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Identification of two novel mutant ANS alleles responsible for inactivation of anthocyanidin synthase and failure of anthocyanin production in onion (Allium cepa L.)

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Abstract

Two genes (DFR-A and ANS) encoding dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) enzymes in the anthocyanin biosynthesis pathway, respectively, are complementarily involved in anthocyanin production in onion (Allium cepa L.). Eleven inactive DFR-A alleles have been reported, with only a single inactive ANS allele previously identified. Two additional inactive ANS alleles are reported in this study. A mutant ANS allele containing a 4-bp insertion at the end of exon1 was identified from yellow bulbs of the F2 population in which the DFR-A genotype was homozygous for an active allele. The 4-bp insertion caused a frame-shift mutation and resulted in creation of a premature stop codon at the start of exon2. This mutant ANS allele was designated ANS PS allele. RT-PCR results showed that transcripts of the ANS PS allele were almost undetectable in yellow F2 bulbs, implying the involvement of nonsense-mediated mRNA decay. A cleaved amplified polymorphic sequence marker was developed for detection of the ANS PS allele. Another inactive ANS allele was identified from the light-red F1 populations showing complementation between DFR-A and ANS genes. A critical amino acid change of the strictly conserved serine residue into leucine was found in this mutant allele designated ANS S188L. In addition, seven variants of active ANS alleles were identified from diverse onion germplasm. A stepwise process consisting of PCR amplification and sequencing of PCR products was devised to identify three inactive (ANS PS, ANS S188L, and ANS G229R), one leaky (ANS P), and two active ANS alleles (ANS L and ANS h1).

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Acknowledgments

This research was supported by the Agriculture Research Center Program, Golden Seed Project (Center for Horticultural Seed Development, No. 213008-04-4-SB910) and a Grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ011034). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.

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Authors and Affiliations

  1. Department of Plant Biotechnology, Biotechnology Research Institute, Chonnam National University, Kwangju, 500-757, Korea

    Eun-Young Kim & Sunggil Kim

  2. National Institute of Horticultural & Herbal Science, RDA, Muan, 534-833, Korea

    Cheol-Woo Kim

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  1. Eun-Young Kim
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  2. Cheol-Woo Kim
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Correspondence to Sunggil Kim.

Electronic supplementary material

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Supplementary material 1 (XLSX 15 kb)

Supplementary material 2 (XLSX 9 kb)

Supplementary material 3 (XLSX 11 kb)

Supplementary material 4 (XLSX 11 kb)

10681_2016_1774_MOESM5_ESM.tif

Supplementary material 5 (TIFF 235) kb Supplementary Fig. 1. Phylogenetic tree constructed using amino acid sequences of 53 ANS enzymes isolated from different species. The GenBank accession numbers are shown in parenthesis. The numbers at the nodes are the bootstrap probability (%) with 1000 replicates. The scale bars indicate nucleotide substitutions per site

10681_2016_1774_MOESM6_ESM.tif

Supplementary material 6 (TIFF 508) kb Supplementary Fig. 2. Onion bulbs of the four red F1 hybrids between a yellow cultivar, ‘Santero’ and four yellow breeding lines. 1145 ‘Santero’ × OB814, 1146 ‘Santero’ × OB251, 1147 ‘Santero’ × OB252, 1148 ‘Santero’ × OB870

10681_2016_1774_MOESM7_ESM.tif

Supplementary material 7 (TIFF 113) kb Supplementary Fig. 3. Alignment of amino acid sequences of ANS enzymes isolated from Arabidopsis thaliana (NP194019) and three onion variants. ‘G229R’, ‘S188L’, and ‘L’ represent ANS G229R, ANS S188L, ANS L alleles, respectively. The positions of G229 and S188 are indicated with vertical arrows. Binding sites of iron and 2-oxoglutarate are indicated with filled and empty circles, respectively, and binding sites of dihydroquercetin and MES/ascorbate are indicated with filled and empty inverted triangles, respectively

10681_2016_1774_MOESM8_ESM.tif

Supplementary material 8 (TIFF 445) kb Supplementary Fig. 4. Alignment of amino acid sequences of 52 ANS enzymes isolated from other plant species and two onion ANS enzymes encoded by ANS P-1 and ANS h1-4 alleles. Only partial alignments around the positions containing amino acid changes among two onion ANS enzymes are presented. The dotted lines indicate the omitted regions. The two positions containing amino acid changes are enclosed with empty rectangular boxes

10681_2016_1774_MOESM9_ESM.tif

Supplementary material 9 (TIFF 350) kb Supplementary Fig. 5. Alignment of nucleotide sequences of the ANS h1, ANS h1-4, and ANS P alleles showing origination of the ANS h1-4 allele by intragenic recombination between ANS h1 and ANS P alleles. Two identical nucleotides at the polymorphic sequences among three ANS alleles are enclosed with empty rectangular boxes. The intron sequences are underlined

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Kim, EY., Kim, CW. & Kim, S. Identification of two novel mutant ANS alleles responsible for inactivation of anthocyanidin synthase and failure of anthocyanin production in onion (Allium cepa L.). Euphytica 212, 427–437 (2016). https://doi.org/10.1007/s10681-016-1774-3

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