Theoretical and Applied Genetics

, Volume 113, Issue 2, pp 331–343 | Cite as

Characterisation of single nucleotide polymorphisms in sugarcane ESTs

  • Giovanni M. Cordeiro
  • Frances Eliott
  • C. Lynne McIntyre
  • Rosanne E. Casu
  • Robert J. Henry
Original Paper


Commercial sugarcane cultivars (Saccharum spp. hybrids) are both polyploid and aneuploid with chromosome numbers in excess of 100; these chromosomes can be assigned to 8 homology groups. To determine the utility of single nucleotide polymorphisms (SNPs) as a means of improving our understanding of the complex sugarcane genome, we developed markers to a suite of SNPs identified in a list of sugarcane ESTs. Analysis of 69 EST contigs showed a median of 9 SNPs per EST and an average of 1 SNP per 50 bp of coding sequence. The quantitative presence of each base at 58 SNP loci within 19 contiguous sequence sets was accurately and reliably determined for 9 sugarcane genotypes, including both commercial cultivars and ancestral species, through the use of quantitative light emission technology in pyrophosphate sequencing. Across the 9 genotypes tested, 47 SNP loci were polymorphic and 11 monomorphic. Base frequency at individual SNP loci was found to vary approximately twofold between Australian sugarcane cultivars and more widely between cultivars and wild species. Base quantity was shown to segregate as expected in the IJ76-514 × Q165 sugarcane mapping population, indicating that SNPs that occur on one or two sugarcane chromosomes have the potential to be mapped. The use of SNP base frequencies from five of the developed markers was able to clearly distinguish all genotypes in the population. The use of SNP base frequencies from a further six markers within an EST contig was able to help establish the likely copy number of the locus in two genotypes tested. This is the first instance of a technology that has been able to provide an insight into the copy number of a specific gene locus in hybrid sugarcane. The identification of specific and numerous haplotypes/alleles present in a genotype by pyrophosphate sequencing or alternative techniques ultimately will provide the basis for identifying associations between specific alleles and phenotype and between allele dosage and phenotype in sugarcane.



This research was undertaken with funding from the Cooperative Research Centre for Sugar Industry Innovation through Biotechnology.

Supplementary material

122_2006_300_MOESM1_ESM.doc (510 kb)
Supplementary material


  1. Ahmadian A, Gharizadeh B, Gustafsson AC, Sterky F, Nyrén P, Uhlén M, Lundeberg J (2000) Single-nucleotide polymorphism analysis by pyrosequencing. Anal Biochem 280:103–110CrossRefPubMedGoogle Scholar
  2. Aitken K, Jackson P, Piperidis G, McIntyre L (2004) QTL identified for yield components in a cross between a sugarcane (Saccharum spp.) cultivar Q165A and a S. officinarum clone IJ76-514. In: Proceedings for the 4th international crop science congress, Brisbane, Australia, 26 September–1 October 2004.
  3. Aitken KS, Jackson PA, McIntyre CL (2005) A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar. Theor Appl Genet 110:789–801CrossRefPubMedGoogle Scholar
  4. Alderborn A, Kristofferson A, Hammerling U (2000) Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. Genome Res 10:1249–1258CrossRefPubMedGoogle Scholar
  5. Bertina RM, Koelemann BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PA (1994) Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369:64–67CrossRefPubMedGoogle Scholar
  6. Bhattramakki D, Rafalski A (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: Henry RJ (ed) Plant genotyping: the DNA fingerprinting of plants. CAB International, Lismore, pp 179–191Google Scholar
  7. Bradbury LMT, Fitzgerald TL, Henry RJ, Jin QS, Waters DLE (2005) The gene for fragrance in rice. Plant Biotechnol J 3:363–370CrossRefPubMedGoogle Scholar
  8. Bremer G (1961) Problems in breeding and cytology of sugarcane. Euphytica 10:59–78CrossRefGoogle Scholar
  9. Bundock PC, Henry RJ (2004) Single nucleotide polymorphism, haplotype diversity and recombination in the Isa gene of barley. Theor Appl Genet 109:543–551CrossRefPubMedGoogle Scholar
  10. Bundock PC, Christopher JT, Eggler P, Ablett G, Henry RJ, Holton TA (2003) Single nucleotide polymorphisms in cytochrome P450 genes from barley. Theor Appl Genet 106:676–682PubMedGoogle Scholar
  11. Carson DL, Botha FC (2000) Preliminary analysis of expressed sequence tags for sugarcane. Crop Sci 40:1769–1779CrossRefGoogle Scholar
  12. Casu RE, Grof CPL, Rae AL, McIntyre CL, Dimmock CM, Manners JM (2003) Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis. Plant Mol Biol 52:371–386CrossRefPubMedGoogle Scholar
  13. Casu RE, Dimmock CM, Chapman SC, Grof CPL, McIntyre CL, Bonnett GD, Manners JM (2004) Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling. Plant Mol Biol 54:503–517CrossRefPubMedGoogle Scholar
  14. Clayton DG (1990) Inference of haplotypes from PCR-amplified samples of diploid populations. Mol Biol Evol 7:111–122PubMedGoogle Scholar
  15. Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff S (2004) Efficient discovery of DNA polymorphisms in natural populations by ecotilling. Plant J 37:778–786PubMedCrossRefGoogle Scholar
  16. Daugrois JH, Grivet L, Roques D, Hoarau JY, Lombard H, Glaszmann JC, Dhont A (1996) A putative major gene for rust resistance linked with a RFLP marker in sugarcane cultivar ‘R570’. Theor Appl Genet 92:1059–1064CrossRefGoogle Scholar
  17. Davignon J, Gregg RE, Sing CF (1988) Apolipoprotein E polymorphism and arteriosclerosis. Arteriosclerosis 8:1–21PubMedGoogle Scholar
  18. Delseny M, Salses J, Cooke R, Sallaud C, Regad F, Lagoda P, Guiderdoni E, Ventelon M, Brugidou C, Ghesquière A (2001) Rice genomics: present and future. Plant Physiol Biochem 39:323–334CrossRefGoogle Scholar
  19. D’Hont A (1994) A molecular approach to unraveling the genetics of sugarcane, a complex polyploid of the Andropogoneae tribe. Genome 37:222–230PubMedCrossRefGoogle Scholar
  20. Excoffier L, Slatkin M (1995) Maximum-likelihood-estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol 12:921–927PubMedGoogle Scholar
  21. Fallin D, Shork NJ (2000) Accuracy of haplotype frequency estimation for biallelic loci, via the EM algorithm for unphased diploid genotype data. Am J Hum Genet 67:947–959CrossRefPubMedGoogle Scholar
  22. Garg K, Green P, Nickerson DA (1999) Identification of candidate coding region single nucleotide polymorphisms in 65 human genes using assembled expressed sequence tags. Genome Res 9:1087–1092CrossRefPubMedGoogle Scholar
  23. Grivet L, D’Hont A, Roques D, Feldmann P, Lanaud C, Glaszmann J-C (1996) RFLP mapping in a highly polyploid and aneuploid interspecific hybrid. Genetics 142:987–1000PubMedGoogle Scholar
  24. Grivet L, Glaszmann JC, Arruda P (2001) Sequence polymorphism from EST data in sugarcane: a fine analysis of 6-phosphogluconate dehydrogenase genes. Genet Mol Biol 24:161–167CrossRefGoogle Scholar
  25. Grivet L, Glaszmann JC, Vincentz M, da Silva F, Arruda P (2003) ESTs as a source for sequence polymorphism discovery in sugarcane: example of the Adh genes. Theor Appl Genet 106:190–197PubMedGoogle Scholar
  26. Gusfield D (2001) Inference of haplotypes from samples of diploid populations: complexity and algorithms. J Comput Biol 8:305–323CrossRefPubMedGoogle Scholar
  27. Gut IG (2001) Automation in genotyping of single nucleotide polymorphisms. Hum Mutat 17:475–492CrossRefPubMedGoogle Scholar
  28. Hawley ME, Kidd KK (1995) HAPLO: a program using the EM algorithm to estimate the frequencies of multi-site haplotypes. J Hered 86:409–411PubMedGoogle Scholar
  29. Heaton MP, Harhay GP, Bennett GL, Stone RT, Grosse WM, Casas E, Keele JW, Smith TPL, Chitko-McKown CG, Laegreid WW (2002) Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle. Mamm Genome 13:272–281CrossRefPubMedGoogle Scholar
  30. Hoarau JY, Grivet L, Offmann B, Raboin LM, Diorflar JP, Payet J, Hellmann M, D’Hont A, Glaszmann JC (2002) Genetic dissection of a modern sugarcane cultivar (Saccharum spp.). II. Detection of QTLs for yield components. Theor Appl Genet 105:1027–1037CrossRefPubMedGoogle Scholar
  31. Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–887CrossRefPubMedGoogle Scholar
  32. Jannoo N, Grivet L, Seguiin M, Paulet F, Domaingue R, Rao PS, Dookun A, D’Hont A, Glaszmann JC (1999) Molecular investigation of the genetic base of sugarcane cultivars. Theor Appl Genet 99:171–184CrossRefGoogle Scholar
  33. Lancia G, Pinotti MC, Rizzi R (2004) Haplotyping populations by pure parsimony: complexity of exact and approximation algorithms. INFORMS J Comput 16:348–359CrossRefGoogle Scholar
  34. McIntyre CL, Casu RE, Drenth J, Knight D, Whan VA, Croft BJ, Jordan DR, Manners JM (2005a) Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance. Genome 48:391–400CrossRefGoogle Scholar
  35. McIntyre CL, Whan VA, Croft B, Magarey R, Smith GR (2005b) Identification and validation of molecular markers associated with pachymetra root rot and brown rust resistance in sugarcane using map- and association-based approaches. Mol Breed 16:151–161CrossRefGoogle Scholar
  36. McIntyre CL, Jackson M, Cordeiro G, Amouyal O, Eliott F, Henry RJ, RE Casu, Hermann S, Aitken KS, Bonnett GD (2006) The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane. Mol Breed (in press)Google Scholar
  37. Ming R, Liu S-C, Moore PH, Irvine JE, Paterson AH (2001) QTL analysis in a complex autopolyploid: genetic control of sugar content in sugarcane. Genome Res 11:2075–2084CrossRefPubMedGoogle Scholar
  38. Ming R, Del Monte TA, Hernandez E, Moore PH, Irvine JE, Paterson AH (2002) Comparative analysis of QTLs affecting plant height and flowering among closely-related diploid and polyploid genomes. Genome 45:794–803CrossRefPubMedGoogle Scholar
  39. Mototani H, Mabuchi A, Saito S, Fujioka M, Iida A, Takatori Y, Kotani A, Kubo T, Nakamura K, Sekine A, Murakami Y, Tsunoda T, Notoya K, Nakamura Y, Ikegawa S (2005) A functional single nucleotide polymorphism in the core promoter region of CALM1 is associated with hip osteoarthritis in Japanese. Hum Mol Genet 14:1009–1017CrossRefPubMedGoogle Scholar
  40. Mullet JE, Klein RR, Klein PE (2002) Sorghum bicolor—an important species for comparative grass genomics and a source of beneficial genes for agriculture. Curr Opin Plant Biol 5:118–121CrossRefPubMedGoogle Scholar
  41. Niu T, Qin ZS, Xu X, Liu JS (2002) Bayesian haplotype inference for multiple linked single-nucleotide polymorphisms. Am J Hum Genet 70:157–169CrossRefPubMedGoogle Scholar
  42. Nurmi J, Kiviniemi M, Kujanpaa M, Sjoroos M, Ilonen J, Lovgren T (2001) High-throughput genetic analysis using time-resolved fluorometry and closed-tube detection. Anal Biochem 299:211–217CrossRefPubMedGoogle Scholar
  43. Nyrén P, Lundin A (1985) Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis. Anal Biochem 151:504–509CrossRefPubMedGoogle Scholar
  44. Pacey-Miller T, Henry R (2003) SNP detection in plants using a single stranded pyrosequencing protocol with a universal biotinylated primer. Anal Biochem 317:165–170CrossRefGoogle Scholar
  45. Pfost DR, Boyce-Jacino MT, Grant DM (2000) A SNPshot: pharmacogenetics and the future of drug therapy. TIBTECH 18:334–338Google Scholar
  46. Quint M, Mihaljevic R, Dussle CM, Xu ML, Melchinger AE, Lubberstedt T (2002) Development of RGA-CAPS markers and genetic mapping of candidate genes for sugarcane mosaic virus resistance in maize. Theor Appl Genet 105:355–363CrossRefPubMedGoogle Scholar
  47. Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94–100CrossRefPubMedGoogle Scholar
  48. Ramsay L, Macaulay M, degli Ivanissevich S, MacLean K, Cardle L, Fuller J, Edwards KJ, Tuvesson S, Morgante M, Massari A, Maestri E, Marmiroli N, Sjakste T, Ganal M, Powell W, Waugh R (2000) A simple sequence repeat-based linkage map of barley. Genetics 156:1997–2005PubMedGoogle Scholar
  49. Rickert AM, Premstaller A, Gebhardt C, Oefner PJ (2002) Genotyping of SNPs in a polyploid genome by pyrosequencing (TM). Biotechniques 32:592–603PubMedGoogle Scholar
  50. Ridker PM, Hennekens CH, Lindpainter K, Stampfer MJ, Eisenberg PR, Miletich JP (1995) Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med 332:912–917CrossRefPubMedGoogle Scholar
  51. Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. J Chromatogr B 739:345–355Google Scholar
  52. Ronaghi M, Karamohamed D, Petterson B, Uhlén M, Nyrén P (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89CrossRefPubMedGoogle Scholar
  53. Ronaghi M, Uhlén M, Nyrén P (1998) Real-time pyrophosphate detection for DNA sequencing. Science 281:363–365CrossRefPubMedGoogle Scholar
  54. Ross P, Hall L, Smirnow I, Haff L (1998) High levelmultiplex genotyping by MALDI-TOF mass spectrometry. Nat Biotechnol 16:1347–1351PubMedCrossRefGoogle Scholar
  55. Rossi M, Araujo PG, Paulet F, Garsmeur O, Dias VM, Chen H, Van Sluys M-A, D’Hont A (2003) Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane. Mol Gen Genet 269:406–419Google Scholar
  56. Sachidanandam R, Weissman D, Schmidt SC, Kakol JM, Stein LD et al (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409:928–933PubMedCrossRefGoogle Scholar
  57. Sills G, Bridges W, Al-Janabi S, Sobral BWS (1995) Genetic analysis of agronomic traits in a cross between sugarcane (Saccharum officinarum L.) and its presumed progenitor (S. robustum Brandes & Jesw. Ex. Grassl). Mol Breed 1:355–363CrossRefGoogle Scholar
  58. da Silva J, Sorrells ME, Burnquist WL, Tanksley SD (1993) RFLP linkage map and genome analysis of S. spontaneum. Genome 36:782–791CrossRefGoogle Scholar
  59. Stephens M, Donnelly P (2003) A comparison of Bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet 73:1162–1169CrossRefPubMedGoogle Scholar
  60. Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68:978–989CrossRefPubMedGoogle Scholar
  61. Storm N, Darnhofer-Patel B, van den Boom D, Rodi CP (2003) MALDI-TOF mass spectrometry-based SNP genotyping. Methods Mol Biol 212:241–262PubMedGoogle Scholar
  62. Syvänen A-C (1999) From gels to chips: “minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 13:1–10CrossRefPubMedGoogle Scholar
  63. Syvänen A-C (2001) Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet 2:930–942CrossRefPubMedGoogle Scholar
  64. Templeton AR, Sing CF, Kessling A, Humphries S (1988) A cladistic analysis of phenotype associations with haplotypes inferred from restriction endonuclease mapping. II. The analysis of natural populations. Genetics 120:1145–1154PubMedGoogle Scholar
  65. Till BJ, Curtner C, Comai L, Henikoff S (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32:2632–2641CrossRefPubMedGoogle Scholar
  66. Tishkoff SA, Goldman A, Calafell F, Speed WC, Deinard AS, Bonne-Tamir B, Kidd JR, Pafstis AJ, Jenkins T, Kidd KK (1998) A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations. Am J Hum Genet 62:1389–1402CrossRefPubMedGoogle Scholar
  67. Venter JC, Adams MD, Myers EW (2001) The sequence of the human genome. Science 291:1304–1351PubMedCrossRefGoogle Scholar
  68. Vettore AL, da Silva FR, Kemper EL, Arruda P (2001) The libraries that made SUCEST. Genet Mol Biol 24:1–7CrossRefGoogle Scholar
  69. Waters DLE, Henry RJ, Reinke RF, Fitzgerald MA (2005) Gelatinisation temperature of rice explained by polymorphisms in starch synthase. Plant Biotechnol J (In press)Google Scholar
  70. Wu K, Burnquist W, Sorrels M, Tew TL, Moore P, Tanksley S (1992) The detection and estimation of linkage in polyploids using single-dose restriction fragments. Theor Appl Genetics 83:294–300CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Giovanni M. Cordeiro
    • 1
    • 3
  • Frances Eliott
    • 1
    • 3
  • C. Lynne McIntyre
    • 2
    • 3
  • Rosanne E. Casu
    • 2
    • 3
  • Robert J. Henry
    • 1
    • 3
  1. 1.Centre for Plant Conservation GeneticsSouthern Cross UniversityLismoreAustralia
  2. 2.CSIRO Plant IndustryQueensland Bioscience PrecinctSt LuciaAustralia
  3. 3.CRC for Sugar Industry Innovation through BiotechnologySt LuciaAustralia

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