Submitted to: Legume Crop Genomics
Publication Type: Monograph
Publication Acceptance Date: 12/1/2003
Publication Date: 3/15/2004
Citation: Ritchie, R.D., Wiley, H., Szymanski, D., Nielsen, N.C. 2004. Targeting induced local lesion in genomes - tilling. Legume Crop Genomics. Champaign, Illinois:AOCS Press. p. 194-203. Interpretive Summary: Impediments to improving soybean remain even after application of modern gene technology. Impediments include a lack of knowledge about individual genes that control important agronomic traits, an inability to define the function of important genes, and the identification of allelic variation for specific genes. To overcome these barriers a versatile set of tools that interact in a complementary way is required. This paper describes one approach in which available tools are combined to study genes that regulate seed development. A large number of genes, most of which have unknown functions but are differentially expressed during seed maturation, were isolated. A technique called 'TILLING' (Targeted Induced Local Lesions IN Genomes) is being used to discover their function. TILLING permits the selection of plants that bear mutations in specific genes to be identified. Once identified, the consequence of the mutation on plant growth and development can be studied. The population of mutant plants generated during TILLING is a valuable resource that can be used by other researchers who wish to study other soybean genes.
Technical Abstract: An approach to study genes that regulate seed development is summarized. cDNA libraries were made from seeds at 7 and 23 days after flowering (DAF). PCR subtraction libraries were created from them in which sequences common to both libraries were removed. This was followed by Mirror Orientation Selection (MOS) to enhance the selection of sequences that are differentially expressed at different developmental stages. Approximately 1000 unique DNA sequences were identified. To define the function of these sequences, a TILLING (Targeted Induced Local Lesions IN Genomes) population was established by chemical mutagenesis using N-nitroso-N-methylurea. Leaf tissue from M2 seedlings were collected for DNA analysis, and M3 seeds harvested and stored. The DNA will be screened to identify mutations in the differentially expressed sequences from the subtraction libraries. Although the soybean TILLING population was established to investigate genes involved in seed development, it is a general resource that can be applied to the study of other genes.