Identification Of Molecular Markers Links To Leaf Rust Resistance Gene RUS In Setaria Italica Beauv. And Isolation Of Disease Resistance Genes

Uromyces setariae-italicae is a destructive pathogen of Setariae-italicae Beauv. all over the world and causes significant yield losses. Resistance cultivars are the most economical and environmentally-friendly way to reduce the damage caused by millet leaf rust. Researches on resistant genes are important and provide the base for the resistance breeding. Not only can our results accelerate the process of plant breeding, but also lay a foundation for the elucidate resistance mechanism and development of rapid and efficient control strategy. Taking Setaria italica Beauv. cultivar Shilixiang (resistant to leaf rust) and Yugu No.1(susceptible to leaf rust) and their F2 progenies as material, research on screening of AFLP marker which closely linked with Setaria italica Beauv. leaf rust resistant gene and cloning of resistant gene was done. Result will contribute to the marker assisted selection (MAS) and identification of resistance mechanism of Setaria italica Beauv. against leaf rust infection. The main results as follows:1. AFLP technology was employed to isolate Setaria italica Beauv. leaf rust resistant gene. 10 primer pairs could reveal the polymorphism of resistance and susceptible parents. Bioinformatics analysis result indicated that two bands had 86% and 92% homology with pol gene of Zea mays cultivar W22 and serine/threonine protein kinase of Oryza sativa respectively. Other bands had no significant similarity with sequence in GenBank database and may be new genes.2. Primers were designed according to known sequences. SCAR marker"SCEM-187"converted from AFLP marker"E+GC/M+TC"which closely linked with leaf rust resistant gene of Setaria italica Beauv. was obtained through the optimized PCR condition. Using SCAR marker"SCEM-187", 118 F2 progenies were employed to analysis separate ratio of resistance and susceptible plants. Results showed that"SCEM-187"linked to resistant gene of Setaria italica Beauv. against leaf rust infection with a distance of 27.4 cM.3. RGA(resistance gene analogs) analysis was employed on genomic DNA and cDNA of Setaria italica Beauv.. Seven NBS-like sequences contained conserved motifs P-loop and kinase 2αwhich were the characteristics of NBS type resistant gene of plant was obtained. These sequences had 47% to 98% homology with protein contained NB-ARC domain of Oryza sativa, NBS-LRR resistance protein of Oryza sativa and so on. Phylogenetic analysis indicated that there were three categories of RGAs. One group included RUS1-1, RUS1-2, RUS1-3 and RUS1-6 which were similar to NBS-LRR type disease resistant gene Pib of Oryza sativa. The second group included RUS1-5 and RUS1 which were similar to LZ-NBS-LRR type disease resistance gene RPP8 and HRT of Arabidopsis. The third group included RUS1-4 which was similar to NBS-LRR type disease resistant gene I2 of tomato.4. Using the primers which designed according to the conserved domain of Fen and Xa21 protein kinase, one STK-like sequence was obtained. BLASTX result indicated that it had 77% to 78% homology with putative receptor serine/threonine kinase PR5K of Oryza sativa, LRK33 and resistance-related receptor-like kinase Lr10 of Triticum aestivum.5. Using the method of Genome Walking and reverse PCR, DNA and cDNA sequence of RUS1 was obtianed. RUS1 included a 2673 bp DNA sequence with a 2193 bp coding region, 3 exons and 2 introns and it encode 731 aa. Molecular mass of RUS1 protein was 82.588 kDa and the pI of it was 8.20. The RUS1 gene could be induced by Uromyces setariae-italicae and was a constitutive gene with low abundance in the genome by semi-quantitative RT-PCR. The expression was increasing with the time of inoculation.6. Bioinformatic result showed that RUS1 contained conserved NBS, LRR and HD domains which were the characteristics of NBS type resistant gene of plant. RUS1 belonged to NBS-LRR class resistant gene and had the most similarity to Pib, HRT and RPP8. Swissplot software analysis result showed that alpha helix was the main type in the predicted secondary structure of RUS1 protein. RUS1 had 58% to 63% homology with NB-ARC domain containing protein of Oryza sativa, NBS-LRR disease resistant protein of Hordeum vulgare and disease resistance protein of Triticum aestivum.7. Southern blotting result displayed that there were multi-copies of RUS1 in the Setaria italica Beauv. genome DNA.8. Promoter sequence of RUS1 gene was obtained. The length of it was 675 bp. RUS1 gene promoter and pCAMBIA1300 vector were fused to construct RUS1∷GUS vector. GUS histochemistry staining result showed that promoter could activate gene expression.9. RUS1 gene (include promoter sequence) was obtained by the method of PCR.RUS1 gene and pCAMBIA1300 vector were fused to construct p1300∶RUS1 vector.10. Technology of SSH was employed to analyze gene express of Setaria italica Beauv. after inoculated with Uromyces setariae-italicae. Eleven sequences related to resistant gene of Setaria italica Beauv. against leaf rust infection were obtained. One sequence had 91% to 97% homology with SGT1 of Oryza sativa, Triticum aestivum, Hordeum vulgare and suppressor of g2 allele of skp1 of Oryza sativa and Zea may. Four sequences had no significant similarity with sequence in GenBank database and may be new genes. Others sequences had 58% to 96% homology with hypothetical protein of Oryza sativa, unnamed protein of Vitis vinifera, photosystem I subunit IX of Oryza sativa and Zea mays, unnamed protein and ribosomal protein S14 of Zea mays.