Major Resistance Candidate Gene Prediction And Dcaps Marker Development Of Head Smut In Maize

Head smut in maize was a never-produced disease, caused by Sphacelotheca reiliana (Kuhn) Clint. Research results showed that the resistance of head smut in maize was controlled by multiple quantitative trait loci (QTL), the major resistance loci of head smut in bin2.09region of maize chromosome was detected, part of the chain and functional markers were developed. However, few studies regarding to cloning and application of resistance gene had been reported. This study on the basis of the resistance candidate sequence mining by using modern bioinformatics methods in major resistance region (bni2.09) of head smut in maize, took near-isogenic lines which only contained major resistance of head smut and their parents as experimental material, used TA-cloning method and DNAMAN software to clone and analysis sequence correlation to predicted candidate resistance sequences, selected the candidate resistance sequences associated with the major resistance gene in maize and developed dCAPS marker, designed to provide technical support for mining resistance candidate gene of head smut in maize and molecular marker-assisted breeding. The main results were summarized as followed:(1)DNA sequence in the2.09region of B73RefGen_v2chromosome was the research object of this study. Monocots in eukaryotes as a reference file, we used genetic forecasting software Fgenesh to predicted gene structure based on Hidden Markov Models (HMM),1356exon structure sequences contained continuous and complete open reading frame (ORF) were obtained in the bin2.09region of chromosome. Used protein structure prediction software InterProScan to predict protein domain of the1356sequences corresponding to the amino acid sequences, the results showed that only452amino acid sequences had conserved domain, and4sequences contained disease-resistant conserved domain NBS/LRR were identified as disease resistance candidate sequences, respectively named YC1, YC2, YC3, YC4.(2)The length of resistance candidate sequences YC1was665bp, ORF was663bp, the sequence BLAST comparison results found the similarity between YC1and disease resistance RPP13of Brachypodium distachyon, disease resistance protein RPM1and LR10of Oryza sativa Japonica Group, stripe rust resistance protein YR10and resistance protein LR10of Triticum aestivum were49%-63%; the length of resistance candidate sequences YC2was1378bp, ORF was1317bp, the similarity between YC2and disease resistance RPP8of Brachypodium distachyon, stripe rust resistance protein Yr10of Triticum aestivum, putative stripe rust resistance protein Yr10/Mlal of Sorghum bicolor were49%-63%; the length of resistance candidate sequences YC3 was959bp, ORF was957bp, the similarity between YC3and disease resistance RPM1of Brachypodium distachyon, disease resistance protein MLA1of Oryza sativa Japonica Group, NBS-LRR-S/TPK stem rust resistance protein of Hordeum vulgare were38%-41%; the length of resistance candidate sequences YC4was890bp, ORF was888bp, the similarity between YC4and disease resistance RPM1and RGH2A of Oryza sativa Japonica Group, disease resistance RPM1of Sorghum bicolor were34%-46%. Meanwhile, the initial predictions of protein physical and chemical properties of4resistance candidate sequences showed all of them had multiple hydrophobic domains, uniformly distributed throughout the amino acid sequence, and contained a large number of random coil structure which constituted protein specific function structural components. It illustrated the4sequences had specific function, rather than had no intention expression.(3)This study took High resistance inbred line Mo17,Qi319, Mo17donor near-isogenic lines L44, Qi319donor near-isogenic lines L272and susceptible inbred line Huang Zaosi as experimental material, successfully cloned4resistance candidate sequences from the five materials by TA-cloning method. Sequence alignment found the similarity between the cloned candidate sequences from4resistance materials and the obtained YC1, YC2, YC3, YC4by bioinformatics respectively were99.25%-99.55%、99.56%-99.78%、99.48%-99.58%and99.33%-99.55%, the similarity between the cloned candidate sequences from susceptible materials and the obtained YC1、YC2、YC3、YC4by bioinformatics respectively were99.40%、99.64%、99.58%and99.55%.(4)Used the DNAMAN sequence alignment software to correlation analysis the4resistance candidate sequences cloned from Mo17, Qi319, L44, L272and Huang Zaosi, the results showed YC1, YC2, YC3between resistant and susceptible materials did not exist different loci.Howerer, YC4between resistant and susceptible materials existed a different locus on808base site,the G locus of resistant materials mutationed C locus of susceptible materials, it was determined a SNP locus regarding to the resistance of head smut in the major resistanceregion.(5)The study had successfully transformed a SNP locus developed from YC4into dCAPS marker. We took65known resistance inbred lines as materials and established BSA bulk, the chi-square test showed dCAPS1marker was effective to screen above the level of highly susceptible materials, however, the internal selection of high resistantce, resistance, medium resistance and susceptible materials was invalid.