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ABSTRACT. A cDNA library of Chinese wild Vitis amurensis, which is the most cold-resistant species in the genus Vitis, was constructed using young leaves of seedlings of the clone Heilongjiang potted and subjected to cold stress. The leaves were harvested at various times after cold stress for total RNA extraction, which was used to generate expressed sequence tags (ESTs). The titer of the original library was 2.67 x 10 6 pfu/mL and the corresponding combination frequency was 98.5%. The test values of the amplified library were 1.53 x 10 9 pfu/ mL and 98.2%, respectively. After randomly choosing, cloning and sequencing, 227 ESTs with high quality were obtained and submitted to GenBank database. Using BLASTX, 79.7% of the ESTs shared homology with known functional DNA sequences and 20.3% shared significant homology with unknown proteins. Some potential hardinessrelated ESTs were obtained, which were involved in signal transduction, Analysis of cold-responsive genes from Vitis amurensis stress inducement, defense reactions, transcription factors, etc. Some functional genes identified from the cDNA library have potential for plant defense. These sequences will be subjected to further researches on hardiness genes and their molecular mechanisms.

The genome of Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) contains many desirable characteristics, such as resistance to biotic and abiotic stresses, which make it suitable for wheat improvement (Kang et al., 2009). Several P. huashanica genes have already been introgressed successfully into the wheat (Triticum aestivum L.) genome (Zhao et al., 2010; Du et al., 2013a,b). In the 1990s, we generated (Chen et al., 1991(Chen et al., , 1996 the F 1 hybrid H881 (2n = 28, ABDNs) and a derived heptaploid H8911 (2n = 49, AABBDDNs) of common wheat cv. 7182 and P. huashanica (GenBank Acc. No. 0503383) via embryo culture, two backcrosses, and selfing. As a result, we generated a large and complex range of wheat-P. huashanica offspring that carried different chromosome number or structure variants, i.e., chromosome additions, deletions, and rearrangements. However, a major problem was the selection of recombinant plants derived from the large interbred population. After several years of screening and identification, a complete set of wheat-P. huashanica disomic addition lines (1Ns -7Ns, 2n = 44 = 22II) was developed. This was a repetitive and difficult work. Although cytogenetic techniques proved to be very useful tools for alien identification, they had limitations because of the small population number, the complexity of the experimental techniques, and envi- AbstractPsathyrostachys huashanica Keng is an endangered species that is endemic to China, which provides an important gene pool for wheat improvement. We developed a quick and reliable PCR-based diagnostic assay to accurately and efficiently detect P. huashanica DNA sequences from introgression lines, which was based on a species-specific marker derived from genomic DNA. The 900-bp PCR-amplified band used as a P. huashanica-specific RAPD marker was tested with 21 different plant species and was converted into a sequence-characterized amplified region (SCAR) marker by cloning and sequencing the selected fragments (pHs11). This SCAR marker, which was designated as RHS-23, could clearly distinguish the presence of P. huashanica DNA repetitive sequences in wheat-P. huashanica derivative lines. The specificity of the marker was validated using 21 different plant species and a complete set of wheat-P. huashanica disomic addition lines (1Ns -7Ns, 2n = 44 = 22II). This specific sequence targeted the Ns genome of P. huashanica and it was present in all the seven P. huashanica chromosomes. Therefore, this SCAR marker is specific for P. huashanica chromosomes and may be used in the identification of alien repetitive sequences in large gene pools. This diagnostic PCR assay for screening the target genetic material may play a key role in marker-assisted selective breeding programs.Additional key words: addition lines; marker-assisted selection; repetitive sequences; RAPD; SCAR.* Corresponding author: cxh2089@126.com; wj2105@163.com Received: 24-03-13. Accepted: 15-10-13.This work has one Supplementary Figure that does not appear in the printed article but that accompan...