesirable genes and had a good compensation effect of A. cristatum chromatin, which further confirmed that the wheat-A. cristatum small alien segments translocations were desirable. Twenty-five small fragment alien translocations were cristatum was labelled with digoxigenin-11-dUTP and visualized with red fluorescence. Chromosomes were counterstained with DAPI and visualized with blue fluorescence. A 2 -R 2 : FISH patterns of wheat-A. cristatum translocation lines. A 2 -N 2 , P 2 -R 2 : The repetitive sequence clone pHvG39 was labelled with biotin-16-dUTP and visualized with green fluorescence; O 2 : The repetitive sequence clone pSc119.2 was labelled with biotin-16-dUTP and visualized with green fluorescence. The repetitive sequence clone pAs1 was labelled with digoxigenin-11-dUTP and visualized with red fluorescence. Chromosomes were counterstained with DAPI and visualized with blue fluorescence. Arrows point to the translocated chromosomes. obtained in the present study by ionizing irradiation and gametocidal chromosome 2C, which would make it possible to utilize desirable genes from A. cristatum 2P chromosome.
Many wheat-alien chromosomal translocation lines carrying desirable genes have been reported in recent years [8,54,55], but some have not been fully utilized in wheat breeding due to the incomplete compensation for the replaced wheat chromosomal segments. Only wellcompensating translocations were beneficial in wheat improvement. Until now, only a few exogenous desirable genes have played an important role in wheat breeding [4,56]. It is of great significance to explore the evolutionary relatedness, homoeologous relationships and degree of colinearity between wheat and alien chromosomes [4,52,57]. Agropyron are cross-pollinating plants. The tetraploid A. cristatum derived from the hybridizations between diploid A. cristatum and A. mongolicum. Although the diploid A. cristatum and A. mongolicum contained the same basic P genome, their P genomes exhibited rearrangements and variation [58,59]. Han et al. [17] detected the genetic rearrangement of P genomes by identifying four different types of wheat-A. cristatum 6P disomic addition lines and speculated that genomic rearrangements may occur in the wheat-A. cristatum addition line. Accordingly, we speculated that A. cristatum chromosome 2P may have genetic arrangement to some extent. Although the A. cristatum chromosome 2P fragment was translocated to wheat homoeologous groups (1, 3, 4, 5, 6 and 7) in this study, some wheat-A. cristatum 2P translocation lines were not agronomically poor in the field environment. Thus, we could not exclude the possibility that the wheat-A. cristatum 2P translocation lines may have a subordinate complementary relationship with wheat homoeologous groups (1, 3, 4, 5, 6 and 7) and that the offspring may have a partial compensation effect because of genomic rearrangement. The present study showed that A. cristatum chromosome 2P was translocated to all three wheat genomes and that the wheat A genome had the highest recombination frequency. The results were inconsistent with previous studies, which showed that the wheat B genome possessed the highest number of chromosome arrangements [60]. The translocation lines obtained in this study differed in breakpoint locations and alien segment lengths. The same 2P chromosome segment could be transferred to different wheat chromosomes/genomes. Different 2P chromosome segments could also be transferred to the same wheat chromosome/homoeologous group. These lines would be useful in studies to better understand recombination, interaction and genetic balance between wheat chromosomes and the A. cristatum 2P chromosome. In addition, they could provide a theoretical basis for the utilization of desirable genes of the chromosome 2P in wheat breeding.
Powdery mildew and leaf rust, caused by Blumeria graminis f. sp. tritici (Bgt) and Puccinia recondita f. sp. tritici, respectively, are two devastating diseases that cause severe yield losses in most of the wheat production areas [61,62]. Powdery mildew can cause yield losses up to 50% [63], whereas leaf rust caused yield losses ranging from 40% [64] to 70% [65]. Breeding resistant varieties is the most economical and effective way to control these diseases. To date, approximately 42 loci with more than 70 alleles conferring resistance to powdery mildew genes [66] and more than 70 loci for leaf rust resistance genes [67] have been reported. Unfortunately, many of these resistance genes usually become ineffective when new pathogen variants emerge due to co-evolution of the host and pathogen [68]. Hence, there is an urgent need to identify new and effective sources of resistance for wheat improvement. It has been observed that the wheat-A. cristatum 2P addition line possessed high resistance to powdery mildew and leaf rust compared with 'Fukuhokomugi' in our laboratory (Submitted). By preliminary phenotypic evaluation, newly developed 2P alien translocation lines 2P-173 and 2P-205 were highly resistant to powdery mildew (S1 Fig) and the translocation line 2P-205 was highly resistant to leaf rust (S2 Fig). This showed that the A. cristatum 2P chromosome harbored genes conferring high resistance to powdery mildew and leaf rust. Furthermore, segregating populations involved in wheat-A. cristatum 2P alien translocation lines with different breakpoints are being developed. These will lay the foundation for locating and cloning novel powdery mildew and leaf rust resistance genes and providing new germplasm accessions for wheat genetic improvement.
In summary, the wheat-A. cristatum 2P alien chromosomal translocations were successfully induced and numerous translocation lines that possessed whole-arm, segmental and intercalary translocations were obtained for the first time in this study. These newly developed translocation lines will not only lay the foundation for mapping and cloning of powdery mildew and leaf rust resistant genes on the A. cristatum 2P chromosome, but also provide excellent germplasm resources and ideal experimental materials for breeding application and basic research.