Arabidopsis TILLING

Overview

We provide two populations to TILL arabidopsis.The first (being implemented) is the diploid A. thaliana population developed by STP in Seattle. The second is a tetraploid population developed at UCD. The choice of system should be helped by the description below.

Diploid arabidopsis TILLING

This is the well proven population from which almost 10,000 mutations have been extracted. Mutants can be either heterozygous (-/+) or homozygous (-/-). The mutation frequency has been described as 1/170kb of diploid DNA, or 5.8 mutations/megabase[1][2]. The background of the population is Col-0 erecta. This population was previously offered by STP in Seattle and screening was performed by the CELI-LiCor assay of mismatches in heteroduplexes. Following STP closure, we will offer it from Davis using the TILLING-by-Sequencing procedure[3]. The DNA is being rearrayed to fit the 3D-pooling.

Tetraploid arabidopsis TILLING

We have developed, in collaboration with Dr. Sundaresan and Dr. Simon Chan, a TILLING population of tetraploid A. thaliana, accession Columbia (Col), and currently have a ATC4X plate ready for screening (528 individuals). This population could be useful to TILL loci that have not yielded mutations through other reverse genetics approaches. For example, loci that affect the gametophyte will be unlikely to be isolated in a diploid because a mutation of that type would not be transmitted. This population has another advantage: it displays a very high density of mutations, about 4 times higher than that in the diploid population used in Seattle. The system also has one disadvantage: mutants are tetraploids. A diploid containing the mutation of interest can be derived easily by two crosses as follows:

Cross 1
mutant -/-/+/+ x diploid +/+
->F1 -/+/+

Cross 2
F1 -/+/+ x diploid +/+
-> -/+

Notes:
  1. the genotypes are provided as an example. Other genotypes are possible.
  2. at least 1/3 of progeny of cross 2 will be diploid, the rest will be aneuploids that can be identified by their aberrant phenotype.
Results with 4x arabidopsis population

We have a set of over 500 lines. We recently tested it on 15 genes targeting amplicons from 1000 to 1500 bases. We obtained 367 high probability mutations (virtually certain), including 19 predicted knock-outs. For an explanation of the F(t) score and its reliability see Tsai et al.[3] Each gene had in average 20-25 mutations.

Table 1. Results from TILLING 16
Publications

[1][2][3]

  1. Greene EA, Codomo CA, Taylor NE, et al. Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics. 2003;164(2):731‐740.
  2. Till BJ, Reynolds SH, Greene EA, et al. Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res. 2003;13(3):524‐530. doi:10.1101/gr.977903
  3. Tsai H, Howell T, Nitcher R, et al. Discovery of rare mutations in populations: TILLING by sequencing. Plant Physiol. 2011;156(3):1257‐1268. doi:10.1104/pp.110.169748
Funding

UCD startup funds and NSF Plant Genome award DBI-0822383, TRPGR: Efficient identification of induced mutations in crop species by ultra-high-throughput DNA sequencing

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