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. 2025 Sep 26;9(9):e70093. doi: 10.1002/pld3.70093

Analysis of Genotypic Variations in the Anthocyanin Biosynthetic Pathway in Potatoes

Chae‐Min Lee 1,2, Seung Yong Shin 1, Su‐Jin Park 1,3, Ji‐Sun Park 1, Changsoo Kim 2, Hyun‐Soon Kim 1,3, Hyo‐Jun Lee 1,4,✉
PMCID: PMC12465227  PMID: 41018493

ABSTRACT

Anthocyanins are pigments that contribute to plant defense and adaptation to environmental stresses. Given their antioxidant properties and positive impacts on human health, enhancing anthocyanin biosynthesis in plants holds significant economic importance. In potato, several genotypes produce a high amount of anthocyanins, but the molecular mechanisms underlying the genotypic variation of anthocyanin content remain poorly understood. Here, key genes that may determine the genotype‐dependent capacity for anthocyanin biosynthesis were analyzed. Anthocyanin content in tubers from five genotypes was measured, and Heimeiren and Desiree, exhibiting high and low anthocyanin content, respectively, were selected. We were unable to identify any evidence of differing activity in anthocyanin biosynthesis enzymes based on single amino acid polymorphism analysis between the two genotypes. However, transcriptome sequencing coupled with prediction of gene function identified 27 candidate genes showing different expression levels in tubers of these genotypes. We additionally verified expression patterns of these genes and found that four genes encoding flavanone 3‐hydroxylase, flavonoid 3′,5′‐hydroxylase, anthocyanin synthase (ANS), and anthocyanin O‐methyltransferase (AOMT) were strong candidates for high accumulation of anthocyanins in Heimeiren. Particularly, ANS and AOMT are strong candidates increasing anthocyanin content in the tuber flesh. These results imply that genotype‐dependent variations of anthocyanin biosynthesis may be due to difference of gene expression, but not enzymatic activities. Our study suggests key anthocyanin biosynthesis genes showing different expression levels in high‐ and low‐anthocyanin genotypes, offering potential for the metabolic engineering of potatoes to increase anthocyanin content.

Keywords: anthocyanins, genotype dependency, potato, tuber

1. Introduction

Anthocyanins belong to the flavonoid group, constituting an important subgroup of secondary metabolites. They are glycosylated polyphenolic compounds derived from anthocyanidins. Anthocyanidin is a flavylium cation, having an oxygenated 2‐phenylbenzopyrilium structure, and is divided into two benzoyl rings, each consisting of six rings (Brouillard 1982; Castañeda‐Ovando et al. 2009). More than 30 natural anthocyanidins have been identified including cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. Among them, petunidin is the most common anthocyanins produced in plants (Ananga et al. 2013). Acylation is a key factor in conferring diversity within anthocyanin molecules (Mazza and Miniati 1993), because the type, number, and position of acyl groups differ the chemical property of anthocyanins (Andersen and Jordheim 2006). Currently, there are over 700 identified anthocyanins, exhibiting diversity in hydroxylation, methylation, glycosylation, and acylation patterns (He and Giusti 2010). They mainly manifest various colors such as red, orange, blue, and purple, serving various functions within plants (Tanaka and Ohmiya 2008).

Anthocyanins play beneficial roles in both plants and humans. In plants, they are synthesized in seeds, fruits, and flowers in many species, promoting reproductive activity by attracting pollinators (Roy et al. 2022). Anthocyanins protect plants from various biotic and abiotic stresses, enhancing their adaptability to climate change (Chalker‐Scott 1999; Ahmed et al. 2014). They absorb excess ultraviolet light to protect the photosynthetic apparatus and facilitate a faster recovery from mechanical damage from oxidation stresses (Gould et al. 2002; Guo et al. 2008). Besides their protective effects during plant growth, anthocyanins can also play a crucial role in enhancing post‐harvest performance in plants. For example, in tomatoes, they act as antioxidants and prevent lipid oxidation to maintain cell membrane integrity and delay cell aging, resulting in reduced over‐ripeness and extended shelf life (Jiao et al. 2012; Bassolino et al. 2013; Y. Zhang et al. 2013).

Antioxidant properties of anthocyanins also contribute to improving human health. Antioxidant capabilities of anthocyanins arise from the multiple hydroxyl groups, providing potent scavenging activities against reactive oxygen and nitrogen species produced in human cells (Martin et al. 2011; Martin and Li 2017; Yashin et al. 2017). Anthocyanins deliver hydrogens and electrons to reactive oxygen and nitrogen species, stabilizing them and generating relatively stable flavonoid radicals. Additionally, flavonoids chelate metal ions involved in free radical generation and inhibit enzymes involved in the production of reactive oxygen and nitrogen species (Heim et al. 2002; Mladenka et al. 2010; Kumar and Pandey 2013). In addition, anthocyanins specifically aid in preventing human cancers, cardiovascular diseases, and other chronic conditions (Joseph et al. 2003; Lee et al. 2005; Achterfeldt et al. 2015; Charepalli et al. 2015). For example, derivatives of delphinidin reduce vascular inflammation, prevent blood clotting, and protect human skin from UV‐B radiation by inhibiting keratinocyte apoptosis (Watson and Schönlau 2015). Moreover, anthocyanins suppress cell cycle and stimulate apoptosis of cancer cells (Lin et al. 2017). In studies with mice, anthocyanin‐containing tomato peel extracts dose‐dependently suppressed the proliferation of human colon and ovarian cancer cells (Zhao et al. 2009), implying the role of plant anthocyanins in contributing various beneficial aspects in mammals.

The content of anthocyanins is significantly influenced by the expression of the anthocyanin biosynthesis pathway genes. Anthocyanin biosynthesis pathway is a part of the flavonoid biosynthesis pathway, with chalcone synthase (CHS) providing the first committed step (Ma et al. 2021). Chalcone serves as a precursor for various types of flavonoids, including flavones, flavonols, flavan‐diols, flavan 4‐ols, proanthocyanidins, isoflavonoids, and anthocyanins. In this context, it synthesizes naringenin chalcone from 4‐coumaroyl‐CoA and malonyl‐CoA. Subsequently, naringenin chalcone is isomerized to naringenin by chalcone isomerase (CHI), and naringenin plays a crucial role as a branching point in the flavonoid pathway. Naringenin acts as a substrate for flavonoid 3′‐hydroxylase (F3′H) and flavonoid 3′,5′‐hydroxylase (F3′5′H) introducing −OH groups at position 3 (F3′H) or positions 3 and 5 (F3′5′H) of the B‐ring, producing eriodictyol and pentahydroxyflavanone, respectively (Alappat and Alappat 2020). All these three flavonoid molecules—eriodictyol, naringenin, and pentahydroxyflavanone—are converted into colored anthocyanins named cyanidin, pelargonidin, and delphinidin, respectively, through the function of several enzymes including flavanone 3‐hydroxylase (F3H), dihydroflavonol 4‐reductase (DFR), and anthocyanin synthase (ANS). Finally, these molecules are further processed by flavonoid 3‐O‐glucosyltransferase (UFGT) and anthocyanin O‐methyltransferase (AOMT) to produce peonidin‐3‐O‐glucoside (POG), pelargonidin‐3‐O‐glucoside (PLG), and petunidin‐3‐O‐glucoside (PTG), respectively.

Various studies have been conducted on key enzymes of the anthocyanin biosynthesis pathway. In potato, tuber skins containing anthocyanin pigments exhibit high expression of genes encoding F3′H, F3′5′H, DFR, ANS, and UFGT (De Jong et al. 2003; Jung et al. 2005; André et al. 2009; Y. Zhang et al. 2009). Transgenic potato plants overexpressing DFR showed an increase of anthocyanin content in their tubers while inhibiting gene expression led to a significant reduction in anthocyanins level (Stobiecki et al. 2003). Additionally, potato tubers overexpressing ANS increased anthocyanin synthesis (H. Zhang et al. 2020). Overexpression of F3′5′H in the red‐skinned potato variety Desiree resulted in purple‐skinned tubers and stems (Jung et al. 2005). These results indicate that anthocyanin content can be modulated by changing expression levels of key genes encoding anthocyanin biosynthesis in potatoes. However, research on molecular basis of genotype‐dependent variations of anthocyanin biosynthesis is limited, and the major genes responsible for determining anthocyanin content in each genotype remain unclear. In this study, we investigated genotype‐dependent differences in anthocyanin biosynthesis pathways through time‐course comparative analyses of anthocyanin contents and transcriptome data and identified four strong candidate genes that showed a high correlation with anthocyanin accumulation in tubers.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

The potato varieties used in this study are Desiree, Nicola, Russet Burbank, Victoria, and Heimeiren. Each variety was propagated in basal Murashige and Skoog (MS) medium (Duchefa, Haarlem, Netherlands) containing 30 g L−1 sucrose and 8 g L−1 plant agar (Duchefa). For in vitro cultivation, plantlets were grown under long‐day conditions (16 h light/8 h dark) at constant temperature of 24°C and light intensity of 100 μmol m−2 s−1. Subsequently, the in vitro cultivated plantlets were transferred to the pots and grown in a greenhouse for 14 weeks. For screening anthocyanin contents in five genotypes, plants were grown from August to November 2021 under an average temperature of 18.2°C. For time‐course analysis of anthocyanin contents in Desiree and Heimeiren, plants were grown from April to July 2022 under an average temperature of 21.4°C. The pots were exposed to sunlight with an average intensity of 585 μmol m− 2 s− 1 during the day. The average time of sunrise and sunset was 05:31 AM and 19:27 PM, respectively.

2.2. Measurement of Anthocyanin Contents

Anthocyanins were analyzed using the HPLC method. Tubers were harvested and separated into the peel and flesh. Plant materials were freeze‐dried and ground. Each powdered sample (100 mg) was extracted with 2 mL of water: formic acid (95:5, v/v) after sonication for 25 min at 25°C. After centrifugation at 10,000 × g for 10 min, the extract was filtered through a 0.45‐μm PTFE syringe filter. The filtrate was then analyzed by an Agilent 1100 HPLC system using a Synergy 4 μm Polar‐RP 80A (150 × 2 mm, i.d.) column with a Security Guard AQ C18 (4 × 3 mm, i.d.; Phenomenex, Torrance, CA). Detection was made at a wavelength of 520 nm, and the column oven temperature was set at 40°C. The injection volume was 10 μL. The solvent system was delivered at a rate of 0.4 mL min−1 and consisted of a mixture of (Solvent A) water/formic acid (95:5, v/v) and (Solvent B) acetonitrile/formic acid (95:5, v/v). The gradient program largely modified from that previously published (N. I. Park et al. 2011) is described as follows: 0 min, 5% B; 8–15 min, 10%–13% B; 15–18 min, 13%–15% B; 18–25 min, 15% B; 25–35 min, 15%–5% B. Quantification of the different anthocyanins was based on peak areas and calculated as equivalents of two representative standard compounds. All contents were expressed as mg per 100 g dry weight.

2.3. Whole Genome Sequencing (WGS) and Single Nucleotide Polymorphism (SNP) Analysis

Genomic DNA from five to seven explants of Desiree and Heimeiren was extracted using the DNeasy Play Mini Kit (QIAGEN, The Netherlands). Whole genome sequencing (WGS) was performed using the Solanum tuberosum DM 1‐3516 R44 reference genome (http://spuddb.uga.edu) on the NovaSeq 6000 platform. DNA libraries were prepared following the TruSeq DNA PCR‐Free Library Prep protocol (Illumina, San Diego, CA). Genomic DNA was randomly sheared using the Covaris S2 system (Covaris, Woburn, MA). The library's quality was verified using Bioanalyzer (Agilent Technologies, Santa Clara, CA). WGS data were analyzed using the nf‐core/sarek pipeline with GATK4 (Garcia et al. 2020). Only variants meeting the following criteria were selected for single nucleotide polymorphism (SNP) analysis: a depth of 20 or more reads and at least 80% alternate chromosome frequency.

2.4. RNA Sequencing (RNA‐seq)

Total RNA was extracted from 50 mg of freeze‐dried tubers using TRIzol (Thermo Fisher Scientific, USA). RNA samples were used to construct libraries with the Illumina mRNA Sample Prep Kit (Illumina). To obtain high‐quality reads, sequencing adapters and low‐quality bases were trimmed from raw reads. High‐quality reads were mapped to the potato reference genome, DM 1‐3 516 R44 v6.1 (http://spuddb.uga.edu/index.shtml). Using R software (https://www.r‐project.org/), genes that were highly or lowly expressed in Heimeiren compared to Desiree at 8, 11, and 14 weeks were visualized as a heatmap. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed by the bioinformatics company (Seeders, Daejeon, Korea).

2.5. Real‐Time Quantitative PCR (RT‐qPCR)

Total RNA was extracted from 50 mg of freeze‐dried tubers using TRIzol (Thermo Fisher Scientific). 1 μg of RNA was synthesized into cDNA using AccuPower CycleScript RT PreMix (Bioneer, Daejeon, South Korea). Gene expression was analyzed by RT‐qPCR using the CFX Connect Real‐Time System (Bio‐Rad, CA, USA) and TOPreal qPCR 2X PreMIX (Enzynomics, Daejeon, South Korea). StEF1α was used as the housekeeping gene for normalizing RT‐qPCR results (Kuster et al. 2017). The primers used in the experiments are listed in Table S1.

2.6. Statistical Analysis

For bar and line graphs, average values were displayed and standard deviations were visualized as whiskers. To determine statistical significance, one‐way analysis of variance (ANOVA) with post hoc Tukey's test and Student's t‐test were performed using RStudio and Excel software, respectively. For ANOVA, letters indicate groups that are statistically significantly different from each other.

2.7. Accession Numbers

UniProt (https://www.uniprot.org) accession numbers: CtF3H, A0A1B0NTT3; VvDFR, P51110; RtPAL1, P11544; Nt4CL1, O24146; MsCHI, P28012; VvUFGT, P51094; AtANS, Q96323; MsCHS, P30074. Spud DB (https://spuddb.uga.edu) accession numbers for potato genes: F3H, Soltu.DM.02G023850; F3′H, Soltu.DM.03G029340; F3′5′H, Soltu.DM.11G020990; DFR, Soltu.DM.02G024900; PAL1, Soltu.DM.03G004870; 4CL1, Soltu.DM.06G024540; CHI, Soltu.DM.05G001950; UFGT, Soltu.DM.07G002000; ANS, Soltu.DM.08G026700; AOMT, Soltu.DM.09G025040; CHS, Soltu.DM.09G028560; TT8, Soltu.DM.09G019660. Accession numbers for other potato genes analyzed in this study were listed in figures and Table S2.

3. Results

3.1. Difference of Anthocyanin Content in Heimeiren and Desiree

We analyzed the content of three key anthocyanin molecules synthesized during the anthocyanin biosynthesis pathways in five representative potato genotypes: Desiree, Nicola, Russet Burbank, Victoria, and Heimeiren. Heimeiren showed an extremely high amount of all molecules, among which the concentration of PTG dominated the sum of the three anthocyanin molecules in both skins and fleshes (Figure 1A). Victoria is the second ranked genotype for accumulating anthocyanins, due to a relatively high amount of PLG and POG in its flesh compared to other genotypes, except Heimeiren. Other genotypes accumulated only marginal levels of anthocyanins, particularly in tuber flesh. Based on these analyses, we selected Heimeiren and Desiree as representative genotypes to explore the molecular reasons for the differences in anthocyanin content. As expected from the anthocyanin content, Heimeiren exhibited purple skin and flesh, whereas Desiree exhibited red skin and ivory flesh (Figure 1B).

FIGURE 1.

FIGURE 1

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Anthocyanin contents in different potato genotypes. (A) Measurement of anthocyanin contents. Contents of the three different chemicals showing different colors were measured (n = 3–4 biological repeats; for Desiree skin, n = 2). Letters indicate groups that are statistically different from each other (p < 0.05, Tukey's test). Note that statistical analysis was separately performed for skin and flesh. Whiskers indicate SD. D, Desiree; H, Heimeiren; N, Nicola; R, Russet Burbank; V, Victoria. (B) Images of tubers from Desiree and Heimeiren. Tubers were harvested from the 14‐week‐old plants grown in soil. The skin and flesh of tubers are displayed. Scale bars, 2 cm.

To analyze in more detail for anthocyanin biosynthesis in different genotypes, we measured anthocyanin content at different growth stages. We harvested whole tubers to examine the overall trend in anthocyanins content throughout different growth stages. Although the content of PTG, PLG, and POG showed no difference in Desiree, the concentration of all three molecules in Heimeiren was highest at the early growth stage (8 weeks) and decreased by 11 weeks (Figure 2A,B). These results suggest that the difference in anthocyanin biosynthetic activity between these two genotypes is greater in the early growth stages than in the late stages.

FIGURE 2.

FIGURE 2

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Time‐course measurement of anthocyanin contents in tubers from Desiree and Heimeiren. Tubers were harvested from the plants grown for the indicated time period. (A) Surface and cross section images. Potato plants were grown for the indicated time periods in soil and then tubers were harvested. Scale bars, 3 cm. w, weeks. (B) Anthocyanin contents during plant growth. Tubers harvested in (A) were used for measuring anthocyanin contents. Whole tubers including skin and flesh were analyzed. Letters indicate groups that are statistically different from each other (p < 0.05, Tukey's test; n = 4 biological repeats). Whiskers indicate SD.

3.2. Conservation of Active Sites in Anthocyanin Biosynthesis Enzymes of Heimeiren and Desiree

Genotype‐dependent differences usually arise from DNA sequence variations in each genotype (J. S. Park et al. 2023). Therefore, we performed WGS for Heimeiren and Desiree to analyze SNPs affecting codon substitutions. Our initial hypothesis was that there would be single amino acid polymorphisms (SAPs) at the active sites of enzymes involved in anthocyanin biosynthesis, leading to different activities in generating anthocyanins between Heimeiren and Desiree. Previous protein structure analyses have identified active sites in key anthocyanin biosynthesis enzymes, such as F3H from Carthamus tinctorius (Sui et al. 2023), DFR from Vitis vinifera (Petit et al. 2007), phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides (Calabrese et al. 2004), 4‐coumarate: CoA ligase (4CL) from Nicotiana tabacum (Li and Nair 2015), CHI from Medicago sativa (Jez et al. 2000), UFGT from V. vinifera (Offen et al. 2006), ANS from Arabidopsis thaliana (Wilmouth et al. 2002), and CHS from M. sativa (Ferrer et al. 1999). We compared amino acid sequences of these reference proteins with homologs found in Heimeiren and Desiree. However, no distinct SAPs were detected between the two genotypes across all analyzed enzymes. For F3H, 4CL1, ANS, and CHS, all active sites were identical to those in the reference proteins (Table 1). For DFR, PAL1, CHI, and UFGT, several amino acids in Heimeiren and Desiree corresponding to active sites in the reference proteins were substituted, but both genotypes exhibited identical substitutions. These results suggest that SNPs at the active sites of anthocyanin biosynthesis enzymes may not be responsible for the differences in anthocyanin content between Heimeiren and Desiree.

TABLE 1.

Analysis of SAPs in active sites of the key anthocyanin biosynthesis proteins. Whole genome sequencing was performed for Desiree and Heimeiren to find SAPs. Reference protein sequences reported in the previous articles were used to align protein sequences. Active sites of the reference proteins and the corresponding amino acids were displayed. Different amino acids in compared to the reference were marked in yellow.

F3H DFR PAL1 4CL1 CHI UFGT ANS CHS
Ct D H Vv D H Rt D H Nt D H Ms D H Vv D H At D H Ms D H
H220 H218 H218 S128 S140 S140 L134 L139 L139 S189 S195 S195 R36 R36 R36 S18 A14 A14 E140 E145 E145 T132 T132 T132
D222 D220 D220 N133 D145 D145 H137 F142 F142 T193 T199 T199 G37 G37 G37 T19 T15 T15 Y142 Y147 Y147 S133 S133 S133
H278 H276 H276 T159 T171 T171 S210 T208 T208 K197 K203 K203 L38 L38 L38 H20 H16 H16 K213 K218 K218 M137 M137 M137
R288 R286 R286 Y163 Y175 Y175 D214 D212 D212 H237 H243 H243 F47 F47 F47 Q84 E81 E81 N215 N220 N220 C164 C164 C164
S290 S288 S288 F164 F176 F176 L215 L213 L213 T336 T342 T342 T48 T48 T48 T141 T138 T138 Y217 Y222 Y222 T197 T197 T197
K167 K179 K179 L266 L263 L263 D420 D426 D426 I50 I50 I50 H150 H147 H147 H232 H237 H237 F215 F215 F215
A218 A230 A230 V269 V266 V266 R435 R441 R441 L101 L101 L101 T280 T279 T279 D234 D239 D239 G216 G216 G216
S221 G233 G233 K468 K463 K463 K437 K443 K443 Y106 Y106 Y106 H350 H349 H349 V235 V240 V240 I254 I254 I254
I222 I234 I234 E496 E491 E491 K441 K447 K447 K109 K109 K109 D374 D373 D373 H288 H293 H293 L263 L263 L263
Q227 Q239 Q239 Q500 Q495 Q495 Q446 Q452 Q452 V110 V110 V110 Q375 H374 H374 R298 R303 R303 F265 F265 F265
K526 K532 K532 N113 N113 N113 F304 F309 F309 H303 H303 H303
T190 S190 S190 E306 E311 E311 N336 N336 N336
M191 I191 I191 P375 P375 P375
D200 A200 A200
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Abbreviations: At, Arabidopsis thaliana; Ct, Carthamus tinctorius; D, Desiree; H, Heimeiren; Ms, Medicago sativa; Nt, Nicotiana tabacum; Rt, Rhodosporidium toruloides; Vv, Vitis vinifera.

3.3. Identification of Anthocyanin Biosynthesis Genes Showing Different Expression Between Heimeiren and Desiree Tubers

Our next hypothesis was that distinct expression of genes related to anthocyanin biosynthesis leads to the variations in anthocyanin content in potato genotypes. To examine our hypothesis, we performed RNA sequencing‐based transcriptome analysis using whole tubers of Heimeiren and Desiree including both skins and flesh. As for the anthocyanin content measurement, differentially expressed genes (DEGs) were analyzed and clustered based on expression patterns during three different growth stages (Figure 3A and Table S2). Among the clusters, genes belonging to cluster 4 (C4) exhibited constitutively low expression levels in Heimeiren; thus, they were designated as putative negative regulators (Figure 3B). On the other hand, genes belonging to Cluster 5 (C5) showed high expression levels in Heimeiren at all growth stages; thus, they were designated as putative positive regulators.

FIGURE 3.

FIGURE 3

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Transcriptome analysis of Desiree and Heimeiren tubers. (A,B) Clustering of DEGs between Desiree and Heimeiren tubers during tuber development. Desiree and Heimeiren plants were grown for the indicated time period and total tubers (mixture of skin and flesh) were harvested for RNA sequencing. Expression levels of DEGs are represented as a heatmap (A). Scale bar, log2 fold change. Clusters containing genes showing decreased expression (C4) or increased expression (C5) in Heimeiren are displayed (B). (C) GO analysis of genes in C5 cluster. Selected GO terms among top 30 significantly overrepresented GO in C5 genes are displayed as a table. The flavonoid biosynthesis‐related GO term is marked in bold. (D) KEGG analysis of genes in C4 and C5 clusters. The number of DEGs belonging to amino acid metabolism pathway is displayed as a table. Flavonoid biosynthesis‐related pathways are marked in bold. (E) List of flavonoid biosynthesis‐related DEGs in C5 cluster. DEGs belonging to flavonoid biosynthesis pathways in KEGG and GO analyses were listed. KEGG numbers refer to flavonoid biosynthetic pathways in (D). GO indicates flavonoid biosynthetic process‐related GO terms in (C). Log2 fold changes at 8, 11, and 14 weeks were represented as a heatmap. Scale bar, log2 fold change.

To identify genes involved in anthocyanin biosynthesis, we performed GO analysis for C4 and C5 genes. Although no GO terms related to anthocyanin biosynthesis were detected for C4 genes, five C5 genes were found to be involved in this term (Figure 3C). Because the number of anthocyanin biosynthesis‐related genes identified by GO analysis was too small, we next performed a KEGG analysis. The 17 genes belonging to C4 were found to be involved in anthocyanin and related biosynthesis pathways, including phenylpropanoid, flavonoid, isoflavonoid, and flavone biosynthesis (Figure 3D). For C5 genes, 18 genes were found to be involved in these biosynthesis pathways. Because our goal is identifying positive regulators that can be applied to increase anthocyanin content, we selected putative positive regulators identified by GO and KEGG analyses. Signal transduction genes encoding TRANSPARENT TESTA 8 (TT8), MYC2, MYC4, and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) were found by GO analysis (Figure 3E). Expression of TT8 was higher in Heimeiren than that in Desiree at all time points, but expression of MYC2 and MYC4 exhibited high expression in Heimeiren only at 8 weeks. Conversely, expression of COP1 was higher at late growth stages. Based on our data showing that anthocyanin content was highest at 8 weeks and then decreased at the later growth stages (Figure 2), we suppose that TT8 and MYCs may play major roles in determining the distinct anthocyanin content in Heimeiren. Among the anthocyanin biosynthesis enzymes found in the KEGG analysis, the expression difference for AOMT, ANS, and F3′5′H was highest at 8 weeks and then decreased at the later growth stages (Figure 3E), similar to the anthocyanin content pattern.

To further analyze the expression of genes involved in anthocyanin biosynthesis, we selected known key genes involved in the biosynthesis of POG, PLG, and PTG (Figure 4A). In our transcriptome analysis, a number of genes belonging to the upstream biosynthesis pathway from naringenin did not show higher expression levels in Heimeiren compared to those in Desiree at 11 and 14 weeks (Figure 4B). On the other hand, genes in the downstream biosynthesis pathway starting from naringenin exhibited higher expression levels in Heimeiren at least until 11 weeks. For verification of transcriptome analysis results, we performed RT‐qPCR using primers specific for each gene (Table S1). Unlike transcriptome analysis, the expression of DFR and TT8 did not show any difference between Heimeiren and Desiree at 8 and 11 weeks (Figure 4C). The expression of F3′H in Heimeiren was higher than that in Desiree, which was not observed in transcriptome analysis. However, other genes showed similar expression patterns to transcriptome analysis results. Notably, the expression fold change for ANS and AOMT was more than eightfold at 8 weeks, suggesting that distinct expression levels of these genes may be critical for high accumulation of anthocyanins in Heimeiren.

FIGURE 4.

FIGURE 4

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Expression of anthocyanin biosynthesis‐related genes in Desiree and Heimeiren. (A) Anthocyanin biosynthesis pathway. Enzymes catalyzing anthocyanin biosynthesis were annotated. PLG, pelargonidin‐3‐O‐glucoside; POG, peonidin‐3‐O‐glucoside; PTG, petunidin‐3‐O‐glucoside. (B) Heatmap of anthocyanin biosynthesis‐related genes analyzed in transcriptome sequencing. Expression levels were represented as a heatmap. Scale bar, log2 fold change. (C) RT‐qPCR of anthocyanin biosynthesis‐related genes. Potato plants were grown in soil for the indicated time periods. Tubers at each time point were harvested for extracting total RNA. Statistical significance was determined using Student's t‐test (*p < 0.05; n = 3 biological repeats). Whiskers indicate SD.

3.4. Expression Analysis of Key Anthocyanin Biosynthesis Genes in the Skin and Flesh of Tubers From Heimeiren and Desiree

Next, we investigated whether the key anthocyanin biosynthesis genes found in our analyses exhibit distinct expression patterns in the skin and flesh. Although the overall expression level decreases over growth periods (Figure 4C), we selected tubers harvested from 14‐week‐old plants to identify genes that express constitutively higher levels in the flesh of Heimeiren compared to those in Desiree. Among the four key genes, ANS and AOMT showed significantly higher expression levels in the flesh of Heimeiren, whereas the expression of F3′5′H and F3H was comparable in the two genotypes (Figure 5A), indicating that expression levels of ANS and AOMT may play a major role of anthocyanin accumulation in the fresh. In the skin, ANS expression in Heimeiren was significantly higher than in Desiree, whereas expression of remaining three genes in Heimeiren was lower (Figure 5B), suggesting that ANS expression may be critical for anthocyanin accumulation in skins. Based on these data, we concluded that the expression levels of two anthocyanin biosynthesis genes, ANS and AOMT, may be critical for the high accumulation of anthocyanins in Heimeiren.

FIGURE 5.

FIGURE 5

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Expression of selected anthocyanin biosynthesis‐related genes in the tuber skin and flesh. Potato plants were grown in soil for 14 weeks. The flesh (A) and skin (B) of tubers were separately harvested for analyzing gene expression. Statistical significance was determined using Student's t‐test (*p < 0.05; n = 8–11 biological repeats). Whiskers indicate SD.

4. Discussion

In the present study, we compared two genotypes exhibiting great differences in anthocyanin content to uncover the molecular reasons for the genotype‐dependent variations. Our genome and transcriptome analyses suggested that the abundance of anthocyanin biosynthesis enzymes may be a key factor rather than enzyme activities, as gene expression levels showed great differences between Heimeiren and Desiree, whereas SAPs at active sites were identical (Table 1 and Figure 3). The genes exhibiting the most pronounced differential expression were ANS and AOMT (Figure 4C), which function in the late steps of anthocyanin biosynthesis. These large expression differences may critically influence anthocyanin contents in the two genotypes, as anthocyanin accumulation was very limited in Desiree tubers, even though the expression levels of anthocyanin biosynthetic genes DFR and UFGT were slightly higher than those in Heimeiren (Figure 4C).

Increasing the expression of genes encoding several anthocyanin biosynthesis enzymes could be effective in producing high‐anthocyanin potatoes. Indeed, overexpression of the 3GT gene elevated anthocyanin content in tuber skins (Wei et al. 2012). Our study suggests that ANS and AOMT would be important targets for overexpression, based on our data showing that the expression fold change of these genes was highest (Figure 4C), and they showed significantly higher expression levels in the flesh of tubers from Heimeiren compared to those from Desiree (Figure 5A).

In previous studies, overexpression of a single gene related to anthocyanin biosynthesis was effective in increasing anthocyanin content only in tuber skins (Jung et al. 2005; Wei et al. 2012; H. Zhang et al. 2020). However, increasing anthocyanin content in tuber flesh is more important because tuber skins are typically not consumed as food. In the present study, we analyzed expression of genes in tubers from different genotypes to identify all genes affecting anthocyanin biosynthesis in both skins and flesh. Based on our data, most of the genes encoding downstream biosynthesis pathways from naringenin showed constitutively high expression levels in the high‐anthocyanin genotype, Heimeiren (Figure 4B,C). Particularly, two anthocyanin biosynthesis genes, ANS and AOMT, sustained high expression in the flesh (Figure 5A). These results suggest that multiple genes are responsible for genotype‐dependent anthocyanin biosynthesis. Therefore, the simultaneous overexpression of these genes would be required to upregulate anthocyanin biosynthesis not only in tuber skins but also in the flesh. In further research, the generation of transgenic plants overexpressing ANS and AOMT together with F3′5′H or F3H could produce high‐anthocyanin tubers similar to Heimeiren.

In conclusion, our results suggest that the difference in anthocyanin content between the high‐anthocyanin genotype Heimeiren and the low‐anthocyanin genotype Desiree would arise from differential expression of genes involved in the anthocyanin biosynthesis pathway, rather than from SNP‐derived differences in enzyme activities. Through WGS, we found no difference in SAPs at the active sites of key anthocyanin biosynthesis enzymes between Heimeiren and Desiree. On the other hand, a number of genes in the anthocyanin biosynthesis pathway showed differential expression levels between the two genotypes. Through GO and KEGG analyses, we identified genes exhibiting high expression levels in Heimeiren but low expression levels in Desiree. Most genes showing constitutively higher expression levels in Heimeiren were downstream pathway genes from naringenin, such as F3′5′H, F3H, ANS, and AOMT. Generation of transgenic plants simultaneously overexpressing these genes in the low‐anthocyanin genotype Desiree will be necessary to verify the role of genes in determining genotype‐dependent variations in anthocyanin content.

Author Contributions

Chae‐Min Lee, Hyun‐Soon Kim, and Hyo‐Jun Lee conceived the experiments. Chae‐Min Lee and Seung Yong Shin primarily performed the experiments. Hyun‐Soon Kim provided plant materials and experimental tools. Su‐Jin Park and Ji‐Sun Park assisted with potato cultivation. Changsoo Kim contributed to scientific discussions. Chae‐Min Lee, Seung Yong Shin, and Hyo‐Jun Lee wrote the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available in the Supporting Information for this article.

Supporting information

Data S1 Peer review.

PLD3-9-e70093-s002.pdf (125.2KB, pdf)

Table S1 Primers used in this study.

Table S2. Differentially expressed genes (DEGs) between Heimeiren and Desiree tubers during plant growth.

PLD3-9-e70093-s001.xlsx (522KB, xlsx)

Acknowledgments

This research was supported by the New Breeding Technologies Development Program (Project No. PJ01653001) provided by the Rural Development Administration of Korea, the Basic Research Program provided by the National Research Foundation of Korea (NRF‐2023R1A2C1003963 and NRF‐2022R1I1A2073565), and the KRIBB Research Initiative Programs (KGM1102313, KGM1082511, and KGM1002521).

Lee, C.-M. , Shin S., Park S.-J., et al. 2025. “Analysis of Genotypic Variations in the Anthocyanin Biosynthetic Pathway in Potatoes.” Plant Direct 9, no. 9: e70093. 10.1002/pld3.70093.

Funding: This work was supported by Rural Development Administration (RDA) (PJ01653001), National Research Foundation of Korea (NRF) (NRF‐2023R1A2C1003963 and NRF‐2022R1I1A2073565), and Korea Research Institute of Bioscience and Biotechnology (KRIBB) (KGM1102313, KGM1082511, and KGM1002521).

Chae‐Min Lee and Seung Yong Shin contributed equally to this study.

Data Availability Statement

Raw RNA‐sequencing data for Desiree and Heimeiren generated in this study were deposited in Korea BioData Station (accession number: KAP230659). Raw whole genome sequencing data for Desiree and Heimeiren were deposited in the same database (KAP230566 for Desiree and KAP230659 for Heimeiren).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1 Peer review.

PLD3-9-e70093-s002.pdf (125.2KB, pdf)

Table S1 Primers used in this study.

Table S2. Differentially expressed genes (DEGs) between Heimeiren and Desiree tubers during plant growth.

PLD3-9-e70093-s001.xlsx (522KB, xlsx)

Data Availability Statement

Raw RNA‐sequencing data for Desiree and Heimeiren generated in this study were deposited in Korea BioData Station (accession number: KAP230659). Raw whole genome sequencing data for Desiree and Heimeiren were deposited in the same database (KAP230566 for Desiree and KAP230659 for Heimeiren).

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