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Thinopyrum intermedium  (2 n  = 6 x  = 42, JJJ S J S StSt) is one of the important resources for the wheat improvement. So far, a few  Th. intermedium  ( Thi )-specific molecular markers have been reported, but the number is far from enough to meet the need of identifying alien fragments in wheat- Th. intermedium  hybrids. In this study, 5,877,409 contigs were assembled using the  Th. intermedium  genotyping-by-sequencing (GBS) data. We obtained 5,452 non-redundant contigs containing mapped  Thi -GBS markers with less than 20% similarity to the wheat genome and developed 2,019 sequence-tagged site (STS) molecular markers. Among the markers designed, 745  Thi -specific markers with amplification products in  Th. intermedium  but not in eight wheat landraces were further selected. The distribution of these markers in different homologous groups of  Th. intermedium  varied from 47 (7/12/28 on 6J/6St/6J S ) to 183 (54/62/67 on 7J/7St/7J S ). Furthermore, the effectiveness of these  Thi -specific markers was verified using wheat- Th. intermedium  partial amphidiploids, addition lines, substitution lines, and translocation lines. Markers developed in this study provide a convenient, rapid, reliable, and economical method for identifying  Th. intermedium  chromosomes in wheat. In addition, this set of  Thi -specific markers can also be used to estimate genetic and physical locations of  Th. intermedium  chromatin in the introgression lines, thus providing valuable information for follow-up studies such as alien gene mining.

Development of Sequence-Tagged Site Marker Set for Identification of J, JS, and St Sub-genomes of Thinopyrum intermedium in Wheat Background