Mutagenesis—A Potential Approach for Crop Improvement - Crop Improvement: New Approaches and Modern Techniques - page 179

Fig. 4.8 Schematic of PCR
and heteroduplex production in
high-resolution melt analysis
(HRM)
to double-stranded DNA during thermal denaturation (Ririe et al. 1997 ). Accurate
control of temperature and continuous monitoring of fluorescence in instruments al-
lows detection of single base mismatches in amplicons up to 500 bp. The method has
been used both for genotyping and SNP discovery in medical genetics (Zhou et al.
2004 , 2005 ), and SNP genotyping in plants has been demonstrated. Mutation scan-
ning by HRM in hexaploid wheat requires a two-step amplification process, first,
using homeologue-specific primers to amplify a larger amplicon containing several
coding regions, followed by HRM analysis using primers specific for each exon or
part thereof; a simple flowchart is shown in Fig. 4.8 . As the melt analysis following
PCR is extremely rapid, the throughput of this technique is equal to or greater than
that of Cel1-based TILLING and is, arguably, easier to establish (Parry et al. 2009 ).
Ecotilling
The genomes of individuals within a single species contain significant genetic vari-
ation that has arisen from spontaneous mutation. The vast majority of this diversity
is in the form of single nucleotide changes commonly referred to as simple nucleo-
tide polymorphisms (SNPs). Such naturally occurring SNPs are of great interest
to scientists because they are useful as genetic markers in mapping, breeding and
genotyping and can provide information concerning gene structure, linkage dis-
equilibrium, population structure or adaptation. A number of different techniques
for identifying SNPs have been developed. Some of these detect differences in de-