Mutagenesis—A Potential Approach for Crop Improvement - Crop Improvement: New Approaches and Modern Techniques - page 178

TILLING was first applied to Arabidopsis thaliana (McCallum et al. 2000a ,
2000b ). A mutagenized population was created by treating seeds with EMS. Proof
of concept was shown by the discovery of novel alleles in two cytosine methyl
transferase genes. Researchers have also developed web based software programs
to calculate the putative effect of induced or natural polymorphisms on gene func-
tion. CODDLE (http://www.proweb.org/input) allows requestors to specifically de-
sign their PCR primers to target the functional domain in which they are interested
or to target the most-conserved domain, which is likely to be the most sensitive
to amino-acid substitutions (Gilchrist and Haughn 2005 ). Also, the conservation-
based SIFT (Sorting Intolerant from Tolerant) software predicts with approximately
75 % accuracy, whether or not an amino acid change is damaging a protein (Ng
and Henikoff 2003 ). By using a reference DNA sequence, an exon/intron position
model and a list of polymorphisms, software reports the effects of these polymor-
phisms on the expressed gene product in a graphical format (Taylor and Greene
2003 ). Perry et al. ( 2003 ) adapted the TILLING method for the model legume Lotus
japonicas . In a pilot experiment, the frequency of point mutations was analyzed in
the symbiosis defective (symbiosis receptor kinase) gene, which is required for root
symbioses (Stracke et al. 2002 ). Using this population, 17 mutations were identified
that relate to six independent alleles, thus demonstrating the concept of Perry et al.
( 2003 ) The applicability of TILLING in a polyploid species for wheat was reported
by Slade and Knauf ( 2005 ). Over 200 mutations were discovered in the pilot screen
and the estimated mutation densities were exceptionally high: 1 mutation/40 kb in
tetraploid and 1/24 kb in hexaploid wheat. The TILLING method was applied to
model crop rice (Till et al. 2007b ). Two different mutagenic treatments provide
a suitably high density of mutations (over 1/500 kb) to consider development of
rice for a high throughput TILLING service. It was shown that high-throughput
TILLING is feasible to maize (an important commercial crop plant with a large ge-
nome but with limited reverse-genetic resources).Screening results from the pools
of DNA samples for mutations in 1 kb segments from 11 different genes, obtaining
17 independent induced mutations from a population of 750 pollens mutagenized in
maize plants. One of the genes targeted was the DMT102chromomethylase gene, in
which an allelic series of three missense mutations were obtained and are predicted
to be strongly deleterious (Till et al. 2004 ).
High-ResolutionMeltAnalysis(HRM):AnAlternative
ScreeningPlatform
Although Cel1-based TILLING is very efficient for detecting mutations in large
(1-2 kb) exon-rich amplicons from target genes, it is less productive when used to
screen genes with multiple small exons separated by larger introns, as mutations in
introns, except those at splice junctions, rarely affect gene function. High-resolution
melt analysis (HRM) has been established as an alternative screening platform for
such targets. HRM depends on the loss of fluorescence from intercalating dyes bound