Abstract
Intact retrotransposon and DNA transposons inserted in a single gene were characterized in onions (Allium cepa) and their transcription and copy numbers were estimated in this study. While analyzing diverse onion germplasm, large insertions in the DFR-A gene encoding dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway were found in two accessions. A 5,070-bp long terminal repeat (LTR) retrotransposon inserted in the active DFR-A R4 allele was identified from one of the large insertions and designated AcCOPIA1. An intact ORF encoded typical domains of copia-like LTR retrotransposons. However, AcCOPIA1 contained atypical ‘TG’ and ‘TA’ dinucleotides at the ends of the LTRs. A 4,615-bp DNA transposon was identified in the other large insertion. This DNA transposon, designated AcCACTA1, contained an ORF coding for a transposase showing homology with the CACTA superfamily transposable elements (TEs). Another 5,073-bp DNA transposon was identified from the DFR-A TRN allele. This DNA transposon, designated AchAT1, belonged to the hAT superfamily with short 4-bp terminal inverted repeats (TIRs). Finally, a 6,258-bp non-autonomous DNA transposon, designated AcPINK, was identified in the ANS-p allele encoding anthocyanidin synthase, the next downstream enzyme to DFR in the anthocyanin biosynthesis pathway. AcPINK also possessed very short 3-bp TIRs. Active transcription of AcCOPIA1, AcCACTA1, and AchAT1 was observed through RNA-Seq analysis and RT-PCR. The copy numbers of AcPINK estimated by mapping the genomic DNA reads produced by NextSeq 500 were predominantly high compared with the other TEs. A series of evidence indicated that these TEs might have transposed in these onion genes very recently, providing a steppingstone for elucidation of enormously large-sized onion genome structure.
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Acknowledgments
This research was supported by a grant (710001-07-4) from the Vegetable Breeding Research Center through the Agriculture, Food and Rural Affairs Research Center Support Program, Golden Seed Project (Center for Horticultural Seed Development, No. 213003-04-1-SB910), Ministry of Agriculture, Food and Rural Affairs, and a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ007992). We thank Ji-wha Hur, Jeong-Ahn Yoo, and Sujeong Kim for their dedicated technical help.
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Communicated by S. Hohmann.
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438_2014_973_MOESM1_ESM.tif
Supplementary material 1 (TIFF 126 kb) Supplementary Fig. 1. Long PCR amplification of the two accessions containing the unclassified DFR-A alleles. The PCR products were amplified using the primer pair DFR-LF1 and DFR-DEL-R1. Primer sequences are shown in Supplementary Table 2. M: 100-bp ladder, 1: control containing the DFR-A R1 allele, 2: PI276324, 3:430C
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Supplementary material 2 (TIFF 334 kb) Supplementary Fig. 2. Identification of TEs isolated from other plant species showing homology with ORF1 of AcCOPIA1, inserted in the DFR-A LTR allele. A. Phylogenetic tree constructed using the deduced amino acids sequences of ORF1 and the TEs isolated from other plant species. The species names are shown in parenthesis. The numbers at the nodes show the bootstrap probability (%) with 1,000 replicates. The scale bars indicate nucleotide substitutions per site. B. Alignment of partial amino acid sequences of the reverse transcriptase domains in AcCOPIA1 and the TEs isolated from other species
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Supplementary material 3 (TIFF 312 kb) Supplementary Fig. 3. Identification of TEs isolated from other plant species showing homology with ORF2 of AcCACTA1, inserted in the DFR-A DTP allele. A. Phylogenetic tree constructed using the deduced amino acids sequences of ORF2 and the TEs isolated from other plant species. The species names are shown in parenthesis. The numbers at the nodes show the bootstrap probability (%) with 1,000 replicates. The scale bars indicate nucleotide substitutions per site. B. Alignment of partial amino acid sequences of the transposases in AcCACTA1 and the TEs isolated from other species
438_2014_973_MOESM4_ESM.tif
Supplementary material 4 (TIFF 310 kb) Supplementary Fig. 4. Identification of TEs isolated from other plant species showing homology with ORF3 of AchAT1, inserted in the DFR-A TRN allele. A. Phylogenetic tree constructed using the deduced amino acids sequences of ORF3 and the TEs isolated from other plant species. The species names are shown in parenthesis. The numbers at the nodes show the bootstrap probability (%) with 1,000 replicates. The scale bars indicate nucleotide substitutions per site. B. Alignment of partial amino acid sequences of the transposases in AchAT1 and the TEs isolated from other species
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Supplementary material 5 (TIFF 170 kb) Supplementary Fig. 5. Flowchart showing the sequential process of PCR amplification and direct sequencing for the genotyping of 15 different DFR-A alleles in onions. The SNPs are shown with the numbers indicating the distances from the start codon of the DFR-A gene. This flowchart is an updated version of the old one presented by Song et al. (2014)
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Kim, S., Park, J.Y. & Yang, TJ. Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.). Mol Genet Genomics 290, 1027–1037 (2015). https://doi.org/10.1007/s00438-014-0973-7
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DOI: https://doi.org/10.1007/s00438-014-0973-7