Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®
© Dong et al; licensee BioMed Central Ltd. 2009
Received: 21 May 2009
Accepted: 4 December 2009
Published: 4 December 2009
TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes.
We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation.
Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.
Detection of SNPs in genes of interest, whether induced or endogenous, is a powerful tool to explore gene function and to identify desired mutations for breeding. TILLING has proven to be a valuable methodology for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection. As a post-genomics tool, TILLING is not only useful for functional genomics , but is also effective for crop improvement . TILLING produces a large chemically mutagenized population with random mutations across the genome, so that an efficient mutation detection method is essential. SNP discovery methods used in TILLING include full sequencing , denaturing high-pressure liquid chromatography (dHPLC)  and heteroduplex mismatch cleavage assay using endonuclease CelI followed by sequencing . Among these, the mismatch cleavage assay has high sensitivity in pooled samples, and is therefore high-throughput and low cost. Other mutation scanning methods such as single-strand conformational polymorphism (SSCP) , denaturing gradient gel electrophoresis (DGGE)  and technologies such as pyrosequencing  and mass spectrometry (MS)  have advantages and disadvantages regarding sensitivity, throughput, cost and simplicity. Heteroduplex mismatch cleavage assay works in any PCR amplicon (usually 0.5-1.5 kb) and any sequence context. The only requirement for heteroduplex assay is the purity of a PCR product. Therefore, PCR reactions for heteroduplex assay are performed using gene-specific primers at high stringency. However, these conditions are sometime difficult to achieve when TILLING a polyploid species. For TILLING in soybean, a recent allotetraploid species , a restriction enzyme digestion of the genomic DNA before PCR was added to the method in an attempt to reduce the homoeologous complexity , but this method would not work without a locus-specific restriction site.
Bread wheat (Triticum aestivum) is an allohexaploid species with three closely related genomes. Most wheat genes are present as three similar sequences of homoeologous loci with high exonic homology and lower homology in introns. Locus-specific primers can usually be designed in intron regions, as shown in wheat waxy genes . However, some wheat genes have high homology among the three homoeologous loci even in introns, so that locus-specific PCR is not easily achievable. Here we report a new method using High Resolution Melting (HRM) analysis and Mutation Surveyor® to screen mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene allowing simultaneous screening of the three homoeologous loci. Conserved regions of a gene which can be identified from multiple sequence alignment of a large number of divergent orthologous genes are believed to have high functional significance http://pfam.sanger.ac.uk/. Mutations in these conserved sequences will have a high likelihood of being deleterious, which is often the purpose of TILLING. For effectively screening mutations in the conserved regions, where locus-specific primers are not easy to obtain, we developed this method allowing simultaneous mutation detection in a functional domain of all three homoeologous genes in hexaploid wheat.
HRM analysis is an extension of previous DNA melting (dissociation) analysis enabled by the new generation of fluorescent dsDNA dyes . These dyes, such as LCGreen and CYTO®9, have low toxicity to PCR and can therefore be used at high concentration to saturate the dsDNA PCR product. Greater dye saturation means there is less dynamic dye redistribution to non-denatured regions of nucleic strands during melting so that the measured fluorescent signals have higher fidelity [12, 13]. The combination of these characteristics provides greater melt sensitivity and higher resolution melt profiles making it possible to detect SNPs in PCR amplicons, even in somatic mutations and methylations [14–18]. Mutation Surveyor® (SoftGenetics, State College, PA, USA) is a commercially available software for DNA variation analysis that allows automatic mutation detection in sequence traces. Mutation Surveyor® is claimed to detect > 99% of mutations, with sensitivity to the mutant allele extending down to 5% of the primary peak (mosaic or somatic mutations) provided the sequence quality meets a minimum Phred score of 20. The method presented here was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population , targeting the SSII genes.
Mutation Surveyor®can detect heterozygous mutations in an ampilcon containing three homoeoloci
The mutation report of Mutation Surveyor® after sequence trace analysis of mutant/non-mutant mixed samples.
Mutant allele in pooled DNA
17 mutations are identified in 192 TILLING lines in ABD6-9 after Mutation Surveyor® analysis of sequence traces and confirmation by HRM analysis.
Mutation Surveyor report
Position in ABD6-9
Position in Gene (SSII-A)
Amino acid change
High Resolution Melting (HRM) analysis of the SSIImutants
Detecting unknown mutations using HRM and Mutation Surveyor®analysis
Comparison of results from independent HRM and Mutation Surveyor® analysisof 140 TILLING lines.
% false positive rate4
Progeny testing and cloning
PCR products ABD3-9 (for 4A7) and ABD6-1 (for 4D7) amplified from homozygous progeny of 4A7 and 4D7 were cloned with the pGEM®-T Easy vector. From eight sequenced clones of 4A7, two had the mutation and the sequence belonged to the A genome. The other six clones were either B genome or D genome lacking the mutation. From seven sequenced clones of 4D7, one had the mutation which was in the A genome. The other six clones were either B genome or D genome lacking the mutation. The locations of both mutations were therefore identified.
TILLING is a reverse genetics tool for studying gene function. The most desirable mutations in TILLING are those causing complete or partial inactivation of the targeted gene product. Screening mutations in a conserved region or functional domain will increase the efficiency and speed for finding such deleterious mutants. The method described in this report is suitable for screening a functional domain of a gene in a polyploid species such as wheat. In plants, polyploidy is very common and many crops are polyploid, e.g. wheat, oats, potato, cotton . TILLING in polyploids, especially autopolyploids can cause complications in mismatch cleavage assays . HRM scanning can be an alternative choice. Although amplicons for HRM analysis are shorter than that used in mismatch cleavage assay, HRM is a closed-tube, low cost and fast assay; no digestion and gel separation steps are required.
The bread wheat SSII gene is very conserved among three homoeoloci, especially within the C-terminal domain. The method presented here is effective in detecting mutations in this region in a TILLING population although false positives are detected by independent HRM analysis or Mutation Surveyor®. It is important to use both assays for confirming a mutation. False positives from HRM analysis may be due to the presence of some non-specific amplification, or differences in PCR amplification between samples. DNA from the TILLING population was extracted with a high-throughput method; therefore, there may be variations among samples in DNA quality, salt and inhibitor concentrations, which may affect PCR performance and HRM analysis . A degree of variation in melting behavior observed within non-mutants of clinical samples was previously reported . With careful DNA extraction and quantitative control, the false positive rate may be reduced to a lower level. False positives from Mutation Surveyor® analysis can be controlled to a low level by using highly stringent criteria to identify mutations.
Amplicon length and sequence content may affect the sensitivity of HRM. Shorter amplicons are preferred for higher sensitivity. However, considering throughput and efficiency of TILLING, relative longer amplicons (200-250 bp) are still practical for TILLING as demonstrated in this report. False positives or negatives from HRM analysis may reduce the mutation detection accuracy. However, further sequence analysis by Mutation Surveyor® will increase the accuracy. Furthermore the cost of sequencing will be largely reduced if HRM is followed by sequencing. Detecting mutations in a TILLING population is not like genotyping of medical samples, which requires 100% accuracy and sensitivity. Missing an occasional mutant will not greatly affect mutant discovery by TILLING. If deleterious mutants are identified, they can be assigned to a particular genome within bread wheat (A, B or D). This can be achieved either by cloning and sequencing the particular PCR products as shown in this report, or by using genome-specific and SNP-specific primers. Because such mutations represent a small percentage of total mutations from EMS mutagenesis, the extra work for such genome assignments should not be large.
HRM can be applied for mutation detection and SNP genotyping in medical research . Application of HRM in plant research is limited. Recent publications in plants demonstrated that HRM is a useful tool for genetic variation discovery and genotyping including SNPs, INDELs and microsatellites [22–24]. To our knowledge, this is the first report of the use of HRM analysis to detect a minor sequence change in mixed PCR fragments of an EMS-treated TILLING population. Among the three different amplicons we studied in this report, HRM of ABD12-22 had the highest sensitivity for detecting mutations. ABD12-22 is the shortest (167 bp) and has the fewest intrinsic SNPs (3 SNPs) between homeoloci. The other amplicons ABD6-1 and ABD2-9 are longer (210 bp and 235 bp respectively) and more complicated (4 SNPs and 8 SNPs respectively). HRM sensitivity is determined by the sequence context, length and divergence in a PCR amplicon containing homoeologous gene fragments. HRM is usually applicable when the melting peaks are clear and distinct in non-mutant samples, which can be tested before large scale experiments, in our experience. However, the maximum fragment length and sequence divergence between homeoloci where HRM remains useful for SNP or mutation detection is unknown and further experiments are required.
HRM analysis is able to detect all single base changes, with greater sensitivity for G/A and C/T changes, and lower sensitivity for A/T and G/C changes . EMS alkylates guanine bases and results in G/C to A/T transitions . HRM is therefore suitable for TILLING, especially EMS-TILLING. Recent development of massively parallel sequencing instruments (Roche 454, Illumina/Solexa, and AB SOLiD) makes it possible to resequence genes of interest in a mutagenized population with relatively low cost [27, 28]. However, the accessibility and affordability to these technologies still needs to be considered by many laboratories. The simplicity and low cost of HRM makes it a good choice for scanning mutations in TILLING or eco-TILLING.
HRM in conjunction with sequence analysis is sensitive enough to detect a heterozygous SNP in a PCR amplicon containing three homoeologous gene fragments of wheat. Genome locations of mutations need only be determined for those are predicted to be deleterious to gene function. This method can be used for screening three homoeologous genes simultaneously, especially in a conserved functional domain or EST sequences. For diploid species, HRM scanning can be used for pooled samples. It may also be useful for SNP marker development and eco-TILLING.
An EMS TILLING population was generated in Australian wheat cultivar Ventura, and DNA samples were prepared as described previously .
Test of Mutation Surveyor®sensitivity
A heterozygous mutant (G1642A in Wx-D1) identified during screening for waxy gene mutants  was used to verify that Mutation Surveyor® is able to detect a heterozygous mutant in a mixed DNA pool. DNA from this heterozygous mutant and a homozygous non-mutant sample were mixed to give mutant:non-mutant DNA ratios of 1:0, 1:1, 1:2, 1:3, 1:4 and 1:5. PCR was performed with these different pools using the primer set Wx7D3  and the PCR products were purified with Wizard® SV Gel and PCR Clean-up system (Promega, Madison, WI, USA) and Sanger-sequenced in both directions (Australia Genome Research Facility, Brisbane, Australia). Mutation Surveyor® software was used for analysis of sequence data with the program set to check 2D (bi-directional) small peaks; the mutation-calling parameters were set to the program default including the overlapping factor and dropping factor. The overlapping factor is calculated by the software from the two different bases in the reference and sample traces on either side of the mutation. The dropping factor is determined from the relative intensities of the four neighboring peaks (two peaks on each side) between samples traces and reference traces. Output reports were displayed in the advanced two direction setting. In this setting the software will search for peaks buried within the baseline and indicate their presence with a short green bar if they are of the same wavelength and are in the same spatial position in both strands of sequence data.
In the analysis of SSII fragments, which has three homoeoloci sequence traces, certain "mutations" were deleted when the same "mutation" appeared multiple times in the same position, because they were SNPs between homoeologous loci or were due to artefacts of sequencing. 2D small peaks identified by the program were checked by examining the GAD (Graphic Analysis Display), the raw sequence chromatographs, and also using the bias of EMS mutagenesis which mutates G/C to A/T .
PCR of SSIIand HRM analysis
PCR primers used to amplify part of the carboxyl terminal domain of the SSII gene (GenBank accessions AB201445, AB201446 and AB201447) were designed using Primer3 version 0.4.0 http://frodo.wi.mit.edu/primer3 and manually justified to avoid regions containing SNPs among the three genes. Primers ABDF6: 5'-CCGTTCACCGAGTTGCCTG-3' and ABDR9: 5'-GGTGCTCCCGCTCGAAGTG-3' amplify a 532 bp fragment of all three homoeologous genes (Figure 2). PCR amplification was carried out in a 50 μl volume containing 2 μl of DNA (~100 ng), 1× PfuUltra®II buffer (Stratagene, La Jolla, CA, USA), 1× enhancer solution (Invitrogen, Carlsbad, CA, USA), 0.2 mM dNTPs, 0.25 μM primers and 1.25 U PfuUltra®II Fusion HS DNA Polymerase (Stratagene, La Jolla, CA, USA). PCR was conducted using a thermal cycler (MasterCycler 5333, Eppendorf, North Ryde, NSW, Australia) as follows: 95°C for 2 min, followed by 6 cycles of touchdown PCR (98°C for 10 s, an annealing step starting at 72°C for 20 s and decreasing 1°C per cycle, a temperature ramp increasing 0.5°C per second to 72°C, and 72°C for 30 min), then 35 more cycles of PCR (98°C for 10 s, 66°C for 20 s and 72°C for 15 s) and finally extension at 72°C for 1 min. PCR products were purified using Promega Wizard® SV 96 PCR Clean-up kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and eluted in 100 μl H2O. The purified PCR products were then sent to AGRF (Australia Genome Research Facility, Brisbane, Australia) for sequencing in both directions, and were used for nested PCR and HRM analysis.
Nested PCR used primers ABDF6 and ABDR1 (5'-ACGATGCCGCGGGTC-3') for a 210 bp amplicon; primers ABDF12 (5'-GGTACCTGTGGGAGCTSAAG-3') and ABDR22 (5'-CAGGGAGAAGTTGGTGTAGC-3') for a 167 bp amplicon; primers ABDF2 (5'-ACGCTGGACTCCGGCAA-3') and ABDR9 for a 235 bp amplicon; and primers ABDF3 (5'-CCTGGACGGGCAGAAGG-3') and ABDR9 for a 137 bp amplicon (Figure 2). PCR was performed in 10 μl reactions under the same conditions as above, except that 2.5 μM CYTO®9 (Invitrogen, Carlsbad, CA, USA) was added to the reactions and 1 μl of a 100× dilution of the first PCR (unpurified) or 1 μl of 20× dilution of purified first PCR product was used as the template. PCR and HRM analysis were carried out in a Rotor-Gene™ 6000 real time PCR machine (Corbett Research, Mortlake, NSW, Australia) set at the following conditions: 1 cycle of 95°C for 3 min; 40 cycles of 95°C for 10 s, 60°C for 15 s, 72°C for 10 s; 1 cycle of 72°C for 90 s and a melt from 72°C to 90°C rising at 0.1°C per step (wait 2 s every step). The amplification was monitored. Significantly early or late amplifications were omitted in HRM analysis, as they may give rise to aberrant melting curves. After the PCR and melting steps, samples were loaded on 2% agarose gels to check whether amplifications were specific.
PCR products of mutant samples were cloned into the pGEM®-T Easy vector (Promega, Madison, WI, USA) according to the manufacturer's instructions. Clones were sequenced to identify the genome locations of mutations.
We thank Corbett Research, Australia for providing a free trial of the Rotor-Gene™ 6000, Dr Bing Yu, Department of Molecular and Clinical Genetics, University of Sydney, for useful discussion, Prof Bob McIntosh and Dr Peng Zhang, Plant Breeding Institute, University of Sydney, for critical reading of the manuscript. This work was supported by the Value Added Wheat Cooperative Research Centre, Australia.
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