What is TILLING? Farmers till their fields and hobbyists till their gardens, but in the world of genetics and molecular biology, TILLING has another meaning.
TILLING stands for Targeting Induced Local Lesions In Genomes. In this case, the local lesions are mutations researchers purposely cause in the DNA of an organism. DNA screening is then used to identify single point mutations, which are changes on only one base pair of the billions of base pairs in the genome of an organism. Such a change might seem insignificant, but because the language of genes is determined by the order of the base pairs, changing only one may change the gene significantly. Researchers focus on genes of interest that have already been identified, such as genes affecting drought tolerance, insect resistance, or herbicide tolerance.
A gene is the molecular unit of heredity for an organism, a group of DNA base pairs that have a function in the organism. Genes vary widely in size and function, and a gene that affects drought tolerance may contain a few thousand base pairs, or a few hundred base pairs. Changing one base pair on a gene related to drought tolerance may make a wheat plant more drought tolerant or less drought tolerant by altering the structure of the protein that is encoded by the gene. Some mutations produce no effect at all due to redundancy in the genetic code.
TILLING uses a chemical to cause mutations, and advanced genomics technologies to detect mutations, but because the traits discovered through the process are introduced into plants using traditional breeding methods, products of TILLING are not considered genetically modified organisms (GMOs). It is an economical method of generating a large genetically diverse population of wheat lines. The CSU wheat breeding program is using TILLING to generate favorable traits in areas such as drought tolerance, wheat stem sawfly resistance, and human health-related properties.
CSU then evaluates these individual mutants’ phenotypes. Phenotypes are the observable properties of plants that are produced by the interaction of the genotype of the plant and the surrounding environment; a few examples being plant height, insect resistance, and disease resistance.
The seeds for the TILLING population are generated by soaking the wheat seeds in a chemical called EMS (Ethyl methyl sulfonate), which causes nucleotide substitution (replacement of single base pairs). After undergoing this process, only about 50 percent of the seeds are still viable and germinate. Those seeds are planted and self-pollinated, and the DNA from the second generation that has self-pollinated is extracted and analyzed, using CSU’s new equipment. Using their new DNA fragment analyzer, CSU wheat researchers look for mutations in genes of interest.
TILLING has been in use at CSU since 2009. In the first year of the project, Dr. Phil Westra and the Department of Bioagricultural Sciences and Pest Management collaborated with Dr. Scott Haley and the wheat breeding team to create a TILLING mutant population of the hard red winter wheat variety Hatcher.
The CSU wheat breeding team has already found some mutants related to drought tolerance, and a provisional patent has been filed on those genes. Those traits are currently being backcrossed into previously adapted Colorado wheat varieties to confirm that the mutants show the desired trait.
CSU is also using TILLING to look for wheat varieties resistant to wheat stem sawfly. This project looks for plants with low volatile production. Volatiles are the scents produced by the plant that attract the wheat stem sawfly.
TILLING is also being used to look for mutants that affect a variety of human-health related traits. This effort fits very well with CSU’s “Crops for Health” strategic initiatives and also the identity-preserved marketing program CWRF and ConAgra have established for the CSU-developed hard white winter wheat varieties Thunder CL and Snowmass.
The first TILLING project at CSU in 2009 was jointly funded by the Colorado Wheat Administrative Committee (CWAC) and Colorado Wheat Research Foundation (CWRF) royalty funds from the sale of certified seed. The CWAC board recognizes the importance of this program and CWAC funding for TILLING has quadrupled to approximately $108,000 annually.
Dr. Harish Manmathan was hired in January 2010 as a post-doctoral associate to lead the TILLING experiments. Dr. Manmathan has a Ph.D. in Plant Pathology from the University of Maine.
“We have expanded our TILLING program considerably since we started a few years ago. As we’ve developed certain traits we’ve realized the immense challenges that face us in transferring these traits to our germplasm, part of which is the need to validate that the traits have benefits for wheat production, disease resistance, or human-health related traits,” said Dr. Scott Haley, CSU wheat breeder.
CWRF royalty funds from the sale of certified seed were used in 2011 to purchase an automated plate reader, an automated liquid handling system, and a DNA fragment analyzer to accelerate the program. CSU Research Associate Rebecca Kottke works with Dr. Manmathan to utilize this new equipment for TILLING mutant identification.
The technology around TILLING changes rapidly. In the first years of the program, CSU sent tissue samples for DNA analysis to Kansas State University (KSU), and now the new equipment purchased with CWRF royalty funds allows that analysis to be done more efficiently on-site at CSU. Recently, CSU began implementing a new marker genotyping method known as KASP (KBiosciences Competitive Allele-Specific PCR) for screening for TILLING mutants and other traits. KASP marker technology is efficient and inexpensive compared to DNA sequencing for screening as traits are transferred to the breeding program. CSU Research Associate Tori Valdez is developing KASP assays for a variety of traits in the breeding program, not only those developed via TILLING.