High resolution melting analysis of almond SNPs derived from ESTs
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High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.
We acknowledge Dr. Yizhou Chen for his helpful discussions and suggestions on HRM analysis. This research was funded by Australian Research Council Grant No. DP0556459.
- CorbettResearch (2006) High resolution melt assay design and analysis CorProtocol™. Corbett Research, SydneyGoogle Scholar
- Felsenstein J (1993) PHYLIP (phylogeny inference package) version 3.6a2. Department of Genetics, University of Washington, SeattleGoogle Scholar
- Griffiths AJF, Wessler SR, Lewontin RC, Carroll SB (2008) Introduction to genetic analysis, 9th edn. W.H. Freeman and Co., New YorkGoogle Scholar
- Gupta PK, Roy JK, Prasad M (2001) Single nucleotide polymorphisms: a new paradigm for molecular marker technology and DNA polymorphism detection with emphasis on their use in plants. Curr Sci 80:524–535Google Scholar
- Hoffmann M, Hurlebaus J, Weilke C (2007) High-resolution melting curve analysis on the LightCycler (R) 480 PCR system. Nat Methods Suppl S:AN17–AN18Google Scholar
- Lehmensiek A, Sutherland MW, McNamara RB (2008) The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley. Theor Appl GenetGoogle Scholar
- Mekuria GT, Collins GG, Sedgley M (1999) Genetic variability between different accessions of some common commercial olive cultivars. J Hortic Sci Biotechnol 74:309–314Google Scholar
- Minch E, Ruiz-Linares A, Goldstein DB, Feldman MW, Cavalli-Sforza LL (1998) Microsat2: a computer program for calculating various statistics on microsatellite allele data. Department of Genetics. Stanford University, StanfordGoogle Scholar
- Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New YorkGoogle Scholar
- Sambrook J, Russell DW, Cold Spring Harbor Laboratory (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
- Toyota T, Watanabe A, Shibuya H, Nankai M, Hattori E, Yamada K, Kurumaji A, Karkera JD, Detera-Wadleigh SD, Yoshikawa T (2000) Association study on the DUSP6 gene, an affective disorder candidate gene on 12q23, performed by using fluorescence resonance energy transfer-based melting curve analysis on the LightCycler. Mol Psychiatry 5:489–494PubMedCrossRefGoogle Scholar