Mutagenesis—A Potential Approach for Crop Improvement - Crop Improvement: New Approaches and Modern Techniques - page 175

bination. However, in higher plants, homologous recombination is not yet efficient
enough for practical use (Wang et al. 2001 ). Another potentially powerful targeting
method uses chimeric RNA/DNA hybrid oligonucleotides to introduce base chang-
es, insertions, or deletions (Rice et al. 2000 ), although, to our knowledge, the broad
applicability of this method remains to be demonstrated.
Nucleotide sequence variation is a major determinant of heritable phenotypic
difference in plant genomes. Variation can either be natural, from divergent popu-
lations, or induced through treatment with mutagens (Till et al. 2007a ). There are
several methods used in discovery mutations, which are natural or induced through
treatment with mutagens in the genomes. TILLING and ECOTILLING are closely
related methods that are useful in the rapid detection of small mutations and natural
polymorphisms, respectively.
TILLING:ABestScreeningToolforMutantPlantPopulation
Genetic variation is a powerful resource that humans have exploited over the mil-
lennia to advance biological knowledge and generate the crops and horticultural
varieties that have become so much a part of everyday life. In recent years, the
availability of genomic sequences from many plant species and the development of
a wide array of molecular-genetic technologies have enhanced our ability to detect
or engineer such variation at specific genetic loci (reverse genetics), greatly expand-
ing our capacity for both probing gene function and genetic engineering. McCallum
et al. ( 2000a ) have introduced a new reverse genetic strategy that combines the high
density of point mutations provided by traditional chemical mutagenesis with rapid
mutational screening to discover induced lesions. TILLING ( T argeting I nduced L o-
cal L esions I N G enomes) combines chemical mutagenesis (Koornneef et al. 1982 )
with a sensitive mutation detection instrument.
The TILLING strategy utilizes traditional mutagenesis followed by high through-
put mutation discovery (Mccallum et al. 2000b ; Colbert et al. 2001 ). The main
steps in TILLING are mutagenesis, the development of a non-chimeric population,
preparation of a germplasm stock, DNA extraction and sample pooling, screening
the population for induced mutations, and the validation and evaluation of mutants
(Fig. 4.6 ). The methods required for each step can be applied to many species, mak-
ing the TILLING process broadly applicable. Mutants discovered by TILLING can
be used for gene-function studies and can be introduced into breeding programs.
In a pilot experiment, DNA from a collection of EMS-mutagenized Arabidop-
sis plants was pooled, subjected to PCR amplification, and screened for mutations
using denaturing HPLC (DHPLC). DHPLC detects mismatches in heteroduplexes
created by melting and annealing of heteroallelic DNA. Among the lesions detected
were base transitions causing missense and nonsense changes that can be used for
phenotypic analyses.
TILLING is suitable for any organism that can be heavily mutagenized, even
those that lack genetic tools. Starting with a homozygous population is desirable,