Abstract：Abstract Psathyrostachys huashanica is an endemic species in China and possesses multi-resistance to biotic and abiotic stresses. In order to develop Ns genome-specific molecular markers of P. huashanica, random amplified polymorphic DNA (RAPD) analysis was performed on genome DNA of P. huashanica and wheat (Triticum aestivum) using 204 arbitrary primers. Primer OP-D15 and OP-F19 could specifically amplify fragments in P. huashanica, but not in China Spring (CS). The two Ns genome-specific sequences, named with Psh-D15 and Psh-F19, were isolated and characterized. Psh-D15 was 1 106 bp containing one copy of CAAAA motif, one pair of direct repeat as well as one pair of invert repeat. Psh-F19 was 1 344 bp long and two copies of CAAAA motif, direct repeats as well as invert repeats were also present in this sequence. The results of BLAST analysis indicated that Psh-D15 had 66% sequence identity to a long terminal repeat (LTR) Gypsy retrotransposon which was part of sequence of T. aestivum (AM932686.1), and Psh-F19 shared 78% homology with LTR Copia retrotransposon which was part of sequence of T. aestivum (HG670306.1). Thus, it could be inferred that both Psh-D15 and Psh-F19 were novel Ns genome-specific repeated sequences of P. huashanica belonging to Gypsy-like and Copia-like retrotransposons, respectively. Based on the sequence information, two pairs of sequence characterized amplified region (SCAR) primers were designed from the low-homologous regions of Psh-D15 and Psh-F19, respectively, and they were designated as Psh-D15 F/R and Psh-F19 F/R. These two pairs of SCAR primers could successfully amplify target bands 270 and 558 bp, respectively, only in P. huashanica, but not in the other genetic stocks, such as Aegilops, Dasypyrum, Leymus、Elytrigia, Agropyron, Hordeum, Secale, Triticum urartu, Triticum durum and T. aestivum. This result indicated that the two novel SCAR markers could reliably and easily distinguish the chromatin of P. huashanica, thus, they could be used as Ns genome-specific markers for monitoring genetic materials of P. huashanica, and designated as Psh-D15270 and Psh-F19558. The development of species-specific markers, Psh-D15270 and Psh-F19558, provides a new tool for characterizing wheat- P. huashanica alien genetic stocks.
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