Identification And Molecular Mapping Of Yellow Rust New Resistance Genes In Wheat-secale Africanum Introgression Lines

Secale africanum is an important species in genus Secale, but it is now at the verge of extinction. S. africanum showed short plant height and excellent resistance to wheat diseases. It is thus essential to conserve the species and to study its potential value for wheat improvement. Wheat yellow rust, caused by Puccinia striiformis Eriks f. sp. tritici, is one of the most severely foliar diseases in wheat production of China. Breeding resistant cultivars is the most economical method to control the disease. Therefore, to develop the new resistant sources, to identify the stripe rust resistance genes, and to utilize continuously resistance genes in wheat breeding are of great significance. In this study, we developed S. africanum specific molecular markers, created and idenfied wheat-S. africanum introgression line, as well as mapped the resistantce genes in these lines. The main results showed as follows:1. Isolation, chromosome location and application of S. africanum specific repetitive sequences. Three repetitive sequences, named pSaP131165, pSaP13662 and pSaO5411 were identified in the S. africanum genome (Ra) by random amplified polymorphic DNA (RAPD). GenBank BLAST search revealed that the sequence of pSaP131165 was highly homologous to Revovler retrotransposon and pSaO5411 to a part of a Ty1-copia retrotransposon. Fluorescence in situ hybridization (FISH) analyses indicated that pSaP131165 and pSaO5411 were significantly hybridized to S. africanum chromosomes of a wheat-S. africanum amphiploid, and they were dispersed along the Secale chromosome arms except the terminal regions, but there was no signal in rye chromosomes with pSaP13662. Based on these sequences, three pairs of sequence-characterized amplified region (SCAR) primers were designed, and the resulted SCAR marker was able to target both cultivated rye and the wild Secale species, which also enabled to identify effectively the S. africanum chromatin introduced into wheat genome.2. Molecular cytogenetic identification of Wheat-S. africanum amphidiploid. Using sequential C-banding and and genomic in situ hybridization (GISH) techniques in the study of Triticum aestivum L. cv. Anyuepaideng-S. africanum (AF) and T. turgidum ssp. carthlicum-S. africanum (BF), we found that the banding patterns of S. africanum chromosomes 1Ra, 2Ra, 3Ra, 5Ra and 6Ra were similar to those of S. cereale chromosomes 1R, 2R, 3R, 5R and 6R, respectively, but the strengths and numbers of telomeric heterochromatin bands were comparatively less. Based on similarities with the standard C-banded karyotype of S. cereale, we recognized the standard C-band in S. africanum. A total of 32 progeny plants from AF and BF were cytologically analyzed, in which there are twelve plants contained breakages of S. africanum chromosomes and nine plants had wheat-Secale chromosome translocations. In particular, we observed non-Robertsonian translocation chromosomes, including intercalary Secale chromosomal segments in wheat chromosomes in three of 16 plants. The result above indicated there were many chromosomal variations and rearrangements between wheat and S. africanum chromosomes.3. Identification of new wheat-S. africanum 1RaS/1BL and small chromosomal segment translocation lines. Among F6 lines from MY11/AF tested by SCAR marker (P13LF/R, P13SF/R and O5F/R), 36 plants have Secale chromatin, in which there are nineteen plants resistant to yellow rust. GISH using S. africanum genomic DNA as probe revealed that the hybridized signals in eleven among nineteen which were resistant to yellow rust. Combining with the C-banding and GISH, we confirmed that line L1 has one 1RaS/1BL translocation chromosome; line L3 has one small segment inserted in wheat chromosome; lines L9-L15 have one pair of 1RaS/1BL chromosome; L16 has a translocation chromosome between S. africanum chromosome 3Ra and wheat chromome 3BS; line L17 is 1Ra substitution line. The rest yellow rust resistant line, there are no hybridized signal by GISH. The diversified wheat-S. africanum introgression lines identified will be of importance for further transferring the S. africanum genes to wheat.4. Molecular mapping for stripe rust resistance genes in wheat-S. africanum derivative line L2. With the help of moleculars and near-isogenic line (NIL), we confirmed that it is Yr17 gene in L2 line. In mapping the Yr17 gene, we developed two SCAR markers, SC-385 and SC-372, and one marker Xbcd348 based on expressed sequence tags (EST), which are tightly linked Yr17 gene and of which SC-385 is by far the nearest marker with 3.4cM distance to Yr17 gene. Using the wheat-rice collinearity, region of Yr17 gene compared with rice was established. We found that the rice AL606450 was possibly close related to the Yr17 gene of wheat, which can be of help to develop new markers for map-based cloning genes Yr17.5. Analysis of S. africanum pyruvate dehydrogenaseα-subunit gene (PDHA1) sequence. In researching second intron sequence in PDHA1 gene of Triticeae, we found a unique MITE insertion in S. africanum and S. sylvestre, which enables to develop specific marker to detect these two wild rye chromatin in wheat background. On phylogenetic analysis of Triticeae with PDHA1 gene, we found that intron polymorphism can be used to study the inter-species within the genus phylogenetic relationships. The localization of the PDHA1 showed that the PDHA1 localized on Dasypyrum villosum chromosome 2V and Aegilops ventricosa chromosome 2N analyzed by the wheat-alien addition lines. The results indicated the marker for PDHA1 gene can be used to trace the unique chromosomes region in Triticeae species. According to the sequence of PDHA1, the expression vector pET28a:PDHA1 was constructed. Prokaryotic expression indicated that the PDHA1 gene can be highly expressed in vitro.In summary, the study focused on the transfer of novel S. africanum chromatin to cultivated wheat. We developed S. africanum species-specific and chromosome specific molecular markers, and identified the wheat-S. africanum introgression lines by molecular cytogenetic techniques, as well as mapped a resistantce gene in the introgression line. The study was helpful for further utilizingthe S. africaum chromatin to the wheat breeding by chromosome manipulation.