W4 Locus Research Articles

Molecular Genetic Analysis of a Novel Recessive White Flower Gene in Wild Soybean

Cultivated soybean [Glycine max (L.) Merr.] flowers are either white or purple, whereas nearly all wild soybean (G. soja Sieb. and Zucc.) accessions have purple flowers. As a result, the soybean flower color phenotype has attracted the attention of plant breeders, biochemists, and population geneticists. Here, we report genetic mapping of a novel recessive white flower gene in YWS415, a wild soybean accession collected in Korea. The W1 locus, which is the major locus determining purple (W1) or white (w1) flower color, encodes flavonoid 3′ 5′‐hydroxylase in YWS415, which was more similar to the common W1‐type sequences than w1‐type sequences. Subsequent mapping results using an F2 population from a cross between the ‘Hwangkeum’ (purple flower) and YWS415 variants indicated that the white flower color was controlled by a recessive allele at the W4 locus that encodes dihydroflavonol‐4‐reductase 2 (DFR2). The w4 allele arose through insertion of a 5584‐bp nonautonomous transposon member of soybean GM_CACTA_33 family into the second intron of DFR2. It was designated as GM_CACTA_33_w4. Results from this study will enhance our understanding of soybean white flower inheritance and facilitate marker‐assisted selection for the antioxidative anthocyanins in soybeans.

Color Variation in a Novel Glycine soja Mutant W4‐S1 with Pinkish‐White Flowers Is Controlled by a Single Recessive Allele at the W4 Locus

Dihydroflavonol‐4‐reductase (DFR) plays a crucial role in anthocyanin biosynthesis. In cultivated soybean [Glycine max (L.) Merr.], the W3 and W4 loci encode DFR1 and DFR2, respectively, and are epistatic to each other. In this study, we discovered a new flower color variant of Glycine soja (L.) Merr., CW13133, which has pinkish‐white flowers, and investigated the genetic and molecular basis of the variation in flower color. We found that the W4 locus encoding DFR2 was responsible for the variation in flower color. Sequence analysis of the DFR2 gene of CW13133 revealed three single‐nucleotide polymorphisms (SNPs). Among these, an SNP (T to C) at nucleotide position 2856 was noteworthy, since it led to a change in an amino acid residue (leucine to proline) at position 326 of DFR2. This amino acid substitution was located in a region that is highly conserved among different plant species. The new allele was designated w4‐s1. A cleaved amplified polymorphic sequence analysis showed cosegregation of the w4‐s1 allele with pinkish‐white flowers. Interestingly, DFR1 was not expressed in the w4‐s1 mutant, indicating that the W3 locus has no association with flower color variation. Based on a randomized comparative analysis using 10 different purple‐flowered accessions of G. soja, we provide evidence for the nonassociation of DFR1 expression with total anthocyanin contents and flower color determination in G. soja. In conclusion, we found that the W3 locus in G. soja does not contribute to flower color determination, and consequently there is no epistatic interaction between the W3 and W4 loci, as shown in G. max.

The endogenous transposable element Tgm9 is suitable for generating knockout mutants for functional analyses of soybean genes and genetic improvement in soybean.

In soybean, variegated flowers can be caused by somatic excision of the CACTA-type transposable element Tgm9 from Intron 2 of the DFR2 gene encoding dihydroflavonol-4-reductase of the anthocyanin pigment biosynthetic pathway. DFR2 was mapped to the W4 locus, where the allele containing Tgm9 was termed w4-m. In this study we have demonstrated that previously identified morphological mutants (three chlorophyll deficient mutants, one male sterile-female fertile mutant, and three partial female sterile mutants) were caused by insertion of Tgm9 following its excision from DFR2. Analyses of Tgm9 insertion sites among 105 independent mutants demonstrated that Tgm9 hops to all 20 soybean chromosomes from its original location on Chromosome 17. Some genomic regions are prone to increased Tgm9-insertions. Tgm9 transposed over 25% of the time into exon or intron sequences. Tgm9 is therefore suitable for generating an indexed insertional mutant collection for functional analyses of most soybean genes. Furthermore, desirable Tgm9-induced stable knockout mutants can be utilized in generating improved traits for commercial soybean cultivars.

Association of New Mutant Allele, w4‐nw, at W4 Locus with Near‐White Flower Color in Soybean

An ethyl methanesulfonate‐induced mutant (PE1033) with near‐white flowers was isolated from the soybean [Glycine max (L.) Merr.] cultivar Pungsannamul with purple flowers that had the W1w3W4 genotype. In the present study, we aimed to investigate the genetic and molecular bases of the flower color variation displayed by the PE1033 mutant. Analyses of genomic sequences and reverse transcription–polymerase chain reaction (RT‐PCR) revealed that the color variation of PE1033 flowers originated from a recessive mutation in the W4 locus that encodes dihydroflavonol 4‐reductase (DFR) 2. The new mutant allele, named w4‐nw, harbored a single‐nucleotide substitution (G to A) at the 5′ spliceosome recognition site in the first intron of DFR2, resulting in the failure of splicing and producing two different abnormal transcripts: w4‐nw‐a and w4‐nw‐b. The former encoded a truncated DFR2 protein because of the occurrence of a premature stop codon in the first intron, which was retained in the transcript; the latter resulted from alternative splicing of the first intron and encoded a DFR2 protein that lacked 11 amino acid residues in the N‐terminus. Derived cleaved amplified polymorphism sequence (dCAPS) analysis revealed a tight cosegregation pattern between the w4‐nw mutant allele and near‐white color phenotype. In conclusion, the novel mutation in the W4 locus, w4‐nw, caused the production of near‐white flowers under the w3 allelic background of PE1033.

Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean.

The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies.

Allelic variation of soybean flower color gene W4 encoding dihydroflavonol 4-reductase 2

BackgroundFlower 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).ResultsComplementation 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.ConclusionsComplete 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.

Excision of an Active CACTA-Like Transposable Element From DFR2 Causes Variegated Flowers in Soybean [Glycine max (L.) Merr.

Active endogenous transposable elements, useful tools for gene isolation, have not been reported from any legume species. An active transposable element was suggested to reside in the W4 locus that governs flower color in soybean. Through biochemical and molecular analyses of several revertants of the w4-m allele, we have shown that the W4 locus encodes dihydroflavonol-4-reductase 2 (DFR2). w4-m has arisen through insertion of Tgm9, a 20,548-bp CACTA-like transposable element, into the second intron of DFR2. Tgm9 showed high nucleic acid sequence identity to Tgmt*. Its 5' and 3' terminal inverted repeats start with conserved CACTA sequence. The 3' subterminal region is highly repetitive. Tgm9 carries TNP1- and TNP2-like transposase genes that are expressed in the mutable line, T322 (w4-m). The element excises at a high frequency from both somatic and germinal tissues. Following excision, reinsertions of Tgm9 into the DFR2 promoter generated novel stable alleles, w4-dp (dilute purple flowers) and w4-p (pale flowers). We hypothesize that the element is fractured during transposition, and truncated versions of the element in new insertion sites cause stable mutations. The highly active endogenous transposon, Tgm9, should facilitate genomics studies specifically that relate to legume biology.

Genetic analysis and molecular mapping of a pale flower allele at theW4locus in soybean

In soybean (Glycine max (L.) Merr.), the w4-mutable line that harbors the w4-m allele was identified in 1983. It was proposed that this line contained an autonomous transposable element at the W4 locus, which is a major locus controlling the biosynthesis of anthocyanin. The w4-m allele can revert to the W4 allele that produces the wild-type phenotype, or sometimes to other alleles that produce intermediate phenotypes. Mutant plants that produce pale flowers were identified among the progeny of a single germinal revertant event from the w4-mutable line. Through genetic analysis, we established that the pale-flower mutation was conditioned by a new allele (w4-p) at the W4 locus. The w4-p allele is dominant to the w4 allele but recessive to the W4 allele, and the w1 allele has an epistatic effect on the w4-p allele. The pale-mutant line (w4-pw4-p) was designated as Genetic Type Collection number T369. An F2 mapping population derived from the cross of Minsoy (W4W4) x T369 (w4-pw4-p) was used to map the W4/w4-p locus, using simple sequence repeat (SSR) markers. The W4 locus was located at one end of molecular linkage group D2, 2.3 cM from the SSR marker Satt386 and close to the nearby telomere.

Genetic and cytological analyses of a partial‐female‐sterile mutant (PS‐1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.

Genetic and cytological analyses of three lethal ovule mutants in soybean (Glycine max; Leguminosae)

Soybean partially sterile mutants 2, 3, and 4 (PS-2, PS-3, and PS-4), recovered from a gene-tagging experiment, were studied to clarify their inheritance, linkage, allelism, and reproductive biology. The PS-2, PS-3, and PS-4 mutants were maintained as heterozygotes and upon self-pollination segregated l fertile: l partially-sterile. For inheritance and linkage tests, all three PS mutants were crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient (CD) mutants CD-1 and CD-5, also recovered from the tagging study. For allelism tests, reciprocal crosses were made among the three partially sterile mutants. Linkage results indicated that the gene for partial sterility in the PS-2, PS-3, and PS-4 mutants was not linked either to the w4 locus or to the genes for chlorophyll deficiency. Studies of pollen development, pollen viability, and pollen-tube germination indicated no difference between normal and partially sterile genotypes. Linkage and allelism tests indicated that the gene in the three partially sterile mutants was not transmitted through the female when they were used as a female parent. A study of megagametogenesis indicated that the ovules from partially sterile plants had normal embryo sac development. Ovule abortion was due to failure of fertilization.

Germinal reversion of an unstable mutation for anthocyanin pigmentation in soybean

Plants of the "w4-mutable" line of soybean [Glycine max (L.) Merr.] are chimeral for anthocyanin pigmentation. Mutable plants produce both near-white and purple flowers, as well as flowers of mutable phenotype with purple sectors on near-white petals. It is established here that the mutable trait is conditioned by an unstable recessive allele of the w4 locus that conditions anthocyanin biosynthesis. The gene symbol w4-m is assigned to the mutable allele. Allele w4-m was derived from a stable, wild-type W4 progenitor allele and reverts at high frequency to a stable, wild-type W4 allele. Reversion occurs both early and late during the development of the germ line. Several experiments give estimates of germinal reversion frequency, indicating that approximately 6% of mutable alleles revert to wild-type from one generation to the next. Allele w4-m exhibits many features typical of an allele controlled by a transposable element.

W4‐Mutable line in soybean

AbstractAn unstable mutation for anthocyanin pigmentation in soybean (Giycine max [L.] Merr.) was identified in 1983. The mutability is conditioned by an allele at the w4 locus that is recessive to wild type. The population containing the mutable allele is known as the w4‐mutable line. Most plants in the line have chimeric flowers with purple sectors on a near‐white background. The mutable allele yields germinal revertants at a rate that varies from 5 to 10% per generation, and the revertant alleles are stable. Approximately 1% of the progenies derived from germinal revertant plants contain mutations at other loci These features, as well as the occurrence of pale flower phenotypes and changes of state, suggest that a transposable element system is producing the unstable phenotype.Several new mutants were isolated in an experiment designed to tag loci. The first three chlorophyll‐deficient mutants found (CD‐1, CD‐2, and CD‐3) are inherited as single‐gene recessives. Each of the mutants lacks the same two mitochondrial malate dehydrogenase (MDH) bands. No recombination has been detected between the MDH phenotype and the chlorophyll‐deficient phenotype. Genetic data indicate that the three mutants are allelic, and additional evidence suggests that each of the CD mutants is the result of a deletion. In the CD‐1, CD‐2, and CD‐3 mutants, the deletions result in the silencing of an MDH locus, atypical chloroplast development, and an altered chlorophyll composition. Additional mutants for root necrosis, partial and near sterility, chlorophyll deficiency, and flower color isolated from the transposon tagging study have provided material for future research.