Dataset Information

Identification and Molecular Analysis of Four New Alleles at the W1 Locus Associated with Flower Color in Soybean.

ABSTRACT: In soybean, flavonoid 3'5'-hydroxylase (F3'5'H) and dihydroflavonol-4-reductase (DFR) play a crucial role in the production of anthocyanin pigments. Loss-of-function of the W1 locus, which encodes the former, or W3 and W4, which encode the latter, always produces white flowers. In this study, we searched for new genetic components responsible for the production of white flowers in soybean and isolated four white-flowered mutant lines, i.e., two Glycine soja accessions (CW12700 and CW13381) and two EMS-induced mutants of Glycine max (PE1837 and PE636). F3'5'H expression in CW12700, PE1837, and PE636 was normal, whereas that in CW13381 was aberrant and missing the third exon. Sequence analysis of F3'5'H of CW13381 revealed the presence of an indel (~90-bp AT-repeat) in the second intron. In addition, the F3'5'H of CW12700, PE1837, and PE636 harbored unique single-nucleotide substitutions. The single nucleotide polymorphisms resulted in substitutions of amino acid residues located in or near the SRS4 domain of F3'5'H, which is essential for substrate recognition. 3D structure modeling of F3'5'H indicated that the substitutions could interfere with an interaction between the substrate and heme group and compromise the conformation of the active site of F3'5'H. Recombination analysis revealed a tight correlation between all of the mutant alleles at the W1 locus and white flower color. On the basis of the characterization of the new mutant alleles, we discussed the biological implications of F3'5'H and DFR in the determination of flower colors in soybean.

SUBMITTER: Sundaramoorthy J

PROVIDER: S-EPMC4956318 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification and Molecular Analysis of Four New Alleles at the W1 Locus Associated with Flower Color in Soybean.

Sundaramoorthy Jagadeesh J Park Gyu Tae GT Chang Jeong Ho JH Lee Jeong-Dong JD Kim Jeong Hoe JH Seo Hak Soo HS Chung Gyuhwa G Song Jong Tae JT

PloS one 20160721 7

In soybean, flavonoid 3'5'-hydroxylase (F3'5'H) and dihydroflavonol-4-reductase (DFR) play a crucial role in the production of anthocyanin pigments. Loss-of-function of the W1 locus, which encodes the former, or W3 and W4, which encode the latter, always produces white flowers. In this study, we searched for new genetic components responsible for the production of white flowers in soybean and isolated four white-flowered mutant lines, i.e., two Glycine soja accessions (CW12700 and CW13381) and t ...[more]

PMID: 27442124

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Project description:BackgroundGlycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat.ResultsB09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants.ConclusionsB09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be a useful tool for studies of the structural and functional properties of F3'5'H genes as well as investigations on the role of flower color in relation to adaptation of G. soja to natural habitats.

Project description:BACKGROUND: Flower color of soybean is primarily controlled by six genes, viz., W1, W2, W3, W4, Wm and Wp. This study was conducted to investigate the genetic and chemical basis of newly-identified flower color variants including two soybean mutant lines, 222-A-3 (near white flower) and E30-D-1 (light purple flower), a near-isogenic line (Clark-w4), flower color variants (T321 and T369) descended from the w4-mutable line and kw4 (near white flower, Glycine soja). RESULTS: Complementation tests revealed that the flower color of 222-A-3 and kw4 was controlled by the recessive allele (w4) of the W4 locus encoding dihydroflavonol 4-reductase 2 (DFR2). In 222-A-3, a single base was deleted in the first exon resulting in a truncated polypeptide consisting of 24 amino acids. In Clark-w4, base substitution of the first nucleotide of the fourth intron abolished the 5' splice site, resulting in the retention of the intron. The DFR2 gene of kw4 was not expressed. The above results suggest that complete loss-of-function of DFR2 gene leads to near white flowers. Light purple flower of E30-D-1 was controlled by a new allele at the W4 locus, w4-lp. The gene symbol was approved by the Soybean Genetics Committee. In E30-D-1, a single-base substitution changed an amino acid at position 39 from arginine to histidine. Pale flowers of T369 had higher expression levels of the DFR2 gene. These flower petals contained unique dihydroflavonols that have not yet been reported to occur in soybean and G. soja. CONCLUSIONS: Complete loss-of-function of DFR2 gene leads to near white flowers. A new allele of the W4 locus, w4-lp regulates light purple flowers. Single amino acid substitution was associated with light purple flowers. Flower petals of T369 had higher levels of DFR2 gene expression and contained unique dihydroflavonols that are absent in soybean and G. soja. Thus, mutants of the DFR2 gene have unique flavonoid compositions and display a wide variety of flower color patterns in soybean, from near white, light purple, dilute purple to pale.

Dataset Information

Identification and Molecular Analysis of Four New Alleles at the W1 Locus Associated with Flower Color in Soybean.

Publications

Identification and Molecular Analysis of Four New Alleles at the W1 Locus Associated with Flower Color in Soybean.

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