Robertsonian translocations (RobTs) in the progeny of triticale (×Triticosecale Wittmack) plants with monosomic substitution of Aegilops kotschyi chromosome 2S k (2R) were investigated by fluorescence in-situ hybridization. Chromosome 2S k of Ae. kotschyi is reported to possess many valuable loci, such as Lr54 + Yr37 leaf and stripe (yellow) rust resistance genes. We used a standard procedure to produce RobTs, which consisted of self-pollination of monosomic triticale plants, carrying 2R and 2S k chromosomes in monosomic condition. This approach did not result in RobTs. Simultaneously, we succeeded in producing 11 plants carrying 2R.2S k compensatory RobTs using an alternative approach that utilized ditelosomic lines of triticale carrying 2RS (short arm) and 2RL (long arm) telosomic chromosomes. Identification of molecular markers linked to Lr54 + Yr37 genes in the translocation plants confirmed that these resources can be exploited in current triticale breeding programmes.Agronomy 2019, 9, 646 2 of 12 from wild relatives into the wheat genetic background [10]. Recently, several attempts were made to transfer rust resistance genes from Aegilops, Agropyron and Triticum species into triticale [11][12][13][14][15].Aegilops species are closely related to wheat (and triticale, per se) and carry a number of valuable traits, which have been effectively incorporated into wheat by developing wheat-Aegilops hybrids and deriving addition, substitution and translocation lines [16]. Aegilops kotschyi Boiss. (2n = 4x = 28 chromosomes, U-and S-genomes) is a wild tetraploid goatgrass native to Northern Africa, the Mid-East, and Western Asia. Ae. kotschyi germplasm is exploited in wheat breeding [17] as a source of high grain protein, iron and zinc [18]. Moreover, Antonov and Marais [19] observed leaf rust resistance that was effective against the infection of Puccinia triticina in Ae. kotschyi. Marais et al. [20] identified the Lr54 and Yr37 leaf rust and stripe rust resistance genes, and developed aT2DS.2S k L wheat-Ae. kotschyi translocation line. The first Lr54 + Yr37 marker was developed by Heyns et al. [21]. Moreover, translocation gene sequences were cloned and specific SSR markers were developed [22].Homoeologous recombination based engineering is the most common way for efficiently utilizing the wild relative gene pool for crop improvement [23]. The generation of translocation lines is the most promising pathway for the exploitation of alien germplasm in crop breeding [23]. In distant hybrids, unpaired chromosomes are present as univalents during meiosis. Monosomic chromosomes are prone to centric breaks at anaphase I of meiosis, which misdivide and the broken ends fuse during the interkinesis of meiosis II [24][25][26]. Fusion of the misdivided products may result in the formation of a Robertsonian translocation (RobT) [27].Several steps are required to generate RobTs (Figure 1a), with self-pollination of double-monosomic plants being the most common method used in the induction of RobTs [26]. Wheat breeders can use a l...