Separation Of The Repeated Sequence Of H. Villosa, And Resistance-related Molecular Markers

To establish an ISSR marker targeted for Dasypyrum villosum specific chromosome, the PCR analysis was performed on D. villosum, Chinese Spring, Mianyang11, Triticum tetres and other materials by using 96 ISSR (inter-simple sequence repeat) primers. A specific segment of 388bp, which was named pDv848/388 (Genbank No. EF411201) was obtained from D. villosum by using primer UBC848. However, this segment was not amplified from the common wheat Chinese Spring and other materials tested. And then, PCR analysis was performed on Aegilops ventricosa, Aegilops tauschii and other related species using UBC848 as primer, the result showed that pDv848/388 was not appeared in any material tested. Furthermore, PCR analysis was performed on a set of T. aestivum-D. villosum addition lines CSDA1V~CSDA7V, the result suggested that pDv848/388 was only obtained from CSDA5V. Moreover, PCR analysis was carried out on D. breviaristatum, wheat-D. breviaristatum amphiploid, wheat-D. villosum amphiploid, wheat- D. breviaristatum partial amphiploid and its derivatives, the results indicated that pDv848/388 existed in all the material tested, suggesting that pDv848/388 can be used to detect Dasypyrum chromosome 5V in wheat background.In order to investigate the disease resistance mechanism of wheat related species, Dasypyrum breviaristatum, several resistance gene analog (RGA) primers were designed to amplify the genomic DNA of D. breviaristatum. A full-length of 5615 bp DNA sequence was isolated, and it was proved to highly homology to the wheat stripe rust resistance gene Yr10. The D. breviaristatum derived Y10-like sequences, named DbRGAYr10, contains 5'-terminal promoter, intron, 3'-terminal sequences and two predicted exon encoding 275 and 496 deduced amino acids respectively. The DbRGAYr10 consists of conserved Nucleotide-binding site (NBS) and Leucine-rich repeats (LRR) domain of plant resistance genes. BLAST search indicated the DbRGAYr10 was high homology to other reported resistance genes or resistance gene analogs in Triticum aestivum, Aegilops tauschii, rice (Oryza sativa), Sorghum bicolor, barley (Hordeum vulgare). In particular, the analysis of 5'-terminal promoter and 3'-terminal sequences of DbRGAYr10 suggests that it is an active putative resistance gene in D. breviaristatum. In addition, one specific primer pairs to target the DbRGAYr10 was successfully developed and used to detect the D. breviaristatum sequences in wheat-Dasypyrum derivatives. It is believed that our study will be beneficial to utilize D. breviaristatum as a donor source to introduce novel disease resistance characteristics into wheat background to enhancing wheat quality and yield.