Theoretical and Applied Genetics

, Volume 115, Issue 7, pp 907–916 | Cite as

Identification, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp.

  • Ana Garcés-Claver
  • Shanna Moore Fellman
  • Ramiro Gil-Ortega
  • Molly Jahn
  • María S. Arnedo-Andrés
Original Paper


A single nucleotide polymorphism (SNP) associated with pungency was detected within an expressed sequence tag (EST) of 307 bp. This fragment was identified after expression analysis of the EST clone SB2-66 in placenta tissue of Capsicum fruits. Sequence alignments corresponding to this new fragment allowed us to identify an SNP between pungent and non-pungent accessions. Two methods were chosen for the development of the SNP marker linked to pungency: tetra-primer amplification refractory mutation system-PCR (tetra-primer ARMS-PCR) and cleaved amplified polymorphic sequence. Results showed that both methods were successful in distinguishing genotypes. Nevertheless, tetra-primer ARMS-PCR was chosen for SNP genotyping because it was more rapid, reliable and less cost-effective. The utility of this SNP marker for pungency was demonstrated by the ability to distinguish between 29 pungent and non-pungent cultivars of Capsicum annuum. In addition, the SNP was also associated with phenotypic pungent character in the tested genotypes of C. chinense, C. baccatum, C. frutescens, C. galapagoense, C. eximium, C. tovarii and C. cardenasi. This SNP marker is a faster, cheaper and more reproducible method for identifying pungent peppers than other techniques such as panel tasting, and allows rapid screening of the trait in early growth stages.



This research was financially supported by INIA-Spanish Ministry of Education and Science, grants SC00-020-C2-1 and RTA2005-00226-00-00 to R.G.-O. and M.S.A.-A., respectively, and grants A16 for consolidated DGA group. M.S.A.-A. was supported by an “INIA-CCAA” research contract from the Spanish MEC and A.G.-C. by “CITA” fellowship from CITA-DGA (Spain). We are grateful to M.R. Navarro and E. Fuertes (CITA, Spain) for technical support. We thank M. Carravedo (CITA, Spain) for providing most of the seeds of C. annuum. We also thank Professor Byung-Dong Kim (Seoul National University, Korea) and Professor Paul W. Bosland (New Mexico State University, NM, USA) for supplying seeds.


  1. Acquadro A, Lee D, Chiapparino E, Comino C, Portis E, Donini P, Lanteri S (2003) Detection and characterisation of SNPs in Capsicum spp. Capsicum Eggplant Newslett 22:37–40Google Scholar
  2. Aluru MR, Mazourek M, Landry LG, Curry J, Jahn M, O’Connell MA (2003) Differential expression of fatty acid synthesis genes, Acl, Fat and Kas, in Capsicum fruit. J Exp Bot 54:165–1664CrossRefGoogle Scholar
  3. Andrews J (1995) Peppers, the domesticated capsicums. University of Texas Press, AustinGoogle Scholar
  4. Arnedo-Andrés MS, Gil-Ortega R, Luis-Arteaga M, Hormaza I (2002) Development of RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in pepper (Capsicum annuum L.). Theor Appl Genet 105:1067–1074PubMedCrossRefGoogle Scholar
  5. Bhattramakki D, Dolan M, Hanafey M, Wineland R, Vaske D, Register JC, Tingey SV, Rafalski A (2002) Insertion–deletion polymorphisms in 3′ regions of maize genes occur frequently and can be used as highly informative genetic markers. Plant Mol Biol 48:539–547PubMedCrossRefGoogle Scholar
  6. Blum E, Liu K, Mazourek M, Yoo EY, Jahn MM, Paran I (2002) Molecular mapping of the C locus for presence of pungency in Capsicum. Genome 45:702–705PubMedCrossRefGoogle Scholar
  7. Blum E, Mazourek M, O’Connell MA, Curry J, Thorup T, Liu K, Jahn MM, Paran I (2003) Molecular mapping of capsaicinoid biosynthesis genes and quantitative trait loci analysis for capsaicinoid content in Capsicum. Theor Appl Genet 108:79–86PubMedCrossRefGoogle Scholar
  8. Carmichael JK (1991) Treatment of herpes zoster and post herpetic neuralgia. Am Fam Physician 44:203–210PubMedGoogle Scholar
  9. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR et al (2000) Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288:306–313PubMedCrossRefGoogle Scholar
  10. Chiapparino E, Lee D, Donini P (2004) Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR. Genome 47:414–420PubMedCrossRefGoogle Scholar
  11. Collins MD, Wasmund LM, Bosland PW (1995) Improved method for quantifying capsaicinoids in Capsicum using high-performance liquid chromatography. HortScience 30:137–139Google Scholar
  12. Curry J, Aluru M, Mendoza M, Nevarez J, Melendrez M, O’Connell MA (1999) Transcripts for possible capsaicinoid biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum spp. Plant Sci 148:47–57CrossRefGoogle Scholar
  13. Daood HG, Illes V, Gnayfeed MH, Meszaros B, Horvath G, Biacs PA (2002) Extraction of pungent spice paprika by supercritical carbon dioxide and sub critical propane. J Supercrit Fluids 23:143–152CrossRefGoogle Scholar
  14. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  15. Fujiwake H, Suzuki T, Iwai K (1982) Capsaicinoids formation in the protoplast from the placenta of Capsicum fruits. Agric Biol Chem 46:2591–2592Google Scholar
  16. Giancola S, McKhann HI, Bérard A, Camilleri C, Durand S, Libeau P, Roux F, Reboud X, Gut IG, Brunel D (2006) Utilization of the three high-throughput SNP genotyping methods, the GOOD assay, amplifluor and TaqMan, in diploid and polyploidy plants. Theor Appl Genet 112:1115–1124PubMedCrossRefGoogle Scholar
  17. Giordano M, Oefner PJ, Underhill PA, Cavalli-Sforza L, Tosi R, Richiardi PM (1999) Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility. Genomics 56:247–253PubMedCrossRefGoogle Scholar
  18. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  19. Hayashi K, Hashimoto N, Daigen M, Ashikawa I (2004) Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theor Appl Genet 108:1212–1220PubMedCrossRefGoogle Scholar
  20. Henry CJ, Emery B (1986) Effect of spiced food on metabolic rate. Hum Nutr Clin Nutr 40:165–268PubMedGoogle Scholar
  21. Iwai K, Suzuki T, Fujiwake H (1979) Formation and accumulation of pungent principle of hot pepper fruits, capsaicin and its analogues, in Capsicum annuum var. annuum cv. karayatsubusa at different stages of flowering. Agric Biol Chem 43:2493–2498Google Scholar
  22. Kim M, Kim S, Kim S, Kim BD (2001) Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization. Mol Cells 11:213–219PubMedGoogle Scholar
  23. Kim MY, Van K, Lestari P, Moon JK, Lee SH (2005) SNP identification and SNAP marker development for a GmNARK gene controlling supernodulation in soybean. Theor Appl Genet 110:1003–1010PubMedCrossRefGoogle Scholar
  24. Kim DS, Kim DH, Yoo JH, Kim BD (2006) Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.). Mol Cells 21:135–40PubMedCrossRefGoogle Scholar
  25. Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410PubMedCrossRefGoogle Scholar
  26. Lai Z, Livingstone K, Zou Y, Church SA, Knapp JS, Andrews J, Rieseberg LH (2005) Identification and mapping of SNPs from ESTs in sunflower. Theor Appl Genet 111:1532–1544PubMedCrossRefGoogle Scholar
  27. Lang Y, Yanagawa S, Sasanuma T, Sasakuma T (2006) A gene encoding a putative acyl-transferase involved in pungency of Capsicum. Breed Sci 56:55–62CrossRefGoogle Scholar
  28. Lee B, Choi D, Lee KW (1998) Isolation and characterization of o-diphenol-O– methyltransferase cDNA clone in hot pepper (Capsicum annuum L). J Plant Biol 41:9–15CrossRefGoogle Scholar
  29. Lee RJ, Yolton RL, Yolton DP, Schnider C, Janin ML (1996) Personal defense sprays: effects and management of exposure. J Am Optom Assoc 67:548–560PubMedGoogle Scholar
  30. Lee SH, Walker DR, Cregan PB, Boerma HR (2004) Comparison of four flow cytometric SNP detection assays and their use in plant improvement. Theor Appl Genet 110:167–174PubMedCrossRefGoogle Scholar
  31. Lee CJ, Yoo EY, Shin JH, Lee J, Hwang HS, Kim BD (2005) Non-pungent Capsicum contains a deletion in the Capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers. Mol Cells 19:262–267PubMedGoogle Scholar
  32. Livingstone KD, Lackney VK, Blauth JR, van Wijk R, Jahn MK (1999) Genome mapping in Capsicum and the evolution of genome structure in the Solanaceae. Genetics 152:1183–1202PubMedGoogle Scholar
  33. Lopez C, Piégu B, Cooke R, Delseny M, Tohme J, Verdier V (2005) Using cDNA and genomic sequences as tools to develop SNP strategies in cassava (Manihot esculenta Crantz). Theor Appl Genet 110:425–431PubMedCrossRefGoogle Scholar
  34. Minamiyama Y, Kinoshita S, Inaba K, Inoue M (2005) Development of a cleaved amplified sequence (CAPS) marker linked to pungency in pepper. Plant Breed 124:288–291CrossRefGoogle Scholar
  35. Moreno-Vázquez S, Ochoa OE, Faber N, Chao S, Jacobs JME, Maisonneuve B, Kesseli RV, Michelmore RW (2003) SNP-based codominant markers for a recessive gene conferring resistance to corky root rot (Rhizomonas suberifaciens) in lettuce (Lactuca sativa). Genome 46:1059–1069PubMedCrossRefGoogle Scholar
  36. Olmstead GR, Sweere JA, Spangler RE, Bohs L, Palmer JD (1999) Phylogeny and provisional classification of the Solanaceae based on chloroplast DNA. Solanaceae 4:111–137Google Scholar
  37. Paris M, Potter RH, Lance RCM, Li CD, Jones MGK (2003) Typing Mlo alleles for powdery mildew resistance in barley by single nucleotide polymorphism analysis using MALDI-TOF mass spectrometry. Aust J Agric Res 54:1343–1349CrossRefGoogle Scholar
  38. Prasad BCN, Kumar V, Gururaj HB, Parimalan R, Giridhar P, Ravishankar GA (2006) Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper (Capsicum sp.) Proc Natl Acad Sci USA 103:13315–13320PubMedCrossRefGoogle Scholar
  39. Schulzeck S, Wulf H (1997) Local therapy with capsaicin or ASS in chronic pain. Der Schmerz 11:345–352PubMedCrossRefGoogle Scholar
  40. Scoville WL (1912) Note Capsicum. J Am Pharm Assoc 1:453Google Scholar
  41. Stewart C, Kang BC, Liu K, Mazourek M, Moore SL, Yoo EY, Kim BD, Paran I, Jahn MM (2005) The Pun1 gene for pungency in pepper encodes a putative acyl-transferase. Plant J 42:675–688PubMedCrossRefGoogle Scholar
  42. St-Pierre B, Laflamme P, Alarco AM, De Luca V (1998) The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl-transfer. Plant J 14:703–713PubMedCrossRefGoogle Scholar
  43. Suzuki T, Fujiwake H, Iwai K (1980) Intracellular localization of capsaicin and its analogues in Capsicum fruit. I. Microscopic investigation of the structure of the placenta of Capsicum annuum var. annuum cv. Karayatsubusa. Plant Cell Physiol 21:839–853Google Scholar
  44. Tanksley SD, Bernatzky R, Lapitan NL, Prince JP (1988) Conservation of gene repertoire but gene order in pepper and tomato. Proc Natl Acad Sci 85:6419–6423PubMedCrossRefGoogle Scholar
  45. Walsh BM, Hoot SB (2001) Phylogenetic relationships of Capsicum (Solanaceae) using DNA sequences from two non-coding regions: the chloroplast atpB-rbcL spacer region and nuclear waxy introns. Int J Plant Sci 162:1409–1418CrossRefGoogle Scholar
  46. Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lipshutz R, Chee M, Lander ES (1998) Large-scale identification, mapping and genotyping of single-nucleotide polymorphisms in the human genome. Science 280:1077–1082PubMedCrossRefGoogle Scholar
  47. Ye S, Dhillon S, Ke X, Collins AR, Day INM (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:E88PubMedCrossRefGoogle Scholar
  48. Yeam I, Kang BC, Lindeman W, Frantz JD, Faber N, Jahn MM (2005) Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum. Theor Appl Genet 112:178–86PubMedCrossRefGoogle Scholar
  49. Zhu YL, Song QJ, Hyten DL, Van Tassell CP, Matukumalli LK, Grimm DR, Hyatt SM, Fickus EW, Young ND, Cregan PB (2003) Single-nucleotide polymorphisms in Soybean. Genetics 163:1123–1134PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Ana Garcés-Claver
    • 1
  • Shanna Moore Fellman
    • 2
  • Ramiro Gil-Ortega
    • 1
  • Molly Jahn
    • 3
  • María S. Arnedo-Andrés
    • 1
  1. 1.Technology for Plant Production DepartmentCentro de Investigación y Tecnología Agroalimentaria (CITA)ZaragozaSpain
  2. 2.Department of Plant Breeding and GeneticsCornell UniversityIthacaUSA
  3. 3.College of Agricultural and Life SciencesUniversity of WisconsinMadisonUSA

Personalised recommendations