The Nipponbare population developed at the USDA ARS laboratory of Tom Tai at UC Davis is described in BMC Plant Biology . From this population, 5,120 individuals have been arrayed and are available for screening. We currently screen units of 512 individuals x 4 or x2. A rice TILLING order starts with 2,048. Deeper screens entail 2,048 + 2,048 + 1,024. Individuals from the population (M3 seed) are distributed by the Dale Bumpers Rice Center in Stuttgart, Arkansas.
Mutation yield and examples of results
Following the demonstration of TILLING by Sequencing described in Plant Physiology, we have developed a TILLING working strategy that queries 2048 individuals pooled in 48 pools. We have run this format reproducibly and with very satisfactory results. For example, in our “TILLING 17” run we processed 28 amplicons (900 – 1600 bp) corresponding to 21 target genes. The results are summarized in Table 1. We found 318 mutations with a CAMBa F(t) score > 4. These are high quality hits most of which are expected to be true. The distribution of the score is shown in Figure 1. The results in Table 1 exemplify what you may expect from a TILLING rice order. Importantly, the yield of mutations will be affected by the choice of the amplicon window CODDLE. Some users with long genes chose to have multiple amplicons TILLED (segm. column) and the effect of that strategy is shown in gene 6 for which 52 good mutations are now available, including two resulting in truncation.
Summary stats for TILLING 17
- searched total per M2: 34,206 bp
- total number M2: 2,048
- total searched bp: 70,053,888
- total high P mutations: 318
- mutations per Mb: 4.54
- Till BJ, Cooper J, Tai TH, et al. Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol. 2007;7:19. Published 2007 Apr 11. doi:10.1186/1471-2229-7-19
- 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 for Rice TILLING and this service is by grant 2004-35604-14265 from USDA Cooperative State Research, Education and Extension Service, NRI Plant Genome Program, and by a grant from the Rockefeller Foundation. TILLING-by-Sequencing is funded by the NSF Plant Genome Research Program.