**3. Results**

### *3.1. Chromosome Segregation in 40* + *M2R* + *M2S<sup>k</sup> Plants of Triticale*

In the control experiment, pollen mother cells (PMCs) of ten triticale plants carrying 40 + M2R + M2S<sup>k</sup> chromosomes were examined to identify metaphase I (MI) and anaphase I (AI) of meiosis (Figure 2a). We observed a 2S<sup>k</sup> univalent in all of 100 analyzed PMCs (Figure 2b). The number of misdivided 2S<sup>k</sup> chromosomes during AI was 9 (Figure 2c). No other misdivided chromosome of triticale was observed. Next, 48 spikes of the monosomic substitution plants (40 + M2R + M2Sk) were allowed to self-pollinate, and 2601 seeds were obtained. A subset of 289 plants were screened using GISH and none were found with the expected RobT. 185 (63.1%) of the derived plants carried a whole *Aegilops* chromosome. In the remaining offspring (104 plants; 36.9%), no *Ae. kotschyi* chromosome fragments were identified.

**Figure 2.** (**a**) Chromosome set of a 40 + M2R + M2S<sup>k</sup> triticale plant (**b**) Segregation of chromosome 2S<sup>k</sup> (green, Atto-488) at anaphase I in a 40 + M2R + M2S<sup>k</sup> triticale plant. (**c**) Centric misdivision of chromosome 2S<sup>k</sup> (yellow, Atto-647) at anaphase I in a 40 + M2R + M2S<sup>k</sup> triticale plant. Scale bar, 10 μm.

### *3.2. Chromosome Segregation in 40* + *M2RS* + *M2RL* + *M2S<sup>k</sup> Plants of Triticale*

The main goal of the experiment was to obtain triticale plants with introgression of a 2S<sup>k</sup> chromosome segmen<sup>t</sup> on the 2R chromosome. Hence, we crossed monosomic substitution (40 + M2R + M2Sk) plants with double ditelosomic D2RSD2RL plants of triticale to obtain triple monosomic triticale plants (40 + M2RS + M2RL + M2Sk). These plants contained a single telosomic 2RS, a single telosomic 2RL and a single monosomic 2S<sup>k</sup> chromosome (Figure 1b). 13,457 flowers of the ditelosomic line were pollinated with pollen collected from monosomic substitution M2S<sup>k</sup> (M2R) plants, and 3464 seeds were obtained (25.74% crossing efficiency). The chromosomal constitution of 100 randomly chosen plants were examined by a combination of genomic and fluorescence in situ hybridization methods (GISH/FISH), using total genomic DNA of rye and *Aegilops* species, and pTa-103 centromere sequence, as probes. 38 plants were found to be triple monosomic (40 + M2RS + M2RL + M2Sk) (Figure 3), 35 lacked the 2R chromosomes (40 + N2R + M2Sk), 15 plants were ditelosomic (40 + M2RS + M2RL) and 12 plants were double telosomic (40 + D2RL).

**Figure 3.** Karyotype of triticale plant carrying 40 + M2RS + M2RL + M2S<sup>k</sup> chromosomes, examined by genomic/fluorescence in situ hybridization (GISH/FISH). Total genomic DNA of rye (red) and *Ae. tauschii* (yellow), and pTa103 centromere sequence (green) were used as probes. Scale bar, 10 μm.

The triple monosomics were selected from further analysis (38 plants; 40 + M2RS + M2RL + M2Sk), and MI and AI were observed in 10 PMCs of each plant. Chromosome 2S<sup>k</sup> was present as a univalent in all PMCs examined. In most cases (35 plants; 92.11%), the *Aegilops* univalent underwent chromosome segregation and was moved randomly to one of the opposite poles of the cell (Figure 4a).

**Figure 4.** (**a**) Chromosome segregation of 2S<sup>k</sup> (green, Atto-488) at anaphase I of triticale line 40 + M2RS +M2RL + M2Sk. (**b**) Centric misdivision of chromosome 2S<sup>k</sup> (green, Atto-488) at anaphase I of triticale line 40 + M2RS + M2RL + M2Sk. Scale bar, 10 μm.

We also observed the misdivision of the 2S<sup>k</sup> chromosome arms in three plants (7.89%) (Figure 4b). Next, a total number of 68 spikes of 38 offspring plants (40 + M2RS + M2RL + M2Sk) were self-pollinated, and 3489 seeds were obtained. The chromosome constitutions of 100 randomly chosen plants were examined by FISH/GISH, using repetitive sequences as probes (pTa-86; pTa-535 and total genomic DNA from rye). The results of chromosome 2S<sup>k</sup> misdivision were observed in 13 plants, including: a double RobT-T2RS.2SkL and T2SkS.2RL (1 plant; Figure 5a, b and c); six plants carrying T2RS.2SkL, four plants with T2SkS.2RL, and two telosomic plants with the 2RS, 2RL and 2SkS chromosome arms (Table 2).

**Figure 5.** (**a**) T2Sk.2R Robertsonian translocations in triticale analysed by fluorescence in situ hybridization (R-genome chromatin—purple, Sk-genome chromatin—blue, pTa-713—green, pTa-465—yellow, pTa-k566—red). Genomic in-situ hybridization pattern of triticale plant carrying Robertsonian traslocations with segments of 2S<sup>k</sup> chromosome (green) at prophase (**b**) and metaphase (**c**) of root meristem cell. Scale bar, 10 μm.


**Table 2.** Chromosome constitutions and Lr54 + Yr37 SSR marker analysis of triticale plants carrying divided 2S<sup>k</sup> chromosome.

### *3.3. Lr54* + *Yr37 SSR Markers Analysis*

Initial analysis of molecular markers S14-297 and S14-410, linked to Lr54 + Yr37 loci [21,22], was conducted on the parental lines; namely the monosomic substitution M2S<sup>k</sup> (M2R) line and double ditelosomic D2RSD2RL triticale line. The same protocol was used to examine the subsequent offspring. An amplicon of 297bp (base pairs) was for obtained for the S14-297 marker, and 410bp for the S14-410 marker, as observed in control samples of *Ae. kotschyi* and double 40 + M2R + M2S<sup>k</sup> monosomic plants (Figure 6). The same 410bp product was amplified form the 6 plants with T2RS.2SkL RobTs and for 1 plant carrying double RobTs-T2RS.2SkL and T2SkS.2RL (Figure 6; Table 2).

**Figure 6.** Amplification products of S14-410 marker linked to loci of *Lr54* + *Lr37* leaf and stem rust resistance genes. (**1**) *Aegilops kotschyi* (UUSkSk); (**2**) triticale 'Sekundo'(AABBRR); (**3**) double 40T + M2R + M2S<sup>k</sup> monosomic; (**4**) T2RS.2SkL and T2SkS.2RL; (**5**) double ditelosomic D2RS + D2RL; (**6**) telosomic M2RS + M2RL + M2SkS; (**7**) T2SkS.2RL; (**8**) T2RS.2SkL.
