AFLP Molecular Markers For Gene RLr1 And Cloning Of Resistance-Related Fragments In Foxtail Millet Against Leaf Rust

Millet leaf rust is one of the most important factors which affects yield of Setaria italica(L.)Beauv.Breeding and applying of disease-resistant varieties in agriculture production has been one of the most effective and economical ways to control Urornyces setariae-italicae(Diet)Yoshino and improve yield.Therefore,Scanning the tightly linkage or linkage molecular markers and cloning millet leaf rust resistance gene should be more important.Actully,it must greatly promote the resistance breeding in foxtail millet.In this study,the parents Shilixiang,Yugu 1 and their F₂ progeny were used to identify the disease resistance to millet leaf rust.Genetic analysis was performed and the resistance gene was mapped by using AFLP.In addition,the degenerate primers were designed based on the conserved domains of the reported resistance genes.The homology-based cloning for the candidate resistance genes from foxtail millet was carried out by combining with genomic walking technique.The main results are summarized as follows:1.Shilixiang,which is highly resistant to millet leaf rust,and Yugu 1,which are all highly susceptible to millet leaf rust,were crossed.F₂ population was obtained through the F₁ generations self-pollination.Inoculation was performed on the Shilixiang,Yugu 1, F₁,and F₂ populations.The result showed that Shilixiang and F₁ were resistant,Yugu 1 was susceptible,but F₂ population segregated significantly.The segregation-ratio in all F₂ generations were 3:1.Therefore,it is confirmed that Shilixiang carries a dominant gene for resistance to millet leaf rust.This resistant gene was provisionally named as RLrl.2.The AFLP analysis was conducted in the two DNA pools.One is from the resistant of F₂ progeny and the other from the susceptible,which were set up by bulked segregate analysis(BSA)in foxtail millet.Using two restriction enzymes MseI and EcoRI,and silver-staining in AFLP technique,33 out of the 102 pairs of randomly selected primers amplified the polymorphic bands.3.However,only two pairs of them(E-CTT/M-ACA,E-CTT/M-TAC)showed the same discriminating results in replications.The primers E-CTT/M-TAC produced 256bp fragment(E2)linked to gene RLrl with a 7.4 cM genetic distance.However,it did not work out that primers E-CTT/M-ACA produced 180bp fragment(E1)link to gene RLrl. The two DNA fragments were reclaimed from the gels.They were cloned and sequenced. The sequences were compared with known sequences with BLAST software in Genbank. E2 showed 62%homology to a part of translocase protein SecY sequence which allowed the translocation of proteins across the plasma membrane and formed part of a channel. The AFLP marker would be facilitated the selection on resistance gene in foxtail millet.4.The primers were designed according to the amino acid conserved regions of the reported plant disease resistance genes encoding proteins containing the nucleotide-binding sites(NBS)and leucine-rich repeats(LRR).Two coding sequences were obtained respectively from the RNA of Shilixiang carrying the Shilixiang genome conferring resistance against millet leaf rust by reverse transcription-polymerase chain reaction(RT-PCR).The promoter sequences were amplified using Genomic walking method.The two prolonged fragments(3 120 bp,1 527 bp)were isolated containing the conserved motifs of NBS-LRR such as P-loop,GLPL domain.BLAST analysis on NCBI showed that the first sequence showed 58 percent identical to the Oryza sativa L. resistance gene RPM1 and 41 percent identical to the wheat leaf rust resistance gene Lr10. What's more,the second one showed a 45 presence rate with the wheat leaf rust resistance gene Lr1.