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. 2013 Jun;126(6):1445-55.
doi: 10.1007/s00122-013-2063-3. Epub 2013 Mar 6.

Allele-specific marker development and selection efficiencies for both flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes in soybean subgenus soja

Yong Guo  1 , Li-Juan Qiu
Affiliations

Allele-specific marker development and selection efficiencies for both flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes in soybean subgenus soja

Yong Guo et al. Theor Appl Genet. 2013 Jun.

Abstract

Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3'-hydroxylase (F3'H; EC1.14.3.21) and flavonoid 3',5'-hydroxylase (F3'5'H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3'H and the W1 locus co-segregated with a gene encoding F3'5'H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3'H and F3'5'H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3'H and one of three alleles for GmF3'5'H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3'H and F3'5'H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3'H allele explained 92.2 % of the variation in tawny and two gmf3'h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3'5'H alleles and one gmF3'5'h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3'h-b and gmf3'5'h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.

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Figures

Fig. 1
Fig. 1
Phylogenetic tree of F3′Hs and F3′5′Hs proteins from soybean and other species. Neighbor-joining tree of F3′Hs and F3′5′H proteins from soybean and other species generated using MEGA 4.0. The numbers on each node are bootstrap values, which indicate the percentage of bootstrap replicates that support this node out of 1,000 samples. Branch lengths are proportional to the estimated number of amino-acid substitutions. Scale bar indicates the estimated amino-acid substitutions per site. F3′H superfamily; Arabidopsis thaliana (AAG16746), Petunia x hybrida (AAD56282), Vitis vinifera (CAI54278), Brassica napus (ABC58723), Antirrhinum majus (ABB53383), Malus × domestica (ACR14867), Zea mays (AEF33624), Sorghum bicolor (AAV74195), and Oryza sativa (AAP52914). F3′5′H superfamily; Petunia × hybrida (CAA80265), Gentiana scabra (BAE86871), Pisum sativum (ADW66160), Antirrhinum kelloggii (BAJ16328), Vitis vinifera (CAI54277), Solanum tuberosum (AAV85470), Solanum lycopersicum (ADC80513), Phalaenopsis hybrid (AAZ79451), Dendrobium moniliforme (AEB96145), Hordeum vulgare (BAK02913)
Fig. 2
Fig. 2
GmF3′H gene structure and polymorphisms in different alleles. The gmf3′h-a1 allele presents a single base deletion of adenine at coding sequence position 973 relative to the start codon. The deletion creates a premature stop codon and a truncated protein of 394 residues compared to the reference GmF3′H protein of 513 residues. The gmf3′h-a2 allele presents a single base insertion of an adenine at position 965 as well as a single base deletion of adenine at position 973, resulting in N322 K and Q324T changes. The gmf3′h-b allele presents a single base deletion of a cytosine at position 1164. This cytosine deletion resulted in a frame-shift that prematurely truncated the protein of 394 residues. Boxes represent exons; lines between boxes represent introns; boxes with dashed lines represent exons with untranslated regions in predicted proteins from different alleles
Fig. 3
Fig. 3
GmF3′5′H gene structure and polymorphisms in different alleles. The GmF3′5′H-b allele had three nucleotide substitutions in exon 3 (C1059A, T1424A, C1509T): two synonymous and one non-synonymous substitution (V475E). The gmf3′5′h allele had a 53-bp fragment insertion at coding sequence position 1352 relative to the start codon, resulting in a prematurely truncated amino-acid sequence of 467 residues compared to the reference GmF3′5′H protein of 501 residues. Boxes represent exons; lines between boxes represent introns; boxes with dashed lines represent exons with untranslated regions in predicted proteins from different alleles
Fig. 4
Fig. 4
Polymorphisms revealed by three gene-tagged markers of GmF3′H. The F3′H-ApoI marker distinguished gmf3′h-a1 and non-gmf3′h-a1 alleles (a); the F3′H-MjaIV marker distinguished gmf3′h-a2 and non-gmf3′h-a2 alleles (b); the F3′H-EcoNI marker distinguished gmf3′h-b and non-gmf3′h-b alleles (c)
Fig. 5
Fig. 5
Polymorphisms revealed by two gene-tagged markers of GmF3′5′H. The F3′5′H-In marker distinguished gmf3′5′h and non-gmf3′5′h alleles (a); the F3′5′H-HphI marker distinguished GmF3′5′H-a and GmF3′5′H-b alleles (b)
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