The annual octoploid partial amphiploids Ganmai 8 and Ganmai 9 (2n = 8x = 56, AABBDDEE) are sister lines that were developed from the cross between common wheat line 91C-9 and the partial amphiploid line Yuan 16-3 in Shanxi Academy of Agricultural Sciences, Taiyuan, Shanxi province. The cold-hardy Th. intermedium (2n = 6x = 42, StStJJJ^SJ^S) accession originated from Russia and is currently maintained in Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang province. An accession of Pseudoroegneria strigose (Schult. A. Löve) (2n = 14, StSt) and Chinese Spring wheat were included as the sources of DNA to be used as a probe and a blocker to detect Th. intermedium chromosomes. Roegneria ciliaris (Trin.) Nevsk (2n = 28, StStYY) (Trin.) Nevsk was used as control in molecular test.

Ganmai 8 and Ganmai 9 as the annual maternal parents were crossed with Th. intermedium. The F₁ plants were perennial but sterile in the first year of growth. A few seeds were obtained from the second year. Similar performance was observed from F₂ to F₆ generations. Seeds obtained from the first harvest of each generation were germinated at 23°C and the plants were transplanted into the fields in April in 2-m rows and 0.2-m spacing between plants. Adjacent rows were spaced 0.3 m apart. From the F₅ to F₆, 30 seeds were sown directly into the field for examining seed germination.

Plots for testing cold-hardiness were established in a farm at Harbin Normal University (126°57′ E, 45°87′ N), Harbin, China. The agricultural climate of Harbin is warm semiarid, with spring drought and a semi-humid summer. The annual average temperature is 3.3°C, −19.4°C in January and 23.1°C in July. The extreme winter temperature is −35°C. Plants were allowed selfing in the fields, seed harvest usually occurred in August, but varied depending upon maturity of individual plants. Grains were harvested in autumn and the above-ground parts were cut at 10 cm above the soil surface. The PHR was visually evaluated from harvest to the onset of winter in October and November. The regrowth of underground rhizomes was also examined. From November to early April in the following year, plants in the field were exposed to the natural cold environment to examine their cold hardiness. If plants could survive during the winter and regrow for at least three consecutive years, they were regarded as cold-hardy perennials. Ganmai 8, Ganmai 9, and the Th. intermedium accession were grown each year in 2-m rows and 0.2-m spacing as the annual and perennial controls, respectively. Morphological observations were as described previously (Li and Li, 2006). Before harvest, about 20 plants of each line were randomly selected for trait investigation, including plant height, tiller number, spike length, spikelet number per spike, underground rhizome, cold hardiness, and growth period.

Cytogenetic analyses for the perennial wheat entries included chromosome counting of the root tip cells, meiotic behaviors of chromosomes, and characterization of chromosome constitutions by genomic in situ hybridization (GISH) and Oligonucleotide in situ hybridization. Seeds were germinated at 23°C for 24 h, 4°C for 48 h, and 23°C for 27.5 h. Root tips were incubated in ice water at 0–4°C for 24 h and fixed in Carnoy’s solution (ethanol: glacial acetic acid = 3:1) for 24 h. They were stained in 1% aceto-carmine for at least 5 h prior to squashing in 45% acetic acid. Chromosome numbers were counted under a light microscope (Leica DM LS2, Mannheim, Germany). Inflorescences were sampled at 8:00 to 9:00 am or 15:00 to 16:00 pm, fixed in Carnoy’s solution for 5–10 h, stored in 75% ethanol, and anthers at appropriate stages were stained in 1% aceto-carmine. Chromosome behaviors were observed under a light microscope.

DNA extraction from fresh leaves was performed using the CTAB method (Doyle and Doyle, 1987). The St-genome DNA as probe can be used for GISH analysis of Th. intermedium chromosome (Chen et al., 1998; Chen, 2005). The probe was prepared by labeling 1 μg (2 μl) Ps. strigose and common wheat “Chinese Spring” genomic DNA in 4 μl of DIG-Nick-Translation mix (Roche, Mannheim, Germany) and 14 μL of ddH₂O at 15°C for 90 min. The reaction was terminated by 1 μL 0.5 mol EDTA (pH 8.0) at 65°C for 10 min. DNA of Chinese Spring wheat and Th. intermedium were sheared to be used as a blocker. Anti-digoxin Rhodamine and DAPI (Roche, Mannheim, Germany) were added prior to incubation in the dark. A Leica DM6000B fluorescence microscope (Leica, Mannheim, Germany) was used for observing the hybridization signals, and images were captured with a Leica digital camera (Model DFC480).

An oligonucleotide (oligo hereafter) multiplex containing oligos pAs1-1, pAs1-3, AFA-4 (GAA) 10, and pSc119.2-1 was used in oligonucleotide in situ hybridization to discriminate wheat chromosomes. The synthetic oligo pAs1-1, pAs1-3, and AFA-4 were 5′ end-labeled with 6-carboxytetramethyl-rhodamine (TAMRA) for red signals. The synthetic oligo pSc119.2-1 and (GAA) 10 were 5′ end-labeled with 6-carboxyfluorescein (6-FAM) for yellow-green signals. Genomic DNA from Ps. strigose was labeled with fluorescein-12-dUTP by the nick translation method as described above and used as a probe for bright green signals. The protocol of GISH/FISH using the synthesized probes was previously described by Wang et al. (2017).

Universal E-genome primers P3 and P4 (5′-GCTGAATCTGCGTATCGTCCC-3′ and 5′-GACTTGTTCTTCGGCGTGTTG-3′), and St-genome primers St₅₄₂ (5′-CTTCCGCAGTCCTGTGTGAAG-3′) and (5′-TAGATGATGTCTCACGCTCAC-3′) were described by You et al. (2002) and Liu et al. (2007) The reaction mixture (25 μL) contained 15.7 μL deionized water, 2.5 μL of 10 × buffer, 2.5 μL of dNTP, 1 μL of primer R and primer L each, 0.3 μL of RT-Taq, and 2 μL (10–30 ng) of DNA. Amplification conditions were 94°C for 5 min and 40 cycles of 94°C for 30 s, 37°C for 45 s, and 72°C for 1 min, followed by 72°C for 10 min. An Eppendorf 9700 thermocycler (Eppendorf, Hamburg, Germany) was used for DNA amplification, and PCR products were examined by agarose gel electrophoresis.

Since 2007, F₁–F₆ hybrids between the two octoploid partial amphiploids, Ganmai 8 and Ganmai 9, and Th. intermedium were grown under the natural cold winter conditions in Harbin. Out of 1200 lines, 34 survived and showed perennial growth habit (Table 1). They were regarded as the wheatgrass type because they resembled their paternal parent Th. intermedium (Figures 1a,c).

Twenty F₁–F₃ lines have grew for 8–11 years under the local conditions. Nine F₁ lines, 1-11-1, 1-11-2, 1-11-3, 1-11-4, 1-12-1, 1-12-2, 1-12-3, 1-12-4, and 1-12-5, four F₂ lines, 1-1-4, 2-19-1, 1-2-10, and 1-6-2, and seven F₃ lines, 1-18-1, 1-15-1, 1-15-2, 1-15-3, 1-15-4, 1-7-4, and 2-11-1, were perennial. The F₁ plants were sterile in the first growth year, because they were heptaploids with poor chromosome pairing during meiosis. However, these plants were able to regrow and survive during the winter. After the second growing year, a few seeds were produced in these plants. Five years later in the field, the chromosome count of the F₁ lines reduced to 2n = 42. The seed set of these plants increased, and the best line, 1-11-3, had a percentage of seed set up to 44% (Table 2), but the seeds were smaller than wheat.

In the first growth year, the F₂ and F₃ plants usually had chromosome numbers ranging from 50 to 56 and were poor in seed setting. After growing in the field for more than 5 years, the chromosome number of all lines reduced from 49 to 42 and the seed set percentage increased to > 80% (Table 3), which was higher than that of the male parent Th. intermedium (∼30%). The highest number of tillers (512) was observed in an F₃ line 1-15-4, and this line had a mean seed set of 93.23% with the similar seed type of wheatgrass. More than three-quarters of the 150 F₁-F₄ plants did not resume growth in spring of 2012 partially because of no snow covering during the winter. Only two F₄ lines, 4-31 and 5-14, survived, and the chromosome numbers and seed set were similar to that of the F₁-F₃ perennial hybrids in the same first growth year. Five years later in the field, the seed set percentages of these lines were > 70% (Table 3).

From 2012 and 2016 90 F₅ hybrids were evaluated for cold hardiness and perennial growth habit. Only four lines, 4-0-8, 4-12-13, 4-14-15, and 4-25-29, survived for more than 3 years and were perennial. These hybrids had more than 2n = 50 chromosomes and low seed set for the first 3 years. The chromosome number was reduced to 2n = 42 with normal meiotic behaviors after grown in the field for 5 years and the seed set increased to more than 90% in later generations (Table 3), which was much higher than Th. intermedium.

During 2013 to 2019, eight F₆ lines survived the cold winter and showed perennial growth habit. Six of them, 42529344, 41213355, 412137111, 42529912, 414156, and 412133204, had 2n = 42 chromosomes. Line 42529912 and 412133204 kept good seed set from the first life cycle to present that was more than 80% in 2017 (Table 3). Meanwhile, the tiller number of line 42529912 (315) was highest in the F₆. The other two lines, 42529356 and 51820443 (Figures 2a,b), were sterile in the first 2 years and seed set was 36.13 and 1.28%, respectively (Table 4). The chromosome number of these lines ranged from 2n = 45 to 50. The tiller number of line 42529356 increased from 81 to 296, but decreased from 118 to 36 for line 51820443 over a 3-year period. These lines were subjected to full characterization in morphological, cytological, and molecular levels. They were the first materials in the F₆ generation to have the cold-hardy perennial trait and keeping more than 2n = 42 chromosomes.

Spikes of the F₁ and F₂ hybrids were classified as wheatgrass type based on their morphologies of spikes (Figure 1b). The common wheat type spikes appeared in the F₃ plants, which had 2n = 42 chromosomes. The wheatgrass-type plants were perennial, but the wheat-type plants were annual. In the F₄ and F₅ generations, a third spike type, designated the tritelytrigia type, was observed. However, most of them were annual and only a few grew in the field for 2–3 years. The regrowth hybrids only produced a few seeds with poor agronomic performances. The chromosome numbers of these lines varied from 2n = 48 to 56. The annual plants resembled the octoploid tritelytrigia parents, Ganmai 8 or Ganmai 9. In the F₆ generation, only the wheatgrass-type plants survived and had perennial growth habit.

From the F₁ to F₆ generations, 34 lines of the wheatgrass-type plants were lush, with an average plant height > 1 m, and except F₆ line 51820443, the number of tillers increased with the number of regrowth seasons (Tables 2–4). All the hybrid plants had PHR after August and the regrowing plants were able to grow up to the end of November (Figure 3a). During December and January, the leaves and stems of such plants remained green under the snow-cover. In March and April, the leaves and stems were withered. After cutting, new leaves regrew from the original crowns and the plants returned to the vegetative state for a new life cycle in May (Figure 3b). The perennial hybrid plants had well-developed roots and almost underground rhizomes after living for more than 3 years (Figures 3c,d). They matured at August, 1 month earlier than the first growth year. Grains obtained from these perennial plants became full and smooth with the increase of life cycles. Some grains, either red or blue in color, were larger than those of the Th. intermedium, but smaller than Ganmai 8 and Ganmai 9 (Figure 1d). The seeds did not germinate in the field during the first 5 years, but a few germinated thereafter.

Sixteen lines of the perennial wheatgrass hybrids from the F₁-F₄ generations were characterized using the molecular markers specific for the E and St genomes. The E genome-specific primers P3 and P4 produced a 982 bp band in 15 lines, except for line 1-12-1 and Chinese Spring (Figure 4a), indicating that they contained the E-genome chromatins. The St-genome-specific primer St₅₄₂ amplified the expected 750- and 450-bp bands in 12 progeny lines, and Th. intermedium, P. strigose, indicating the presence of the St-genome chromatins in these materials (Figure 4b and Table 5). Four lines, F₁ 1-12-1, F₃ 1-18-1, F₃ 1-15-2, and F₄ 4-31, failed to produce the expected bands, indicating the absence of the St-genome chromatins. During the 2016 and 2017 cropping seasons, 8 F₆ and 4 F₅ perennial lines were genotyped with the E- and St-genome specific molecular markers. All the lines examined produced the 982-bp product with P3 and P4 markers (Figure 4c) and the 750-bp and 450-bp bands with the St₅₄₂ marker (Figure 4d and Table 5). These results indicate that the perennial hybrids from the F₅ and F₆ generation had retained the genomic components of Th. intermedium.

Using the Th. intermedium-genome DNA probe, GISH analysis demonstrated that Ganmai 8 and 9 with 2n = 56 chromosomes were composed of 42 wheat chromosomes and 14 Th. intermedium chromosomes (Figure 5a). Using the St-genome DNA probe, examination of F₅ plants that had only survived for 2–3 years, indicated that these plants contained 28 to 42 wheat chromosomes and 14 to 24 Th. intermedium chromosomes, resulting in 48 to 56 chromosomes in total. Such as line 425294, GISH analysis showed that 24 chromosomes showing hybridizing signals with St-genomic probe. 20 chromosomes belonged to the St genome with the whole signals and four belonged to the J chromosome with the terminal signals. The rest of chromosomes from wheat displayed the blue were 32 (Figure 5b).

The perennial wheatgrass type of hybrids (2n = 42 = 46) varied in their chromosome compositions. Using St-genome and common wheat “Chinese spring” DNA probe, GISH analysis demonstrated that 30 lines of wheatgrass with 2n = 42 chromosomes from F₁ to F₆ has no wheat chromosomes and their genomes were consisted of Th. intermedium chromosomes. Only four lines from F₆, 412133204, 414156, 42529356 and 51820443, showed a difference of genome. Line 412133204 with 2n = 42 chromosomes mainly consisted of Th. intermedium chromosomes with 2 wheat chromosomes and 2 wheat-wheatgrass translocated chromosomes (Figure 5c). Line 414156 with 2n = 42 chromosomes consisted of Th. intermedium chromosomes with 2 wheat chromosomes and 4 wheat-wheatgrass translocated chromosomes (Figure 5d).

In order to determine the genome composition of the perennial hybrids, both St genome and oligo-probes were used to distinguish between wheat and Th. intermedium chromosomes. Based on the fluorescent signal patterns, the chromosome compositions of two lines with cold-hardiness were analyzed in detail. The F₆ line 42529356 (2n = 46) consisted of 11 St-genome chromosomes with bright pink fluorescence hybridization signals along the entire chromosomes; 4 J-genomes chromosomes that displayed a light-blue fluorescence signals over most of their lengths except for the terminal regions. The remaining chromosomes originated from common wheat. So, this line had 15 complete chromosomes from Th. intermedium and 31 complete wheat chromosomes (Figure 2c). Another F₆ line 51820443 (2n = 45) contained 13 St-genome chromosome, 2 J-genome chromosome, 2 J^s-genome and 2 St-wheat genome translocated chromosome, St-4D and St-1A, and 26 wheat chromosomes. Oligo-probes showed that wheat chromosomes contained 10 A-genome chromosome (1A, 3A, 4A, 6A, and a pair of 2A, 5A, and 7A), 5 B-genome chromosome (2B, 3B, 5B, 6B, and 7B), and 11 D-genome chromosome (1D, 3D, 4D and a pair of 2D, 5D, 6D, and 7D) (Figure 2d). This indicates that wheat chromosomes can be maintained in high numbers in the new wheatgrass hybrids up to the F₆ generation.