Molecular Markers For Lr38, Lr45 And Their Resistance-related Analysis Against Wheat Leaf Rust

Leaf rust, caused by Puccinia triticina, is one of the most severe diseases of common wheat (Triticum aestivum L.) all over the world. Breeding and application of resistant cultivars are the most economical and environment-friendly ways to reduce the damage caused by wheat leaf rust. Lr38 and Lr45 are the effective resistance genes to wheat leaf rust currently in the world. Related studies were carried out on both genes for molecular markers, and analysis of expression sequence as well as proteome, laying a solid foundation for the marker assisted selection (MAS) and identification of resistance mechanism for Lr genes.1. A new specific molecular marker disgnated as Y38SCAR982 for wheat leaf rust resistance gene Lr38 was developed by using a SCAR marker derived from E-chromosome of Thinopyrum, and it was found that Y38SCAR982 was co-segregated with gene Lr38 in the tested F2 progeny and can be used in MAS feasibly.2. A RAPD marker for Lr45 was acquired and converted into a stable SCAR marker named as YpSc20H1272. A SCAR marker, named YpSc20H750, for Lr45 was obtained based on specific RAPD fragment pSc20H.2 of Secale cereale. A new specific molecular marker for Lr45 was developed based on the fragment amplified with a SCAR marker primers (H11R/F) derived from S. cereale, and named as YpScH650, with a long genetic distance to Lr45. The three SCAR markers can be used to detect 1R to 7R chromosomes loci of S. cereale, except for YpSc20H750 in which no alleles loci were detected on 5R. These markers can be used in MAS feasibly based on the detection of 100 wheat cultivars used in this paper.3. Resistant genes of wheat leaf rust, Lr38 and Lr45, were investigated first time by TRAP, combined with SRAP and SSR or EST-SSR. A 277 bp specific fragment was acquired in TcLr38. The specific fragment has no homologous sequence in GenBank, but the same size fragment was amplified in most of the 100 wheat cultivars. The genes Lr38 and Lr45 were studied by TRAP as well, with a combination of SRAP with RGA. A specific fragment was acquired in TcLr45, which showed highly homology with EST sequence, but no homology with DNA sequences in GenBank database. One sequence of the specific fragment had 90% homology with Lr10, indicating that it may closely related to disease resistance. 4. An elongated fragment of 1213 bp from the 3′end of Y38SCAR982 in Lr38 gene was obtained first time by chromosome walking NASS-PCR. A full length of 2195 bp sequence with a complete ORF, encoding 313 amino acid of retro-transposons rve, has 67% homology with rve integrase core domain sequence of Hordeum.5. The 2-D maps of TcLr38 and Thatcher were analyzed first time. One different expression protein spot was identified in Tathcher, and was analyzed and searched its homologous proteins in the database based on NCBI by using MALDI-TOF-MS. Oxygen-bursting complex precursor (OEC), a kinase-like and beta galactosidase-like protein showed highly homology with a part of the differential protein polypeptide.6. A pair of specific primers were designed based on the OEC, and a fragment of 750 bp expected product was acquired when amplification was carried out on cDNA of TcLr38, Thatcher and DNA of TcLr38. 1000 bp products was acquired just in DNA of TcLr38 and Thatcher. So, a conclusion can be drawn that there were two kinds of OEC genes in TcLr38 genome, one with introns, and the other with protein sequence of oxygen-bursting complex.