Constructing An EMS Mutant Population And Applicating TILLING, EcoTILLING Technology In Brassica Napus

A mutant is an individual, organism, or new genetic character arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the wild type. With mutants, genetic researchers are to find the rules of development of organisms, and breeders are to discover new genetic resources. In past, researchers mainly found mutants in nature and they found there were not enough natural mutants for their researches. In the post-genomic era, lots of genomic sequences are released and many genes are being to be annotated. Thus, constructing mutant population for special plants is very important. According to the characters of Brassica genome, the best way of constructing mutant population is physical or chemical mutagenized way. However, it is very difficult to find where the mutations are in genome. TILLING (Targeting Induced Local Lesions In Genome) is a new reverse genetics technology which was first reported on 2 000. It has been successfully applied on functional genomic and crop development. Since 2 000, TILLING has been reported on the model plant Arabidopsis and rice and it has been to be the important tool such as T-DNA inserting in those model plants. EcoTILLING is also a reverse genetics tool which was developed from TILLING. It can be used to screen variations in natural population; and these variations can be applied for genetic resource discovery, molecular marker development, association mapping and molecular evolution analysis. However, due to the complex genome and not enough sequence information, there was no report about TILLING or EcoTILLING applied on Brassica. Erucic acid was one component of lipid in seeds of Brassica, erucic acid content in seeds is one of the most important traits for crops in Brassica family. FAE1 (fatty acid elongase1) was considered to be the key gene to control erucic acid content synthesis in seeds. Low erucic acid content in seeds was one aim for Brassica breeding. The first B. napus variety with low erucic acid content in seeds was developed in 1960s and then there was no new low erucic acid content resource was found for B. napus. Therefore, investigation variations of erucic acid content in seeds for natural Brassica varieties and regenerating new low erucic acid content resource are very important.Therefore, in this research, a B. napus EMS mutant population was constructed. The genomic information of gene FAE1 was investigated with DNA library and genetic mapping population. Some mutations of FAE1 were screened with TILLING. At the same time, gene variations of FAE1 in Brassica natural population were also identified with EcoTILLING The following are the main results of this research.In the experiment of constructing B. napus mutant population, one DH line of B. napus cv. Ningyou7 was used as the wild parent to construct EMS mutant population. The mutant population was treated with two dosages of EMS solutions, 0.6% EMS concentration and 0.3% EMS concentration. In M2 generation, 0.6% EMS population contained 7 110 plants and 0.3% EMS contained 3 926 plants. All M2 plants were scrutinized for visible variant phenotypes. These included key developmental characters throughout the growing season, such as plant architecture, leaf, floral, and reproductive morphology. Oil content of part of M3 seeds was also measured. There were lots of mutants through the forward genetic screening.In the experiment of TILLING, 13 BAC clones of FAE1 were identified in B. napus cv. Tapidor DNA BAC library. And then it was found there were two paralogous FAE1 genes in B. napus genome. With sequences obtained from these BAC clones and TN genetic mapping population, molecular markers for these two FAE1 were developed. Then, the two FAE1 genes were mapped on A8 and C3 chromosomes. At the same time, two major erucic acid content QTL in seeds were detected on the map. The two FAE1 genes were found to be located on the peaks of the two QTL separately. 19 mutations were screened in 1 344 M2 plants in the mutant population with TILLING. The mutation loaded for 0.3% EMS population was one mutation is on every 130.8 kb in each 0.3% EMS M2 plant and one mutation for 41.5 kb in each 0.6% EMS M2 plant. The erucic acid content in M3 seeds of 4 plants showed to be reduced among these 19 plants which contained mutations in FAE1 gene. Two of the 4 plants were chose to be scrutinized for further genetic analysis and they were found that the mutations in FAE1 gene were the cause of reducing erucic acid content in seeds.In the experiment of EcoTIILING, 128 accessions of Brassica varieties were collected for EcoTILLING analysis. 8 were B. rapa, 9 were B. oleracea and 101 were B. napus. FAE1 polymorphisms in these 101 B. napus accessions were discovered with EcoTILLING Association analysis was done for these polymorphisms and erucic acid content variations in seeds. The possible cause of reducing erucic acid content in seeds for B. rapa and B. oleracea was also found. The deduced sequences of B. napus FAE1 with EcoTILLING and the FAE1 sequence obtained for B. rapa and B. oleracea. The polymorphisms between A and C genomes were found with these sequence and the molecular evolution relationship for Brassica also were analyzed.