*2.2. Chromosome Preparation*

Accumulation of cells at mitotic metaphase and fixation was carried out according to Kwiatek et al. [35]. The root meristemes were digested at 37 ◦C for 2 h and 40 min in an enzymes solution containing 0.2% (*v*/*v*) Cellulase Onozuka R-10 and Calbiochem cytohelicase (1:1 ratio) and 20% pectinase (Sigma), in 10 mM citrate buffer (pH 4.6). Chromosome preparations were made according Heckmann et al. [36]. Digested root tips were placed on slides with a drop of ice-cold 60% acetic acid. The mixture was spread on a slide using metal needle for 2 min on a heating table (Medax) set at 48 ◦C. The slides were washed with 200 μL of ice-cold ethanol-acetic acid (3:1, *v*/*v*) and placed in 60% acetic acid for 10 min, washed in 96% ethanol, and air dried.

Meiotic chromosome spreads from pollen mother cells were prepared from flower buds fixed with ethanol-acetic acid (3:1, *v*/*v*) according to Zwierzykowski et al. [37]. Anthers were transferred to a slide with a drop of ice-cold 60% acetic acid and dispersed with a metal needle. The slide was placed on a hot plate (45 ◦C) for 2 min, during which the drop was spread using a needle. The slide was removed from the hot plate, a drop of 60% acetic acid added and covered with a cover slip. The slide was frozen in liquid nitrogen, and cover slip was removed with a razor blade.

### *2.3. Probe Preparation and Fluorescence in Situ Hybridization*

Total genomic DNA was isolated using GeneMATRIX Plant & Fungi DNA Purification Kit (EURx, Gdansk, Poland). DNA of *Aegilops sharonensis* Eig. (a progenitor of the S-genome of *Ae. kotschyi*; PI 551020, U.S. National Plant Germplasm System, Aberdeen, ID, United States of America) was labeled by nick translation with Atto-488 dye (Atto-488NT kit; Jena Bioscience, Jena, Germany) to investigate *Aegilops* chromosomes behavior during meiosis in the hybrid. Blocking DNA from triticale (Sekundo, Lamberto and Bogo) was sheared by boiling for 30-45 min and used at a ratio of 1:50 (probe:block). Genomic in-situ hybridization (GISH) or multicolor GISH was carried out according to previously published protocols [35]. Mitotic chromosomes were identified using fluorescence in situ hybridization (FISH) with the repetitive sequences from pTa-86, pTa-535, pTa-465, pTa-k-566 and pTa713 clones characterized by Komuro et al. [38]. They were amplified from genomic DNA of wheat (Chinese Spring) according to Kwiatek et al. [39], and labelled with Atto-488, Atto-550, and Atto-647 dyes using a nick translation kit (Jena Bioscience). FISH was performed according to Kwiatek et al. [35]. Slides were examined with the Olympus BX 61 automatic epifluorescence microscope equipped with Olympus XM10 CCD camera. Image processing was carried out using Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH: Münster, Düsseldorf, Germany) imaging software and PaintShop Pro X5 software (version 15.0.0.183; Corel Corporation, Ottawa, ON, Canada). Chromosomes of *Aegilops* and triticale were identified by comparing the signal patterns of the probes [39].

### *2.4. Lr54* + *Lr37 SSR Marker Screening*

Genomic DNA of parental forms and offspring plants were isolated using Plant DNA Purification Kit (EurX Ltd., Gda ´nsk, Poland). All primers (Table 1) were manufactured by Sigma-Aldrich (Merck).

PCR reactions were performed in a LabCycler thermal cycler (SensoQuest Biomedizinische Elektronik, Goettingen, Germany). The 20 μL PCR reaction consisted of 150 nM each primer, 0.2 mM of each nucleotide, 1.5 mM MgCl2, 0.2 units of Taq-DNA hot-start polymerase (TaqNovaHS, Blirt, Poland), and 50 ng of genomic DNA as a template. A typical PCR procedure was as follows: 5 min at 95 ◦C, then 35 cycles of 30 s at 94 ◦C, 30 s at 50–60 ◦C (depending on the primer, Table 1), 1 min at 72 ◦C, and 5 min at 72 ◦C. 0.5μL Midori Green Direct (Nippon Genetics Europe) was added to each amplification product, ran on 2% agarose gel (Sigma), and then visualized and photographed (EZ GelDoc System, BioRad).

**Table 1.** Primer sequences and PCR conditions used for *Lr54* + *Lr37* marker identification on 2S<sup>k</sup> chromosome.

