The seeds of K. hirsuta were collected individually from 7 different locations in Qinghai, China. Three to five individuals were randomly selected for cytological investigation in each population. Detailed information on collection sites is listed in Table 1 and Fig. 1.

Figure 1.

Map of sample collection sites.

Fourteen cDNA clones were selected for single-gene probes, which were previously mapped to the short and long arms of the seven homoeologous chromosomes in common wheat, respectively, by Danilova et al. (2014) (Table 2). The cDNA clones were developed and kindly supplied by the National BioResource Project-Wheat, Japan. cDNA sequences were amplified with PrimeSTAR Max DNA Polymerase (Takara Bio Inc., Kusatsu, Shiga, Japan Cat. # R001B) and standard primers T3/T7 by polymerase amplification reactions (PCR). The amplification products were purified with an Omega MicroElute Cycle-Pure Kit (Omega Bio-tek, Inc., Norcross, Georgia, USA Cat. # D6293-02).

All cDNA probes were labeled by nick translation as described previously, with minor modifications (Kato et al. 2004, 2006). Briefly, all cDNA amplifications were labeled with tetramethyl-rhodamine-5-dUTP (Roche Diagnostics GmbH, Mannheim, Germany Cat. # 11534378910). After the addition of EDTA (500 mM, pH 8.0) to terminate the nick translation reaction, the probes were purified using the Omega DNA Probe Purification Kit (Cat. # D6538-02) following the manufacturer’s recommendations.

The oligonucleotide probes were used for 5S rDNA, 45S rDNA, pAs1, and (AAG)_10. The designated oligonucleotides pAs1-1 plus pAs1-2, 5Sg, Oligo-pTa71-2 representing pAs1, 5S and 45S rDNA respectively (Danilova et al. 2012; Tang et al. 2014). All oligonucleotides were end-labeled using either fluorescein amidite (FAM; green) or carboxy tetramethyl rhodamine (TAMRA; red) (Sangon Biotech Co., Ltd., Shanghai, China).

Genomic DNAs of Pseudoroegneria stipifolia (Nevski, 1934) Á. Löve, 1984 (2n = 2x = 14; St genome) and Aropyron cristatum Gaertner, 1770, (2n = 4x = 28; P genome) were fragmented by autoclaving following the procedures of Dou et al. (2009). The treated genomic DNAs were labeled with tetramethyl-rhodamine-5-dUTP (red) or fluorescein-12-dUTP (green) (Roche Diagnostics, Germany) by a random primer labeling method.

The seeds were germinated on moist filter paper in Petri dishes at room temperature. Root tips with a length of 1–2 cm were collected and pretreated with nitrous oxide at a pressure of 7–8 atm for 2 h at room temperature following the method of Kato (1999). The pretreated root tips were fixed in ethanol-glacial acetic acid (3:1, v/v) for at least 30 min at room temperature, subsequently; each root tip was squashed in a drop of 45% acetic acid and observed with a phase contrast microscope (Olympus BX43). The slides with good chromosome spread were selected for further FISH.

The chromosome preparations were denatured in 0.2M NaOH and 70% ethanol for 10 minutes at room temperature; subsequently, they were rinsed in cold 70% ethanol for 1 hour and quickly air dried. The hybridization mixture per slide (total volume = 10 μl) contained 100 ng labeled probe DNA, 50% v/v formamide, 2 × SSC, 10% w/v dextran sulfate, and 0.1 μg salmon sperm DNA. The hybridization mixture with single-gene probes or labeled genomic DNAs was denatured in the boiled water for 5 minutes, and immersed in ice-water for at least 10 minutes. The hybridization with oligo-based probes was conducted directly without denaturation. The hybridization was carried out overnight at 37 °C. A sequential FISH technique with multiple rounds of hybridization on the same chromosome preparation was adopted in this study. The first hybridization was conducted with single-gene probes. The slide was washed in 2 × SSC at 42 °C three times in this round, and at least 10 cells with distinct signals were captured. Subsequently, the slide was washed by tap water. The second and third round hybridizations were carried out using the probe combination 45S rDNA and 5S rDNA, and the combination of pAs1 and (AAG)₁₀, respectively. After each of the second and third rounds of hybridization, the hybridization signals were removed by heating at 55 °C for 10 minutes on hot plate, followed by washing in tap water. The last round was genomic hybridization in situ hybridization (GISH) with genomic DNA probes of P. stipifolia and A. cristatum. Images were captured with a cooled CCD camera (DP80) under a fluorescence microscope (Olympus BX63). Finally, images were adjusted with Adobe Photoshop 6.0 for contrast and background optimization.

Fourteen selected single-gene probes are evenly distributed on the chromosomes of the seven homoeologous groups in wheat, two of which were mapped on the short arm and the long arm on each chromosome, respectively. Thus, two single-gene probes on each chromosome were used in the present study to identify the corresponding species-crossed homoeologous chromosomes in K. hirsuta. The individuals of the population HCZ (Table 1) were randomly selected for single-gene mapping. The first round of hybridization using single-gene probes showed that most of the single-gene probes produced the expected six hybridization signals in K. hirsuta, each genome of which had two hybridization signals on the pair of the homologous chromosomes (Fig. 2a). Exceptionally, the single-gene probe 6L-1 produced eight signals (Fig. 2 a5), whereas the single-gene probe 7S-1 yielded 10 signals in the chromosomes (Fig. 2 a6). Further, the single-gene mapped chromosomes were clearly identified by hybridizations with 5S rDNA, 45S rDNA, pAs1, and (AAG)₁₀ after the second and third rounds of hybridization (Fig. 2b and 2c). Lastly, the characterized target chromosomes were allocated to the different genomes as St, Y and P by GISH (Fig. 2d).

Figure 2.

Sequential FISH-GISH on mitotic chromosomes of K. hirsuta with single-gene and repetitive sequence probes a single-gene probes (arrowed) b 45S rDNA (green) and 5S rDNA (red) c pAs1 (red) and (AAG)₁₀ (green) d genomic DNA probes of P. stipifolia (red) and A. cristatum (green). Scale bars: 10 μm.

After multiple hybridizations with 14 single-gene probes, each of the K. hirsuta chromosomes was not only characterized with distinct chromosomal markers, but also its homoeology was determined (Fig. 3; Table 3). The St genome chromosome sizes ranged from 6.60 to 8.76 μm with an average of 7.68 μm; the Y genome chromosome sizes ranged from 5.54 to 7.56 μm with an average of 6.55 μm; and the P genome chromosome sizes ranged from 11.70 to 14.21 μm with an average of 12.96 μm. The relative chromosome arm ratio (long arm to short arm) ranged from 2.13 for the largest chromosome, 5St, to 1.07 for the smallest, 7St, in the St genome; the corresponding ratios ranged from the 2.08 for the largest chromosome, 5Y, to 1.09 for the smallest, 6Y in the Y genome; and from 1.90 for the largest chromosome, 4P, to 1.12 for the smallest, 7P in the P genome (Table 3). A referenced karyotype idiogram of K. hirsuta was suggested with the designated chromosome number from 1–7 corresponding to those in common wheat (Fig. 4). The features of chromosomes in K. hirsuta are as follows.

Figure 3.

Molecular karyotype of K. hirsuta with 14 single-gene probes and repetitive sequences probes a single-gene probe b 45S rDNA (green) and 5S rDNA (red) c pAs1 (red) and (AAG)₁₀ (green). Scale bar: 10 μm.

Chromosomes 1St and 5St were distinguished by 5S rDNA and 45S rDNA sites on the short arm, respectively. Both 2St and 6St have one major (AAG)₁₀ hybridization site, differing from 3St and 4St which both included two discrete (AAG)₁₀ hybridization sites. However, 2St was associated with (AAG)₁₀ on the short arm near the centromere, distinct from 6St with (AAG)₁₀ around the centromere, while 3St showed an additional (AAG)₁₀ minor signals in the long arm rather than 4St showed an additional major signal in the short arm. 7St showed none or one major (AAG)₁₀ hybridization site in the telomeric region of the short arm, and thus differed from the others (Fig. 4).

Figure 4.

Idiogram for chromosomes of K. hirsuta showing the distribution of 45S rDNA, 5S rDNA, pAs1, (AAG)₁₀ and single-genes. The names of single-gene probes that hybridized to more than one chromosome, and to non-homoeologous chromosome are highlighted in red. The color scheme (bottom right) shows the color of each probe as represented in this idiogram.

Chromosome 1Y was the smallest, with one major or minor (AAG)₁₀ site on the short arm. Chromosomes 2Y and 5Y harbored a 5S rDNA site in the intercalary and subtelomeric regions on the short arm, respectively. Both 3Y and 6Y had one distinct major (AAG)₁₀ site around the centromere, but 6Y showed stronger hybridization intensity than 3Y. Chromosome 4Y showed the two (AAG)₁₀ sites similar to 5Y, but 5Y was with the 5S sites. Chromosome 7Y exhibited one major (AAG)₁₀ site near the centromere on the long arm and one or two minor (AAG)₁₀ sites in the intercalary region on the short arm (Fig. 4).

Chromosome 1P was distinct with one major 45S rDNA in the telomeric region of the short arm. Chromosome 2P was identified as a metacentric chromosome with a distinct pAs1 hybridization on the intercalary part of the short arm, and a weak (AAG)₁₀ around the centromere. The single gene probe 4L-4 and 2L-1 produced hybridizations on the short and the long arms, respectively, in this chromosome. Chromosome 4P was characterized as a sub-metacentric chromosome with more pAs1 hybridizations on the long arm than on the short arm. The single gene probes 2S-1 and 4S-6 produced hybridizations on the respective short and long arms in this chromosome. Therefore, 2P and 4P could be reciprocal translocation chromosomes. Tentatively, 2P and 4P were respectively designated as 4PL/2PL and 2PS/4PS. Chromosome 3P was identified as a metacentric chromosome with opulent pAs1 hybridizations on the short arm. Chromosome 5P was characterized by the segregated 5S rDNA and 45S rDNA sites on the short arm. Chromosome 6P was characterized by weakly dispersed pAs1 hybridizations mostly distributed in the half of the short arm near the telomere, whereas 7P was identified as including dispersed pAs1 hybridizations on both arms (Fig. 4).

Comparison between the single-gene probes on K. hirsuta and the homoeologous groups of common wheat showed that most of the probes hybridized to the corresponding homoeologous chromosome arms of K. hirsuta, generally in their corresponding positions (Danilova et al. 2014). This indicates the chromosomal synteny across species. However, the degree of synteny varied between the genomes and between the chromosomes. The St genome chromosomes were revealed to be most strongly conserved, with each of the 14 single-gene probes in the corresponding positions as described in the wheat chromosomes. Most of the Y genome chromosomes maintained the same synteny as the St chromosomes, except that 3Y was detected as having an additional hybridization site at 7S-1 in the interstitial regions of the short arm. Both 7S-1 and 7L-4 mapped on the long arm of 7Y, suggesting the pericentric inversion in this chromosome. The collinearity of the P genome chromosomes was more distorted. Besides the translocation between 2P and 4P, additional hybridization of 6L-1 in the subtelomeric region of the 1PL and additional site of 7S-1 in the telomeric regions of 2PS were revealed in P genome (Figs 3 and 4; Table 2).

The same original cDNA probes were used for karyotype and chromosome structural analysis in Agropyron cristatum Gaertner, 1770, (2n = 14; P genome) (Said et al. 2018). Though fewer single-gene probes were used in K. hirsuta than in A. cristatum, chromosomal collinearity of the P genome across species was compared by shared common single-probes. Well-conserved single-probe hybridization positions were revealed in 3P, 5P and 7P across both species. Chromosome 1P in both K. hirsuta and A. cristatum showed conserved 1S-1 and 1L-1 hybridizations, but 1P in A. hirsuta included an additional hybridization 6L-1 in the subtelomeric region of the long arm. Chromosome 1P in A. cristatum was characterized as metacentric, whereas 1P in K. hirsuta was submentacentic. This indicates that 1P in A. hirsuta was structurally different from that in A. cristatum. Coincidentally, the reciprocal translocations between 2P and 4P were identified in both K. hirsuta and A. cristatum. The hybridization positions of single-gene probes 4L-4 and 2L-1 in 2P of K. hirsuta were corresponding to those in 2P of A. cristatum. Physical mapping more single-genes on 4P of A. cristatum revealed more complicated structural variations such as inversion. Though the limited single-gene probes were mapped in 4P of K. hirsuta, the single-gene probe 4S-6 in the long arm on 4P of K. hirsuta was accordant to those 4S-1, 4S-2, 4S-3, and 4S-4 in the long arm on 4P of A. cristatum. This observation indicates that although chromosomes 2P and 4P in K. hirsuta may be modified from those in A. cristatum, they are still considered to include an ancient common rearrangement. A paracentric inversion on the long arm of 6P was identified in A. cristatum (Said et al. 2018). However, the 6L-1 hybridization position in 6P of K. hirsuta was equivalent to the position of the homoeologous wheat chromosome This indicates no major chromosome arrangements in 6PL in K. hirsuta.

Karyotyping was conducted on 29 individuals of K. hirsuta from 7 different populations, by using repetitive sequences as chromosomal landmarkers. Chromosomal homoeology were detenmined referring the above reference karyotype derived from HCZ population. The polymorphisms of each chromosome and the karyotype of each individual were well described (Fig. 5; Table 4). The results identified a total of 47 chromosomal variants in the 29 individuals (Table 4). The St genome showed the highest number of chromosome variants (19), whereas the Y genome had 16 and the P genome had 12. Chromosomes 1St, 5Y, 6Y, 4P, 6P, and 7P were the most stable, with identical variants across populations, while 7St was the most variable with more than 3 variants. The majority of individuals showed FISH patterns in the homozygous state, although some were in the heterozygous state (34.48%, ten of 29 plants; Table 4).

Figure 5.

Molecular karyotypes of 29 K. hirsuta samples. The patterns of chromosomes were characterized by probe combinations A: 45S rDNA (green) and 5S rDNA (red); and B: pAs1 (red) and (AAG)₁₀ (green). Different variants are annotated by different letters. The translocated chromosomes are underlined, and indicated by Roman numerals I–II. Scale bar: 10 μm.

Most of the variants were characterized by the absence or presence of additional hybridization signals due to duplications, or deletions of repeats of pAs1 and (AAG)₁₀, as well as the absence or presence of hybridizations of 45S rDNA (Fig. 5 5St c, 5P b). Moreover, translocations involved in 3Y, 4Y, and 3P were detected in a few individuals. A tentative pericentric inversion was identified in 5P (Fig. 5 5P a) in different populations.

Chromosomal structural variations were detected in 3Y, 7Y, 1P, 2P, and 4P by using single-gene probes in the individuals of the population HCZ. Furthermore, the chromosomal polymorphisms were revealed in above chromosomes across different populations. The polymorphisms of 1P were detected as the hybridization intensity variation of 45S rDNA in the terminal part of the short arm (Fig. 5 1P a and b), whereas those of 2P were detecet as an additional 5S hybridization in the intrcalary region of the long arm (Fig. 5 2P a and b). However, the structural variation detected by single-gene FISH was in the distal part of the long arm of 1P and the distal part of the short arm of 2P, respectively. No polymorphisms of 4P were detected between populations. It suggests that the chromosomal structural variation in 1P, 2P, and 4P might be species-specific. Since the polymorphism sites on the short arm of 3Y and those on the long arm of 7Y were corresponding to the arms involving structural variation detected by single-gene probes, it indicates the structural variations in 3Y and 7Y might be population-specific.

Although the number of samples was not the same in the different populations, the results indicate that the populations QL, HCZ, and GCN included more variants than the others (Table 4). In particular, one specific inter-genomic translocation variant (3Y c) with the P genome segment was identified in the population GCN; one specific reciprocal translocation (4Y c and 3P b) was identified in the population HCZ (Fig. 5); and the individuals of QL showed a high heterozygous state of 80% (Table 4).