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A Rearrangement Resulting in Small Tandem Repeats in the F3'5'H Gene of White Flower Genotypes Is Associated with the Soybean W1 Locus

Dep. of Crop Sciences, Univ. of Illinois, Urbana, IL 61801

View larger version (23K): [in this window] [in a new window]   Figure 1. Anthocyanin metabolic pathway in Glycine max. Enzyme abbreviations and the flower and seed coat color loci (in uppercase letters) show the three branches leading to the synthesis of the purple delphinidins, pink pelargonidins, red cyanidins, and the flavonols. The flavonol kaempferol derived from dihydrokaempferol was not included to simplify the scheme. Wp, W3, and Wm are the flower color markers associated with the indicated enzymes in the pathway based on molecular and genetic evidence (Zabala and Vodkin, 2005; Fasoula et al., 1995; Takahashi et al., 2007). The suggested W1 assignment (marked by a circle) as F3'5'H was made by Buzzell et al. (1987) based on biochemical evidence. The I and T are the well-characterized loci that control seed pigmentation and that encode CHS (Todd and Vodkin, 1996) and F3'H (Toda et al., 2002; Zabala and Vodkin, 2003), respectively. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3'H, flavonoid 3'-hydroxylase; F3'5'H, flavonoid 3'5'-hydroxylase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol-4-reductase; ANS, anthocyanidin synthase (also called LDOX, leucoanthocyanidin dioxygenase); UFGT, UDP-flavonoid glucosyltransferase; FLS, flavonol synthase.

View larger version (63K): [in this window] [in a new window]   Figure 2. Illustration of the effect of w1 on flower and seed coat phenotypes. (A) Purple flower of plants with W1 genotype (left panel) or white flower of plants with w1 genotype (right panel) in lines L79-908 (W1,ii,R,T,Wp) and Williams (w1,ii,R,T,Wp) respectively, both of which have yellow seed coats because of silenced ii allele. (B) Seed coats of plants L83-930 with W1,i,r,t,Wp genotype (left panel) compared with seed coats of plants of the isoline L82-2669 with w1,i,r,t,Wp genotype (right panel). It shows little difference in color revealing the very small contribution delphinidins and prodelphinidins make to seed coat pigmentation.

View larger version (22K): [in this window] [in a new window]   Figure 3. (A) Schematic representation of F3'5'H genomic sequences. Top graphic represents the components of the F3'5'H genomic sequence isolated from the L79-908 (W1) line. The three solid blocks are three exons predicting a cDNA 1801 bp in length. The two blocks representing introns of 754 and 2102 bp in length are shown in hatched lines. Bottom scheme is a graphic summary of the F3'5'H genomic sequence isolated from the ‘Williams’ isoline with the w1 allele. The three solid blocks represent the three exons predicting a slightly larger cDNA of 1854 bp. Exon 3 is 53 bp longer in the F3'5'H gene of the w1 white-flowered line than in the F3'5'H gene of the W1 purple-flowered line. The small block above exon 3 represents the extra 53 bp that contains part of the tandem repeats (arrows) resulting from the 65-base insertion (53 additional bases and 12 bases of sequence substitution). The two introns (755 bp and 2104 bp respectively) in the F3'5'H alleles isolated from both the w1 and W1 isolines are very similar in size and sequence. (B) Small tandem repeats in the F3'5'H gene of plants with the w1 white flower allele. Nucleotide numbering is from the Williams (w1) genomic sequence. The nucleotide sequence and translated amino acids flanking the insertion are shown. Nucleotides in lowercase italic letters denote the 53 extra bases of the insertion. Highlighting denotes two identical 37-bp repeats found at positions 4242 to 4278 and 4289 to 4325 that are separated by 10 nucleotides. Bold letters indicate the 12 nucleotides of the uninterrupted F3'5'H allele that are replaced by the 65-bp insertion. Also marked is the 21 nucleotide repeat found at position 4189 to 4209 in both the F3'5H alleles of the W1 line and w1 isolines that matches a portion of the tandem repeat unit of the insertion. The conserved P450 heme-binding domain is underlined. * denotes the premature stop codon created by the insertion.

View larger version (66K): [in this window] [in a new window]   Figure 4. Alignment of three Glycine max F3'5'H cDNAs derived amino acid sequences. The three amino acid sequences were derived from (i) the cDNA annotated as F3'5'H in GenBank with accession number AY117551 and isolated from the cultivar Chin-Ren-Woo-Dou (W1) by R.-M. Liao, T.-M. Chu, and C.-S.Wang (http://www.ncbi.nlm.nih.gov); (ii) the cDNA sequence predicted from the L79-908 (W1) F3'5'H genomic sequence reported here; and (iii) the cDNA sequence predicted from the ‘Williams’ (w1) genomic sequence described here. The alignment was done with Multalin, a multiple sequence alignment on-line tool by Corpet (1988). The consensus sequence shows identical amino acids in the three sequences; divergences between two of the amino acid sequences are highlighted. The amino acid sequence derived from the Williams w1 recessive allele shows the amino acid substitutions introduced by the 65-bp insertion. This sequence terminates earlier (stop codon indicated by *) and is missing the last 44 amino acids. Overall, it is a predicted 42 amino acids shorter than the polypeptide derived from the wild-type sequence in the dominant W1 line. Underlined is the motif that distinguishes P450s from other heme-binding enzymes.

View larger version (33K): [in this window] [in a new window]   Figure 5. Correlation between the F3'5'H 65-bp insertion and independently introduced w1 alleles. Polymerase chain reaction (PCR) fragments of 579 and 526 bp amplified from genomic DNAs isolated from multiple soybean lines (Table 1) with independently introduced W1 alleles were separated in a 3% NuSieve 3:1 agarose gel. The 53-bp longer PCR fragment was amplified from DNAs of cultivars and isolines with the w1 allele while the smaller 526 PCR fragment correlated with cultivars and lines with the W1 allele. Primers used were F35H-3980F and F35H-4558R. (A) Ethidium bromide stained gel showing the PCR fragments amplified from DNAs isolated from two independent plants of two isolines, L68-2056 and L66-14, which were used as parents for the segregating population shown in Table 2. (B) Ethidium bromide stained gel with PCR products amplified from DNAs isolated from 10 different soybean lines varying at the W1 locus (Table 1).

View larger version (81K): [in this window] [in a new window]   Figure 6. Glycine max F3'5'H gene polymorphisms and copy number. A DNA gel blot showing the hybridization of a genomic F3'5'H probe (3.5 kb) to DNAs isolated from three pairs of W1 isolines digested with HindIII and/or PstI. The smaller size HindIII fragment shows the 53-bp size difference between the W1 and w1 lines both in the single (HindIII) and double (HindIII and PstI) digested samples. HindIII cuts the F3'5'H gene in exon 3; the hybridization pattern shows the resulting two fragments in samples digested with this enzyme. PstI does not cut the F3'5'H gene.

View larger version (38K): [in this window] [in a new window]   Figure 7. Expression of F3'5'H by reverse transcriptase–polymerase chain reaction (RT–PCR) in purple- and white-flowered plants. Ethidium bromide stained gel showing the RT–PCR amplified cDNAs from flower buds of three W1 locus isolines, L79-908 (W1,ii,R,T,Wp), ‘Williams’ (w1,ii,R,T,Wp), and L76-2023 (w1,ii,R,t,Wp). The 1.6-kb amplified cDNAs shown in the (+) lanes are evidence that the two F3'5'H alleles in W1 and w1 lines are expressed at low levels in the flowers of these soybean lines. The (-) lanes are the result of parallel PCR reactions that were allowed in the absence of superscript to assess the extent of DNA contamination (negative controls).