Thinopyrum intermedium (2n = 6x = 42, JrJrJvsJvsStSt) and Th. ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs, but their chromosomal composition remains elusive. In current study, a complete set of wheat-Th. intermedium disomic addition lines (DALs) derived from Zhong4 (a wheat-Th. intermedium partial amphidiploid) were created by nullisomic backcross method. Based on sequential FISH-GISH, mc-GISH, as well as molecular marker selection, ES-01 (DA1St), ES-02 (DA2St), ES-03 (DA3St), ES-05 (DA5St-W), ES-06 (DA6Jvs-W), and ES-07 (DA7Jvs) were accurately characterized. Combined with cytogenetic identification of Zhong4, the determined genome composition was 14A + 16B + 14D + 2 (1St) + 2 (2St) + 2 (3St) + 2 (5St-W) + 2 (6Jvs-W) + 2 (7Jvs) + 2t. Furthermore, based on L4 (DA4St) and L7 (DA6St) derived from TAF46 (14A + 14B + 14D + 2 (1J) + 2 (2St) + 2 (3J) + 2 (4St) + 2 (5J) + 2 (6St) + 2 (7J)), individual St-chromosome of Th. intermedium was obtained by comparative FISH analysis. And the utility of the constructed FISH karyotypes in identification of two different 3St (3D) disomic substitution lines (DSLs) suggested that it could be a valuable tool for effective detection of St-chromosome in diverse common wheat background.

In addition, compared with the alien chromosome FISH karyotypes detected in Zhong4, genome composition of the wheat-Th. ponticum partial amphidiploid Xiaoyan 784 was analyzed. And it was 14A + 14B + 15D + 2 (1St) + 2 (2St) + 2 (3St) + 2 (4J) + 2 (5St-W) + 2 (6Jvs-W) + 2 (7JvsS). Four wheat–Thinopyrum DSLs, ES-09 (2St (2A)) and ES-10 (3St (3D)) derived from Xiaoyan784, as well as, ES-23 (2St (2A)) and ES-24 (3St (3D)) derived from Zhong4, were characterized to confer the same chromosomal compositions. According to the identical alien chromosome FISH patterns and similar specific agricultural performances, it was speculated that the alien chromosome (2St or 3St) derived from Th. intermedium and Th. ponticum conferred close homoeologous relationships. And it was further confirmed by meiotic chromosome pairing and genomic polymorphism. Based on SLAF-seq, four transferable St-chromosome-specific molecular markers from Th. intermedium to Th. ponticum were developed. The utility of 3St-chromosome-specific markers, PTH-113 and PTH-135, in a BC1F2 population showed that the newly developed markers could be useful tools for efficient identification of St-chromosome in a common wheat background.