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

Plant Mol Biol. 2002 Sep;50(2):187-96. doi: 10.1023/a:1016087221334.

Abstract

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 T locus in a F2 population segregating for the T locus. The above results strongly suggest that sJ3'h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Base Sequence
  • Chromosome Mapping
  • Cloning, Molecular
  • Crosses, Genetic
  • Cytochrome P-450 Enzyme System / genetics*
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • DNA, Complementary / isolation & purification
  • DNA, Plant / genetics
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Plant
  • Genes, Recessive / genetics
  • Genotype
  • Mixed Function Oxygenases / genetics*
  • Molecular Sequence Data
  • Mutation
  • Pigments, Biological / genetics*
  • Polymorphism, Restriction Fragment Length
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Soybeans / enzymology
  • Soybeans / genetics*

Substances

  • DNA, Complementary
  • DNA, Plant
  • Pigments, Biological
  • RNA, Plant
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • flavonoid 3'-hydroxylase

Associated data

  • GENBANK/AB061212