Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species

Abstract

In the beginning of mutation research, mutations could only be detected indirectly through the analysis of the phenotypic alterations that they caused. The detection of mutations at the DNA level became possible with the development of sequencing methods. Nowadays, there are many different methods and strategies that have been created for mutation detection, both in natural and mutagenised populations. The strategies differ in accuracy and sensitivity, as well as in the laboratory facilities, time, costs and efforts that are required. The majority of them involve the pooling of DNA samples and the amplification of a gene (fragment) of interest followed by heteroduplex formation. One of the popular strategies for mutation identification takes advantage of the specific endonuclease (e.g. CEL I) that recognises and cuts heteroduplexes precisely at the 3 0 position of the mismatch site. The cleaved fragments are usually visualised through electrophoresis in a polyacrylamide gel using LI-COR sequencers, but agarose electrophoresis may also be used for this purpose, although with less sensitivity. A different mutation identification strategy, which is based on the high-resolution melting (HRM) technique, may be the method of choice when working with a short gene or a gene fragment whose length optimally does not exceed 400 bp.

The dmc1 Mutant Allows an Insight Into the DNA Double-Strand Break Repair During Meiosis in Barley (Hordeum vulgare L.)

“…TILLING was performed on DNA of 5,376 M 2 plants of the Hor TILLUS population. The method of mutational screening applied in this study was performed according to the protocol described elsewhere (Szurman-Zubrzycka et al, 2017; Jost et al, 2019). Briefly, the eight-fold pools were used for PCR reaction with IRDye-700 and IRDye-800 labeled and unlabeled primers (Supplementary Material 3).…”

The genetic relationships of Indian jujube (Ziziphus mauritiana Lam.) cultivars using SSR markers

“…Both systems employ fluorescent labelling and automated detection, which when compared, obtained the same results. The gel and fluorescence-based fragment analysis system of the LI-COR 4300 DNA analyzer is particularly useful and suitable for crop breeding research due to the sensitive detection of mutations and data visualization for genotyping [ 32 ]. Breeders could easily identify those cultivars and potential mutations based on the high-resolution genotype images from LI-COR 4300 DNA analyzer.…”

Forward Genetics Approach Reveals a Mutation in bHLH Transcription Factor-Encoding Gene as the Best Candidate for the Root Hairless Phenotype in Barley

“…One SNP marker could not be transformed into CAPS and it was genotyped using HRM method ( Supplementary Table S1 ), followed by the protocol described by Szurman-Zubrzycka et al (2017) , which allows to differentiate homozygotes and heterozygotes. The reaction was prepared in 20 μl volume with a ready-made High Resolution Melting Master Kit version 06 (Roche Diagnostics, Germany) according to manufacturer’s protocol, with 0.2 μM of each primer, 0.75 mM MgCl 2 and 20 ng of template DNA.…”

“…After the PCR reaction, the products were subjected to heteroduplex formation and digestion by CelI enzyme, followed by the purification of digestion products and polyacrylamide gel electrophoresis, according to the protocol described by Szurman-Zubrzycka et al (2017) .…”