Mapping Of SA1 Gene Resistant To Sitobion Avenae And Expression Analysis Of Candidate Genes In Wheat (Triticum Aestivum L.)

Wheat, one of the most important crops worldwide, is always seriously damaged by Sitobion avenae F., resulting in yield and quality decline significantly. Breeding and utilizing resistant cultivars are the most economical, environment-friendly, simple and efficient approach to control the aphids S. avenae. In this study, the aphid S. avenae resistance of wheat germplasm resources was evaluated. The resistant gene to S.avenae was identified using SSR（Simple Sequence Repeats） molecular markers and China spring nulli-tetrasomic lines, and mapped on chromosome 7DL in wheat. Also, differential expression cDNA library of resistant genes was constructed by using DDRT-PCR （Differential Display Reverse Transcriptase-PCR）. The candidate genes of S.avenae resistance were cloned from the differential expression cDNA library and sequenced. The gene functions of the candidates were also predicted in present study. The main results are as follows.1. On the basis of field and release investigation, we applied gray system theory, TOPSIS method （technique for order preference by similarity to ideal solution method） and the cluster analysis to evaluate 377 copies of wheat germplasm resources. Results indicate that there are no immune types and 168 resistant germplasm resources occupied 44.56% of the materials, among which 14.06% are of high and moderate resistance germplasm resources with a number of 53 copies. The correlation degree analysis of agronomic characters and aphid index reveal that the resistance breeding should obey the prior selection principle: first, the resistance, and then characters related to yield and quality should be taken into account. Results of TOPSIS and clustering analysis confirmed that the excellent S.avenae-resistant germplasm resources are Yumai 70, Amigo, 186 Tm, Xiaoyan 22, PI Gao, Donghan 1 hao and 98-10-35, and the highly susceptible germplasm resources are 1376 and Datang 991.2. Fourteen wheat germplasm resources were analyzed by using 175 SSR markers. Results showed that 36 SSR markers had polymorphisms in the three groups of wheat homologous chromosomes A, B and D, and group D homologous chromosomes had the highest genetic diversity. Among 21 pairs of chromosomes （1A～7D）, 7D chromosome consisted the highest genetic diversity while 7A consisted the lowest genetic diversity. Based on these results, the 36 polymorphic SSR markers were used for PCR amplification in 14 wheat germplasm resources. A total of 255 clear bands were obtained and among them, 244 bands were polymorphic. Each pair of primer could be amplified 3 to 13 SSR bands with average of 6.8 bands each. The percentage of polymorphic site was from 40% to 100%. Nei’s Genetic diversity and Shannon information index was in the range of 0.3264～0.4243 and 0.5078～0.6154 respectively. Cluster analysis showed that SSR classification results were consistent with that of traditional classification. Fourteen wheat germplasm resources were divided into three categories and the germplasm resources with the same resistance were classified to the same group as parent of resistant- germplasm resources.3. Genetic analyses of the F1, F2, F3 and BC1 populations bred from 98-10-35/1376 and Amigo/1376 demonstrated that aphid-resistance was controlled by one pair of dominant gene. By using 180 plants of F2, 71 plants of F3 and 8 plants of BC1 mapping population, it was observed that the S.avenae-resistant genes of 98-10-35 was linked to the four SSR markers, Xgwm350, Xbarc70, Xwmc702 and XCfd46 on wheat chromosome 7DL with genetic distances of 6.3cM, 15.1cM, 34.1cM and 19.1cM. According to the results of Chinese Spring nulli-tetrasomic lines location and pedigree analysis of 98-10-35, S.avenae-resistant genes which is new are located on chromosome 7DL, temporarily named as Sa1.4. Difference segments between 98-10-35/1376 F3 resistant plants and resistant parents of 98-10-35 as well as that between F3 resistant and susceptible population are selected through DDRT-PCR. Paired t test results were not significant while the probability value of 0.9158 indicating that gene expression on the RNA level is consistent with appearing in F3 resistant individuals and resistant parents, F3 resistant and susceptible population. Five differential expression patterns were detected between the progeny and its resistant parents: I. The gene silenced in one of the parents,Ⅱ. Special expression showed in progeny, III. Express consistent showed with resistant parents, IV. Up expression only showed in progeny but not in both parents,Ⅴ. Down expression only showed in progeny but not in both parents.5. Two cDNA segments of 308bp and 500bp were identified in the resistant population of F3 and F5 respectively by comparing cloning and sequencing of difference segments of resistant and sensitive plants. Blast analysis showed that the 308 bp difference segment is homologous to the presumed sorghum drought-resistant protein SORBIDRAFT₀5g025690, hypothetical protein OsJ₁7396, hypothetical protein Os05g0188500, hypothetical protein SCC3 and asparagine synthase protein in grape with the identities of 91%, 83%, 83%, 83%, and 67% in protein level respectively. The different segments have homology with binding protein of sister chromatid and protein related to hypothetical stromal antigen. The identities of different segments the protein encoded of 500bp to hypothetical photosynthetic system protein are respectively 51%. Thus, we presumed both of the candidate genes are new and related to S.avenae-resistance.