Abstract
Developed recently, high resolution melting (HRM) analysis is an efficient, accurate and inexpensive method for distinguishing DNA polymorphisms. HRM has been used to identify mutations in human genes, and to detect SNPs, INDELs and microsatellites in plants. However, its capacity to discriminate DNA variants in the context of complex haplotypes involving INDEL as well as SNP variants has not been examined until now. In this study, we genotyped an almond (Prunus dulcis (Mill.) D. A. Webb, syn. Prunus amygdalus Batsch) pseudo-testcross mapping population that showed segregation of complex haplotypes associated with CYP79D16 promoter sequence. The 175 bp region in question included a 7 bp INDEL and 3 SNPs, and manifested as three different haplotypes in the parents. Thus, with one homozygous and one heterozygous parent, two relevant genotypes were identified in the mapping population. Although the population displayed monomorphism with respect to the INDEL and one of the SNPs, HRM was sufficiently sensitive to distinguish genotypes on the basis of the two informative SNPs, and the resulting data were used to map CYP79D16 to linkage group 6 of the almond genome. Thus the capacity of HRM to resolve genotypes arising from complex haplotypes has been demonstrated, and this has important implications for the design of efficient HRM markers for various genetic applications including mapping, population studies and biodiversity analyses.
References
Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27:617–631. doi:10.1007/s00299-008-0507-z
Andersen MD, Busk PK, Svendsen I, Moller BL (2000) Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin—cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes. J Biol Chem 275:1966–1975. doi:10.1074/jbc.275.3.1966
Bennett CD, Campbell MN, Cook CJ, Eyre DJ, Nay LM, Nielsen DR, Rasmussen RP, Bernard PS (2003) The LightTyper™: high-throughput genotyping using fluorescent melting curve analysis. BioTechniques 34:1288–1295
Bliss FA, Arulsekar S, Foolad MR, Becerra V, Gillen AM, Warburton ML, Dandekar AM, Kocsisne GM, Mydin KK (2002) An expanded genetic linkage map of Prunus based on an interspecific cross between almond and peach. Genome 45:520–529. doi:10.1139/G02-011
Brandstrom M, Ellegren H (2007) The genomic landscape of short insertion and deletion polymorphisms in the chicken (Gallus gallus) genome: a high frequency of deletions in tandem duplicates. Genetics 176:1691–1701. doi:10.1534/genetics.107.070805
Chagne D, Gasic K, Crowhurst RN, Han Y, Bassett HC, Bowatte DR, Lawrence TJ, Rikkerink EH, Gardiner SE, Korban SS (2008) Development of a set of SNP markers present in expressed genes of the apple. Genomics 92:353–358. doi:10.1016/j.ygeno.2008.07.008
Ching A, Caldwell KS, Jung M, Dolan M, Smith OS, Tingey S, Morgante M, Rafalski AJ (2002) SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines. BMC Genet 3:19. doi:10.1186/1471-2156-3-19
Croxford AE, Rogers T, Caligari PD, Wilkinson MJ (2008) High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar. New Phytol 180:594–607. doi:10.1111/j.1469-8137.2008.02588.x
Dudley JW (1993) Molecular markers in plant improvement: manipulation of genes affecting quantitative traits. Crop Sci 33:660. doi:10.1111/j.1471-8286.2007.01857.x
Flot JF (2007) Champuru 1.0: a computer software for unraveling mixtures of two DNA sequences of unequal lengths. Mol Ecol Notes 7:974–977. doi:10.1111/j.1471-8286.2007.01857.x
Forslund K, Morant M, Jorgensen B, Olsen CE, Asamizu E, Sato S, Tabata S, Bak S (2004) Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus. Plant Physiol 135:71–84. doi:10.1104/pp.103.038059
Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV (2007) Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 53:1544–1548. doi:10.1373/clinchem.2007.088120
Kennerson M, Nicholson G, Kowalski B, Krajewski K, El-Khechen D, Feely S, Chu S, Shy M, Garbern J (2009) X-linked distal hereditary motor neuropathy maps to the DSMAX locus on chromosome Xq13.1–q21. Neurology 72:246–252. doi:10.1212/01.wnl.0000339483.86094.a5
Koch BM, Sibbesen O, Halkier BA, Svendsen I, Moller BL (1995) The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of l-tyrosine to P-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. Arch Biochem Biophys 323:177–186. doi:10.1006/abbi.1995.0024
Kosambi DD (1944) The estimation of map distances from recombination values. Ann Eugen 12:172–175
Lamboy WF, Alpha CG (1998) Using simple sequence repeats (SSRs) for DNA fingerprinting germplasm accessions of grape (Vitis L.) species. J Am Soc Hortic Sci 123:182–188
Lehmensiek A, Sutherland M, McNamara R (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 Genet 117:721–728. doi:10.1007/s00122-008-0813-4
Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50:1156–1164. doi:10.1373/clinchem.2004.032136
Mackay JF, Wright CD, Bonfiglioli RG (2008) A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods 4:8. doi:10.1186/1746-4811-4-8
Nielsen JS, Moller BL (2000) Cloning and expression of cytochrome P450 enzymes catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glucosides in Triglochin maritima. Plant Physiol 122:1311–1321. doi:10.1104/pp.122.4.1311
Olsen KM, Hsu S-C, Small LL (2008) Evidence on the molecular basis of the Ac/ac adaptive cyanogenesis polymorphism in white clover (Trifolium repens L.). Genetics 179:517–526. doi:10.1534/genetics.107.080366
Pertoldi C, Bijlsma R, Loeschcke V (2007) Conservation genetics in a globally changing environment: present problems, paradoxes and future challenges. Biodivers Conserv 16:4147–4163. doi:10.1007/s10531-007-9212-4
Sanchez-Perez R, Howad W, Dicenta F, Arus P, Martinez-Gomez P (2007) Mapping major genes and quantitative trait loci controlling agronomic traits in almond. Plant Breed 126:310–318. doi:10.1111/j.1439-0523.2007.01329.x
Schlötterer C (2004) The evolution of molecular markers—just a matter of fashion? Nat Rev Genet 5:63–69. doi:10.1038/nrg1249
Studer B, Jensen L, Fiil A, Asp T (2009) “Blind” mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis. Mol Breed. doi: 10.1007/s11032-009-9291-x
The-Arabidopsis-Genome-Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815. doi:10.1038/35048692
Tindall EA, Petersen DC, Woodbridge P, Schipany K, Hayes VM (2009) Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments. Hum Mutat 30:876–883
Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74. doi:10.1093/nar/gkm306
Van Ooijen JW, Voorrips RE (2001) JoinMap® 3.0, Software for the calculation of genetic linkage maps. Plant Research International, Wageningen
Vignal A, Milan D, SanCristobal M, Eggen A (2002) A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol 34:275–305. doi:10.1051/gse:2002009
Weeden NF (1994) Approaches to mapping in horticultural crops. In: Gresshoff P (ed) Plant genome analysis. CRC Press, Boca Raton, pp 57–68
Wirthensohn MG, Sedgley M (2002) Almond breeding in Australia. Acta Hortic 591:245–248
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860. doi:10.1373/49.6.853
Wu S-B, Wirthensohn MG, Hunt P, Gibson JP, Sedgley M (2008) High resolution melting analysis of almond SNPs derived from ESTs. Theor Appl Genet 118:1–14. doi:10.1007/s00122-008-0870-8
Wu S-B, Tavassolian I, Rabiei G, Hunt P, Wirthensohn M, Gibson JP, Ford CM, Sedgley M (2009) Mapping SNP anchored genes using high resolution melting analysis in almond. Mol Genet Genom. doi: 10.1007/s00438-009-0464-4
Zhou L, Vandersteen J, Wang L, Fuller T, Taylor M, Palais B, Wittwer CT (2004) High-resolution DNA melting curve analysis to establish HLA genotypic identity. Tissue Antigens 64:156–164. doi:10.1111/j.1399-0039.2004.00248.x
Acknowledgments
We are grateful to Dr Jennifer Guerin for her contribution to this work by originally isolating the almond CYP79D16 promoter sequence by genomic walking. This research was funded by Australian Research Council (ARC) grant DP0556459, and ARC grant LP0560480 in conjunction with the Almond Board of Australia. Supplementary funding was also provided by The University of Adelaide through the Faculty of Sciences Research Development Scheme (2008).
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Wu, SB., Franks, T.K., Hunt, P. et al. Discrimination of SNP genotypes associated with complex haplotypes by high resolution melting analysis in almond: implications for improved marker efficiencies. Mol Breeding 25, 351–357 (2010). https://doi.org/10.1007/s11032-009-9324-5
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DOI: https://doi.org/10.1007/s11032-009-9324-5