**1. Introduction**

Rye (*Secale cereale* L.) is a useful gene source supporting disease and insect resistance, stress resistance, and higher yield for wheat (*Triticum aestivum* L.) breeding programs [1–6]. In fact, rye 5R chromosome contains some elite genes that can be used to improve wheat cultivars. The wheat-rye 4BL/5RL translocation chromosome indicates that the 5RL arm carried loci that can increase copper efficiency [7]. The wheat-rye 5R(5A) and 5R(5D) substitution line exhibits some favorite traits, including big spike, multispikelet, high satiation seed, high protein content, and positive effects on somatic embryogenesis [8–10]. The rye 5RS arm carries the resistance gene to Russian wheat aphid (RWA) [11]. The 5RaS arm that was derived from *S. africanum* Stapf. carries genes for stripe rust resistance, increasing spike length and reducing grain hardness [12]. The gene *Ddw1* that can reduce plant height in rye is located on the 5RL arm and its close linkage markers have been found [13]. It was also reported that the 5R chromosome harbors genes that determine its growth habit [14]. However, the elite genes on 5R chromosomes have not been successfully used in wheat cultivars because of linkage drag and the non-compensating and low recombination frequency of 5R with its wheat homologues [15,16]. Elite genes can be utilized effectively when segments of 5R chromosomes have been transferred into wheat backgrounds and been accurately identified. Rye chromosome-specific markers are conducive to identifying rye chromatin in wheat backgrounds and help in the effective application of rye elite genes

in wheat breeding programs. So far, few 5R-specific markers have been developed. Although terminal deletions of 5RL (T5RS.5RL-del) were obtained, they were not used to physically map 5R-specific markers [17]. In this study, the 5RKu chromosome derived from rye Kustro (*S. cereale* L.) was proven to carry stripe rust resistance gene(s), and some new PCR-based and 5RKu-specific markers were developed using specific length amplified fragment sequencing (SLAF-seq) technology. These new markers were mapped to six regions on 5RKu using 5RKu dissection lines in a wheat background. Additionally, the stripe rust resistance gene(s) was also physically located on a segmen<sup>t</sup> of 5RKu.
