, Volume 184, Issue 3, pp 335–344 | Cite as

Development of DNA markers that discriminate between white- and blue-flowers in Japanese gentian plants

  • Takashi Nakatsuka
  • Misa Saito
  • Yuka Sato-Ushiku
  • Eri Yamada
  • Takashi Nakasato
  • Nobue Hoshi
  • Kazumichi Fujiwara
  • Takashi Hikage
  • Masahiro NishiharaEmail author


We developed molecular markers for discrimination of white and blue flower color in Japanese gentian plants. White-flowered gentians can be classified into two types, based on genetic and physiological features. One type includes four allelic variations (gtmyb3-1, gtmyb3-2, gtmyb3-3, and gtmyb3-4) of an anthocyanin biosynthetic regulator gene (GtMYB3), distinguished by three PCR-based molecular markers. The other type contains a newly identified inactive allele (ans1) of the anthocyanidin synthase (ANS) gene with a premature stop codon generated from a 4-bp deletion in the second exon. The ans1 allele was distinguished from the active ANS allele by a cleaved amplified polymorphism sequence (CAPS) marker. The genotypes of 12 white-flowered gentian cultivars/lines could be identified and classified as either ans1 or gtmyb3 using these four molecular markers. No white-flowered gentians contained ans1 and gtmyb3 alleles simultaneously. The mutated ANS gene co-segregated with white flower color in an F2 population, demonstrating that the CAPS marker is useful to discriminate between white and blue flowers in gentian. Markers to discriminate flower color in Japanese gentian will be useful for early selection of progeny and for breeding management.


Flower color Japanese gentian Marker-assisted selection Molecular marker 



We thank Mses. Y. Abe, C. Yoshida, and R. Horikiri, Iwate Biotechnology Research Center, for technical support. We also thank Dr. K. Kobayashi, Ehime University, for designing markers. This research was supported by a ‘Research Project for Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries’ (no. 2040) from the Ministry of Agriculture, Forestry and Fisheries of Japan (MAFF), and by the Japan Science and Technology Agency (JST).


  1. Andersen JR, Lubberstedt T (2003) Functional markers in plants. Trends Plant Sci 8:554–560PubMedCrossRefGoogle Scholar
  2. Ashikari M, Matsuoka M (2006) Identification, isolation and pyramiding of quantitative trait loci for rice breeding. Trends Plant Sci 11:344–350PubMedCrossRefGoogle Scholar
  3. Azuma A, Udo Y, Sato A, Mitani N, Kono A, Ban Y, Yakushiji H, Koshita Y, Kobayashi S (2011) Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes. Theor Appl Genet. doi:10.1007/s00122-00011-01542-00127
  4. Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol 48:958–970PubMedCrossRefGoogle Scholar
  5. Bang H, Kim S, Leskovar D, King S (2007) Development of a codominant CAPS marker for allelic selection between canary yellow and red watermelon based on SNP in lycopene β-cyclase (LCYB) gene. Mol Breed 20:63–72CrossRefGoogle Scholar
  6. Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394PubMedCrossRefGoogle Scholar
  7. Chagné D, Carlisle CM, Blond C, Volz RK, Whitworth CJ, Oraguzie NC, Crowhurst RN, Allan AC, Espley RV, Hellens RP, Gardiner SE (2007) Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple. BMC Genomics 8:212PubMedCrossRefGoogle Scholar
  8. Fleury D, Jefferies S, Kuchel H, Langridge P (2010) Genetic and genomic tools to improve drought tolerance in wheat. J Exp Bot 61:3211–3222PubMedCrossRefGoogle Scholar
  9. Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53:814–827PubMedCrossRefGoogle Scholar
  10. Haliassos A, Chomel JC, Grandjouan S, Kruh J, Kaplan JC, Kitzis A (1989) Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers. Nucleic Acids Res 17:8093–8099PubMedCrossRefGoogle Scholar
  11. Hikage T, Kogusuri K, Tanaka-Saito C, Watanabe S, Chiba S, Kume K, Doi H, Saitoh Y, Takahata Y, Tsutsumi K (2011) W14/15 esterase gene haplotype can be a genetic landmark of cultivars and species of the genus Gentiana L. Mol Gen Genomics 285:47–56CrossRefGoogle Scholar
  12. Jones N, Ougham H, Thomas H, Pasakinskiene I (2009) Markers and mapping revisited: finding your gene. New Phytol 183:935–966PubMedCrossRefGoogle Scholar
  13. Kakizaki Y, Nakatsuka T, Kawamura H, Abe J, Abe Y, Yamamura S, Nishihara M (2009) Development of codominant DNA marker distinguishing pink from blue flowers in Gentiana scabra. Breed Res 11:9–14 (Japanese)Google Scholar
  14. Kawasaki S, Murakami Y (2000) Genome analysis of Lotus japonicus. J Plant Res 113:497–506CrossRefGoogle Scholar
  15. Ke X, Collins A, Ye S (2001) PIRA PCR designer for restriction analysis of single nucleotide polymorphisms. Bioinformatics 17:838–839PubMedCrossRefGoogle Scholar
  16. Kim S, Jones R, Yoo KS, Pike LM (2004) Gold color in onions (Allium cepa): a natural mutation of the chalcone isomerase gene resulting in a premature stop codon. Mol Gen Genomics 272:411–419CrossRefGoogle Scholar
  17. Kim S, Yoo KS, Pike LM (2005) Development of a PCR-based marker utilizing a deletion mutation in the dihydroflavonol 4-reductase (DFR) gene responsible for the lack of anthocyanin production in yellow onions (Allium cepa). Theor Appl Genet 110:588–595PubMedCrossRefGoogle Scholar
  18. Kim S, Bang H, Yoo KS, Pike LM (2006) Identification of the fourth allele of the ANS (anthocyanidin synthase) gene and its effect on red color intensity in onions (Allium cepa). Euphytica 149:45–51CrossRefGoogle Scholar
  19. Kim S, Baek D, Cho DY, Lee ET, Yoon MK (2009) Identification of two novel inactive DFR-A alleles responsible for failure to produce anthocyanin and development of a simple PCR-based molecular marker for bulb color selection in onion (Allium cepa L.). Theor Appl Genet 118:1391–1399PubMedCrossRefGoogle Scholar
  20. 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
  21. Kump KL, Bradbury PJ, Wisser RJ, Buckler ES, Belcher AR, Oropeza-Rosas MA, Zwonitzer JC, Kresovich S, McMullen MD, Ware D, Balint-Kurti PJ, Holland JB (2011) Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population. Nat Genet 43:163–168PubMedCrossRefGoogle Scholar
  22. Kunihisa M, Fukino N, Matsumoto S (2003) Development of cleavage amplified polymorphic sequence (CAPS) markers for identification of strawberry cultivars. Euphytica 134:209–215CrossRefGoogle Scholar
  23. Landjeva S, Korzun V, Börner A (2007) Molecular markers: actual and potential contributions to wheat genome characterization and breeding. Euphytica 156:271–296CrossRefGoogle Scholar
  24. Lee J, Yoon JB, Han JH, Lee WP, Kim SH, Park HG (2010) Three AFLP markers tightly linked to the genic male sterility ms3 gene in chili pepper (Capsicum annuum L.) and conversion to a CAPS marker. Euphytica 173:55–61CrossRefGoogle Scholar
  25. Matsubara K, Chen S, Lee J, Kodama H, Kokubun H, Watanabe H, Ando T (2006) PCR-based markers for the genotype identification of flavonoid- 3′, 5′-hydroxylase genes governing floral anthocyanin biosynthesis in commercial petunias. Breed Sci 4:389–397CrossRefGoogle Scholar
  26. Nakajima T, Matsubara K, Kodama H, Kokubun H, Watanabe H, Ando T (2005) Insertion and excision of a transposable element governs the red floral phenotype in commercial petunias. Theor Appl Genet 110:1038–1043PubMedCrossRefGoogle Scholar
  27. Nakatsuka T, Nishihara M, Mishiba K, Yamamura S (2005) Two different mutations are involved in the formation of white-flowered gentian plants. Plant Sci 169:949–958CrossRefGoogle Scholar
  28. Nakatsuka T, Nishihara M, Mishiba K, Hirano H, Yamamura S (2006) Two different transposable elements inserted in flavonoid 3’, 5’-hydroxylase gene contribute to pink flower coloration in Gentiana scabra. Mol Gen Genomics 275:231–241CrossRefGoogle Scholar
  29. Nakatsuka T, Haruta KS, Pitaksutheepong C, Abe Y, Kakizaki Y, Yamamoto K, Shimada N, Yamamura S, Nishihara M (2008) Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers. Plant Cell Physiol 49:1818–1829PubMedCrossRefGoogle Scholar
  30. Nakatsuka T, Abe Y, Kakizaki Y, Kubota A, Shimada N, Nishihara M (2009) Over-expression of Arabidopsis FT gene reduces juvenile phase and induces early flowering in ornamental gentian plants. Euphytica 168:113–119CrossRefGoogle Scholar
  31. Nakatsuka T, Saito M, Yamada E, Nishihara M (2011) Production of picotee-type flowers in Japanese gentian by CRES-T. Plant Biotechnol 28:173–180CrossRefGoogle Scholar
  32. Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14:387–392PubMedCrossRefGoogle Scholar
  33. Nishihara M, Nakatsuka T, Mizutani-Fukuchi M, Tanaka Y, Yamamura S (2008) Gentians: from gene cloning to molecular breeding. In: Jaime A, da Silva T (eds) Floricultural and ornamental biotechnology v. Global Science Books, Middlesex, pp 57–67Google Scholar
  34. Ribaut JM, Hoisington D (1998) Marker-assisted selection: new tools and strategies. Trends Plant Sci 3:236–239CrossRefGoogle Scholar
  35. Shimada N, Nakatsuka T, Nakano Y, Kakizaki Y, Abe Y, Hikage T, Nishihara M (2009) Identification of gentian cultivars using SCAR markers based on intron-length polymorphisms of flavonoid biosynthetic genes. Sci Hort 119:292–296CrossRefGoogle Scholar
  36. Takahashi R, Dubouzet JG, Matsumura H, Yasuda K, Iwashina T (2011) A new allele of flower color gene W1 encoding flavonoid 3’5’-hydroxylase is responsible for light purple flowers in wild soybean Glycine soja. BMC Plant Biol 10:155CrossRefGoogle Scholar
  37. Tanaka M, Takahata Y, Nakayama H, Yoshinaga M, Kumagai T, Nakatani M (2010) Development of cleaved amplified polymorphic sequence (CAPS)-based markers for identification of sweetpotato cultivars. Sci Hort 123:436–442CrossRefGoogle Scholar
  38. Taylor DR, Ingvarsson PK (2003) Common features of segregation distortion in plants and animals. Genetica 117:27–35PubMedCrossRefGoogle Scholar
  39. Tian F, Bradbury PJ, Brown PJ, Hung H, Sun Q, Flint-Garcia S, Rocheford TR, McMullen MD, Holland JB, Buckler ES (2011) Genome-wide association study of leaf architecture in the maize nested association mapping population. Nat Genet 43:159–162PubMedCrossRefGoogle Scholar
  40. Wang X, Wadl PA, Rinehart TA, ScheZer BE, Windham MT, Spiers JM, Johnson DH, Trigiano RN (2009) A linkage map for fowering dogwood (Cornus forida L.) based on microsatellite markers. Euphytica 165:165–175CrossRefGoogle Scholar
  41. Yagi M, Onozaki T, Taneya M, Watanabe H, Yoshimura T, Yoshinari T, Ochiai Y, Shibata M (2006) Construction of a genetic linkage map for the carnation by using RAPD and SSR markers and mapping quantitative trait loci (QTL) for resistance to bacterial wilt caused by Burkholderia caryophylli. J Japan Soc Hort Sci 75:166–172CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Takashi Nakatsuka
    • 1
  • Misa Saito
    • 1
  • Yuka Sato-Ushiku
    • 1
  • Eri Yamada
    • 1
  • Takashi Nakasato
    • 2
  • Nobue Hoshi
    • 2
  • Kazumichi Fujiwara
    • 2
  • Takashi Hikage
    • 3
  • Masahiro Nishihara
    • 1
    Email author
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.Iwate Agricultural Research CenterKitakamiJapan
  3. 3.Hachimantai City Floricultural Development Research CenterHachimantaiJapan

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