Plant Molecular Biology

, Volume 50, Issue 2, pp 187–196 | Cite as

A single-base deletion in soybean flavonoid 3′-hydroxylase gene is associated with gray pubescence color

  • Kyoko Toda
  • Daijun Yang
  • Naoki Yamanaka
  • Satoshi Watanabe
  • Kyuya Harada
  • Ryoji Takahashi


The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3′-hydroxylase (F3′H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3′H gene. A putative full-length cDNA, sf3′h1 was isolated by 3′ and 5′ RACE. Sequence analysis revealed that sf3′h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3′H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3′H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3′h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3′h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the Tlocus in a F2 population segregating for the T locus. The above results strongly suggest that sf3′h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.

deletion F3′H flavonoid flavonoid 3′-hydroxylase P450 pubescence color soybean 


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  1. Altschul, S.F., Madden, T.L., Schaffer A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucl. Acids Res. 25: 3389–3402.Google Scholar
  2. Brugliera, F., Barri-Rewell, G., Holton, T.A. and Mason, J.G. 1999. Isolation and characterization of a flavonoid 3?-hydroxylase cDNA clone corresponding to the HT1 locus of Petunia hybrida. Plant J. 19: 441–451.Google Scholar
  3. Buttery, B.R. and Buzzell, R.I. 1973. Varietal differences in leaf flavonoids of soybeans. Crop Sci. 13: 103–106.Google Scholar
  4. Buttery, B.R. and Buzzell, R.I. 1976. Flavonol glycoside genes and photosynthesis in soybean. Crop Sci. 16: 547–550.Google Scholar
  5. Buzzell, R.I. and Buttery, B.R. 1982. Genetics of black pigmentation of soybean seed coats / hila. Soybean Genet. Newsl. 9: 26–29.Google Scholar
  6. Daar, I.O. and Maquat, L.E. 1988. Premature translation termination mediates triosephosphate isomerase mRNA degradation. Mol. Cell Biol. 8: 802–813.Google Scholar
  7. Dixon, R.A. and Steele, C.L. 1999. Flavonoids and isoflavonoids-a gold mine for metabolic engineering. Trends Plant Sci. 4: 394–400.Google Scholar
  8. Dooner, H.K. and Robbins, T.P. 1991. Genetic and developmental control of anthocyanin biosynthesis. Annu. Rev. Genet. 25: 173–199.Google Scholar
  9. Durst, F. and O'Keefe, D.P. 1995. Plant cytochromes P450: an overview. Drug Metabol. Drug Interact. 12: 171–187.Google Scholar
  10. Fasoula, D.A., Stephens, P.A., Nickell, C.D. and Vodkin, L.O. 1995. Cosegregation of purple-throat flower color with dihydroflavonol reductase polymorphism in soybean. Crop Sci. 35: 1028–1031.Google Scholar
  11. Forkmann, G. 1991. Flavonoids as flower pigments: the formation of the natural spectrum and its extension by genetic engineering. Plant Breed. 106: 1–26.Google Scholar
  12. Holton, T.A. and Cornish E.C. 1995. Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7: 1071–1083.Google Scholar
  13. Kouchi, H. and Hata, S. 1993. Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol. Gen. Genet. 238: 106–119.Google Scholar
  14. Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Day, M.J., Lincoln, S.E. and Newberg, L. 1987. Mapmaker: an interactive computer package for constructing primary genetic linkage map of experimental and natural populations. Genomics 1: 174–181.Google Scholar
  15. Mizutani, M., Ward, E. and Ohta, D. 1998. Cytochrome P450 superfamily in Arabidopsis thaliana: isolation of cDNAs, differential expression, and RFLP mapping of multiple cytochrome P450. Plant Mol. Biol. 37: 39–52.Google Scholar
  16. Murray, M.G. and Thompson, W.F. 1980. Rapid isolation of high molecular weight plant DNA. Nucl. Acid Res. 8: 4321–4325.Google Scholar
  17. Nicholas, C.D., Lindstrom, J.T. and Vodkin, L.O. 1993. Variation of proline rich cell wall proteins in soybean lines with anthocyanin mutations. Plant Mol. Biol. 21: 145–156.Google Scholar
  18. Palmer, R.G. and Kilen, T.C. 1987. Qualitative genetics and cytogenetics. In: J.R. Wilcox (Ed.) Soybeans: Improvement, Production, and Uses, 2nd ed. (Agronomy Monograph 16), ASA, CSSA and SSSA, Madison, WI, pp.135–209.Google Scholar
  19. Ross, J. 1995. mRNA stability in mammalian cells. Microbiol. Rev. 59: 423–450.Google Scholar
  20. Schoenbohm, C., Martens, S., Eder, C., Forkmann, G. and Weisshaar, B. 2000. Identification of the Arabidopsis thaliana flavonoid 3?-hydroxylase gene and functional expression of the encoded P450 enzyme. Biol. Chem. 381: 749–753.Google Scholar
  21. Schuler, M.A. 1996. Plant cytochrome P450 monooxygenases. Crit. Rev. Plant Sci. 15: 235–284.Google Scholar
  22. Shah, D.M., Hightower, R.C. and Meagher, R.B. 1983. Genes encoding actin in higher plants: intron positions are highly conserved but the coding sequences are not. J. Mol. Appl. Genet. 2: 111–126.Google Scholar
  23. Shirley, B.W. 1996. Flavonoid biosynthesis: new functions for an old pathway. Trends Plant Sci. 1: 377–382.Google Scholar
  24. Stephens, P.A. and Nickell, C.D. 1992. Inheritance of pink flower in soybean. Crop Sci. 32: 1131–1132.Google Scholar
  25. Stewart, R.T. and Wentz, J.B. 1930. A defective seed-coat character in soybeans. J. Am. Soc. Agron. 22: 658–662.Google Scholar
  26. Sunada, K. and Ito, T. 1982. Soybean grain quality as affected by low temperature treatments in plants (color of hilum, seed coat cracking) (in Japanese). Rep. Hokkaido Branch, Crop Sci. Soc. Jpn./Hokkaido Branch, Jpn. Soc. Breed. 22: 34.Google Scholar
  27. Takahashi, R. 1997. Association of soybean genes I and T with low-temperature-induced seed coat deterioration. Crop Sci. 37: 1755–1759.Google Scholar
  28. Takahashi, R. and Asanuma, S. 1996. Association of T gene with chilling tolerance in soybean. Crop Sci. 36: 559–562.Google Scholar
  29. Takahashi, R. and Shimosaka, E. 1997. cDNA sequence analysis and expression of two cold-regulated genes in soybean. Plant Sci. 123: 93–104.Google Scholar
  30. Todd, J.J. and Vodkin, L.O. 1993. Pigmented soybean (Glycine max) seed coat accumulate proanthocyanidins during development. Plant Physiol. 102: 663–670.Google Scholar
  31. Todd, J.J. and Vodkin, L.O. 1996. Duplications that suppress and deletions that restore expression from a chalcone synthase multigene family. Plant Cell 8: 687–699.Google Scholar
  32. Xu, D.H., Abe, J., Kanazawa, A. and Shimamoto, Y. 2001. Identification of sequence variations by PCR-RFLP and its application to the evaluation of cpDNA diversity in wild and cultivated soybeans. Theor. Appl. Genet. 102: 683–688.Google Scholar
  33. Yamanaka, N., Ninomiya, S., Hoshi, M., Tsubokura, Y., Yano, M., Nagamura, Y., Sasaki, T. and Harada, K. 2001. An informative linkage map of soybean reveals QTLs for flowering time, leaflet morphology and regions of segregation distortion. DNA Res. 8: 61–72.Google Scholar
  34. Yoshikura, K. and Hamaguchi, Y. 1969. Anthocyanins of black soybeans. Jpn. Soc. Food Nutr. J. 22: 15–18.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Kyoko Toda
    • 1
  • Daijun Yang
    • 1
  • Naoki Yamanaka
    • 2
  • Satoshi Watanabe
    • 2
  • Kyuya Harada
    • 2
  • Ryoji Takahashi
    • 1
  1. 1.National Institute of Crop ScienceTsukuba, IbarakiJapan
  2. 2.Graduate School of Science and TechnologyChiba UniversityMatsudo, ChibaJapan

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