Genome-Wide Identi cation and Expression Analysis of Anthocyanin Biosynthesis Pathway Genes PAL and CHS in Colored Potato Tuber

Lina Shang College of Agronomy and Biotechnology, Southwest University, Chongqing Mengyuan Wan College of Agronomy and Biotechnology, Southwest University, Chongqing Yi Fu College of Agronomy and Biotechnology, Southwest University, Chongqing Rongrong Liu College of Agronomy and Biotechnology, Southwest University, Chongqing Zhonghui Jin College of Agronomy and Biotechnology, Southwest University, Chongqing Jichun Wang College of Agronomy and Biotechnology, Southwest University, Chongqing Baigeng Hu National Engineering Research Center for Potato, Dezhou Hongju Jian College of Agronomy and Biotechnology, Southwest University, Chongqing Dianqiu Lyu (  smallpotatoes@126.com ) College of Agronomy and Biotechnology, Southwest University, Chongqing

are formed [20]. Anthocyanin glycosides can be transported to vacuoles via vacuolar transporters such as glutathione S-transferase (GST), and collected and stored in vacuoles [21].
Potato, one of the top four major food crops, is widely cultivated in the world [22]. Potato tubers are rich in nutrients, including starch, sugar and protein, as well as a variety of antioxidants, such as polyphenols, vitamin C, carotenoids, selenium and so on [23]. Anthocyanidin, a water-soluble natural pigment, belongs to polyphenols. Anthocyanin is a natural antioxidant since its antioxidant activity, anti-aging and anti-vascular hardening functions [11]. It is a potential anticancer compound, which are largely abundant in colored potatoes. As an important secondary metabolite in potatoes, anthocyanin has attracted more and more attention, and become one of the hot spots in the development of functional food. However, the research eld of potato anthocyanin is relatively backward and still in its infancy.
PAL and CHS genes played key roles in anthocyanin synthesis pathway identi ed in Arabidopsis thaliana, so we used the protein sequences of PAL and CHS genes in A. thaliana as the query sequences to search the corresponding homologous gene sequences in the genomes of 18 species including potatoes in this study and the evolutionary relationship were analyzed based on the evolutionary tree. The molecular characteristics of potato PAL and CHS genes were also predicted. The tissue expression characteristics, stress response, hormone response were analyzed based on the existing expression data. Further, expression differences of colored potato PAL and CHS genes were analyzed by qRT-PCR. The function of potato anthocyanin biosynthetic pathway genes was preliminarily analyzed, which laid a foundation for further functional analysis.

Identi cation and Evolution Analysis of PAL and CHS Genes in 18 Plant Species
Genome-wide identi cation of PAL and CHS genes in 18 plant species, which not only included major classes like monocots and dicots, but also represent diverse groups such as cereals (Sorghum bicolor, Oryza sativa, Setaria italic and Zea mays), fruits (Vitis vinifera), trees (Populus trichocarpa, Amygdalus persica, Citrus sinensis and Malus pumila), vegetables (Brassica oleracea, Brassica rapa, Cucumis sativus and Lycopersicon esculentum), legumes (Glycine max), Gossypium, Solanum tuberosum and model dicot and monocot species (Arabidopsis thaliana and Brachypodium distachyon). Shown in Table 1, the 13 dicots used in this study have an average genome size of 618.86 Mb compared with that of 780.54 Mb average genome size of the 5 monocots. In addition, higher number of chromosomes were observed in 13 dicots (14.2 in average) compared with that in 5 monocots (9.2 in average). In this study, 135 PAL and 479 CHS genes were identi ed in the 18 plant species (Table 1 and Table S2). For PAL genes, the maximum number of PAL genes was found in Zea mays (12) and Vitis vinifera (11), respectively. More number of PAL genes were found in monocots (9.4 in average) than that in dicots (6.8 in average). Similar results were also obtained in CHS genes, namely more gene number in monocots (38.2 in average) than that in dicots (22.2 in average). The maximum number of CHS genes was detected in Glycine max (43) and Sorghum bicolor (55) in dicots and monocots, respectively. Notably, 12 CHS and 8 PAL genes were identi ed in Solanum tuberosum. Based on protein sequence similarities, the evolutionary trees of PAL and CHS gene family were constructed using MEGA 7.0 software (Fig. 1). For the PAL family, 135 protein sequences were divided into 8 subgroups (A-H). The members of subgroups A and B were monocots speci c, while the others belonged to dicots (Fig. 1A). For the CHS family, 479 CHS protein sequences were divided into six subgroups, of which B and D branches belonged only to monocotyledons, while the other subgroups were common in both monocotyledons and dicotyledon (Fig. 1B  StuPALs and StuCHSs genes were unevenly located on chromosomes and they were distributed on four (Chr3. Chr5, Chr9 and Chr10) and ve chromosomes (Chr1. Chr2, Chr5, Chr9 and Chr12), respectively (Fig. 2). StuPALs and StuCHSs genes were mainly distributed on chromosome Chr9 and Chr12, respectively (Fig. 2). Tandem and segmental duplication were the major duplication patterns, which led the expansion of gene families in the process of plant evolution [24,25]. In this study, tandem duplicated genes of StuPALs (StuPAL3 and StuPAL4) and StuCHSs (StuCHS2, StuCHS3, StuCHS4, StuCHS6, StuCHS7 and StuCHS8) were indicated by yellow blocks in Fig. 2, which suggested that gene duplication played critical roles in the expansion of StuPALs and StuCHSs gene families.
To better explore the structural features of the StuPAL and StuCHS genes, the coding domain sequences and corresponding genomic sequences of the same StuPALs and StuCHSs gene were submitted to GSDS website together to display their exon/intron features. The results revealed the number of introns per gene varied from zero to a maximum of two (Fig. 3). All StuPAL and StuCHS genes had only one or zero intron except StuCHS1, which had two introns. Six StuPAL and nine StuCHS genes had one intron and two StuPAL (StuPAL4 and StuPAL8) and two StuCHS (StuCHS3 and StuCHS7) genes had no intron.
To further understand the potential functions of StuPAL and StuCHS genes, 10 motifs were screened within each protein sequence using the MEME website. The detailed information of predicted motifs was showed in Fig. 4 and Table 3. The length of predicted motifs was 21-50 aa and 8-50 aa for StuPAL and StuCHS proteins, respectively. Among them, motif 1, 2, 3, 4, 10 were detected in all eight StuPAL genes, StuPAL1 and StuPAL4 lacked motif 5 as well as StuPAL4 and StuPAL8 lacked motif 6, 7, 8, 9. Interestingly, the same motif appears more than once in the same StuPAL member, such as motif 1, 4 and 6. The same patterns were also detected in StuCHS proteins. To be noteworthy, only three motifs (motif 2, 4, 10) were screened in StuCHS5 (Fig. 4). represented the Chal-sti-synt-N domain, and motifs 3 and 4 possessed the Chal-sti-synt-C domain and no known domain was detected in the rest motifs among StuCHSs (Table 3).

MicroRNA Targeting Prediction of StuPAL and StuCHS Genes
To explore the potential roles of miRNAs involved in regulation of StuPALs and StuCHSs genes, the software psRNATarget Server was used to predict possible miRNAs based on the genomic sequences of all StuPALs and StuCHSs genes. In total, 68 and 61 putative miRNAs targeting all eight StuPALs and twelve StuCHSs genes, respectively, were detected and constructed the relationship network using Cytoscape software (Fig. 5). We further analysis the regulation network and found that StuPAL1, StuPAL6 and StuPAL7 were targeted by the top three miRNAs. The stu-miR8015-3p targeted StuPAL1, StuPAL2, StuPAL3 and StuPAL4 (Fig. 5A). For StuCHS genes, StuCHS10 was targeted by 23 miRNAs and stu-miR8040-3p targeted StuCHS8, StuCHS10 and StuCHS12. StuCHS6 and StuCHS7 are targeted by some common miRNAs. (Fig. 5B).

StuPAL s and StuCHSs Genes Cis-acting Element Analysis
Gene expression was mainly regulated at the transcriptional level and cis-acting elements regulated the precise initiation and transcriptional e ciency of gene transcription by binding to transcription factors. Cis-acting elements in StuPAL and StuCHS gene promoters were analyzed using the PlantCARE database. In our study, promoter sequences (1500 bp from the coding start) of StuPALs and StuCHSs genes were used to screen putative cis-acting elements, which involved in auxin (TGA-element and AuxRR-core), salicylic acid (TCA-element), abscisic acid responsiveness (ABRE), gibberellin (TATC-box, GARE-motif and P-box), MeJA (CGTCA-motif and TGACG-motif), defense and stress responsiveness (TC-rich repeats), drought-inducibility (MBS), low-temperature responsiveness (LTR), MYBHv1 binding site (CCAAT-box), endosperm expression (GCN4), cell cycle regulation (MSA-like) and the anaerobic induction (ARE). In total, 70 and 116 cis-acting elements were detected in StuPAL and StuCHS gene promoters (Fig. 6). Among them, 3 (StuPAL2)-12 (StuPAL5 and 7) and 4 (StuCHS8) -13 (StuCHS11) cis acting regulatory elements were varied in two gene families. In StuPAL gene promoters, 14 ABREs and 13 AREs counted the top two elements (Fig. 6A). And 21 ABA responsive and 31 MeJA responsive elements were detected in StuCHS promoters, indicating that StuCHS genes may be regulated by the two hormones (Fig. 6B).

Expression Analysis of StuPALs and StuCHSs Genes in Various Tissues of Potato
The expression patterns of genes are usually correlated with their functions. To analysis the expression patterns of StuCHSs and StuPALs genes in various tissues and organs, RNA-Seq data from PGSC were downloaded and analyzed. A heatmap was generated using the FPKM data of all StuCHS and StuPAL genes by the software Mev 11.0 (Fig. 7). Thirteen different tissues and organs were included in the analysis. All StuPALs genes showed high expression levels in all tissues except StuPAL1, which showed very low expression level in all tissues. Notably, StuPAL4 had the highest expression levels in all tissues except in stamens. StuPAL5 had relatively low expression levels in stamens (Fig. 7A). For StuCHS genes, their expression patterns were very diverse (Fig. 7B). StuCHS5, StuCHS9 and StuCHS11 (except in callus and stolons) showed constitutive expression in all detected tissues. StuCHS1 was only expressed in roots and tubers, while StuCHS6 and StuCHS7 were only highly expressed in shoots, sepals and petioles. StuCHS3, StuCHS4, StuCHS8 and StuCHS10 showed little expression in detecting tissues. Highly expressed in shoots, sepals, stolons, petioles, petals and fruits was detected in StuCHS2, while StuCHS12 showed high expression level in sepals, stamens, owers, petals, carpels and fruit (Fig. 7B).

Expression Analysis of StuPAL and StuCHS Genes in Potato under Environmental Stresses
To further insight into the roles of StuPAL and StuCHS genes in response to abiotic (salt, drought and heat), biotic (PIL, BTH and BABA) and hormone (ABA, BAP, GA 3 and IAA) stresses, we analysis the fold changes of FPKM using the expression data obtained from the Spud DB database (Fig. 8). For abiotic stresses, seven of eight StuPAL genes (except StuPAL8) were down-regulated after salt and drought stresses and StuPAL6 and StuPAL7 were strongly up-regulated in response to heat stress (Fig. 8A). The expression patterns were varied in StuCHS genes in response to abiotic stresses (Fig. 8B). StuCHS3 and StuCHS12 were up-regulated (log 2 fold change>1) in both salt and drought stresses, while only StuCHS8 was signi cantly down-regulated in salt stress. StuCHS9 StuCHS11 and StuCHS12 were strongly upregulated in heat stress. The rest StuCHS gene members have little response (|log 2 fold change|<1). For biotic stresses, all StuPAL genes were down-regulated in response to PIL (except StuPAL4 and StuPAL8) and BTH (except StuPAL7) stresses while StuPAL4 and StuPAL6 were signi cantly up-regulated in response to BABA stress (Fig. 8A). All StuCHS genes were down-regulated in response to BABA and PIL (except StuCHS1) stress, while ve and four StuCHS genes were up-and down-regulated in response to BTH stress and the rest three StuCHS genes had no response to BTH stress (Fig. 8B). For exogenous hormone treatments, all StuPAL genes had no express in response to ABA, GA3 (except StuPAL1) and IAA (except StuPAL6 and StuPAL7), while all except StuPAL1 were down-regulated in response to BAP treatment (Fig. 8A). No StuCHS genes were induced by BAP and IAA, while StuCHS11 and StuCHS12 were strongly up-regulated after GA 3 treatment. For ABA treatment, ve StuCHS genes, namely StuCHS1, StuCHS3, StuCHS8, StuCHS11 and StuCHS12, were up-regulated while StuCHS2, StuCHS4 and StuCHS10 were down-regulated (Fig. 8B).

Anthocyanin content analysis in three colored potato varieties
Three colored varieties of potato were selected in this study. Among them, JYS are purple peel with purple pulp, while both peel and pulp of 1-11 is red and 16-A2 shows yellow both peel and pulp (Fig. 9A). The content of anthocyanins in the peel and pulp were signi cant difference among the three materials. The anthocyanin content was the highest in JYS peel while it was the lowest in 16-A2 pulp (P < 0.05) (Fig. 9B).

Expression Analysis of StuPAL and StuCHS Genes in Peel and Pulp of Colored Potato
To further analysis the functions of StuPAL and StuCHS genes in anthocyanin biosynthesis pathways, the expression levels of StuPAL and StuCHS genes in three colored potato varieties were detected using qRT-PCR (Fig. 10). For StuPAL genes, StuPAL1-StuPAL8 were mainly expressed in the peel of the three potato varieties, but almost not expressed in the pulp (Fig. 10A). Among them, StuPAL2, StuPAL3, StuPAL4 and StuPAL6 had the highest expression in JYS peel, followed by 1-11 peel, and the lowest expression in 16-A2 peel.
The expression patterns of the rest genes are different. StuPAL1, StuPAL5, StuPAL7 and StuPAL8 had the lowest expression in 1-11 peel. Among them, StuPAL1 had the highest expression in JYS peel, followed by 16-A2 peel. The expression levels of StuPAL5 and StuPAL8 in JYS and 16-A2 peel were basically the same, while StuPAL7 had the highest expression in 16-A2 peel, followed by JYS peel. However, the expression of StuCHS genes in the peel and pulp of the three varieties changed greatly (Fig. 10B). StuCHS1, StuCHS4, and StuCHS6 had almost no changes in the three different colored potatoes peel. StuCHS3 was highly expressed in JYS peel, followed by 1-11 peel, and almost no expression in 16-A2 peel. StuCHS5 had the highest expression in JYS peel, and there was little difference between 1-11 and 16-A2 peel. StuCHS7 had the highest expression in 16-A2 peel and almost no expression between JYS and 1-11 peel. However, StuCHS9 and StuCHS11 had the highest expression in 1-11 peel, followed by JYS peel, and almost no expression in 16-A2 peel. Most of the StuCHS genes were expressed at low levels in the pulp, except StuCHS1 was clearly expressed in JYS pulp. StuCHS3, StuCHS9 and StuCHS11 were hardly expressed in the pulp among three potato varieties. The expression of StuCHS5 in JYS, 1-11 and 16-A2 pulp did not change much. StuCHS4, StuCHS6 and StuCHS7 was obviously expressed in JYS and 16-A2 pulp, but almost not expressed in 1-11 pulp. The expression of StuCHS2, StuCHS8, StuCHS10 and StuCHS12 were lower in the three varieties, and the data was not shown.

Discussion
Bioinformatics analysis of PAL and CHS genes in higher plants As the rst and rate-limiting enzyme in the phenylpropanoid pathway, PAL plays a key role in plant growth, development and environmental adaptation [26]. In this study, the BLAST alignment program was performed and eight StuPAL genes were obtained (Table  1). Our results showed that signi cant differences of the PAL gene family members among various species were observed (Fig. 1). The genome duplication may occur in the evolution of potato because the number of this gene in potato genome is twice that in A. thaliana genome. Duplication events including genome duplications, segmental duplications and tandem duplications may contribute to the expansion and evolution of gene families [27]. StuPAL3 and StuPAL4 were identi ed tandem duplication gene pair based on their chromosomal distribution and phylogenetic relationships (Fig. 2). In previous studies, the CHS gene family acts key roles in plant growth and development and contains smaller gene family in most plant genomes [28]. For example, eight CHS genes in Petunia hybrid [29], six in Ipomoea [30], four in Arabidopsis thaliana [31] and fourteen in Zea mays [28] were detected. In our study, 12 CHS genes were identi ed in potato and it was considered that tandem duplication was the major force for the gene family expansion during evolutionary process.
Six tandemly duplicated genes were found in 12 StuCHS genes (Fig. 2). Gene structure and conserved motif analysis provide more evidences for the evolutionary relationship of multigene family and gene members with close evolutionary relationships have similar gene structure and motif composition [32,33]. In this study, gene structure and motif characteristics of PAL and CHS gene members in potato also accorded with this pattern (Fig. 3 and Fig. 4). In the PAL family, except that StuPAL4 and StuPAL8 do not contain introns, the other members all contain one intron (Fig. 3). Similarly, the motif characteristics of StuPAL4 and StuPAL8 are different from those of other members, but both contain L-Aspartase-like feature domains. In the CHS gene family, StuCHS3 and StuCHS7 do not contain introns, and the other members all contain 1-2 introns. The motif characteristics of StuCHS3 and StuCHS7 are different from other members, and StuCHS5 only contains three motifs (Fig. 4). All gene members contain Chal_sti_synt_C or Chal_sti_synt_N characteristic structures (Table  3). In addition, the software psRNATarget Server was used to predict the potential miRNA of StuPAL and StuCHS genes. The results showed that StuPAL and StuCHS genes had 68 and 61 miRNA respectively, indicating that their functions were accurately regulated. StuCHS2, StuCHS5, StuCHS10 and StuPAL genes were regulated by multiple miRNAs (Fig. 5). As binding sites of transcription factors, cisacting elements determine the expression pattern of genes to some extent [34]. A large number of cis-acting elements in response to environmental stresses were identi ed in the promoter sequences of StuPAL and StuCHS genes, indicating that their expression level was precisely regulated by environmental stresses (Fig. 6).

Expression pro les of StuPAL and StuCHS genes in response to environment stresses and different tissues
Gene expression pattern is an important aspect of gene function. High throughput sequencing provides a convenient way to study the gene expression patterns. As the rst key genes in the phenylpropanoid pathway, PAL genes were precisely regulated in all respects, such as the pre-and post-transcriptional levels [35]. Different expression patterns in different organs were observed in many species. However, very few studies on the PAL genes in potatoes were reported. In this study, expression data were downloaded from PGSC. As shown in Fig. 7A, StuPAL1 was speci c expressed in carpels. StuPAL2, StuPAL3, StuPAL5 and StuPAL8 highly expressed in all detected organs except in stamens. The rest members, StuPAL4, StuPAL6 and StuPAL7 were highly expressed in all detected organs. In previous study, PAL genes play key roles in response to drought stress after being treated for 24 and 36 h in Salvia miltiorrhiza [26], while there is no response to drought stress for all StuPAL gene members (Fig. 8A). This may be due to the different simulators (PEG-6000 and mannitol for S. miltiorrhiza and potatoes, respectively) of the treatments, the different treatment time and the different phase of plant growth and development. Heat treatment prior to storage alleviated chilling injury and enhanced PAL activity in banana fruit [36]. StuPAL6 and StuPAL7 were strongly up-regulated after heat stress in this study. In S. miltiorrhiza, all SmPALs were down-regulated after 12 h MeJA treatment, while SmPAL1 was up-regulated against after 48 h treatment [26]. However, all eight StuPAL genes except StuPAL1 were downregulated after BAP treatment and no response to the rest three hormones (IAA, ABA and GA 3 ) treatments in our study (Fig. 8A). For StuCHS genes, the expression patterns among different tissues were very diverse (Fig. 7B). StuCHS5, StuCHS9 and StuCHS11 (except in callus and stolons) showed constitutive expression in all detected tissues. While the rest StuCHS genes were expressed in speci c tissues, suggesting functional diversi cation of StuCHS genes (Fig. 7B). Plant growth and development are strongly in uenced by environmental factors, such as salt, drought, heat, diverse pathogens and chemical inducers during their life cycles. Many stress-related genes were induced or restrained to adapt the adverse environments [37,38]. The CHS family has been reported to be regulated by hormone treatment [39] and light [40]. However, few studies about CHS genes responding to environmental stresses have been reported in potatoes. Thus, the expression changes of StuCHS genes in response to environmental stresses based on RNA-Seq data have been explored in this study. StuCHS3 and StuCHS12, induced by salt, drought, ABA, GA 3 and BTH, were identi ed as key CHS genes in response to environmental stresses (Fig. 8B).
The contents of anthocyanin are varied in colored potato peel and pulp As one of the most important food crops, potato is regarded as an important source of antioxidants for the human diet. Potato tubers contain a lot of polyphenols. Anthocyanins, the major count of the visible polyphenols, are very rich in different colored potatoes [41].
Anthocyanin not only plays important physiological roles in plant growth, development and stress response, but also have functions in the treatment of cardiovascular diseases as an antioxidant in human body [11]. Therefore, cultivating potato varieties with high anthocyanin content is an important measure to enhance people's health. In purple and red potatoes, the levels of anthocyanins are signi cantly higher than those in white and yellow tubers [42]. As shown in Fig. 9B, the levels of anthocyanin in peel of colored potato is signi cantly higher than that in pulp. Among them, purple potato peel had the highest anthocyanin level, followed by red potato peel, and yellow peel had the lowest anthocyanin level.

Expression analysis of StuPAL and StuCHS genes in colored potato tubers
Detecting the expression levels of StuPAL and StuCHS genes in potato tubers will help to further understand their functions in anthocyanin biosynthesis pathways. For StuPAL genes, the expression levels of all StuPAL genes were mainly expressed in the peel of the three potato varieties, but almost not expressed in the pulp. It was speculated that they were mainly involved in the accumulation of anthocyanin in peel, but not in pulp. The expression changes of StuPAL2, StuPAL3, StuPAL4 and StuPAL6 were consistent with the changes in anthocyanin content (Fig. 10A). It is speculated that these four genes were more likely to be involved in the anthocyanin synthesis. However, the StuCHS genes were quite different. StuCHS family members were expressed in both peel and pulp. The expression levels of StuCHS3, StuCHS4, StuCHS5, StuCHS6, StuCHS9and StuCHS11 were higher in peel than pulp among three potato varieties, while StuCHS1 is only highly expressed in purple pulp (Fig. 10B). These results suggested that StuCHS genes could be involved in both peel and pulp anthocyanin accumulation. This also showed that StuPAL and StuCHS genes had different functions in the regulation of anthocyanin synthesis in different colored potato varieties. In conclusion, these ndings provided in this study lay an important basis to further explore the biological functions of the StuPAL and StuCHS gene family in colored potato.

Conclusion
Anthocyanins are the most abundant class of plant avonoid pigments. In our study, 135 PAL and 479 CHS genes were comprehensively identi ed in the 18 plant species. Among them, Eight StuPAL and 12 StuCHS genes were analysised in potato for the rst time. These genes were mainly distributed on chromosome Chr9 and Chr12, respectively. StuPAL genes mainly contained L-Aspartase-like feature domains and StuCHS genes members contained Chal_sti_synt_C or Chal_sti_synt_N characteristic structures. They are fairly conserved and their gene structures were related to their functions. StuPAL genes were mainly expressed in the peel of colored potato, StuPAL2, StuPAL3, StuPAL4 and StuPAL6 had the highest expression, consistent with the content of anthocyanins. StuCHS family genes were expressed in the peel and pulp. These results suggested that StuPAL genes could be involved in the synthesis of anthocyanins in the colored potato tuber peel, while StuCHS genes might performed functions both in peel and pulp. These researches provided insights into the characteristics of PAL and CHS genes and could facilitate to further explore the biological functions of the StuPAL and StuCHS gene family in colored potato.

Materials And Methods
Sequence Retrieval and Phylogenetic Trees Constructed of PAL and CHS Genes in 18 Plant Species Protein sequences of PAL and CHS genes of A. thaliana were downloaded from the website TAIR (https://www.arabidopsis.org/). These protein sequences were used as query sequences and Blastp program was performed in Phytozome 12  [49]. To predict these genes-targeted miRNAs, genomic DNA sequences of these genes were searched against the published Solanum tuberosum miRNAs sequences using psRNATarget Server with default parameters (http://plantgrn.noble.org/psRNATarget/) [50]. The interaction networks of the predicted miRNAs and the corresponding target genes were visualized using Cytoscape software (https://cytoscape.org/).

Plant samples and Determination of anthocyanin content
Three potato cultivars were selected in this study based on the color of tuber peel and pulp: JYS, 1-11 and 16-A2 (Fig. 9A). These varieties were grown in elds located in Wulong, Chongqing, during the 2020/2021 season and tubers were harvested at their physiological maturity.
Anthocyanin content of tuber peel and pulp of three potato cultivars was measured using sodium phosphate, dibasic-citric acid buffer method [51]. All samples were ground to powder and about 2 g powder was placed into 40 mL of extraction buffer (Sodium phosphate, dibasic-Citric Acid buffer) and incubated at 60℃ for two hours. After cooling, keep volume to 100 mL and let stand for 2 hours.

qRT-PCR analysis
Total RNA of skin and tuber esh from three potato cultivars was extracted using the Pure Plant RNA Kit (Sangon, China) and convert it to cDNA by reverse transcription. In addition, primers for qRT-PCR of StuPAL and StuCHS genes were designed using Premier 5.0 (Table S1)  Availability of data and materials The gene expression data were downloaded from the PGSC website with accession number SRA030516 in NCBI.

Competing interest
The authors declared no con ict of interest in the authorship and publication of this document.   Exon-intron structures of StuPAL and StuCHS genes. The phylogenetic tree was constructed using MEGA 7 software. Yellow rectangles represent CDS, blue rectangles represent the upstream and downstream, black thin lines represent intron. A: StuPAL genes; B: StuCHS genes.

Figure 4
Phylogenetic relationships and architecture of the conserved protein motifs in StuPAL and StuCHS genes. The motifs, numbered 1-10, are displayed in different colored boxes. The length of the protein can be estimated using the scale at the bottom. A: StuPAL genes; B: StuCHS genes.  The promoter cis-elements analysis of StuPALs and StuCHSs. The 1500 bp DNA fragments upstream of the ATG staring code of genes were analyzed using online analysis software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). A: StuPAL genes; B: StuCHS genes.

Supplementary Files
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