Mutagenesis—A Potential Approach for Crop Improvement - Crop Improvement: New Approaches and Modern Techniques - page 180

naturation or single stand structure that result from changes in nucleotide sequence
but such techniques fail to identify the number or position of mutations within the
DNA fragment examined (DeFrancesco and Perkel 2001 ) so detection must be fol-
lowed by sequencing to distinguish between different polymorphisms. The more
direct methods of array hybridization or sequencing are currently expensive when
applied to multiple loci in large numbers of individuals. TILLING provides an alter-
nate approach to identification of naturally occurring SNPs in large populations that
is both robust and relatively inexpensive. This application of TILLING has been
termed ECOTILLING (Comai et al. 2004 ). Allowing of forceful discovery of mu-
tations, high throughput TILLING technology is ideal for the detection of natural
polymorphisms: CEL I cut with partial efficiency, allowing the display of multiple
mismatches in a DNA duplex. Therefore, interrogating an unknown homologous
DNA by heteroduplexing to a known sequence reveals the number and position of
polymorphic sites. Both nucleotide changes and small insertions and deletions are
identified, including at least some repeat number polymorphisms. This method is
called ECOTILLING.
As with TILLING, ECOTILLING is general, and should be applicable to most
species. The ECOTILLING allows the rapid detection of variation in many indi-
viduals and is cost effective because only one individual for each haplotype need to
be sequenced. The technology is applicable to any organism including those that are
heterozygous and polyploid (Comai et al. 2004 ). Sixty-three novel SNPs were iden-
tified in 9 target genes, for 41 tree accessions. The ECOTILLING method also was
applied to sugarcane ( Saccharum sp.), a complex polyploidy species, as a model to
develop and test new protocols for high throughput ECOTILLING using capillary
electrophoresis (Eliot et al. 2008 ). If SNPs in a population occur relatively rarely
(less than one polymorphic individual per pool), the DNA of up to eight such in-
dividuals can be pooled, as is done in TILLING. However, when most individuals
within a population differ at one or more base pairs in any given specific target
sequence, 8-fold pooling will complicate genotyping. For this reason, in highly het-
erozygous species, the genomic DNA from each individual is usually pooled only
with DNA from a reference individual for which the target has been sequenced.
In addition, to detect those loci that were heterozygous prior to pooling, unpooled
genomic DNA from an individual is Ecotilled separately.
ECOTILLING detects the number and relative position of all SNP's, includ-
ing point mutations, and small insertions and deletions, within a target sequence in
each individual tested. Thus both the spectrum of natural variation within the target
sequence and the distribution of that variation throughout the population can be
established. If knowledge of the specific nucleotide changes is required then DNA
sequencing must be done following ECOTILLING (Fig. 4.7 ). However, since the
number of different genotypes will normally be much smaller than the number of in-
dividuals examined, the target DNA from only a few representative individuals will
need to be sequenced to establish the exact array of genotypes thus reducing the cost
of SNP detection relative to direct sequencing (Simek and Novoselovi 2012 ). The
efficacy of ECOTILLING has been demonstrated by two recent studies involving