Construction Of EMS Mutagenesis Mutant Library And TILLING Platform In Cucumber(Cucumis Sativus L.)

Cucumber(Cucumis sativus L.) is one of important the main vegetables in China and all over the world. With the rapid development of high-throughput sequencing technology, a large number of cucumber transcriptome, expression profile data and a large number of genetic resources have been identified in many institutes, Currently being analyzed gene function. However, in order to determine the function of the cucumber gene as early as possible, researchers tends to choose Arabidopsis or tobacco as receptor, due to the low efficiency of cucumber transgene, which has delayed the understanding of cucumber gene function and its mechanism of action, and also delayed the cucumber molecular breeding speed in a certain extent. According to the above situation, Ethyl methane sulfonate(EMS) combined with reverse genetics targeting induced local lesions in genomes(TILIING) technology for coverage of the cucumber genome mutant library construction and gene function studies based on the known genomic information of cucumber, should be an effective supplementary way for the low transformation efficiency in cucumber, but also enrich the existing cucumber germplasm resources.In this study, the cucumber cultivar 649, which was provided by the cucumber research group of Northeast Agricultural University, was used as the experimental material. EMS mutagenesis technique was used to create the cucumber mutant library. TILLING platform was constructed by using TILLING technology and the gene sequence information. Cucumber female gene F(Cs ACS1G), bitterness gene Bt and a low nitrogen tolerance-related gene CsNAC were used as as target genes t in the present study. The main results were as follows.(1) In this study, the cucumber cultivar 649 was induced by 5 groups of EMS concentration(0.5%、1%、1.5%、2%�'� 2.5%) gradient, According to the seedling rate and other comprehensive consideration, A cucumber mutant library was constructed by using 1.5% and 2% EMS concentration mutagenesis cucumber seeds. After two generations of self pollination, A total of 421 M2 families and 1807 M3 seeds were obtained in this study, which showed a mutant frequency of 19.1% and 15.2%. In addition, some important mutant traits were obtained in the M2 phenotype identification, such as having a few spines mutant, fruit co lor mutant, short fruits mutant, round leaf mutant, no flower mutant, dwarf mutant, loss of green leaf margin mutant, long leaf mutant, long fruits mutant, leaf crimple and androecious mutant.(2) In this study, An average of 6-8 plants per M2 family were sampledand mixed. DNA was extracted. DNA of every eight families was mixed to obtain DNA mixed pool after standardization, 53 DNA pools were obtained. Primers were designed by using sequence information of cucumber female gene F(Csa6G496450), bitterness gene Bt(Csa5G157230) and low nitrogen tolerance-related gene CsNAC(Csa6G127320), Amplified by nested PCR, a total of 5 amplified target fragment screened, screening the sequence length is about 8440 kb, Cucumber TILLING technology platform was tried construction by using a point mutation detection kit and the genetic analysis system.