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bioRxiv · 10.1101/2025.09.20.677491

Expansion of the DFR Gene Family in Fagopyrum

Samsolnizam, S.·Pucker, B.
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Abstract

BackgroundGene duplication is a fundamental evolutionary mechanism that contributes to genetic innovation, such as in the diversification of plant metabolic pathways. This study investigates the duplication of dihydroflavonol-4-reductase (DFR) genes in Fagopyrum, focusing on their evolutionary origin and implications for flavonoid biosynthesis. DFR is a key enzyme in the flavonoid pathway, specifically involved in the biosynthesis of anthocyanins and proanthocyanidins. DFR can accept three main substrates: dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM), and they exhibit variable substrate preferences. ResultThrough comparative genomic analysis, this study identified three unique DFR gene copies in Fagopyrum, designated DFR1, DFR2a, and DFR2b. DFR2a and DFR2b show unique mutations affecting the 26-amino acid substrate-binding region, including substitution of the conserved asparagine at position 3 with valine or isoleucine, and an insertion between positions 5 and 6 involving glycine, arginine, or lysine. Synteny analysis reveals that DFR1 is located in a conserved region across related species, while DFR2a and DFR2b lie at distinct genomic loci. Gene expression analysis shows DFR1 is universally expressed, whereas DFR2a and DFR2b expression is primarily restricted to seeds and roots. Promoter analysis supports this, revealing the absence of MYB recognition elements in DFR2a and DFR2b that are present in the DFR1 promoter. Selective pressure analysis indicates that DFR2 copies are under strong purifying selection relative to ancestral DFR1 but appear slightly relaxed between DFR2a and DFR2b in a way suggesting functional differentiation, particularly in conserved regions. ConclusionBased on multiple lines of evidence, we propose DNA duplication associated with inversion (DDAI) as a mechanism to explain the emergence of the DFR2 copies, involving a staggered single-strand break, followed by inversion and non-homologous end joining repair mechanism.

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Samsolnizam, S., Pucker, B.. 2025-09-21. Expansion of the DFR Gene Family in Fagopyrum. https://doi.org/10.1101/2025.09.20.677491

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