Germplasm Identification And Evaluation Of Resistance To TuMV And EIf(iso)4E Gene Cloning And Sequence Analysis In Brassica Campestris L. Ssp. Chinensis

No-heading Chinese cabbage(Brassica campestris L. ssp. Chinensis) is one of the most important vegetable crops in China. Virus disease, caused mainly by turnip mosaic virus(Tu MV), seriously affects its yield and quality. The most economic, effective and safe way to prevent viral disease is marker assisted breeding by screening different kinds of resistant materials and excavating resistant gene further. In this study, in order to obtain different kinds of resistant materials, identification of the resistance to Tu MV of no-heading Chinese cabbage germplasms at seedling stage was firstly conducted. In addition, the genetic analysis of resistance to Tu MV was performed using F2 populations constructed by parents with obviously different resistance. Furthermore, we cloned e IF(iso)4E.a and e IF(iso)4E.c gene in no-heading Chinese cabbage, analyzed the polymorphic loci and their influence on coding protein and the correlation with Tu MV resistance. These results could lay the foundation for further understanding the molecular mechanism of cabbage resistance to Tu MV in no-heading Chinese. Main results were as followed:1. Identification of the resistance to Tu MV of 127 no-heading Chinese cabbage germplasms was conducted with the method of artificial inoculation identification and ELISA test respectively at seedling stage. The result showed that the DI value ranged from 3.55 to 95.68, which expressed significant difference of resistance to Tu MV among accessions. Distribution plot for different resistance level to Tu MV basically corresponded to normal distribution and the peak was slightly partial to susceptible region. 6 and 13 accessions showing highly resistant and resistant to Tu MV were screened by the method of seedlings inoculation identification and expressed relatively abundant diverse of main agronomic characters. The result of ELISA test showed that the amount of virus among 117 materials was significantly different with P/N value ranging between 3.10 and 25.37, but it had no significant correlation with DI value. ELISA test could be used as a supplementary identification method to screening materials with highly resistance.2. 3 F2 populations were constructed crossed by 4 lines with different resistance. Identification of groups showed that the F1 plants were susceptible to Tu MV-C4, and the segregation data of resistant plants and susceptible plants in F2 generation were nearly 1: 3, fitting for the expected segregation of Mendelian model based on the action of one recessive allele.3. According to Bra035393 and Bra035531 sequence of B.rapa reference genome information, we cloned and sequenced e IF(iso)4E.a gene and e IF(iso)4E.c gene from 65 and 38 non-heading Chinese cabbage accessions respectively. Comparative analysis using DNAAMN and DNASP softwares revealed that totally 33 SNPs and 11 In Dels were characterized from allele of e IF(iso4E.a gene producing 7 haplotypes and 19 SNPs and 5 In Dels were characterized from allele of e IF(iso)4E.c gene producing 8 haplotypes. Compared with the reported sequences of heading Chinese cabbeage and oilseed rape, only 13 and 15 polymorphism sites existed in non-heading Chinese cabbage respectively. The results of Pi value and Sliding-window analysis both displayed that nucleotide polymorphism in non-coding region was far higher than that in coding region. 8 SNPs in coding sequence of e IF(iso)4E.a gene produced 3 different CDS, coding 3 kinds of proteins. Three missense mutation sites resulted in amino acid changes(Asp27His、Thr79Ser and Phe108Ty). 4 SNPs of e IF(iso)4E.c produced 5 kinds of CDS, coding 4 kinds of proteins. Two missense mutation sites led to amino acid changes(Ile80Thr and Pro150Gln). Three-dimensional structure of coding proteins predicted by SWISS-MODEL software indicated that amino acid substitutions mostly located near the cap binding pocket, leading to the change in part of the space structure, which possibly influenced on the location and amount of protein-protein binding sites.4. 5’-flanking and 3’-flanking sequences were analyzed by UCSC/Ensembl, NNPP, PLACE, Plant CARE and ARNold software. The results showed that the transcription start site of e IF(iso)4E.a gene was located at 52 bp of ATG upstream, and the gene expression may be regulated by light and stress. Some SNPs or In Dels had effect on the existence or the amount of regulatory elements. There was an terminator independent on ρ factor at 543 bp after stop codon. However, no terminator was predicted in the 3’flanking sequences of e IF(iso)4E.c gene.5. Through the correlation analysis between haplotypes, variation sites and Tu MV resistance,it resulted that different haplotypes of e IF(iso)4E.a and e IF(iso)4E.c had no correlation with Tu MV resistance, only In Del-6 of e IF(iso)4E.a gene were significantly related with Tu MV resistance. But the correlation may be a false positive because In Del-6 only e exist in one resistant material. Therefor, we deduced that e IF(iso)4E.a and e IF(iso)4E.c might not be the primary gene controlling the resistance to Tu MV in no-heading Chinese cabbage.