A single-base deletion in soybean flavonoid 3'-hydroxylase gene is associated with gray pubescence color.
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.
Toda
Kyoko
K
National Institute of Crop Science, Tsukuba, Ibaraki, Japan.
Yang
Daijun
D
Yamanaka
Naoki
N
Watanabe
Satoshi
S
Harada
Kyuya
K
Takahashi
Ryoji
R
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005808:Genes, Recessive / Q000235:genetics; D005838:Genotype; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010860:Pigments, Biological / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013025:Glycine max / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D010860:Pigments, Biological; D018749:RNA, Plant; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C038888:flavonoid 3'-hydroxylase