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Tilling
In
the TILLING
process (Colbert
et al., 2001), DNA is collected from a population of
plants with random point mutations induced by ethylmethanesulphonate
(EMS). By selectively pooling the DNA and amplifying with fluorescently
labelled primers, mismatched heteroduplexes can be generated between
wild type and mutant DNA. Heteroduplexes are incubated with the
plant endonuclease, CEL I, from celery, which cleaves heteroduplex
mismatched sites, and the resultant products are visualised on
a sequencing gel (see image). Subsequent analysis of the individual
plant DNAs from the pool DNA is used to identify the plant bearing
the mutation.
We
have set up such a population for Lotus japonicus (Perry
et al., 2003) which is now used as part of a community resource for TILLING (RevGenUK). Using our Lotus population we have shown that there was a significant bias
for replacements of glycine residues in functionally defective alleles (Perry et al., 2009) isolated by TILLING.
Perry, J. A., Wang, T.L., Welham, T.J., Gardner, S., Pike, J.M., Yoshida, S., Parniske, M. A Tilling reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiology 131: 866-871, 2003.
Perry J, Welham T, Brachmann A, Binder A, Charpentier M, Groth M, Haage K, Markmann K, Wang TL, Parniske M. Tilling in Lotus japonicus identified large allelic series for symbiosis genes and revealed a bias in functionally defective ems alleles towards glycine replacements. Plant Physiology (2009), Doi:10.1104/Pp.109.142190. |
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