Master of Science / Genetics Interdepartmental Program / Bikram S. Gill / TILLING (Targeting Induced Local Lesions IN Genomes) is a high-throughput, reverse
genetics strategy for scanning mutagenized populations for point mutations in loci of interest.
Originally, TILLING was used to investigate gene function in Arabidopsis and has since been
similarly applied for gene functional analysis in other organisms. TILLING also allows the
generation of novel genetic variation in specific genotypes and, thus, has been implemented as a
tool for crop improvement.
Ethyl methanesulfonate (EMS) is a widely used mutagen to induce point mutations in
most TILLING protocols. M1 plants are then self-pollinated and M2 seed harvested. A single
seed is grown from each M2 progeny and tissue taken for DNA isolation. M3 seed is cataloged.
DNA is pooled to increase the efficiency and aid in mutation detection. Polymerase chain
reaction (PCR) is used to amplify a locus of interest using the M2 DNA pools as a template. The
PCR products are digested with an endonuclease that cleaves mismatched, mutant DNA, and the
digested products are visualized. The pools for which PCR products are positive for a mutation
are deconvoluted to determine which individual plant of the pool was responsible for the
mutation. DNA from the positive individual is sequenced to determine the type of mutation
(missense, nonsense, synonymous). Individuals with mutations that are more likely to disrupt
gene function (nonsense and certain missense) are studied further by growing the corresponding
M3 generation.
In bread wheat, Triticum aestivum, TILLING is complicated by polyploidy: genes that
have homoeologs require that the functionality of each be studied. If functional homoeologs are
present for all three genomes, mutants must be identified for each homoeolog, followed by
successive intercrossing to produce a triple mutant plant. As a model for wheat genetics, we
propose TILLING in diploid wheat.
EMS mutant populations were created in diploid wheat (Triticum monococcum ssp.
monococcum) and the hexaploid bread wheat cultivar ‘Jagger’. The diploid and hexaploid wheat
populations were screened for mutations at the waxy locus, GBSS1, as a validation of our
population and for comparative analysis of mutation rates in 2x and 6x wheat. For diploid
wheat, GBSSI was screened in 716 M2 plants, and one mutant was found for 1.9 Mb screened.
3
For hexaploid wheat, GBSSI was screened in 518 M2 plants, and 30 mutants were identified
within a total of 657 Kb screened, giving a mutation frequency of one mutation per 22 Kb. The
reasons for this vast difference in mutation frequency between diploid and hexaploid wheat are
discussed. The diploid wheat population was further examined by screening for mutations
within four lignin biosynthesis candidate genes, for a total of 2 Mb screened. A single mutant
was discovered for both of the lignin genes PAL6 and HCT, giving a mutation frequency of one
mutation per 1 Mb screened.
| Identifer | oai:union.ndltd.org:KSU/oai:krex.k-state.edu:2097/4125 |
| Date | January 1900 |
| Creators | Rothe, Nolan |
| Publisher | Kansas State University |
| Source Sets | K-State Research Exchange |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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