Expansion of the DFR Gene Family in Fagopyrum

Expansion of the DFR Gene Family in Fagopyrum | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Expansion of the DFR Gene Family in Fagopyrum View ORCID Profile Syafiq Samsolnizam , View ORCID Profile Boas Pucker doi: https://doi.org/10.1101/2025.09.20.677491 Syafiq Samsolnizam 1 Plant Biotechnology and Bioinformatics, IZMB, University of Bonn , Kirschallee 1, 53115 Bonn, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Syafiq Samsolnizam Boas Pucker 1 Plant Biotechnology and Bioinformatics, IZMB, University of Bonn , Kirschallee 1, 53115 Bonn, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Boas Pucker For correspondence: pucker{at}uni-bonn.de Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Background Gene 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. Result Through 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. Conclusion Based 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. Background Gene duplication is one of the most important evolutionary mechanisms generating functional diversity. Theoretical studies suggest that it is far easier to evolve new functions from pre-existing ones through neofunctionalization than to develop them de novo [ 1 ]. After duplication, each gene copy can evolve independently, accumulating mutations that can potentially lead to functional divergence and innovation [ 1 ]. Gene duplication can occur through several molecular mechanisms, each producing different outcomes in terms of genomic structure. In general, gene duplication refers to the duplication of a genomic segment, which may then be inserted at the same or a different location within the genome [ 2 ]. Gene duplication thus provides raw genetic material for the evolution of novel traits and functions [ 3 ]. This is particularly relevant in plants, where duplicated genes are highly abundant [ 2 ]. Plant genomes have undergone multiple rounds of whole-genome duplication (WGD) events over the past 200 million years of angiosperm evolution, which have significantly contributed to the complexity and adaptability of plant lineages [ 2 ]. In addition to their history of WGD, plant genomes tend to evolve more rapidly and exhibit greater structural variation than those of many other eukaryotes. This has resulted in larger genome sizes and a higher degree of genetic redundancy. The high abundance of gene duplicates in plants is especially interesting because of their frequent contribution to lineage-specific innovations [ 2 ]. The most extensive form of duplication is WGD, where the entire set of chromosomes is duplicated. Ancient WGD events have been detected in the ancestors of seed plants (approximately 340 million years ago) and angiosperms (around 170 million years ago) [ 4 ]. This type of duplication leads to the presence of multiple copies of every gene in the genome and is referred to as polyploidization. Polyploidy can result from hybridization between species (allopolyploidy) or occur within a single species (autopolyploidy) [ 5 ]. On a smaller scale, tandem duplications involve the replication of genes in close physical proximity, forming tandemly arrayed genes (TAGs). These local duplications typically arise through unequal crossing over during meiosis. The size of the duplicated region can vary depending on the site of chromosomal breakage. Such events may be caused by homologous recombination between similar sequences on homologous chromosomes or sister chromatids, or by non-homologous recombination resulting from replication-dependent chromosome breakage [ 6 , 7 ]. In maize, for example, thousands of tandem duplicates have been identified, representing approximately 10% of all annotated genes [ 8 ]. These duplications contribute to genetic diversity and often play roles in gene family expansion and adaptive evolution. Transposable elements (TEs) can also drive sequence duplication through various mechanisms. TEs are repetitive DNA sequences with the ability to move themselves or even nearby genes to new locations within the genome. This process, called transposition, can occur within the same chromosome or between different chromosomes. The proportion of TEs varies widely across organisms, from about 3% of the genome in yeast to more than 80% in maize [ 9 , 10 ]. In Fagopyrum esculentum , TEs occupy a particularly large portion of the genome [ 11 ]. This includes both gradually accumulated elements and several classes of recently amplified TEs that underwent rapid multiplication between 0.5 and 1 million years ago. This transposon activity has significantly contributed to the threefold increase in genome size of F. esculentum compared to its close relative Fagopyrum tataricum [ 11 ]. There are two primary mechanisms by which TEs can cause gene duplication: retroduplication and transduplication. Retroduplication occurs when the mRNA of a host gene is reverse-transcribed into complementary DNA and inserted into a different genomic location by retrotransposon enzymes. These retrocopies are characterized by the absence of introns, the presence of a poly-A tail at the 3’ end, and flanking target site duplications (TSDs) [ 12 ]. Transduplication is facilitated by DNA transposons, particularly those with terminal inverted repeats (TIRs). These elements use a “cut-and-paste” mechanism to excise themselves and often adjacent gene sequences. They then insert into a new location in the genome, also flanked by TSDs. Unlike retrocopies, the duplicated fragments from DNA transposons can retain intron-exon structures [ 7 , 13 ]. Helitrons, another type of DNA transposon, operate through a “peel-and-paste” mechanism. In this process, the positive DNA strand is peeled off to form a circular DNA molecule, which is then replicated through rolling circle replication. This mechanism can also mobilize gene fragments that may contain introns, contributing to structural genome variation [ 7 , 13 ]. In addition to TE-driven duplications, segmental duplications represent another important form of gene duplication. Samonte and Eichler define segmental duplication as long stretches of duplicated sequences ranging from 1 to 200 kilobases and sharing more than 95% sequence identity [ 14 ]. Segmental duplications can arise from the transposition of chromosomal regions and often include multiple genes with intron-exon structures. Unlike tandem duplications, these segments are typically dispersed throughout the genome. This distribution suggests that mechanisms other than unequal crossing over, such as duplicative transposition, are responsible. Additional research is required to find a definitive explanation for the frequency, organization, and distribution of segmental duplications, particularly in large and complex genomes like the human genome [ 14 ]. After gene duplication, the newly formed duplicates can follow several evolutionary trajectories: (1) pseudogenization and loss, (2) neofunctionalization, or (3) subfunctionalisation. The most common fate is pseudogenization often followed by complete loss of one copy [ 15 ]. Typically, only one gene copy remains under purifying selection to maintain its original function, while the other is free to accumulate deleterious mutations that can cause it to be nonfunctional [ 7 ]. If two duplicate genes are functionally redundant and completely identical, there is generally no selective disadvantage in deleting either copy [ 2 ]. Pseudogenes are identified based on their sequence similarity to functional genes and the presence of disabling mutations. Interestingly, pseudogenes are not always rapidly eliminated. In species such as Arabidopsis thaliana and rice, thousands of pseudogenes are conserved within the genome [ 16 ]. One possible explanation is that some pseudogenes were only recently pseudogenized and are still in the early stages of degeneration [ 2 ]. Another possible fate is neofunctionalization, where the duplicate acquires a novel function. If this novel function provides a selective advantage, natural selection may act to preserve the gene. Ohno originally proposed the neofunctionalization model, in which one copy retains the ancestral function while its paralog evolves a new one [ 2 , 3 ]. A notable example is the evolution of flavone synthase I ( FNS I ) in the Apiaceae , which arose through duplication of flavanone 3-hydroxylase ( F3H ), followed by neofunctionalization, as revealed by phylogenetic and comparative genomic analyses [ 17 ]. In contrast, subfunctionalisation occurs when both duplicates partition the functions of the original gene due to the accumulation of degenerative mutations. This process, also known as the duplication-degeneration-complementation (DDC) model [ 18 ], involves each copy losing a subset of ancestral functions. As a result, both gene copies must be retained to maintain the full spectrum of original functions. Subfunctionalisation does not require the evolution of new functions, but rather a division of pre-existing ones [ 2 ]. Another important trajectory is escape from adaptive conflict (EAC). Here, a single-copy gene is constrained by pleiotropy from optimizing both its ancestral and novel functions. After duplication, one copy can specialize in the ancestral role, while the other improves the novel function [ 19 ]. Unlike neofunctionalization, which involves adaptive change in only one copy (with the other maintained by purifying selection), EAC involves adaptive changes in both duplicates, with the ancestral function itself also being improved. EAC may also involve a combination of both neo- and sub-functionalisation mechanisms as it invokes both the neofunctionalization and DDC models to explain duplicate retention [ 2 ]. Although theoretically important, EAC remains understudied and can be difficult to distinguish from neofunctionalization. Other potential fates include gene dosage retention, where both duplicates do not diverge much, remain functional and are expressed to increase the overall dosage of gene product [ 3 ]. This can be advantageous for meeting metabolic demands. Additional mechanisms that influence duplicate gene retention include dosage balance and paralog interference, and these processes are not necessarily mutually exclusive [ 2 ]. Flavonoids are one of the most important classes of plant secondary metabolites, playing key roles in plant survival, adaptation, and interactions with the environment, as well as having nutritional and medicinal value for humans [ 20 – 27 ]. Flavonoids are a diverse family of aromatic molecules derived from phenylalanine and malonyl-coenzyme A and they comprise six major subgroups commonly found in higher plants: chalcones, flavones, flavonols, flavandiols, anthocyanins, and condensed tannins (also known as proanthocyanidins). Among them, anthocyanins are widely known for their roles as red, purple, or blue pigments, contributing to the vivid coloration of flowers and fruits [ 20 ]. In addition to their pigmentation roles, flavonoids function as phytoalexins and antioxidants, possessing reactive oxygen species (ROS) scavenging abilities [ 21 ]. They protect plants from various biotic and abiotic stresses, including UV irradiation, cold stress, pathogen infection, and herbivory [ 22 – 25 ]. Flavonoids also have widespread applications in food such as natural colourant [ 26 ]. Anthocyanins for example, are edible pigments that influence the taste and color of food and wine [ 20 ]. Flavones play important roles in signaling and defense responses against pathogens [ 27 ], while proanthocyanidins contribute to seed coat pigmentation [ 28 ]. Anthocyanin biosynthesis involves several enzymatic steps. A key branch-point enzyme in this pathway is dihydroflavonol 4-reductase (DFR, EC 1.1.1.219), which catalyses the NADPH-dependent reduction of dihydroflavonols into leucoanthocyanidins [ 29 , 30 ]. These leucoanthocyanidins are subsequently converted into anthocyanidins by anthocyanidin synthase (ANS) and anthocyanin-related glutathione-S-transferase (arGST) and finally into stable anthocyanins via glycosylation by glycosyltransferases ( Figure 1 ) [ 31 ]. DFR catalyses the reduction of dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM) into their corresponding leucoanthocyanidins, which are leucopelargonidin, leucocyanidin, and leucodelphinidin, respectively [ 32 ]. Since these dihydroflavonols differ only in the number of hydroxyl groups on the B-ring (which is not the site of enzymatic action), DFR enzymes in most plants can use all three substrates. Dihydroflavonols serve as substrates not only for DFR but also for FLS, which produces flavonols such as kaempferol, quercetin, and myricetin depending on substrate availability [ 33 ]. Based on currently available information, angiosperm FLS appears to prefer DHK, while DFR appears to favor DHQ and DHM [ 34 ]. Hydroxylation of dihydrokaempferol into dihydroquercetin or dihydromyricetin is catalysed by flavonoid 3’-hydroxylase (F3’H) and flavonoid 3’,5’-hydroxylase (F3’5’H), respectively [ 35 ]. Substrate specificity is a critical factor in determining the balance between flavonol and anthocyanin production. A 26-amino-acid region involved in DFR substrate specificity has been identified through multiple sequence alignments [ 36 , 37 ]. In particular, position 133 relative to Arabidopsis thaliana DFR (3rd position in the 26-amino acid region) has been shown to influence substrate preference: asparagine (N) allows acceptance of all dihydroflavonols; leucine (L) restricts substrate usage to DHK; aspartic acid (D) permits the use of DHQ and DHM; and alanine (A) results in high affinity for DHK [ 34 ]. Download figure Open in new tab Figure 1: Simplified illustration of the flavonoid biosynthesis pathway. The enzyme responsible for each step is indicated next to the corresponding arrow. The activity of different branches in flavonoid biosynthesis is tightly regulated at the transcriptional level e.g. to prevent simultaneous activity of competing branches like flavonol and anthocyanin biosynthesis [ 34 ]. In particular, DFR expression is activated by the MBW complex, a transcription factor complex composed of MYB, bHLH, and WD40 proteins [ 38 ]. Fagopyrum is a small genus comprising less than 30 species, most of which are endemic to southern China [ 39 ]. It includes two orphan crops: common buckwheat ( F. esculentum ) and tartary buckwheat ( F. tataricum ), both of which were domesticated independently from distinct wild progenitors in southwest China, outside the major centers of domestication. While F. tataricum is primarily cultivated in China, F. esculentum is grown globally, especially across Eurasia, and is valued for its rapid growth at high altitudes and its ability to thrive in low-fertility, marginal lands [ 40 ]. Fagopyrum is classified into two major lineages: the cymosum group (e.g., F. esculentum , Fagopyrum homotropicum , F. tataricum , and Fagopyrum dibotrys [also known as F. cymosum ]) and the urophyllum group (e.g., F. urophyllum , F. gracilipes , and F. leptopodum ), based on morphological characteristics and molecular phylogenetics [ 39 , 41 ]. The tribe Fagopyreae , which contains only the genus Fagopyrum , is part of the family Polygonaceae , which is part of the order Caryophyllales . Polygonaceae consists of about 48 genera and over 1200 species, distributed worldwide but most diverse in temperate regions [ 42 ]. The plants are typically herbaceous but can also be shrubs or small trees. Within the subfamily Polygonoideae , major tribes include Fagopyreae (e.g., Fagopyrum ), Rumiceae (e.g., Rumex , Oxyria ), Persicarieae (e.g., Persicaria ), and Polygoneae (e.g., Fallopia , Polygonum ) [ 43 ]. Polygonaceae is considered a non-core family of Caryophyllales , a hyperdiverse order encompassing approximately 12,500 species in 39 families, accounting for about 6% of extant angiosperm diversity. The order exhibits remarkable life-history variation, from tropical trees to temperate annual herbs, from desert-adapted succulents (e.g., Cactaceae ) to carnivorous plants [ 44 ]. This diversity makes Caryophyllales a compelling system for studying relationships between WGD, diversification, and adaptation to varying climates [ 44 ]. A notable feature of core Caryophyllales is the presence of betalain pigments that are derived from tyrosine and incorporate betalamic acid. These pigments replace the more widespread anthocyanins in some other core Caryophyllales lineages [ 45 , 46 ]. The mutually exclusive distribution of betalains and anthocyanins has sparked decades of debate and research, revealing several mechanisms to explain this exclusivity including wholesale gene loss, modified gene function, altered gene expression, and degeneration of the MBW (MYB-bHLH-WD40) transcription factor complex regulating anthocyanin biosynthesis [ 47 ]. However, non-core Caryophyllales families, such as Polygonaceae , including the buckwheat genus Fagopyrum , retain the ability to synthesize anthocyanins rather than betalains [ 45 ]. Continued research into flavonoid biosynthesis within this family could provide insights into the evolutionary trajectory of pigment biosynthesis in Caryophyllales , and into how non-core lineages may have retained or modified ancestral anthocyanin pathways. Katsu et al. [ 48 ] identified two copies of DFR genes in buckwheat using two degenerate PCR primers. These primers were designed based on conserved regions of DFR proteins, one of which had previously been used to isolate DFR homologs in other plants [ 49 ]. The two genes were named FeDFR1a and FeDFR2 , where FeDFR1a exhibited high sequence identity (99%) to a previously deposited F. esculentum DFR sequence (ACZ48698), whereas FeDFR2 showed only 80% identity [ 48 ]. Phylogenetic analysis placed FeDFR2 in the same major clade as FeDFR1a , rather than with the DFR -like genes such as BEN1 , suggesting that both enzymes may be involved in catalyzing the conversion of dihydroflavonols to leucoanthocyanidins . At the protein level, FeDFR1a has an asparagine residue at the third position within the substrate specificity-determining region, while FeDFR2 has an uncommon valine at the same position and contains an additional glycine residue between positions 6 and 7 (position 136 in FeDFR2) [ 48 ]. Functional assays of recombinant DFR proteins, expressed in Escherichia coli , were conducted to evaluate enzyme activity against three dihydroflavonol substrates and both FeDFR1a and FeDFR2 accept all three substrates; however, FeDFR1a exhibited similar catalytic efficiency for DHM and DHK, with lower efficiency for DHQ [ 48 ]. In contrast, FeDFR2 displayed approximately twice the catalytic efficiency for DHQ compared to DHM, and the lowest activity for DHK [ 48 ]. The primary goal of this study is to investigate the duplication of DFR genes in F. esculentum . By leveraging phylogenetic analysis, synteny analysis, gene expression analysis, cis-regulatory element analysis, and selective pressure analysis, this study explores the evolutionary history and mechanism of DFR duplication. In addition, the research examines the role of the different DFR genes in the anthocyanin biosynthesis pathway, which is particularly interesting in F. esculentum as a member of Caryophyllales , a lineage better known for betalain production. Methods Datasets Genome sequences of various Polygonaceae species were obtained from the National Center for Biotechnology Information (NCBI). The species and their corresponding GenBank accession numbers or other sources are listed in the Additional file 1. Gene Prediction Since annotations for most of the retrieved Polygonaceae genome sequences were not publicly available, GeMoMa v1.9 [ 50 ] was used for homology-based gene prediction. Well-annotated genome sequences and corresponding annotations from the same order of Caryophyllales were retrieved from the reference sequence (RefSeq) database at NCBI [ 51 ] to be used as reference in the GeMoMa pipeline. The genome sequences are Beta vulgaris (RefSeq: GCF_026745355.1), Chenopodium quinoa (RefSeq: GCF_001683475.1) and Spinacia oleracea (RefSeq: GCF_ 020520425.1). RNA-seq data was incorporated into the prediction to utilise intron position conservation and accurately predict the splice site for the genes. Alignment and Phylogenetic Tree Construction Candidate DFR sequences in the Polygonaceae genome sequences were identified using KIPEs v3.2.6 [ 52 ] with its existing collection of bait sequences v3.3.7z. In addition, a set of DFR coding sequences, including DFR -like sequences, was retrieved from the NCBI database for phylogenetic validation. The sequences are publicly available on Github [ 53 ]. The initial candidate DFR sequences from Polygonaceae were validated through phylogenetic analysis using FastTree v2.2 [ 54 ], based on amino acid alignment generated with MAFFT v7.505 [ 55 ] using the --auto parameter. Sequences that were clustered within the main clade together with confirmed DFR sequences from NCBI were retained for further analysis. In contrast, sequences clustering outside of this group, such as within the DFR -like clade containing BAN (BANYULS/ANR) (NM_104854.4) [ 56 ], BEN1 (NM_130102.5) [ 57 ], and DRL1 (NM_119708.3) [ 58 ] from A. thaliana , were excluded. The amino acid sequences and CDS from the filtered Polygonaceae DFR , with representatives from other Caryophyllales families as outgroups, were used for phylogenetic tree construction (available on Github) [ 53 ]. A codon-aware alignment approach was used for CDS sequence alignment, where the amino acid sequence was first aligned, and then substituted by their corresponding codons using a Python script [ 59 ]. The sequences were aligned using MAFFT v7.505 [ 55 ] using --maxiterate 1000 and --genafpair parameter. Maximum likelihood trees were then constructed by FastTree v2.2 [ 54 ] using standard parameters, IQ-TREE v2.4.0 [ 60 ] using the -m MFP -B 1000 parameter, and RAxML-NG v1.2.2 [ 61 ] using the --model TIM3+G4 --bs-trees 1000 --tree pars{25},rand{25} parameter. All resulting phylogenetic trees were visualised using Interactive Tree of Life (iTOL) v7 [ 62 ] and inspected for their topologies and the positions of DFR in Polygonaceae clades. Sequence Analysis The retrieved Fagopyrum DFR sequences were analyzed for protein sequence similarity. A custom Python script was used to calculate sequence similarities by running multiple sequence alignment with MAFFT v7.505 [ 55 ], generating a pairwise sequence identity matrix, and visualizing the results as a heatmap [ 53 ]. The 26–amino acid conserved region was manually examined based on the MAFFT alignment and visualized using Jalview v2.11.5 [ 63 ]. Synteny Analysis Synteny of DFR in the Polygonaceae family was investigated using MCScan from JCVI v1.5.7 [ 64 , 65 ]. Regions surrounding the DFR genes were manually selected for visualization in both macro- and microsynteny analyses using the JCVI toolkit. Macrosynteny visualization was performed to compare the chromosomal regions containing DFR in Fagopyrum and its relatives, using the standard parameters of MCScan. Microsynteny visualization was carried out to compare the DFR gene regions at the gene level, where DFR and its flanking genes were manually chosen for visualization. The locations of DFR in the macrosynteny analysis were identified manually using Jbrowse2 v3.6.4 [ 66 ]. Connections of genes between the species were manually validated and revised based on phylogenetic trees. Gene Expression Analysis RNA-seq datasets were retrieved from the NCBI Sequence Read Archive (SRA) using fasterq-dump v3.0.3 [ 67 ]. Transcript quantification was performed with kallisto v0.51.1 [ 68 ] using default parameters, executed through the Python wrapper script kallisto_pipeline3.py [ 69 ]. Merged transcript TPM values and read counts were generated using merge_kallisto_output3.py [ 69 ]. Low quality or atypical datasets were removed based on TPM and read count distributions with filter_RNAseq_samples.py [ 69 ]. A custom Python script was then used to generate boxplots of DFR expression across tissues [ 53 ]. Because the genes of interest are highly similar and located in close proximity, RNA-seq reads were also aligned to the genome sequence with STAR v2.7.11b [ 70 ]. The results were compared with kallisto to check consistency and reduce ambiguity. Threshold parameters such as --outFilterMultimapNmax, --alignIntronMax, --outFilterScoreMin, --outFilterMatchNminOverLread, and --outFilterMismatchNoverLmax were applied. The resulting SAM files were converted into BAM format, sorted, and indexed using Samtools v1.21 [ 71 ], and manually inspected in JBrowse2 v3.6.4 [ 66 ]. Transcript quantification from the alignments was performed with the STAR --quantMode GeneCounts function. Promoter Region Analysis RNA-seq reads were mapped to the genome sequence using STAR and visualized in JBrowse2 to identify the TSS. To extract the promoter regions of each DFR gene in Fagopyrum , a 1000 bp sequence upstream of the predicted transcription start site (TSS) of each gene was retrieved. The upstream regions preceding the mapped reads of each DFR duplicate were then extracted for further analysis. The promoter sequences were analyzed using the PLACE v30.0, PlantPan v4.0, and PlantCARE databases [ 72 – 74 ]. Results from the three databases were compared to identify motifs associated with DFR gene expression. In addition, a custom Python script was developed to visualize the output of PlantPan [ 53 ]. Selective Pressure Analysis The aligned coding sequences of DFR from Fagopyrum that were previously used for the alignment and phylogenetic tree construction were analysed to estimate the synonymous substitution rate (Ks) and the nonsynonymous substitution rate (Ka). The aligned sequences were first converted into an AXT file, where each sequence was compared with its orthologs or paralogs. KaKs Calculator 3.0 [ 75 ] was used to calculate the Ka/Ks ratio for each sequence pair, providing insights into the selective pressures acting on DFR gene duplicates. The selective pressure on duplicate gene pairs can be inferred from the Ka/Ks ratio. A Ka/Ks ratio close to 1 indicates neutral selection, which may suggest pseudogenization. A Ka/Ks ratio less than 1 (Ka/Ks 1) suggests positive selection, which may imply that the gene duplicates are evolving to acquire new functions [ 76 ]. The Ka/Ks calculations were performed using the Model Selection (MS) method [ 77 ]. Duplication Mechanism Analysis Several analyses were performed to investigate the mechanism behind the duplication of DFR in Fagopyrum . Extensive de novo TE Annotator (EDTA) v2.2.2 [ 78 ] was used on each Fagopyrum genome to identify TEs located near the duplicates that could have contributed to the duplication. In particular, terminal inverted repeats (TIRs) and helitrons flanking the duplicates were examined [ 13 ]. To test for segmental duplication, dot plots were generated with ModDotPlot v0.9.0 [ 79 ]. Both DFR1 and DFR2 loci were compared against each other and against themselves. Evidence for segmental duplication would appear as dense linear arrays of dots, either parallel or inverted relative to the main diagonal, but displaced from it [ 80 ]. Results Three DFRs are specific to the genus of Fagopyrum Gene Position and Structures A total of 14 DFR gene copies have been identified across four Fagopyrum species, with three copies each in F. esculentum , F. homotropicum , and F. dibotrys , and 5 copies in F. tataricum . The three DFR copies in each Fagopyrum species show a conserved pattern and syntenic arrangement within the same chromosome ( Figure 2 ). Notably, these patterns of duplicates were not observed in other species of the Polygonaceae family, suggesting that this gene duplication is specific to the genus of Fagopyrum . Additionally, F. tataricum possesses two extra DFR copies, which are located at the same genomic locus as the other DFR copies and remain on the same chromosome, but differ in their sequence and are assumed to be pseudogenes. FtDFR2a and FtDFR2b are located at another locus but are assumed to be caused by a genome inversion. This finding differs from the previous study by Katsu et al. [ 48 ], which identified only two DFR copies in F. esculentum , named FeDFR1a and FeDFR2 . Download figure Open in new tab Figure 2: (A) Positions of each DFR duplicate in the chromosome and (B) their positions with arrow shape direction showing their orientation. (C) The exon and intron structure for each DFR . Blocks indicate the positions of exons, while lines indicate connecting introns. Different colors were used to highlight DFR1, DFR2a, DFR2b, and DFR3, respectively. The DFR genes identified here were renamed based on their syntenic positions and placement in the constructed phylogenetic tree. They are referred to as DFR1 , DFR2a , DFR2b , DFR3 , and DFR4 . DFR2a and DFR2b are located in close proximity, with a distance ranging from 40 Kbp in F. dibotrys to 400 Kbp in F. esculentum ( Figure 2A ). In contrast, DFR1 is positioned at a distant locus of 4 to 27 Mbp away from the DFR2 locus. This scattered pattern of gene duplication across the chromosome in Fagopyrum suggests a unique form of duplication that differs from the previously studied DFR duplications such as those reported in Lotus japonicus , where the DFR copies are arranged in tandem repeats [ 81 ]. The orientation of each duplicate is also conserved between orthologs across all Fagopyrum species with an exception of F. tataricum which is assumed to be caused by a secondary event such as an inversion ( Figure 2B ). DFR1 in Fagopyrum maintains a conserved intron-exon structure, consisting of six exons and five introns, a pattern also observed in morning glories and onions [ 82 , 83 ]. The DFR2 genes generally have the same number of exons, except for FeDFR2b with five exons. In comparison, FtDFR3 has eight exons and FtDFR4 has four exons ( Figure 2C ). Enzyme Similarities and Substrate Binding Region Sequence similarities between the DFR proteins revealed a high degree of conservation within each ortholog group ( Figure 3A ). DFR1 share at least 99.4% similarity between species. Similarly, DFR2a and DFR2b show high similarity to their ortholog group with more than 91.9% for DFR2a and more than 90.7% for DFR2b. Notably, DFR2a and DFR2b are more similar to each other with at least 88.0% similar, than to DFR1 , suggesting a closer evolutionary relationship. Interestingly, DFR3 and DFR4 in F. tataricum appear to be distinct from both DFR2 and DFR1 which strengthens the assumption that they might be pseudogenes. Download figure Open in new tab Figure 3: (A) Protein sequence similarities between each DFR. (B) The aligned sequences correspond to the 26-amino acid regions that determine substrate specificity. The first * on top of the alignment corresponds to the 3 rd position that is studied to be responsible for substrate specificity and the second * corresponds to the insertion in DFR2 duplicates between position 5 and 6. The full sequence alignment of each protein can be found in Additional file 2. When the sequences were aligned, variations in the substrate binding domain were observed between DFR1 and DFR2 ( Figure 3B ). The substrate specificity is determined by a conserved region comprising 26 amino acids that has been well studied in other plants such as Petunia , Gerbera and Cymbidium [ 36 , 37 , 84 , 85 ]. All DFR1 proteins contain an asparagine (N) at position 3, which permits acceptance of all three dihydroflavonols as substrates. In contrast, DFR2a and DFR2b show a substitution at this position, where all members have valine (V), except for FtDFR2b, which has isoleucine (I). This differs from other well-studied aspartic acid (D) substitution, which increases the preference for DHQ and DHM, and the alanine (A) substitution, which favours DHK while opposing DHM [ 34 ]. In addition, DFR2a and DFR2b contain a unique insertion at position 5-6, which is glycine (G) in all DFR2b, arginine (R) in FhDFR2a, FtDFR2a, FdDFR2a, and lysine (K) in FeDFR2a. This insertion is absent in DFR1 and other well-studied conserved DFR regions in plants. There exist also other substitutions in the conserved region of DFR2 genes when compared to DFR1, but they are still not yet known or studied for their consequences. Phylogeny of DFR A tree classified DFR candidates from the buckwheat family by determining whether they were placed within the clade of known DFR genes in the buckwheat family or in the DFR -like clade. Some species within the family harbor at most one DFR -like gene. The classification of true DFR genes is further supported by comparison with other families in the Caryophyllales . Other species in the Polygonaceae family also exhibit multiple DFR duplications, particularly in the genera Persicaria . Persicaria maculosa has three DFR copies, while Persicaria tinctoria possesses four. Fallopia convolvulus contains two copies, whereas only one was identified in Fallopia multiflora and Reynoutria japonica has 3 DFR copies. All these DFR duplicates are found on different chromosomes, except for R. japonica that has two copies that are located on the same chromosome. In contrast, other species such as Oxyria digyna , the genus Rheum and the genus Rumex each have a single DFR copy. The DFR genes in Fagopyrum form two distinct clades. One clade harbours known DFR sequences from databases and is positioned near related species such as Rheum , Oxyria , Polygonum , and Fallopia . This group is designated as DFR1 , as it is assumed to be the original copy that maintained the ancestral function (canonical DFR ). In contrast, Persicaria species form a separate clade outside of the Fagopyrum - Rheum - Polygonum grouping, which is expected given their evolutionary divergence [ 86 , 87 ]. F. esculentum is clustered closely with F. homotropicum , while F. tataricum is clustered closely with F. dibotrys (also known as F. cymosum ), as they are closely related to each other, respectively. These four Fagopyrum species in this study are placed within the major cymosum group of the tribe Fagopyreae , in the subfamily Polygonoideae of the buckwheat family [ 39 , 41 ]. An interesting finding is that the DFR duplicates in the Fagopyrum genus, including the FeDFR2 sequences identified in a previous study [ 48 ], are positioned at the base of the Polygonaceae rather than clustering closely with DFR of other family members. This distinct clade, referred to as the DFR2 clade, can be further divided into two subclades: DFR2a and DFR2b . Each Fagopyrum species possesses one DFR copy in each subclade except for F. tataricum with 3 DFR copies in DFR2a subclade ( FtDFR2a , FtDFR3 , and FtDFR4 ). FeDFR1a (LC216398.1) from Katsu et al. is clustered within the DFR1 clade, while FeDFR2 (LC216399.1) is clustered within the DFR2a clade ( Figure 4 ). Download figure Open in new tab Figure 4: Maximum likelihood tree of DFR by IQ-TREE v2.4.0. Sequences retrieved from NCBI databases are labeled with their accession numbers and the other sequences were retrieved using KIPEs from each genome dataset. Sequences from the other family in the core Caryophyllales were used as outgroups. DFR from Fagopyrum are clustered into 3 main clades: DFR1 (blue), DFR2a (green), and DFR2b (Red). See Additional file 3 for additional support. Synteny of DFR Synteny analysis was performed using MCScan from JCVI to study the positions and order of genes on chromosomes across species, as well as to investigate their evolutionary origin. All four Fagopyrum genome sequences, together with those of closely related species such as Rheum palmatum , Polygonum aviculare , and Fallopia multiflora , were used to visualize the synteny of DFR . Macrosynteny analysis ( Figure 5A ) showed that the syntenic block harbouring DFR1 is highly conserved among species of the family, with DFR1 located in the middle of the block. This observation is also supported by the microsynteny analysis ( Figure 5B ). In contrast, the synteny of DFR2 is more complex. There is no clear syntenic block that connects the DFR2 locus in Fagopyrum with other family members. In macrosynteny, the DFR2 locus is found outside of the syntenic blocks that harbour DFR1 and lacks collinear or conserved flanking genes between Fagopyrum and other species. Interestingly, in F. tataricum , DFR2a and DFR2b do not follow this pattern. They are located at a different locus, which is instead replaced by DFR3 and DFR4 . Nevertheless, the names of the duplicates in F. tataricum remain as FtDFR2a and FtDFR2b , because their sequences are more similar ( Figure 3A ) and also closer to other DFR2 orthologs in the phylogenetic tree ( Figure 4 ). This difference in synteny pattern is likely due to another mechanism, most probably a chromosomal inversion that occurred after duplication during evolution. Microsynteny of DFR2 ( Figure 5C ) further supports this, as the upstream region of DFR2 genes is conserved across all species, but the region containing DFR2 itself exists only in Fagopyrum and not in other family members. Download figure Open in new tab Figure 5: (A) Macrosynteny of the chromosome harboring DFR among Fagopyrum species and other species of buckwheat family Rheum palmatum , Polygonum aviculare , and Fallopia multiflora . The accession number of each chromosome is listed along with the species name. Black lines connecting species indicate the syntenic locus containing DFR , while gray waves represent other syntenic blocks between species. (B & C) Microsynteny of DFR1 (blue), DFR2a (green), and DFR2b (red) among the species with the location of each locus in the chromosome displayed. The microsynteny of DFR2 in F. tataricum is divided into two loci of different locations in its chromosome, divided by dotted lines. Dotted lines in R. palmatum , P. aviculare , and F. multiflora indicate that the genes DFR2 and its flanking genes do not exist in these species. DFR Expression RNA-seq datasets were retrieved from the SRA based on the availability of clear labels indicating the plant tissue of origin. For F. esculentum and F. tataricum , samples were available from root, leaf, flower, seed, stem, and seedling. For F. dibotrys , datasets were available for root, leaf, stem, rhizome, and tuber, but not for flower or seedling. F. homotropicum , on the other hand, had no RNA-seq datasets in the database. The SRA run IDs for each species, grouped by tissue, are listed in Additional file 4. Boxplots were generated to visualize transcript abundance in transcripts per million (TPM) for each gene ( Figure 6 ). Across all species, DFR1 consistently shows the highest expression across tissues. In F. esculentum , DFR1 expression is strongest in the root, with a median TPM of ∼600, while other tissues range between 200–300 TPM. In F. tataricum , the highest expression is found in the flower (∼200 TPM), followed by the root (∼170 TPM). In F. dibotrys , which had fewer RNA-seq datasets, the highest expression is observed in the tuber (∼600 TPM), with other tissues showing lower expression between 50–200 TPM. In contrast, DFR2a and DFR2b show divergent and generally lower expression compared to DFR1 . In F. esculentum , only FeDFR2a is expressed, mainly in root and seed, with values below 50 TPM. In F. tataricum , both FtDFR2a and FtDFR2b are expressed, with FtDFR2a showing relatively high expression in seed, and FtDFR2b in root. FtDFR4 shows low expression in some tissues, but this is likely due to background noise. In F. dibotrys , both FdDFR2a and FdDFR2b show no noticeable expression in any tissue. Download figure Open in new tab Figure 6: Boxplot diagrams illustrating the expression levels of DFR duplicates in F. esculentum , F. tataricum , and F. dibotrys . TPM = transcripts per million. Cis-regulatory Element To further investigate the expression differences between DFR copies, promoter sequences were analyzed for major transcription factor binding motifs. The search focused on MYB and bHLH binding sites, specifically: MYBCORE (CNGTTR), MYBPLANT (MACCWAMC), MYBPZM (CCWACC), and MYCCONSENSUS (also known as the E-box or bHLH binding site, CANNTG). MYBCORE, MYBPLANT, and MYBPZM are proposed MYB transcription factor binding sites [ 88 – 91 ], while MYCCONSENSUS is recognized as a bHLH binding site [ 92 ]. These motifs are crucial for MYB and bHLH transcription factors to bind and form the MBW complex with WD40, which regulates DFR expression [ 38 , 93 ]. The analysis revealed an abundance and diverse pattern of MYB and bHLH recognition elements in the promoter regions of DFR genes across all species ( Figure 7 ). In particular, several MYB binding sites are conserved across all DFR1 copies, including two MYBPLANT and one MYBPZM element located within the proximal 400 bp upstream of the putative TSS. MYBCORE sites are more dispersed throughout the promoter but show partial conservation in the FeDFR1 and FhDFR1 promoters, likely reflecting their close relationship. The MYCCONSENSUS (bHLH) binding site located ∼200 bp upstream of the TSS is conserved across all DFR1 promoter sequences. Three such bHLH sites were identified, two of which are located close together, possibly facilitating bHLH transcription factor dimerization [ 94 ]. Beyond the 400 bp region upstream of the TSS, binding sites appear more randomly and are not conserved across species. In contrast, DFR2 promoters generally lack MYB binding sites, especially within the proximal 400 bp. An exception is FtDFR2a , which contains one MYBCORE and two MYBPLANT motifs. FeDFR2b and FdDFR2a each contain two MYBCORE motifs located around -400 bp. Despite the overall scarcity of MYB sites, a conserved pair of bHLH binding sites is present in nearly all DFR2a and DFR2b promoters, except for FtDFR2a , FtDFR3 , and FtDFR4 . These conserved bHLH sites may have originated from those found in DFR1 and could still support gene expression, even in the absence of MYB motifs. Download figure Open in new tab Figure 7: Schematic representation of transcription factor binding motifs in the promoter regions. The putative transcription start site (TSS), identified based on RNA-seq alignment to the genome, is set as position +1. Selective Pressure Analysis To examine the selection pressures acting on the DFR duplicates, the Ka and Ks values were calculated for each orthologous and paralogous gene pair within the genus Fagopyrum . Gene pairings were determined based on their orthologous partners between species ( Table 1 ) and their paralogous pairs within the same species ( Table 2 ). This analysis was performed on both the entire DFR coding sequence and on the conserved 26–amino acid region to assess whether selection pressures differ between the two regions ( Table 1 , Table 2 , Additional file 5). View this table: View inline View popup Download powerpoint Table 1: Comparison of substitution rates between orthologous DFR duplicates among Fagopyrum species. The comparison is done for the whole coding sequence (CDS) and their corresponding 26-amino acid conserved region. The full table can be found in Additional file 5. View this table: View inline View popup Download powerpoint Table 2: Comparison of substitution rate between paralogous DFR duplicates among Fagopyrum species. The comparison is done for the whole coding sequence (CDS) and their corresponding 26-amino acid conserved region. The full table can be found in Additional file 5. Orthologous Pair Table 1 shows that DFR1 orthologs are generally well conserved, both across their entire sequence and in the conserved region. The Ka/Ks values were below 0.04 for the full coding sequence and below 0.003 for the conserved region. F. esculentum and F. homotropicum show few substitutions (Additional file 5) when compared, as do F. tataricum and F. dibotrys , which are expected given their close phylogenetic relationships. Notably, the conserved region of DFR1 in F. esculentum and F. homotropicum shows no substitutions at all. The number of nonsynonymous substitutions is also low, with a maximum of two for the full gene and none in the conserved region. In contrast, DFR2a orthologs show generally higher Ka/Ks values than DFR1 . For example, comparison between FeDFR2a and FhDFR2a gives a Ka/Ks value of 0.07 for the full CDS but an unusually high value of 50 for the conserved region, because all four substitutions in this region are nonsynonymous. However, this small number of substitutions also results in a higher p-value, making the result less reliable. Other pairwise comparisons of DFR2a and DFR2b orthologs generally show similar Ka/Ks values for both the full CDS and the conserved region, with the exception of FeDFR2b vs. FhDFR2b and FtDFR2b vs. FdDFR2b , where the conserved region shows higher values. Paralogous Pair Across all paralogous comparisons in Table 2 , DFR1 consistently shows low Ka/Ks ratios when compared to DFR2a or DFR2b . Specifically, Ka/Ks values remain below 0.05 for the full CDS and below 0.08 for the conserved region. The total number of substitutions is also relatively consistent, ranging from 218–242 in the full CDS, and 28–31 in the conserved region. Another clear trend is that the number of substitutions between DFR1 and DFR2b is lower than between DFR1 and DFR2a in all species. In contrast, comparisons between DFR2a and DFR2b show higher Ka/Ks values, though still below 0.5 for the full CDS. In the conserved region, however, the ratios can be much higher, for example, 1.787 for FeDFR2a vs. FeDFR2b and 50 for FhDFR2a vs. FhDFR2b . Interestingly, comparisons of FtDFR2a vs. FtDFR2b and FdDFR2a vs. FdDFR2b show lower Ka/Ks values for the conserved region, only 0.616 and 0.175, respectively. Discussion Hypothetical Mechanism of Duplication It can be assumed that DFR1 represents the gene copy with the original ancestral function, while DFR2 genes are more recent duplicates that are diverging from the main function of bona fide DFR . This assumption is supported by three lines of evidence. First, DFR1 is located in a conserved syntenic region with DFR genes in other plant species, both within the Polygonaceae family and the broader Caryophyllales order. Second, DFR1 consistently shows the highest expression levels compared to the other duplicates, strongly suggesting that it retains the ancestral function, whereas the duplicates with low or no expression may be pseudogenes or in the process of pseudogenization. Third, DFR1 clusters phylogenetically closer with DFR genes from related species ( Figure 4 ). The observation that DFR2a and DFR2b are located at different genomic loci than DFR1 raises questions about the mechanism of their origin, since this pattern does not fit the typical case of gene duplication. WGD was considered as a possible origin for DFR2 , however, comparisons with other Polygonaceae species indicate that DFR duplicates often reside on different chromosomes. Chromosome number variation and polyploidy patterns in Fallopia and Persicaria species suggest historical WGDs in some taxa [ 95 – 97 ], but the genomic distribution of DFR2 in Fagopyrum does not support a WGD origin. Transposable element (TE)-mediated duplication was also considered, given the high TE content and evidence of recent TE proliferation in F. esculentum [ 11 , 98 ]. While TEs are present near DFR1 and DFR2 loci, the retention of introns in DFR2a and DFR2b rules out retrotransposon-mediated duplication. TE annotations using EDTA [ 78 ] show no TIR or DNA transposons including helitrons that directly flank the DFR duplicates, suggesting that TEs may not have directly mediated the duplication [ 13 ]. Segmental duplication was further evaluated using dot plot analyses through ModDotPlot [ 79 ] (Additional file 6). No patterns indicative of segmental duplication was observed between DFR1 and DFR2 loci in Fagopyrum , although repetitive sequences are present near both loci. These repeats may represent TEs or other repetitive elements, but they do not appear sufficient to explain the duplication mechanism. Therefore, while DFR2 likely arose through a mechanism independent of WGD or retrotransposition, the exact process potentially involving TE-associated rearrangements remains unclear. DNA Duplication Associated with Inversion This case of duplication from DFR1 to DFR2 appears to deviate from typical duplication mechanisms and was therefore examined for alternative explanations. Several clues support this: first, the DFR2 copies are assumed to be located at the breakpoints of genome inversions or rearrangements ( Figure 5A ); second, the orientation of DFR2 is inverted relative to DFR1 and the two loci are separated by a considerable genomic distance ( Figure 2 ). Additional observations include frequent genome rearrangements both within Fagopyrum species and between Fagopyreae and other members of the Polygonaceae family. These patterns suggest that the DFR gene duplications may be associated with structural variations, such as inversions, rather than arising from tandem or segmental duplications. These observations point towards a mechanism of gene duplication associated with chromosomal inversions, first described by Ranz et al. [ 99 ] in the genome of Drosophila melanogaster , and later observed in other species such as Drosophila buzzatii [ 100 ], Drosophila mojavensis [ 101 ] and even in the bacterium Helicobacter pylori [ 102 ]. This mechanism has also been discussed in a review on chromosomal inversions by Casals and Navarro [ 103 ] that differentiate between ectopic recombination and this staggered break model. Furuta et al. referred to this process as “DNA duplication associated with inversion” (DDAI), a term that will be used from this point on in this study [ 102 ]. According to them, DDAI involves the duplication of a DNA segment at one chromosomal locus and its reinsertion at a distant site on the same chromosome in an inverted orientation, accompanied by an inversion of the sequence in between [ 102 ]. Ranz et al. described it as an inverted duplication associated with inversion breakpoint regions resulting from staggered breaks [ 99 ]. The mechanism of this type of gene duplication will serve as the working hypothesis for the duplication of DFR1 to DFR2 in this study ( Figure 8 ). In this model, a segment of DNA on a chromosome undergoes two pairs of staggered single-strand breaks at its ends, resulting in long 5’ overhangs. The segment is then inverted and joined into the opposite breakpoints. The breaks are then repaired by non-homologous end joining (NHEJ), in which the 5’ overhangs are filled in and ligated to restore DNA continuity through replication. If a gene lies within the region affected by the staggered break and is successfully retained during inversion and repair, this results in a duplicated copy of the gene in an inverted orientation relative to the ancestral copy - without disrupting the original gene. A possible alternative to this mechanism involves only one pair of staggered single-strand breaks at one end and a double-strand break at the other. A hallmark of this mechanism is that gene copies are found in an inverted orientation, near chromosomal inversion breakpoints, and are present only in species that have the specific chromosomal rearrangement. Using both experimental and computational analysis, Ranz et al. found that 17 out of 29 chromosomal inversion breakpoint regions between D. melanogaster and its close relatives D. simulans and D. yakuba are associated with inverted gene duplications resulting from these inversions [ 99 ]. The average nucleotide identity (± standard deviation) between the duplicated gene pairs was approximately 88% ± 5.4%, which closely resembles the similarity observed in this study ( Figure 3A ). In the case of the DFR2 duplication, all the key features supporting the DDAI mechanism are present. The formation of DFR2a and DFR2b can be further explained as resulting from a subsequent tandem or proximal duplication of the initial DFR2 copy generated through DDAI ( Figure 8E,F ). A comparable example is observed in D. melanogaster , where the genes CG31286 and CG34034 , located at two separate breakpoints, underwent tandem duplication following a DDAI event associated with inversion 3R(7) when compared to D. simulans [ 99 ]. Furuta et al. studied the “birth” and “death” of genes linked to chromosomal inversions in H. pylori , a bacterium with high genome plasticity. By comparing multiple genomes, they found that gene gains and losses often occurred at inversion breakpoints, providing evidence for DNA duplication associated with inversion (DDAI). They reported cases of complete and inverted gene duplication, partial duplication, and gene decay. Importantly, they showed that the near-identical homology created by DDAI events promotes subsequent homologous recombination, although this recombination decreases as sequence divergence accumulates over time [ 102 ]. Calvete et al. analyzed the 2m and 2n inversions in D. buzzatii . Inversion 2m was associated with a 13 kb duplicated segment generated by staggered single-strand breaks and repair via NHEJ. The segment contained five genes, but some copies became pseudogenes or were lost. Inversion 2n was instead linked to double-strand breaks and NHEJ repair, and both inversions may have shared breakpoints [ 100 ]. Guillén and Ruiz reported two inversions in D. mojavensis (2h and 2q), both involving inverted duplications of non-repetitive DNA created by staggered single-strand breaks and NHEJ repair. The 2h inversion contained a 7.1 kb segment with three genes, while 2q involved a smaller 1 kb duplicated segment [ 101 ]. Download figure Open in new tab Figure 8: Proposed mechanism of DFR duplication in Fagopyrum . (A) The hypothetical ancestral state in the common ancestor of Fagopyrum and its family members. (B) A pair of staggered single-strand breaks, containing the DFR sequence, results in a long 5’-overhang at one end of the segment, with a double-strand break at the other end. (C) The segment is inverted, and (D) the 5’-overhang is filled and the end of the segments are ligated to the corresponding DNA segments by the NHEJ repair mechanism (depicted with white arrows). (E) The intermediate state following the repair process, and (F) the tandem or proximal gene duplication event leading to DFR2 being duplicated into DFR2a and DFR2b . Preexisting Gene Duplications at Breakpoints An important finding in the study of Guillén and Ruiz concerns the presence of preexisting gene duplications at inversion breakpoints [ 101 ]. They observed four cases where inversion breakpoints were located between preexisting duplicated genes. Their statistical analysis showed that the breakpoints were significantly more likely to be located between duplicated genes than would be expected by chance. Guillén and Ruiz proposed two possible explanations for this observation. First, they suggested that duplicated genes might cause genomic instability, leading to an increased rate of double-strand breaks (DSBs) or may create “permissive” regions where breakage is more likely. Alternatively, they hypothesized that the mobilization of a duplicated gene could provide beneficial positional effects, supporting in the fixation of the inversion within the species. When duplicated genes are co-located in the same chromosomal domain, their evolution may be constrained due to the shared regulatory sequences, and sequence homogenization by frequent conversion or ectopic recombination events [ 101 ]. Relocating one of the copies to a different chromosomal region could relieve these constraints and potentially lead to advantageous regulatory changes [ 101 ], which could also be the case for DFR1 and DFR2 . In a comparative study between Drosophila arizonae and D. mojavensis , Runcie and Noor identified two inversion breakpoints that were located near an inverted gene duplication and a common repetitive element [ 104 ]. However, they found that the inverted gene duplication likely predated the inversion event and therefore rejected the DDAI-like hypotheses. The duplication was hypothesized to have originally been present in the non-inverted ancestral chromosome, although the ancestral copy appeared to have degenerated [ 104 ]. In this study, it remains unclear whether the inverted gene duplication predated the inversion, as no dataset from a primitive Fagopyrum species or a most recent common ancestor without the inversion is available. This finding presents an alternative hypothesis for the DFR2 duplication in Fagopyrum . In this scenario, a tandem gene duplication likely occurred first, leading from DFR1 to DFR2 . The inversion event would have occurred later, resulting in the original or its derived copies being translocated to a distant locus by chromosomal inversion resulting in an inverted orientation. The further tandem gene duplications of DFR2 into DFR2a and DFR2b could have occurred either before or after the inversion event. Evolutionary Implications of DFR Gene Duplications Duplication Mechanism and Timing The duplication of DFR genes in Fagopyrum species is hypothesized to have occurred via DDAI, followed by a tandem gene duplication, resulting in three DFR copies. An alternative hypothesis would be that a tandem gene duplication occurred first, followed by an inversion that relocated one copy to a distant locus and another gene duplication resulting in DFR2a and DFR2b . The origin of FtDFR3 and FtDFR4 , however, remains unclear. It is assumed to have arisen via a DDAI event, as it is located at a putative chromosomal breakpoint ( Figure 5 ). Notably, the intermediate chromosomal state between the ancestral synteny and the current arrangement following the hypothesized DDAI event has not yet been verified. Genome sequences of only four Fagopyrum species have been assembled to date, and all belong to the same cymosum subgroup within the Fagopyreae tribe and no genomes from the urophyllum subgroup have yet been sequenced. Extending the genome assembly of more species in both subgroups would be very informative to search for tracing the origin and evolutionary history of the DFR duplication. Nonetheless, evidence for chromosomal inversions involving the DFR locus in Fagopyrum exists. When macrosynteny between Fagopyrum and other Polygonaceae members (e.g., F. multiflora and P. aviculare ) is examined, DFR2a and DFR2b in F. dibotrys are found near one end of a chromosomal breakpoint ( Figure 5 ). This breakpoint may have been reused again during the DDAI event that duplicated DFR . It is also possible that multiple inversions occurred after the initial DDAI event, potentially driven by homologous recombination between regions of high sequence identity generated during the duplication process [ 102 ]. The exact breakpoints responsible for the duplication and inversion remain unidentified and require further investigation. However, dot plot comparisons between the duplicated loci (Additional file 6) reveal homologous regions that may result from the presence of TEs. These elements could contribute to the sequence similarity necessary to facilitate recombination, further supporting the possibilities of ectopic recombinations involving the syntelog containing DFR following the DDAI [ 102 ]. The gene sequence similarity, structural conservation, substrate-binding regions, and phylogenetic placement all confirm that the observed gene copies are indeed authentic DFRs, rather than unrelated genes or pseudogenes mistakenly identified. However, there remains ambiguity regarding the phylogenetic placement of the DFR2 copies. In the constructed phylogenetic tree, they cluster near the base of the Polygonaceae family ( Figure 4 ), rather than grouping closely with the known clade of Fagopyrum DFR1 . This suggests that the duplication event leading to DFR2 may have occurred before the divergence of species within the family. However, this scenario is less parsimonious. If the duplication had occurred prior to the diversification of the Polygonaceae family, one would expect other family members to retain at least one additional DFR copy resulting from the same duplication event. This is, however, not observed except for R. japonica that has 2 DFR duplicates on a single chromosome. The Fate of Fagopyrum DFR Duplicates The 26-amino-acid region responsible for substrate specificity [ 37 ] displays distinct features in DFR2a and DFR2b , with all duplicates containing valine at the third position, except for FtDFR2b , which contains isoleucine. Genes with valine at this position have been reported to have high affinity toward DHQ over other dihydroflavonols [ 48 , 105 ] but the functional implications of isoleucine at this site in FtDFR2b remain unknown. Additionally, all derivative DFR genes possess an extra amino acid between positions 5 and 6 ( Figure 3B ), which is glycine in all DFR2b , arginine in DFR2a , except in FeDFR2a with lysine. The insertion of glycine in FeDFR2 is hypothesised by Katsu et al. to increase the flexibility and alter the conformation of the loop spanning residues 4 to 18, which overlays the catechol ring of DHQ [ 48 ]. Although none of the residues in this loop directly interact with the substrate, such conformational changes may influence substrate selection [ 48 ]. Their docking models of the two proteins in complex with the three substrates suggested that FeDFR1a , with asparagine at the third position, could form more hydrogen bonds with each substrate than FeDFR2 , which has valine at the same site [ 48 ]. The steric hindrance from a bulky Phe133 residue in FeDFR2 may account for its preferential activity toward DHQ over DHM [ 48 ] and thus, both the identity of the amino acid at the third position and the surrounding residues forming the substrate-binding pocket contribute significantly to the substrate specificity of DFR enzymes in F. esculentum . The functional impact of the alternative inserted residues (arginine or lysine) at this position remains unclear and requires further investigation. The sequence divergences suggest that the duplicated DFR genes have undergone structural evolution in their substrate-binding regions, potentially altering their functional roles in flavonoid biosynthesis. Differential gene expression data indicate that some DFR2 genes are only expressed in seeds and roots, whereas DFR1 is broadly expressed across tissues. This is not unusual among duplicates in other organisms, as approximately 70% of gene pairs in Arabidopsis exhibit significant differences at the transcriptional level [ 2 ]. This tissue-specific expression pattern likely results from differences in cis-regulatory elements in their promoters. Notably, the promoter regions of DFR2 contain fewer MYB-binding sites and predominantly feature bHLH-binding motifs, which may also explain the overall lower expression levels of DFR2 [ 38 ]. This is also supported by the promoter analysis from Katsu et al. [ 48 ], that FeDFR2 lacks the MYBPLANT and MYBPZM motifs, known binding sites for MYB transcription factors. Their gene expression analysis via real-time PCR across six organs (roots, stems, leaves, buds, flowers, and seeds) showed that FeDFR1a was expressed in all examined tissues while FeDFR2 was predominantly expressed in roots and seeds, with expression levels in these organs approximately two orders of magnitude higher than those of FeDFR1a [ 48 ]. In contrast, our analysis shows DFR2 expression is lower than DFR1 ( Figure 6 ), likely due to methodological differences. Further research is needed to clarify the regulatory elements driving tissue specificity, the DHQ preference of DFR2, and the functional effects of the different amino acid insertions. Selective pressure analysis supports the functional relevance of the DFR copies. Orthologous comparisons of DFR1 across species show very low Ka/Ks values, indicating strong purifying selection and conservation as the primary functional DFR gene in Fagopyrum . DFR2a and DFR2b orthologs exhibit slightly higher, yet still <0.5 Ka/Ks values, suggesting that they too are under purifying selection after the duplication event, albeit slightly relaxed. Paralogous comparisons reveal that DFR2a and DFR2b versus DFR1 have Ka/Ks values below 0.1, indicating strong purifying selection and retention of DFR structure. Interestingly, when DFR2a and DFR2b are compared with each other, higher Ka/Ks values are observed, particularly within conserved regions, implying divergence in different functional directions. This could suggest that while both copies maintain the ancestral sequence and structure, they may be evolving distinct roles to reduce functional redundancy. The absence of pseudogenization or genetic drift features (Ka/Ks ≈ 1) in any of the pairwise comparisons further support the idea that all DFR copies remain under selective pressure and are likely beneficial to the genome. After gene duplication, the rate of sequence evolution is generally expected to increase, at least initially, due to relaxed selection against previously deleterious mutations. Consistent with this, the DFR duplicates exhibit relaxed purifying selection and sequence divergence. However, this increase in evolutionary rate is not always symmetric between duplicates [ 2 ], as shown here where DFR2 exhibits a higher mutation rate than the ancestral DFR1 . The most common fate of a duplicated gene is partial or complete deletion, reverting the locus to a singleton state [ 15 ]. Duplications that are retained may diverge to provide new functions (neofunctionalization) or split the ancestral function between copies (subfunctionalization). Whether the case of DFR duplicates represents neofunctionalization or subfunctionalization remains unclear, as changes in expression levels or tissue specificity alone do not conclusively determine either outcome [ 106 ]. Subfunctionalization or duplication-degeneration-complementation (DDC) involves the retention of both duplicates to preserve the full spectrum of ancestral functions which is here the ability to accept all three dihydroflavonols. A possible explanation is that after DFR duplication, each copy randomly lost part of its functional capacity (degeneration), and because the losses affected different subfunctions, both duplicates were retained to complement each other. Subfunctionalization does not always partition ancestral functions symmetrically, as one copy typically retains significantly more subfunctions than its paralog [ 2 ]. This aligns with the findings of Katsu et al., who showed that FeDFR1a has higher activity toward DHM and DHK but lower efficiency for DHQ, while FeDFR2 shows high catalytic efficiency toward DHQ but lower activity for DHM and DHK. A study by Choudhary and Pucker [ 34 ] speculated that through a yet unknown mechanism, DFR with N at their 3rd position in the conserved region might become more specific for the substrate that is not favoured by the additional DFR copy. On the other hand, a simpler explanation is that the DFR2 copies are undergoing neofunctionalization, evolving toward a yet unknown novel function under positive selection, while DFR1 is maintained for its ancestral role under purifying selection. This idea is supported by the observation that the conserved region of DFR2 copies has diverged from the ancestral DFR , including the insertion of a new amino acid between positions 5 and 6 ( Figure 3B ), along with several additional substitutions. However, it is still difficult to conclude that neofunctionalization is the fate in this case, as the specific new function of DFR2 has not yet been identified. An alternative explanation is escape from adaptive conflict (EAC). In this model, the ancestral single-copy DFR in Fagopyrum may have been under selection to perform both a novel function and the ancestral DFR function. Because improvements to one function would negatively affect the other due to pleiotropic constraints, duplication was favoured [ 19 ]. This resulted in a DFR2 copy free to optimize the novel function, and a DFR1 copy maintained for the ancestral function. However, this case does not fully meet the criteria set by Des Marais and Rauscher [ 19 ], which require that both duplicates undergo adaptive changes and that the ancestral function itself is improved. Here, only DFR2 shows adaptive changes in the conserved region, while DFR1 appears to be maintained under purifying selection, with no evidence that the ancestral function has been enhanced. In conclusion, the fate of the DFR duplicates cannot yet be determined with certainty. The most plausible hypothesis may be EAC, supported by Des Marais and Rauscher who concluded that EAC is clearly a feature of DFR evolution [ 19 ]. However, further experimental studies are needed to confirm whether DFR2 has acquired a novel function and if Fagopyrum DFR1 indeed has an improved ancestral function. Following duplication, some level of functional redundancy is expected. However, redundancy may persist not because it is beneficial, but because insufficient time has passed for its loss. Whether the duplicates like FeDFR2b , FdDFR2a , and FdDFR2b that show no expression, can be considered a pseudogene remains unresolved. The gene does not contain any inactivating mutations such as premature stop codons or frameshifts. Nonfunctional duplicates are not always deleted immediately as some may have only recently begun pseudogenisation and are in the early stages of decay [ 2 ]. Alternatively, the duplicates may still be retained because not enough time has passed for deleterious mutations to accumulate and eliminate it fully. Maere et al. [ 107 ] suggested that genes involved in secondary metabolism and biotic stress responses decay more slowly after both small- and large-scale duplication events. This could explain why DFR duplicates are still retained, as DFR is part of the flavonoid pathway and may provide adaptive diversity in secondary metabolites. List of Abbreviations DFR dihydroflavonol-4-reductase DHK dihydrokaempferol DHQ dihydroquercetin DHM dihydromyricetin DDAI DNA duplication associated with inversion WGD whole-genome duplication TE Transposable element EAC escape from adaptive conflict TSS transcription start site NHEJ non-homologous end joining Declaration Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets supporting the result and conclusions of this article are included in the article and additional files or are available from public databases. All the sequences and scripts used in the different analyses have been deposited in the author’s GitHub repository ( https://github.com/SyafiqSamsolnizam/DFR-of-Fagopyrum ). Competing interests The authors declare that they have no competing interests. Funding This work received no external funding. Authors’ contributions SS and BP conceptualized the research project. SS carried out the analyses and prepared figures. SS wrote the manuscript. SS and BP revised the manuscript. The authors read and approved the final manuscript. Additional File Additional file 1 (.pdf) : List of datasets used in this study to retrieve the gene of DFR . Additional file 2 (.pdf) : Extended version for the amino acid and CDS multiple sequence alignment of DFR. Additional file 3 (.pdf) : Supporting maximum likelihood tree of DFR sequences constructed using FastTree and RAxML-NG. Additional file 4 (.xls) : SRA run IDs for gene expression analysis. Additional file 5 (.xls) : Comparison of substitution rates between orthologous and paralogous DFR genes among Fagopyrum species. Additional file 6 (.pdf): Dot plots of the genomic loci of DFR copies in F. dibotrys and F. homotropicum . Acknowledgements We thank the members of the Plant Biotechnology and Bioinformatics group at the University of Bonn for their helpful discussions and valuable comments during the analysis and revision of this manuscript. This work was supported by the de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI) and ELIXIR-DE (Forschungszentrum Jülich and W-de.NBI-001, W-de.NBI-004, W-de.NBI-008, W-de.NBI-010, W-de.NBI-013, W-de.NBI-014, W-de.NBI-016, W-de.NBI-022). Footnotes https://github.com/SyafiqSamsolnizam/DFR-of-Fagopyrum References 1. ↵ Kondrashov FA . Gene duplication as a mechanism of genomic adaptation to a changing environment . Proc R Soc B Biol Sci . 2012 ; 279 : 5048 – 57 . doi: 10.1098/rspb.2012.1108 . OpenUrl CrossRef PubMed 2. ↵ Panchy N , Lehti-Shiu M , Shiu S-H . Evolution of Gene Duplication in Plants . Plant Physiol . 2016 ; 171 : 2294 – 316 . doi: 10.1104/pp.16.00523 . OpenUrl Abstract / FREE Full Text 3. ↵ Ohno S . 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Share Expansion of the DFR Gene Family in Fagopyrum Syafiq Samsolnizam , Boas Pucker bioRxiv 2025.09.20.677491; doi: https://doi.org/10.1101/2025.09.20.677491 Share This Article: Copy Citation Tools Expansion of the DFR Gene Family in Fagopyrum Syafiq Samsolnizam , Boas Pucker bioRxiv 2025.09.20.677491; doi: https://doi.org/10.1101/2025.09.20.677491 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17680) Bioengineering (13888) Bioinformatics (41927) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13373) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22494) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)