Systematic Analysis of Galactinol Synthase and Raffinose Synthase Gene Families in Potato and Their Expression Patterns in Development and Abiotic Stress Responses

Raffinose family oligosaccharides (RFOs) are very important for plant growth, development, and abiotic stress tolerance. Galactinol synthase (GolS) and raffinose synthase (RFS) are critical enzymes involved in RFO biosynthesis. However, the whole-genome identification and stress responses of their coding genes in potato remain unexplored. In this study, four StGolS and nine StRFS genes were identified and classified into three and five subgroups, respectively. Remarkably, a total of two StGolS and four StRFS genes in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. Subsequent analysis revealed that StGolS4 exhibited significantly high expression levels in transport-related tissues, PEG-6000, and ABA treatments, with remarkable upregulation under salt stress. Additionally, StRFS5 showed similar responses to StGolS4, but StRFS4 and StRFS8 gene expression increased significantly under salt treatment and decreased in PEG-6000 and ABA treatments. Overall, these results lay a foundation for further research on the functional characteristics and molecular mechanisms of these two gene families in response to ABA, salt, and drought stresses, and provide a theoretical foundation and new gene resources for the abiotic-stress-tolerant breeding of potato.


Introduction
Potato (Solanum tuberosum L.) is globally recognized as the foremost non-cereal food crop, and it belongs to the genus Solanum of family Solanaceae [1,2]. It is widely cultivated and essential for food security around the world [3]. Regrettably, potato production is frequently plagued by an array of abiotic stresses, with salt and drought stresses representing the primary factors that restrict yields. Salt stress inhibits tuber growth and causes yield losses of up to 60% in potato [4]. In a recent study, 103 commercial potato cultivars were found to have a decreased fresh tuber weight by 54% under drought stress, with the cultivar Connantre showing the greatest reduction [5,6]. Therefore, improving the salt and drought tolerance of potato is a viable pathway to ensure agricultural sustainability.
Some osmolytes, including proline, mannitol, trehalose, and raffinose, are commonly accumulated in various plant species in response to different abiotic stresses [7,8]. Raffinose family oligosaccharides (RFOs) are small and soluble molecules that serve various functions in plant biology. They act as primary transport carbohydrates in some plant species, serve as signaling molecules after pathogen attack and physical injury, and accumulate in vegetative tissues as a response to diverse abiotic stresses [9]. The accumulation of raffinose is considered as an important indicator of the plant stress response, particularly under drought and salt stresses. Several studies have shown that drought and salt stresses significantly increase raffinose levels [10,11]. Some researchers propose that raffinose's natural hygroscopic properties might protect plants from drought stress [12]. Additionally, the from this study lay a foundation for further research on the functional characteristics and molecular mechanisms of the StGolS and StRFS genes in response to ABA, salt, and drought stresses in potato, and they provide a theoretical basis and new gene resources for the development of potato varieties tolerant to abiotic stress.

Plant Material and Abiotic Stress Treatments
The doubled monoploid potato clone DM1-3 516 R44 (DM, 2n = 2x = 24) was used in this study and its tissue-cultured plants were grown in test tubes with 10 mL Murashige and Skoog (MS) solid medium containing 3% (w/v) sucrose (pH 5.6~5.8). The photoperiod in the light incubator for DM tissue-cultured plants was 16 h daylight at 22 • C and 8 h darkness at 18 • C [27,28].
The concentrations of NaCl, PEG-6000, and abscisic acid (ABA) in the treatments were the same as those in previous studies [29][30][31]. Here, 45-day-old test tube plantlets were used for ion balance with 10 mL MS liquid medium with or without 400 mM NaCl (the final concentration of NaCl was 200 mM) for 24 h, and subsequently all of the liquid media were removed immediately from the test tubes. The aboveground parts of the test tube plantlets with 200 mM NaCl treatment were sampled at 3 h (S1), 6 h (S2), 12 h (S3), and 24 h (S4) after the 24 h ion balance, and the 24 h samples without 200 mM NaCl treatment (after the 24 h ion balance with 10 mL MS liquid medium) were considered as the controls (CK). All of the collected samples were placed into liquid nitrogen immediately and stored at −80 • C.
Similar to the previous methods, 30% PEG-6000 + MS liquid medium (simulating drought stress) and 0.2 mM abscisic acid + MS liquid medium (ABA) were added to the test tube plantlets, respectively. After balancing for 24 h, the liquid medium was removed. At 3 h, 6 h, 12 h, and 24 h (P1, P2, P3, and P4; A1, A2, A3, and A4), the aboveground parts of the test tube plantlets were collected and stored at −80 • C. The 24 h samples without any stress (after the 24 h ion balance with 10 mL MS liquid medium) were the controls (CK). Each experiment was repeated three times with three biological replicates and four test tube plantlets for each replicate.

Identification of the StGolS and StRFS Genes
The whole-genome data used in this study were downloaded from the Spud DB (http://spuddb.uga.edu, accessed on 11 January 2022), which contains the newest potato genome DM v6.1 [32]. Amino acid sequences of the previously identified genes from Arabidopsis were used to perform a BLASTp search to obtain homologs with an E-value cutoff of 10 −5 [33]. The profile hidden Markov model of the HMMER3.0 program was applied to verify the identification results [34]. The amino acid sequence information of each gene was further verified by the Pfam [35] and SMART [36] online tools. The physicochemical properties of each protein, including the molecular weight (MW), theoretical isoelectric point (pI), and hydrophilicity mean (GRAVY), were calculated by the online tool ProtParam [37].

Chromosome Localization of the StGolS and StRFS Genes
The position information of potato genes on chromosomes can be obtained from the Spud DB. Based on the relative position of each gene on each chromosome, the chromosome localization map of the genes was drawn using the Mapinspect software (http://www. softsea.com/download/MapInspect.html, accessed on 11 January 2022).

Phylogenetic Tree Construction of the StGolS and StRFS Genes
Using the MEGA7.0 software, amino acid sequences from potato, Arabidopsis, and tomato were aligned by Clustal W, and a phylogenetic tree was generated based on the neighbor-joining method (NJ) [38]. The phylogenetic tree was drawn and beautified using the iTOL software (https://itol.embl.de/, accessed on 3 April 2023).

Structure Drawing and Motif Analysis of the StGolS and StRFS Genes
Gene structure diagrams were drawn using the software TBtools [39]. The conserved functional motifs of the genes were predicted by the MEME software [40].

Cis-Element Analysis in Promoters of the StGolS and StRFS Genes
In order to identify the cis-elements in the promoters of the StGolS and StRFS genes, 1500-bp sequences before the initiation codons (ATG) were extracted from the potato genome database. We submitted the promoter sequences to the website of the PlantCARE database and analyzed the types, positions, and numbers of cis-elements [41,42]. The results were visualized using the software TBtools [39].

Syntenic Analysis of the GolS and RFS Genes
For the gene synteny among different genomes, we selected potato, tomato, and A. thaliana genomes for syntenic analysis. The assemblies of tomato and A. thaliana were obtained from TAIR (http://www.arabidopsis.org, accessed on 3 April 2023) and Phytozome (http://www.phytozome.net, accessed on 3 April 2023), respectively. The determination of syntenic blocks among potato, tomato, and A. thaliana was performed using the TBtools software [39].

qRT-PCR Analysis
Gene expression analysis was conducted using the quantitative real-time PCR (qRT-PCR) technique, as described previously [43,44]. Total RNA was extracted using the RNAprep Pure Plant Plus Kit (DP441, TianGen, Beijing, China), following the manufacturer's instructions, and was reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, Takara, Dalian, China). Subsequently, qRT-PCR was performed in triplicate for each sample on the CFX96 Touch TM Real-Time PCR Detection System (Bio-Rad, Washington, DC, USA) using the TB Green ® Premix Ex Taq™ I (RR820A, Takara, Dalian, China). The reference gene StActin97 was used to normalize the gene expression levels. The relative expression levels of the genes were calculated using the Gene Expression Macro software, version 1.1 (Bio-Rad Laboratories, Washington, DC, USA). The sequences of the specific primers used for qRT-PCR are listed in Supplementary Table S1.

Identification of the GolS and RFS Gene Families in Potato
Based on the amino acid sequences of the AtGolS and AtRS genes, a comprehensive analysis was conducted to identify the GolS and RFS genes in potato. The identification process involved the utilization of SMART and Pfam, which confirmed the presence of conserved domains. Four GolS genes were identified and designated as StGolS1 to StGolS4, while nine RFS genes were identified and labeled as StRFS1 to StRFS9 (Table 1).
Subsequently, an in-depth investigation was carried out to assess the physicochemical properties of the StGolS and StRFS genes. The analysis revealed that the StGolS proteins exhibited variations in protein length, molecular weight, and theoretical isoelectric point (pI), spanning a range of 322 to 348 aa, 36.74 to 39.68 kDa, and 5.44 to 6.67, respectively (Table 1). Conversely, the StRFS proteins demonstrated a wide range of amino acid lengths, ranging from 110 to 874 aa, with molecular weights varying between 12.51 and 97.37 kDa.
Notably, all StGolS and StRFS proteins exhibited acidic characteristics, as indicated by pI values below 7.00.

Chromosomal Distribution of the StGolS and StRFS Genes
The genomic distribution of the identified StGolS genes in potato was examined, revealing their presence on two distinct chromosomes ( Figure 1a and Table 1). Specifically, genes StGolS1 and StGolS2 were found on chromosome 1, while genes StGolS3 and StGolS4 were located on chromosome 2. These results provide valuable insights into the spatial arrangement of the StGolS genes within the potato genome. In the case of the StRFS genes, their distribution across the 12 chromosomes of the potato genome was found to be non-uniform ( Figure 1b and Table 1). Four StRFS genes were identified on chromosome 3, indicating a relatively higher density in this region. In contrast, the number of StRFS genes observed on chromosomes 1 and 2 was minimal, with only one gene present on each of these chromosomes. Chromosome 7 exhibited an intermediate pattern, housing three StRFS genes. These findings shed light on the heterogeneous distribution of the StRFS genes throughout the potato genome.

Phylogenetic Analysis of the GolS and RFS Genes from Several Differnet Species
A phylogenetic analysis was conducted to investigate the relationships of GolS and RFS family genes among three different plant species, namely potato, tomato [45,46], and Arabidopsis [47]. The protein sequences of four StGolSs, seven AtGolSs, and four SlGolSs were aligned, and a phylogenetic tree was constructed (Figure 2a and Supplementary Ma-

Phylogenetic Analysis of the GolS and RFS Genes from Several Differnet Species
A phylogenetic analysis was conducted to investigate the relationships of GolS and RFS family genes among three different plant species, namely potato, tomato [45,46], and Arabidopsis [47]. The protein sequences of four StGolSs, seven AtGolSs, and four SlGolSs were aligned, and a phylogenetic tree was constructed ( Figure 2a and Supplementary Materials, Table S2). Based on their homology relationships, the four StGolS proteins could be classified into three subgroups. StGolS2 and SlGolS1 belonged to subgroup I and showed homology with AtGolS5 and AtGolS6, respectively. The branches of the phylogenetic tree indicated a close genetic relationship between the potato and tomato GolS genes in subgroup III, which contained StGolS3 and StGolS4. Subgroup V comprised StGolS1 and SlGolS3, which displayed homology with AtGolS1. Subgroups II and IV consisted exclusively of AtGolS proteins, with no corresponding proteins found in potato and tomato.
For the RFS genes, multiple sequence alignment was performed on the protein sequences from potato, tomato, and Arabidopsis, followed by the construction of a neighborjoining tree using MEGA7.0 ( Figure 2b and Supplementary Materials Table S2). The RFS genes were categorized into five distinct groups based on their homology relationships. Group I encompassed StRFS3, StRFS4, and SlRS1, which exhibited homology with AtRS4. Group II included StRFS5, StRFS6, StRFS7, SlRS2, and SlRS4, showing homology with AtRS5. StRFS1 and SlRS6 comprised group III, displaying homology with AtRS1. Group IV consisted of StRFS2 and SlRS3, which exhibited homology with AtRS2. Finally, group V was composed of StRFS8, StRFS9, and SlRS5, demonstrating homology with AtRS6.

Gene Structure and Motif Composition of the StGolS and StRFS Genes
The arrangement of exons and introns plays a crucial role in the evolution of gene families [48]. A phylogenetic tree was constructed using full-length protein sequences (Figures 3 and 4) to establish relationships between gene sequences and their corresponding exon/intron distributions and motif compositions. Gene structure and motif analyses of the StGolS and StRFS genes were performed to gain insights into their evolutionary characteristics.
Regarding the StGolS genes, there was observed variation in the exon/intron organization across different members of the family (Figure 3a,b). Specifically, StGolS1 consisted of four exons, whereas the remaining three StGolS genes contained three exons within their coding regions. In order to obtain a deeper understanding of the diversity of StGolS proteins, conserved motifs were identified using the MEME program. A total of eight conserved motifs were discovered, with motif 8 being the shortest and consisting of eight aa (Supplementary Materials, Table S3). The distribution of these motifs varied among different subgroups (Figure 3c). Motifs 1, 2, 3, 4, 7, and 8 were found to be shared among all StGolS members, suggesting their conserved nature. On the other hand, motifs 5 and 6 were deficient in StGolS2.

Gene Structure and Motif Composition of the StGolS and StRFS Genes
The arrangement of exons and introns plays a crucial role in the evolution of gene families [48]. A phylogenetic tree was constructed using full-length protein sequences (Figures 3 and 4) to establish relationships between gene sequences and their corresponding exon/intron distributions and motif compositions. Gene structure and motif analyses of the StGolS and StRFS genes were performed to gain insights into their evolutionary characteristics.
Regarding the StGolS genes, there was observed variation in the exon/intron organization across different members of the family (Figure 3a,b). Specifically, StGolS1 consisted of four exons, whereas the remaining three StGolS genes contained three exons within their coding regions. In order to obtain a deeper understanding of the diversity of StGolS proteins, conserved motifs were identified using the MEME program. A total of eight conserved motifs were discovered, with motif 8 being the shortest and consisting of eight aa (Supplementary Materials, Table S3). The distribution of these motifs varied among different subgroups (Figure 3c). Motifs 1, 2, 3, 4, 7, and 8 were found to be shared among all StGolS members, suggesting their conserved nature. On the other hand, motifs 5 and 6 were deficient in StGolS2. of the StGolS and StRFS genes were performed to gain insights into their evolutionary characteristics.
Regarding the StGolS genes, there was observed variation in the exon/intron organization across different members of the family (Figure 3a,b). Specifically, StGolS1 consisted of four exons, whereas the remaining three StGolS genes contained three exons within their coding regions. In order to obtain a deeper understanding of the diversity of StGolS proteins, conserved motifs were identified using the MEME program. A total of eight conserved motifs were discovered, with motif 8 being the shortest and consisting of eight aa (Supplementary Materials, Table S3). The distribution of these motifs varied among different subgroups (Figure 3c). Motifs 1, 2, 3, 4, 7, and 8 were found to be shared among all StGolS members, suggesting their conserved nature. On the other hand, motifs 5 and 6 were deficient in StGolS2. The exon-intron architecture of the StRFS genes was thoroughly investigated by aligning their genomic and coding sequences, as depicted in Figure 4a,b. The comprehensive analysis unveiled a pattern wherein the majority of StRFS genes exhibited multiple introns, ranging from 3 to 13, with the sole exception of StRFS7, which possessed a single intron. A distinct characteristic was observed in StRFS9, which lacked introns entirely and solely comprised exonic regions within their coding sequences. This analysis provides  Table S4). Notably, except for StRFS7 and StRFS9, all other StRFS proteins encompassed the aforementioned eight identified motifs. StGolS9 exclusively contained motif 1, while StGolS7 only possessed motif 8. This meticulous motif analysis contributes valuable information concerning the conservation and potential functional elements within the StRFS proteins.

Cis-Element Analysis in Promoters of the StGolS and StRFS Genes
To gain further insights into the potential functions of the StGolS and StRFS genes in potato, an analysis of the cis-acting elements within their promoter regions was conducted. For the StGolS genes, cis-acting elements were predicted in the 1500-bp sequences upstream of the transcription start site (TSS). Some significant cis-acting elements were identified in the promoters of the StGolS genes, including four elements associated with plant hormone responses, three MYB binding sites, and one cis-acting element involved in defense and stress responses (Figure 5a). It should be noted that, except for the StGolS3 gene, multiple cis-acting elements involved in abscisic acid responsiveness were found in the promoter regions of all StGolS genes, suggesting their potential involvement in ABAmediated processes. The promoter of the StGolS4 gene contained the highest abundance of cis-acting elements, with a maximum of nine, encompassing six different types. Within the cis-acting elements of the StGolS4 gene, there were four cis-acting elements involved in abscisic acid responsiveness and one auxin-responsive element.
A total of 61 cis-elements were detected in the promoter regions of the StRFS genes, belonging to five hormone-related elements, four MYB-related sites, and two stress-related elements (Figure 5b). The hormone-related elements encompassed an ABA response element, salicylic acid (SA) response element, gibberellin (GA) response element, and two auxin response elements. Among the hormone-related elements, the cis-acting element

Cis-Element Analysis in Promoters of the StGolS and StRFS Genes
To gain further insights into the potential functions of the StGolS and StRFS genes in potato, an analysis of the cis-acting elements within their promoter regions was conducted. For the StGolS genes, cis-acting elements were predicted in the 1500-bp sequences upstream of the transcription start site (TSS). Some significant cis-acting elements were identified in the promoters of the StGolS genes, including four elements associated with plant hormone responses, three MYB binding sites, and one cis-acting element involved in defense and stress responses (Figure 5a). It should be noted that, except for the StGolS3 gene, multiple cis-acting elements involved in abscisic acid responsiveness were found in the promoter regions of all StGolS genes, suggesting their potential involvement in ABA-mediated processes. The promoter of the StGolS4 gene contained the highest abundance of cis-acting elements, with a maximum of nine, encompassing six different types. Within the cis-acting elements of the StGolS4 gene, there were four cis-acting elements involved in abscisic acid responsiveness and one auxin-responsive element.
A total of 61 cis-elements were detected in the promoter regions of the StRFS genes, belonging to five hormone-related elements, four MYB-related sites, and two stress-related elements (Figure 5b). The hormone-related elements encompassed an ABA response element, salicylic acid (SA) response element, gibberellin (GA) response element, and two auxin response elements. Among the hormone-related elements, the cis-acting element involved in abscisic acid responsiveness was the most abundant, with a total of 18, distributed in the promoter regions of seven StRFS genes, excluding StRFS4 and StRFS6. Furthermore, cis-acting elements involved in defense and stress responsiveness were found in the promoter regions of the StRFS2, StRFS7, StRFS8, and StRFS9 genes. The cis-element analysis provides valuable information regarding the potential regulatory mechanisms and functional implications of the StGolS and StRFS genes in potato, particularly their involvement in hormone responses and stress-related processes.

Interspecific Synteny of the GolS and RFS Genes from Several Different Species
To further explore the evolutionary relationship between the GolS and RFS genes in potato and the genes in Arabidopsis and tomato, synteny analysis was performed. The results demonstrated a syntenic relationship between four of the StGolS genes and the GolS genes from tomato, followed by two StGolS genes with Arabidopsis ( Figure 6 and Supplementary Materials, Tables S5 and S6). Additionally, a syntenic relationship was observed between six StRFS genes and the RFS genes from tomato, along with four StRFS genes and Arabidopsis (Figure 6 and Tables S5 and S6). Remarkably, a total of two StGolSs (StGolS1 and StGolS2) and four StRFSs (StRFS1, StRFS4, StRFS5, and StRFS8) in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. The number of collinear pairs of StGolS and StRFS genes localized at highly conserved syntenic regions from tomato, belonging to the Solanum family, was greater than that from Arabidopsis, belonging to the dicot species. These results underscore the homology among the GolS and RFS genes of Arabidopsis, potato, and tomato, thus implying potential functional similarities in their respective gene functions.

Interspecific Synteny of the GolS and RFS Genes from Several Different Species
To further explore the evolutionary relationship between the GolS and RFS genes in potato and the genes in Arabidopsis and tomato, synteny analysis was performed. The results demonstrated a syntenic relationship between four of the StGolS genes and the GolS genes from tomato, followed by two StGolS genes with Arabidopsis ( Figure 6 and Supplementary Materials, Tables S5 and S6). Additionally, a syntenic relationship was observed between six StRFS genes and the RFS genes from tomato, along with four StRFS genes and Arabidopsis (Figure 6 and Tables S5 and S6). Remarkably, a total of two StGolSs (StGolS1 and StGolS2) and four StRFSs (StRFS1, StRFS4, StRFS5, and StRFS8) in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. The number of collinear pairs of StGolS and StRFS genes localized at highly conserved syntenic regions from tomato, belonging to the Solanum family, was greater than that from Arabidopsis, belonging to the dicot species. These results underscore the homology among the GolS and RFS genes of Arabidopsis, potato, and tomato, thus implying potential functional similarities in their respective gene functions.
(StGolS1 and StGolS2) and four StRFSs (StRFS1, StRFS4, StRFS5, and StRFS8) in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. The number of collinear pairs of StGolS and StRFS genes localized at highly conserved syntenic regions from tomato, belonging to the Solanum family, was greater than that from Arabidopsis, belonging to the dicot species. These results underscore the homology among the GolS and RFS genes of Arabidopsis, potato, and tomato, thus implying potential functional similarities in their respective gene functions.

Expression Characteristics of the StGolS and StRFS Genes in Different Tissues of Potato
To gain insights into the expression characteristics of the StGolS and StRFS family genes across different tissues (leaf, tuber, stamen, flower, shoot tip, stem, stolon, petiole, and root), we analyzed their gene expression patterns using potato RNA-seq data available in the Spud database (Figure 7). Among the StGolS genes, StGolS2 showed minimal expression throughout development, while StGolS3 displayed only slight expression in tubers. Regarding transport-related tissues (stem, petiole, and root), StGolS1 and StGolS4 were prominently expressed, whereas the expression of other genes in this family was comparatively weak.

Expression Characteristics of the StGolS and StRFS Genes in Different Tissues of Potato
To gain insights into the expression characteristics of the StGolS and StRFS family genes across different tissues (leaf, tuber, stamen, flower, shoot tip, stem, stolon, petiole, and root), we analyzed their gene expression patterns using potato RNA-seq data available in the Spud database (Figure 7). Among the StGolS genes, StGolS2 showed minimal expression throughout development, while StGolS3 displayed only slight expression in tubers. Regarding transport-related tissues (stem, petiole, and root), StGolS1 and StGolS4 were prominently expressed, whereas the expression of other genes in this family was comparatively weak.
Among the nine StRFS genes, StRFS4 and StRFS7 displayed relatively low expression levels across all tissues. StRFS5 and StRFS6 were solely expressed in tubers and roots, while StRFS9 showed expression in leaves and petioles. StRFS1, StRFS2, and StRFS8 exhibited high expression levels in all tissues (Figure 7). By elucidating the expression profiles of the StGolS and StRFS genes in diverse tissues, our analysis provides valuable insights into their tissue-specific functions in potato development.

Expression Patterns of the StGolS and StRFS Genes in Potato under Abiotic Stress
To investigate the involvement of the StGolS and StRFS genes in the response to abiotic stress, we collected qRT-PCR data from various treatments, including salinity, drought, and abscisic acid (ABA), at different time points (3 h, 6 h, 12 h, and 24 h). Importantly, despite similar sequences within subfamilies, not all genes exhibited identical expression levels (Figure 8). Under salt stress conditions, StGolS1, StGolS4, StRFS2, Among the nine StRFS genes, StRFS4 and StRFS7 displayed relatively low expression levels across all tissues. StRFS5 and StRFS6 were solely expressed in tubers and roots, while StRFS9 showed expression in leaves and petioles. StRFS1, StRFS2, and StRFS8 exhibited high expression levels in all tissues (Figure 7). By elucidating the expression profiles of the StGolS and StRFS genes in diverse tissues, our analysis provides valuable insights into their tissue-specific functions in potato development.

Expression Patterns of the StGolS and StRFS Genes in Potato under Abiotic Stress
To investigate the involvement of the StGolS and StRFS genes in the response to abiotic stress, we collected qRT-PCR data from various treatments, including salinity, drought, and abscisic acid (ABA), at different time points (3 h, 6 h, 12 h, and 24 h). Importantly, despite similar sequences within subfamilies, not all genes exhibited identical expression levels ( Figure 8). Under salt stress conditions, StGolS1, StGolS4, StRFS2, StRFS3, StRFS4, StRFS5, StRFS8, and StRFS9 showed significant upregulation. In the presence of PEG-6000, StGolS4 and StRFS5 displayed notable increases in their expression levels. Likewise, the expression of StGolS4, StRFS2, and StRFS5 was prominently upregulated in response to ABA treatment. Furthermore, we observed that the expression levels of StGolS4 and StRFS5 were significantly elevated after exposure to NaCl, PEG-6000, and ABA. Additionally, salt treatment led to a significant increase in the expression of StRFS4 and StRFS8, while PEG-6000 and ABA treatments resulted in decreased expression. Genes with low expression levels (Ct > 35) across all treatments were excluded from the analysis. The expression patterns of the StGolS and StRFS genes in response to salinity, drought, and ABA treatments shed light on their potential roles in mediating plant adaptation to abiotic stresses.

Discussion
RFOs have various functions, such as seed maturation and transporting and storing sugars, and are considered to be related to plant drought and salt tolerance. The enzymes galactinol synthase (GolS) and raffinose synthase (RFS) are crucial for the biosynthesis of raffinose, a key component of RFOs. However, there is limited knowledge regarding the GolS and RFS gene families in potato, making it imperative to investigate these genes from

Discussion
RFOs have various functions, such as seed maturation and transporting and storing sugars, and are considered to be related to plant drought and salt tolerance. The enzymes galactinol synthase (GolS) and raffinose synthase (RFS) are crucial for the biosynthesis of raffinose, a key component of RFOs. However, there is limited knowledge regarding the GolS and RFS gene families in potato, making it imperative to investigate these genes from a comprehensive whole-genome perspective. Our results lay a foundation for further study of the functional characteristics and molecular mechanisms of the StGolS and StRFS genes in response to ABA, salt, and drought stresses in potato.

Analysis of the Number, Structure, and Evolution of the GolS and RFS Genes in Potato
Variation in the number of genes may be related to the evolutionary diversity, genome duplication events, and genome sizes in plants [49]. In potato, only four StGolS genes located on two chromosomes were identified, and the number of these genes is comparatively lower than in both kiwifruit [50] and sesame [46], but is consistent with findings in tomato [45]. The structural disparities in exons and introns may arise from specific splicing events and serve as informative markers in investigating their evolutionary mechanisms [51]. The observed limited variation in the exon-intron structures of the StGolS genes, with three to four exons across different species, aligns with previous reports in other plants [49,52,53]. Furthermore, the four StGolS genes can be classified into three subfamilies, which aligns with the findings in tomato [45]. In this study, nine StRFS genes were found to be distributed on four chromosomes in potato, consistent with the findings in the tea genome [54]. Phylogenetic analysis revealed that all StRAFS genes could be categorized into five subgroups. Amino acid and nucleotide sequence analysis indicated that members of the StGolS and StRFS gene families within the same subgroup exhibited similar structural features and conserved motifs, suggesting a close evolutionary relationship, as reported in sesame [46].

StGolS and StRFS Genes Play a Key Role in Potato Growth and Development
The expression patterns of GolS genes in different tissues of potato indicate that different GolS proteins may fulfill distinct functions. Previous studies have linked GolS expression in leaves to phloem export, carbon storage, and plant defense against abiotic stress and oxidative damage [55]. Specifically, the orthologs of Pa×gGolSI [56] and members of the BnGolS3 subfamily [57] were found to be highly expressed in leaves. In contrast, no significant expression of GolS genes was observed in tobacco leaves [57]. Additionally, the expression of GolS in the root tissues has been linked to the response to osmotic stress [58]. In our study, StGolS4 displayed high expression levels in transport-related tissues, suggesting that StGolS4 may play a crucial role in material transport in potato. The expression profiles of the StRFS genes in different tissues were also examined using RNA-seq data. In cotton, GhRFS1A, GhRFS2D, GhRFS8A, and GhRFS8D displayed consistently high expression levels across all tissues, indicating their potential role in promoting vegetative growth in upland cotton [51]. In the case of potato, StRFS1, StRFS2, and StRFS8 exhibited high expression levels, suggesting their significance during potato growth. In addition, relatively low expression levels were observed for StRFS4 across all tissues, while StRFS5 exhibited exclusive expression in tubers.

Differential Expression of Potato StGolS and StRFS Genes under Salinity, Drought, and Abscisic Acid
Raffinose plays important roles in plant seed development and participates in the plant response to different external stresses [51]. The raffinose content increases significantly in plants in response to abiotic stress [59]. In this study, the expression levels of several StGolS and StRFS genes were found to be upregulated under salinity, drought, and abscisic acid (ABA) treatments. Studies on GolS genes in Verbascum phoeniceum [60] and cassava [49] have shown different expression characteristics under salt and drought stresses, suggesting that each member of the gene family may have distinct physiological roles. The induction of GolS genes by salt stress has also been observed in other plants, such as AtGolS1 in Arabidopsis [11] and StGolS4 in the same subgroup, which displayed significant increases in expression under salt treatment. Phylogenetic analysis revealed that StRFS2, StRFS5, and StRFS8, which exhibited high expression levels under salt stress in potato, belonged to three distinct subgroups. The results suggest that not all StRFS genes within the same subgroup and with similar sequences exhibit the same expression levels.
StGolS4 and StRFS5 were found to be activated under salinity, drought, and ABA treatments. The upregulation of these genes in response to NaCl treatment may be induced by osmotic stress caused by salt. Furthermore, due to its high expression in transportrelated tissues, it is highly likely that StGolS4 plays a key role in potato material transport and osmotic regulation. Its upregulated expression under salt stress might be attributed to the influence of osmotic stress induced by salt. Notably, an analysis of the promoter region of StGolS4 revealed the existence of ABA-responsive motifs, which are regulated by ABA. While the expression levels of StRFS4 and StRFS8 significantly increased under salt stress, they decreased under PEG-6000 and ABA treatment, suggesting that their responses to salt stress may involve other mechanisms, such as ion toxicity.

Conclusions
In this study, a total of four StGolS and nine StRFS genes were identified in the whole genome of DM potato and were systematically analyzed by bioinformatics, and their expression patterns in different tissues, under ABA and abiotic stresses, were investigated. The two gene families were classified into three and five subgroups, respectively. Remarkably, a total of two StGolS and four StRFS genes in potato were identified to form collinear pairs with those in both Arabidopsis and tomato, respectively. StGolS4 exhibited significantly high expression levels in transport-related tissues and in PEG-6000 and ABA treatments, with remarkable upregulation under salt stress. Similar to StGolS4, StRFS5 exhibited comparable responses to the treatments. Intriguingly, StRFS4 and StRFS8 demonstrated increased expression under salt treatment but decreased expression in PEG-6000 and ABA treatments. Taken together, the results lay a foundation for further study of the functional characteristics and molecular mechanisms of the StGolS and StRFS genes in response to ABA, salt, and drought stresses in potato, and they provide a theoretical basis and new gene resources for the development of potato varieties with tolerance to abiotic stresses.