Precise Identification of Chromosome Constitution and Rearrangements in Wheat–Thinopyrum intermedium Derivatives by ND-FISH and Oligo-FISH Painting

Thinopyrum intermedium possesses many desirable agronomic traits that make it a valuable genetic source for wheat improvement. The precise identification of individual chromosomes of allohexaploid Th. intermedium is a challenge due to its three sub-genomic constitutions with complex evolutionary ancestries. The non-denaturing fluorescent in situ hybridization (ND-FISH) using tandem-repeat oligos, including Oligo-B11 and Oligo-pDb12H, effectively distinguished the St, J and JS genomes, while Oligo-FISH painting, based on seven oligonucleotide pools derived from collinear regions between barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), was able to identify each linkage group of the Th. intermedium chromosomes. We subsequently established the first karyotype of Th. intermedium with individual chromosome recognition using sequential ND-FISH and Oligo-FISH painting. The chromosome constitutions of 14 wheat–Th. intermedium partial amphiploids and addition lines were characterized. Distinct intergenomic chromosome rearrangements were revealed among Th. intermedium chromosomes in these amphiploids and addition lines. The precisely defined karyotypes of these wheat–Th. intermedium derived lines may be helpful for further study on chromosome evolution, chromatin introgression and wheat breeding programs.


Introduction
As a perennial grass with a native distribution throughout the Mediterranean and Eastern Europe [1], Thinopyrum intermedium (Host) Barkworth & D.R. Dewey has undergone the most direct selection for hay and pasture grass improvement [2]. As an allohexaploid (2n = 6x = 42), Th. intermedium chromosomes have been previously identified and allocated into three tentatively designated sub-genomes sets as J, J S and St, where St is closely related to the genome of Pseudoroegneria strigosa and J is closely related to that of Th. bessarabicum. The J S genome is a modified version of the J and St genomes [3,4]. Th. intermedium consists of two subspecies, namely, intermediate wheatgrass, ssp. intermedium and a pubescent variant ssp. trichophorum that carries novel and high levels of resistance to several wheat fungal diseases, such as rusts and powdery mildew, and many useful agronomic traits, including novel glutenins, and contributes to the tertiary gene pool for wheat improvement [4,5]. The production and identification of amphiploids or partial amphiploids between wheat and Th. intermedium is an important intermediate step for gene transfer [5]. To date, a number of wheat-Th. intermedium amphiploids, Otrastsyuskaya (OT), TAF46, Zhong1 to Zhong5, 78829, TAI7044, TE-3, TE253-1, TE257, TE267, TE346, SX12-787, SX12-1150 and SX12-1269, have been obtained [4,[6][7][8][9][10]. The precise identification of individual Th. intermedium chromosomes is essential for comparative molecular and cytogenetic analysis of Th. intermedium species and the introgression of diverse Th. intermedium chromatin for breeding purposes of wheat. Due to the complexity of the genomic composition of Th. intermedium, the precise recognition of individual Th. intermedium chromosomes in wild grass itself and wheat-Th. intermedium partial amphiploids is rather difficult by current molecular cytogenetic methods including C-banding, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with the reported probes [4,5,[10][11][12]. Recently, we developed seven bulked pools for an oligo painting system, which enabled the assignment of the chromosomes to the Triticeae linkage groups and the characterization of wheat-alien chromosome derivatives [13]. The powerful Oligo-FISH painting methods [14], in combination with non-denaturing fluorescent in situ hybridization (ND-FISH) using multiple oligo probes [15][16][17][18][19][20], have clearly improved the efficiency and accuracy of distinguishing Th. intermedium chromosome segments and their introgression in a common wheat background.
In the present study, we established a standard karyotype of individual Th. intermedium chromosomes, which show the genomic heterogeneity of wheatgrass. Comprehensive FISH and Oligo-FISH painting enabled the precise identification of individual chromosomes and chromosome rearrangements in a wheat-Th. intermedium partial amphiploid and derived lines.

Identification of Individual Thinopyrum intermedium Chromosomes
All 21 pairs of Th. intermedium chromosomes have been previously identified by ND-FISH using the repetitive sequences Oligo-pSc119.2 and Oligo-pTa535 as probes. Subsequently, ND-FISH using the probes Oligo-B11 and Oligo-pDb12H was performed to further characterize the mitotic metaphase chromosomes of the Th. intermedium accession PI440043. The Oligo-pDb12H was able to clearly classify the 14 J S chromosomes of Th. intermedium, while the abundant signals associated with Oligo-B11 revealed the St chromosomes, and the mostly telomeric and sub-telomeric signals with Oligo-B11 on the J chromosomes [17,18]. As shown in Figure 1a, all the 42 Th. intermedium chromosomes were clearly distinguished into three subgenomes designated as J, St and J S , each with 14 chromosomes by using ND-FISH. The sequential ND-FISH patterns using probes Oligo-pSc119.2 and Oligo-pTa535, combined with the FISH patterns by Oligo-B11 and Oligo-pDb12H, were able to distinguish the individual Th. intermedium chromosomes in three J, St and J S sub-genomes ( Figure 1b).
The wheat-barley bulked oligo pools Synt1 to Synt7 were then used by sequential FISH for validation of the signal distribution and specificity for the chromosomes of Th. intermedium PI440043 (Figure 1). Each chromosome plate was first analyzed by ND-FISH with probe combinations of Oligo-B11 + Oligo-pDb12H ( Figure 1a) and then Oligo-pSc119.2 + Oligo-pTa535 (Figure 1b,e,g,i). For example, the probe Synt5 produced strong hybrid green signals on each of three homoeologous chromosome pairs along the entire lengths of these groups of six chromosomes (Figure 1c). According to ND-FISH results, the three pairs of homoeologous chromosomes belong to each St, J and J S subgenome. They are thus designated as 5St, 5J and 5J S (Figure 1c). In the same metaphase cell, the probe Synt7 generated distinct red signals on three chromosomes pairs of 7St, 7J and 7J S (Figure 1c), and Synt3 identified another three chromosomes pairs 3St, 3J and 3J S (Figure 1d), respectively. Similarly, the bulked probes Synt1, Synt2, Synt4 and Synt6 also hybridized to mitotic chromosomes of Th. intermedium, and they all produced distinct signals on their individual three homoeologous chromosomes pairs ( Figure 1). Therefore, all seven painting probes resulted in distinct hybridization signals covering chromosome arms along their entire lengths for each linkage group, suggesting that the bulked Oligo-based FISH painting can be used to identify each of the Th. intermedium chromosomes or chromosome segments of particular homoeologous groups. The 21 chromosome pairs were finally assigned to seven homoeologous groups among St, J and J S -subgenomes (Figure 1k).
Based on the hybridization patterns of Oligo-pSc119.2 and Oligo-pTa535 on the chromosomes of PI440043, some homologous chromosomes pairs showed different hybridization signal patterns for Oligo-pSc119.2, including 2St, 6St and 3J S with the presence or absence of signals at their terminal regions. Furthermore, the chromosome pairs 7J S and 5J displayed different Oligo-pTa535 signals on the pericentromeric regions ( Figure 1). Importantly, the different ND-FISH patterns were visible only on one of the homologous pairs, indicating structural chromosome heterozygosity in Th. intermedium plants.

Chromosome Identification of Wheat-Th. intermedium Amphiploids
The wheat-Th. intermedium partial amphiploid TAF46 was selected to characterize the Thinopyrum chromosome compositions. Sequential ND-FISH using Oligo-pSc119. using the probes Oligo-B11 and Oligo-pDb12H for TAF46 showed the absence of Oligo-pDb12H hybridization signals, indicating that TAF46 did not possess J S chromosomes. A total six St and eight J chromosomes were observed based on the hybridization patterns of Oligo-B11 (Figure 2a,b). This is consistent with the previous reports of St-genome based GISH [3]. The Oligo-FISH painting by seven probes gave rise to distinct hybridization signals covering wheat and Th. intermedium chromosome arms along their entire lengths for each homoeologous group. The probe Synt1 produced strong green signals on the chromosome pairs of 1A, 1B and 1D (Figure 2c), in addition to another a pair of Th. intermedium group 1 chromosomes, which was defined as chromosomes 1J compared to the standard karyotype (Figure 2b,d). Similarly, the probe Synt3 generated distinct red signals on eight chromosomes including the previously identified 3A, 3B and 3D by Oligo-pSc119.2 + Oligo-pTa535 (Figure 2c), and a pair of Th. intermedium 3J-chromosomes (Figure 2c). Similarly, the bulked probes Synt2 + Synt5 ( Figure 3e) and Synt4 + Synt6 (Figure 2g) also hybridized specifically to chromosomes of TAF46, compared to the sequential ND-FISH of Oligo-pSc119.2 + Oligo-pTA535 (Figure 2f,h). These results show that Th. intermedium chromosomes of TAF46 consisted of J genome chromosomes of the homoeologous groups 1, 3, 5, 7 and St genome chromosomes of groups 2, 4 and 6 ( Figure 2). The karyotype for individual chromosomes of the subgenomes and linkage groups of Th. intermedium of TAF46 is shown in Figure 2j. Our results are consistent with the chromosome assignment of Th. intermedium addition lines by GISH and molecular analysis of the TAF46-derived wheat-Thinopyrum addition lines L1 to L7 [21]. . FISH using the probes Oligo-B11 and Oligo-pDb12H for TAF46 showed the absence of Oligo-pDb12H hybridization signals, indicating that TAF46 did not possess J S chromosomes. A total six St and eight J chromosomes were observed based on the hybridization patterns of Oligo-B11 (Figure 2a,b). This is consistent with the previous reports of St-genome based GISH [3]. The Oligo-FISH painting by seven probes gave rise to distinct hybridization signals covering wheat and Th. intermedium chromosome arms along their entire lengths for each homoeologous group. The probe Synt1 produced strong green signals on the chromosome pairs of 1A, 1B and 1D (Figure 2c), in addition to another a pair of Th. intermedium group 1 chromosomes, which was defined as chromosomes 1J compared to the standard karyotype (Figure 2b,d). Similarly, the probe Synt3 generated distinct red signals on eight chromosomes including the previously identified 3A, 3B and 3D by Oligo-pSc119.2 + Oligo-pTa535 (Figure 2c), and a pair of Th. intermedium 3J-chromosomes ( Figure 2c). Similarly, the bulked probes Synt2 + Synt5 ( Figure 3e) and Synt4 + Synt6 ( Figure 2g) also hybridized specifically to chromosomes of TAF46, compared to the sequential ND-FISH of Oligo-pSc119.2 + Oligo-pTA535 (Figure 2f,h). These results show that Th. intermedium chromosomes of TAF46 consisted of J genome chromosomes of the homoeologous groups 1, 3, 5, 7 and St genome chromosomes of groups 2, 4 and 6 ( Figure 2). The karyotype for individual chromosomes of the subgenomes and linkage groups of Th. intermedium of TAF46 is shown in Figure 2j. Our results are consistent with the chromosome assignment of Th. intermedium addition lines by GISH and molecular analysis of the TAF46-derived wheat-Thinopyrum addition lines L1 to L7 [21].  Figure S1a), we constructed the karyotype of the 14 Th. intermedium chromosomes of 78829, as linkage groups 3St, 4St, 5St, 6J S -J, 7J S , 1St-J S and 2St-J S , respectively. The Th. intermedium chromosome karyotype is shown in Figure  3j. Therefore, the bulked Oligo-based FISH painting was effective in identifying each of the wheat chromosomes or chromosome segments for particular linkage groups in the wheat-Th. intermedium amphiploid. Figure 3. Sequential FISH probes Oligo-B11 + Oligo-pDb12H (a), Oligo-pSc119.2 + Oligo-pTa535 (b,d,f,h) and Oligo painting using specific bulked oligo probes Synt1+ Synt7 (c), Synt3 + Synt6 (e) Figure 3. Sequential FISH probes Oligo-B11 + Oligo-pDb12H (a), Oligo-pSc119.2 + Oligo-pTa535 (b,d,f,h) and Oligo painting using specific bulked oligo probes Synt1+ Synt7 (c), Synt3 + Synt6 (e) and Synt2 + Synt5 (g) for wheat-Th. intermedium partial amphiploid 78829, respectively. Karyotypes of wheat chromosomes (i) and Th. intermedium (j) are shown. Chromosomes were counterstained with DAPI (blue). Arrows showed the wheat-Thinopyrum small translocation chromosomes.
We used a similar approach to precisely identify the Th. intermedium chromosomes in 78,829 (2n = 56). The ND-FISH using Oligo-pSc119.2 and Oligo-pTa535 revealed that 78,829 has complete wheat chromosomes, each of 14 A, B, and D chromosomes. The sequential ND-FISH by probes Oligo-B11 and Oligo-pDb12H revealed the Thinopyrum chromosomes included six St, four St-J S , two J-J S and two J S chromosomes (Figures 1 and 3b). It is also interesting to observe that an Oligo-B11 signal appeared in the terminal region of 4D, indicating a small translocation between 4D and a Th. intermedium chromosome (Figure 3a). In comparison with ND-FISH patterns using Oligo-pSc119.2 + Oligo-pTa535 and in combination with bulked pool probes Synt1 + Synt7 (Figure 3c Figure S1a), we constructed the karyotype of the 14 Th. intermedium chromosomes of 78829, as linkage groups 3St, 4St, 5St, 6J S -J, 7J S , 1St-J S and 2St-J S , respectively. The Th. intermedium chromosome karyotype is shown in Figure 3j. Therefore, the bulked Oligo-based FISH painting was effective in identifying each of the wheat chromosomes or chromosome segments for particular linkage groups in the wheat-Th. intermedium amphiploid.

Revisiting the Karyotype of Wheat-Th. intermedium Additions Z3
The wheat-Th. intermedium chromosome addition line Z3 contained a pair of shortsatellited chromosomes with clear nucleolus regions. The ND-FISH using probes Oligo-B11 + Oligo-pDb12H, showed that the added Th. intermedium chromosomes were a J-St-JS translocation, and the sequential ND-FISH using probes Oligo-pSc119.2 + Oligo-pTa535 showed that these chromosomes had weak signals (Figure 5a). The Oligo-FISH painting using probes Synt1 and Synt5 was used to screen the metaphase spreads of Z3. The probe Synt1 produced strong green signals on the chromosomes 1A, 1B and 1D, and the probe Synt5 generated the red signals on chromosomes 5A, 5B and 5D, respectively, while the added Th. intermedium chromosomes showed the green and red signals in each arm ( Figure 5b). Therefore, the Oligo-FISH painting results indicated that the added Thinopyrum chromosomes in Z3 were a pair of translocated chromosomes involving the short arms of group 5 and long arms of group 1.  The sequential ND-FISH with probes Oligo-5SrDNA, Oligo-pTa71, Oligo-3A1 and Oligo-pSt122 was performed to confirm the karyotype of the added Th. intermedium chromosomes of Z3. The signals from Oligo-5SrDNA, Oligo-pTa71 were observed in the interstitial regions of the satellite regions of Th. intermedium chromosomes in Z3 ( Figure S3). The alien chromosome in Z3 has strong Oligo-3A1 hybridization signals on the short arm closed to centromere, and Oligo-pSt122 signals at the telomeric ends of long arms. In addition, the PCR analysis by molecular markers of group 5S (CINAU1462, CINAU1463, CINAU1472, CINAU 1489) and group 1L (TNAC1021, TNAC1042, TNAC1076, TNAC1088) produced the Th. intermedium specific amplification of Z3 ( Figure 6). The evidence provided by molecular markers and the standard Th. intermedium karyotype (Figure 1) indicates that the Th. intermedium chromosome in Z3 was 5JS.1St-J S L. Therefore, the Oligo-FISH painting combined with molecular markers provide an opportunity for the precise identification of a complex chromosome rearrangement.

Discussion
Assigning a chromosome to a specific linkage group in polyploid Triticeae species with extremely large genomes traditionally relies on aneuploid analysis combined with individual chromosome recognition [22]. However, such aneuploid stocks are difficult to maintain and complete sets of aneuploids are not available for all Triticeae species [23]. Chromosome-banding techniques have long been considered as a fast, reliable, and economical means for the identification of chromosomes for wheat and related species; however, chromosome banding represents uninformative heterochromatin blocks [24][25][26][27][28][29][30][31][32][33][34][35]. Previous studies revealed that Th. intermedium possessed a large amount of cytogenetic polymorphism and structural modifications of chromosomes as revealed by C-banding [24]. Moreover, conventional FISH for chromosome identification was principally based

Discussion
Assigning a chromosome to a specific linkage group in polyploid Triticeae species with extremely large genomes traditionally relies on aneuploid analysis combined with individual chromosome recognition [22]. However, such aneuploid stocks are difficult to maintain and complete sets of aneuploids are not available for all Triticeae species [23]. Chromosomebanding techniques have long been considered as a fast, reliable, and economical means for the identification of chromosomes for wheat and related species; however, chromosome banding represents uninformative heterochromatin blocks [24][25][26][27][28][29][30][31][32][33][34][35]. Previous studies revealed that Th. intermedium possessed a large amount of cytogenetic polymorphism and structural modifications of chromosomes as revealed by C-banding [24]. Moreover, conventional FISH for chromosome identification was principally based on the dispersed or tandem repetitive DNA sequences, which are mostly not linkage group-specific [16,20,23,26]. Synthesizing short single-copy oligonucleotide pools has provided a new and affordable method to develop chromosome specific painting probes for FISH [27,28]. We first applied seven bulked oligos Synt1 to Synt7 as permanent resources, which enabled us to identify the homoeologous groups of chromosomes from Triticeae species fast, at a low cost and with high efficiency [13,14]. In the present study, we conducted the sequential ND-FISH probed by Oligo-B11 and Oligo-pDb12H to distinguish each of the three genomes of St, J and J S in Th. intermedium. After that, Oligo-FISH painting by probes Synt1 to Synt7 was used to identify the seven individual linkage groups. In the present study, we first set up all the 21 pairs of Th. intermedium chromosomes with their precise assignment of subgenomes and linkage groups based on the Oligo-FISH painting probes and ND-FISH with subgenome-related probes (Figure 1). Our protocols can be used to karyotype the different accessions of Th. intermedium. The heterozygosity of each linkage group of the Th. intermedium was revealed (Figure 2). The results indicate that the heterozygous FISH patterns were observed in seven chromosome pairs, indicating the complexity of the karyotype of Th. intermedium, which is difficult to be identified by previous cytogenetic methods. Recombination among inter-genomic and non-homologous chromosome pairs was not found in the present Th. intermedium accession. The appropriate multiplex probes could be applied for studying chromosome constitution and chromosome variation for a large number of wild wheatgrass genetic resources.
Cauderon [31] and Cauderon et al. [32] reported the production of a wheat-Th. intermedium partial amphiploid TAF46 with 56 chromosomes, and it was firstly identified to have seven pairs of alien chromosomes and wheat 5B-7B reciprocal translocation by C-banding [24]. GISH using a genomic DNA from Ps. strigosa, Chen et al. [33] clearly determined that TAF46 has six S and eight J genome chromosomes in a wheat background, and also found that chromosome 6A was missing in an aneuploid of TAF46. In the present study, we precisely identified the individual St and J chromosomes in TAF46 and confirmed that four 6D chromosomes substituted for 6A of wheat ( Figure 3). Zhong 5 has the Thinopyrum chromosome composition, including four S, two J S and eight S-J S or S-J translocation chromosomes [33]. The present study defined the linkage group of the individual Thinopyrum chromosome in Zhong 5, which is consistent with the previous ND-FISH results from the Zhong 5-derived addition lines [24]. The Robertsonian translocation between 1St and 1J S , 2J S and 2St, as well as two small translocations, occurred between 4D and Th. intermedium chromosomes in 78,829 (Figure 3). In the present study, the Oligo-FISH painting probes facilitated the study of inter-species chromosome homologous relationships and visualized non-homologous chromosomal rearrangements in some wheat-Thinopyrum derivatives. Our results provide a precise recognition of individual Th. intermedium chromosome and their rearrangement of the 13 wheat-Th. intermedium partial amphiploids, which will be helpful for the subsequent transfer of Thinopyrum chromatin from amphiploid to wheat. The high transmission rate of chromosome 7J S in the identified amphiploids may be partially due to the presence of novel disease-and insect-resistance genes [4,34,35].
The wheat-Th. intermedium partial amphiploid contained stable karyotypes, since the homoeologous group of three subgenome chromosomes have high complementary [4]. However, their chromosome structures were easily rearranged following their hybridization with wheat [5]. The wheat-Th. intermedium introgression lines have been developed to transfer novel gene(s) from the diversified gene-pool of Thinopyrum to wheat [5,8]. Tang et al. [34] revealed that the addition line Z3 derived from Zhong 5 contained a pair of Th. Intermedium-derived small-satellite chromosomes by GISH. Hu et al. [36] revealed that the C-banding patterns and the FISH signals by pTa71 hybridization of 1St#2 chromosomes in AS1677 differed from those in Z3. In the present study, the ND-FISH with multiple probes and the Oligo-FISH painting methods indicated that the added Th. intermedium chromosome in Z3 is a 5JS.1St-J S L translocation ( Figure 5). The Th. intermedium chromosomes in Z3 are derived from the chromosome set in the parent Zhong 5 ( Figure 4). Our previous study identified that the line Hy37, which originated from Zhong 5, contained a 5JS.3StS chromosome [24], indicating frequent chromosome modifications after the wheat-Th. intermedium amphiploid was crossed to wheat. Therefore, the oligo-painting probe-based FISH proved to be a useful tool for visualizing the occurrence of chromosome rearrangements during early and later generations of wheat-alien transfer subsequent to wide hybridization and chromosome engineering.
High molecular weight glutenin subunits (HMW-GSs) are encoded by the Glu-1 loci located on the long arm of group-1 chromosomes of Triticeae species [37,38]. Several genes for HMW-GS have been identified in the subgenomes of Th. intermedium [39]. Niu et al. [40] systematically characterized the HMW-GS composition of several wheat-Th. intermedium partial amphiploids and derivatives, and found that TAF46 and Zhong 2 expressed the Thinopyrum-specific HMW-GS. Hu et al. [36] confirmed that the Th. intermedium ssp. Trichophorum-derived partial amphiploid and substitution line AS1677 expressed the 1St-specific HMW-GS. The present study found that both TAF46 and Zhong 2 have the 1J chromosome, while other amphiploids contained the 1StS.1J S L-rearranged chromosome ( Figure 4). The line Z3 with 5JS.1J S L did not express Thinopyrum-specific HMW-glutenin subunits [40]. It is worthwhile to investigate HMW-GS expression in newly developed Th. intermedium 1J S substitution lines [41,42], and also to reveal the structure of the 1J S L in Z3 and determine the absence of the expression of HMW-GS. The HMW-GS gene expansion and expression will be studied in detail to reveal the mechanism of the changes during allo-polyploidization and introgression [43]. The ongoing precise genome sequencing of Th. intermedium species at a chromosomal level and the re-sequencing of wheat-Th. intermedium derivatives will be essential to dissect the complex evolutionary history of Triticeae species [43][44][45][46].

Probe Preparation
Seven oligo-FISH pools of probes Synt1 to Synt7 corresponded to each of the seven Triticeae homoeologous groups, respectively [13]. These oligo probes were selected from the single-copy sequences derived from 1H to 7H barley chromosomes with a 96% sequence homology with wheat linkage groups 1 to 7 chromosomes, which enabled us to distinguish the chromosomes for specific linkage groups. The bulked oligo libraries Synt1 to Synt7 were synthesized by MYcroarray (Ann Arbor, MI, USA). Probe preparation from the synthesized oligo library was performed as described by Han et al. [29]. The tandem repeat-based oligo-nucleotide probes for ND-FISH are listed in Table S1. Labeled Oligonucleotide probes were synthesized by Shanghai Invitrogen Biotechnology Co. Ltd. (Shanghai, China). The labeling and preparation of bulk painting oligos was conducted following the description of Li and Yang [14].

Fluorescence In Situ Hybridization
Root tips from germinated seeds were collected and treated with nitrous oxide followed by enzyme digestion, using the procedure of Han et al. [47]. The synthetic oligonucleotides were either 5 end-labelled with 6-carboxyfluorescein (6-FAM) for green or 6carboxytetramethylrhodamine (Tamra) for red signals. The protocol of non-denaturing FISH (ND-FISH) by the synthesized probes was described by Fu et al. [19]. After the oligobased FISH, the sequential FISH with bulk painting with oligos was conducted following the description by Li and Yang [14]. Photomicrographs of FISH chromosomes were taken with an Olympus BX-53 microscope equipped with a DP-70 CCD camera.

Molecular Marker Analysis
The PLUG markers [48] and CINAU primers [49] for location on specific chromosomes were obtained by searching the database of Wheat Genome Assembly ref. v1.0. The PCR protocol used the 8% PAGE gel separation was as described by Hu et al. [36].

Conclusions
The present oligo-bulked pool FISH visualized homoeologous regions directly on Triticeae chromosomes in a simple and fast experimental procedure. Our present comparative genomic-based oligo-painting FISH studies provide new insights into the evolution of the Th. intermedium genomes. The protocol of FISH with multiple types of oligos has great potential for the high-throughput karyotyping of wheat-Th. intermedium introgression lines for effective wheat breeding by chromosome manipulation.