Genome-Wide Identification and Characterization of the Potato bHLH Transcription Factor Family

Plant basic/helix–loop–helix (bHLH) transcription factors participate in a number of biological processes, such as growth, development and abiotic stress responses. The bHLH family has been identified in many plants, and several bHLH transcription factors have been functionally characterized in Arabidopsis. However, no systematic identification of bHLH family members has been reported in potato (Solanum tuberosum). Here, 124 StbHLH genes were identified and named according to their chromosomal locations. The intron numbers varied from zero to seven. Most StbHLH proteins had the highly conserved intron phase 0, which accounted for 86.2% of the introns. According to the Neighbor-joining phylogenetic tree, 259 bHLH proteins acquired from Arabidopsis and potato were divided into 15 groups. All of the StbHLH genes were randomly distributed on 12 chromosomes, and 20 tandem duplicated genes and four pairs of duplicated gene segments were detected in the StbHLH family. The gene ontology (GO) analysis revealed that StbHLH mainly function in protein and DNA binding. Through the RNA-seq and quantitative real time PCR (qRT-PCR) analyses, StbHLH were found to be expressed in various tissues and to respond to abiotic stresses, including salt, drought and heat. StbHLH1, 41 and 60 were highly expressed in flower tissues, and were predicted to be involved in flower development by GO annotation. StbHLH45 was highly expressed in salt, drought and heat stress, which suggested its important role in abiotic stress response. The results provide comprehensive information for further analyses of the molecular functions of the StbHLH gene family.


Introduction
Transcription factors are important in regulating gene expression on the transcriptional level. Transcription factors play essential roles in plant growth and development, and activate component syntheses and responses to environmental changes [1]. Most biological processes in eukaryotic cells or organisms are finely controlled by transcription factors on the transcriptional level.
Basic/helix-loop-helix (bHLH) transcription factors are widely found in animals and plants [2]. The bHLH superfamily is the second largest transcription factor family in plants [3]. Basic/helix-loop-helix transcription factors are named for their highly conserved alkaline/helix-loop-helix domains [4]. Transcription factors usually contain two different functional domains involved in DNA binding and protein interactions, which may be regulated by a variety of mechanisms, including the formation of dimers [5]. A bHLH transcription factor consists of two conserved motifs, a basic region and helix-loop-helix region (HLH region). The basic region is located at the N-terminal end, while the

Gene Structure and Conserved Motif Characterization
The potato bHLH protein sequences were uploaded to ExPASy [45] to calculate the number of amino acids, molecular weights and isoelectric points. The intron numbers and chromosomal locations of potato bHLH genes were retrieved from the PGSC. The conserved motifs were predicted by the MEME program [46]. The parameters were set as any number of repetitions, optimum motif width of 10-200 residues, and searching for 20 motifs, with all other parameters in default. The predicated motif sequences were annotated by CDD [43], SMART [44] and InterPro [47]. The Gene Structure Display Server [48] was used to show the exon-intron structures of potato bHLH genes.

Chromosomal Location and Gene Duplication
The chromosome positional data for bHLH genes were retrieved from the PGSC genome browser. bHLH gene mapping was performed using MapChart software [49]. Gene duplication was confirmed using two criteria: (a) the shorter aligned sequence covered >70% of the longer sequence in length; (b) the similarity of aligned sequences was >70% [49,50]. Two genes located in the same chromosomal fragment of less than 100 kb and separated by five or fewer genes were identified as tandem duplicated genes [51]. The duplicated potato bHLH gene segments were confirmed by searching the Plant Genome Duplication Database [52].

Phylogenetic Analysis and Classification
The phylogenetic tree contained full-length amino acid sequences of bHLH from Arabidopsis [28] and potato (Table S1). All of the full-length amino acid sequences were aligned with ClustalX [53] using default parameters. MEGA6 [54] was used to generate the unrooted Neighbor-joining phylogenetic tree with the following parameters: 1000 times bootstrap test, Poisson model and pairwise deletions. The classifications of potato bHLH proteins were based on the topology and bootstrap values of the phylogenetic tree.

GO Annotation and RNA-Seq Data Analysis
The GO analysis of potato bHLH genes was performed using the Blast2GO program. The full-length amino acid sequences of potato bHLH proteins were uploaded to the program, and Arabidopsis was chosen as the reference database. The analysis contained three parts: molecular function, cellular component and biological process.
The RNA-seq data (DM_v4.03) [55] represented as fragments per kilobase per million (FPKM) values of StbHLHs in various tissues were downloaded from PGSC. The raw data of FPKM values were equalized by the mean of whole data. Subsequently, the processed data were transformed by log2. After then, HemI [56] was used to generate the expression heatmap of StbHLH genes.

Plant Materials, Growth Conditions and Stress Treatments
The potato cultivar Desiree was planted in a greenhouse of our laboratory at Northwest A&F University. Various tissues, including flower, stolon and mature tuber, were sampled [57]. There were three biological replicates for each sampled tissue.
A doubled monoploid potato variety (DM) with low levels of heterozygosity genome was used in the stress treatments. The plantlets were grown in Murashige and Skoog (MS) medium that contained 3% sucrose and 0.8% agar at pH 5.9, and sustained in an artificial climate chamber with a photoperiod of 16 h light/8 h dark at 22 ± 1 • C. The four-week-old plantlets were transferred into containers of 1/2 MS liquid medium and sustained for a week with the same growth conditions as before. The abiotic stress conditions were referred to previous reports [55]. The heat stress was conducted at 35 • C. For salt and drought stresses, the plantlets were treated with 150 mM NaCl and 260 mM mannitol, respectively. The aboveground parts of plantlets were collected at 0 and 24 h after treatment. All of the collected materials were immediately frozen in liquid nitrogen and stored at −80 • C.

RNA Extraction and qRT-PCR Analysis
An RNAsimple Total RNA Kit (BioTeke, Beijing, China) was used to extract the total RNA of the sampled plant materials. The cDNA was synthetized using a First Strand cDNA Synthesis Kit. The cDNA was diluted 10 times with nuclease-free water. All of the procedures were conducted according to the manufacturers' protocols.
The specific potato bHLH gene primers for quantitative real-time PCR (qRT-PCR) were designed with Primer Premier 5 (Table S2). The qRT-PCR was conducted in a 20-µL reaction system with the following components: 10 µL KAPA SYBR FAST qPCR Kit Master Mix (2×) Universal, 1 µL 10 µM forward primer, 1 µL 10 µM reverse primer, 1 µL diluted cDNA and 7 µL ddH 2 O. A Bio-Rad CFX96 Real Time PCR System was used for the qRT-PCR. The procedure was: 95 • C for 2 min and 40 cycles of 95 • C for 5 s and 60 • C for 30 s. A melt curve was generated from 65 • C to 95 • C with increments of 0.5 • C every 5 s. Each gene was detected in three biological replicates and two technical replicates. The internal reference gene was ef1α. The relative expression was calculated using the 2 −∆CT method for the tissue expression profile and 2 −∆∆CT method for expression profile under stress [58].

Identification and Characterization of bHLH Proteins in Potato
In total, 190 putative bHLH transcription factors were identified by HMMER [59]. Then, the bHLH protein sequences encoded by non-representative transcripts were excluded. The remaining sequences were checked for the existence of complete bHLH domains using CDD, Pfam and SMART. In total, 124 sequences were confirmed as potato bHLH proteins. Based on their chromosomal locations, the proteins were assigned from StbHLH1 to StbHLH124 (Table S3). The number of potato bHLH proteins was similar to that of tomato, a sibling species of potato belonging to Solanaceae, in which 159 bHLH proteins were identified [36]. The length of StbHLH proteins varied from 62 (StbHLH67) to 708 (StbHLH12) amino acids. Generally, the conserved bHLH domain contains~60 amino acids [33]. The divergent lengths of potato bHLH proteins were mainly the result of other domains not the bHLH domain. The molecular weights ranged from 7.5 kDa (StbHLH67) to 75.9 kDa (StbHLH92). StbHLH genes were randomly distributed on 12 chromosomes, while three genes (StbHLH1, StbHLH2 and StbHLH3) could not be anchored on any of the potato chromosomes. Only 623 Mb (86%) of the assembled genome are genetically anchored, and the constructed pseudomolecules for the 12 potato chromosomes harbor 90.3% of the predicted genes [55], which means 9.7% of the predicted genes could not be anchored on any of the 12 chromosomes. The predicted isoelectric point values of StbHLH proteins were between 4.66 (StbHLH51) and 10.42 (StbHLH67).

Gene Structure and Motif Analysis of StbHLH
Gene organization plays a vital role in the evolution of multiple gene families [60]. A Neighbor-joining phylogenetic tree was constructed with MEGA6 [61] ( Figure 1A). The genomic sequence and corresponding cDNA sequence of the same StbHLH gene were submitted to GSDS [48] together to show its gene structure. The number of introns varied from zero to seven (Table S3, Figure 1B). In addition, 12 (9.7%) genes were intronless, while 19 (15.3%) genes contained one intron. The remaining genes had two or more introns. Genes with few or no introns are considered to show lower expression levels in plants, which is different from that in animals [62]. However, the compact gene structure may contribute to the rapid expression of genes in response to endogenous and/or exogenous stimuli [63]. For example, intronless genes, StbHLH65 and StbHLH76, were expressed with low levels in most tissues, but were upregulated in response to heat stress. Owing to the closely related duplication relationship, the tandem duplicated genes contained the same amounts of introns. For example, the tandem duplicated genes StbHLH15, StbHLH16, StbHLH17 and StbHLH18 have two introns. Three intron phases are generally acknowledged, phases zero, one and two, in which the splicing occurred after the third, first, and second nucleotide, respectively [64]. Also, a conserved intron phase was observed through the StbHLH gene family ( Figure 1B). The number of introns with intron phases of zero was 351, which accounted for 86.2% of the introns.
The MEME [46] program was used to identify the conserved motifs of StbHLH proteins. Meanwhile, the predicted motifs were annotated. Twenty conserved motifs were identified and varied from 10 to 138 residues in length ( Figure 1C). Details of the 20 motifs were listed in Table 1. Each StbHLH protein contained different numbers of conserved motifs, ranging from one to seven. StbHLH23, StbHLH59 and StbHLH111 only had one conserved motif, while seven StbHLH proteins each contained seven conserved motifs. All of the predicted motifs were identified only once in each StbHLH protein sequence. In general, closely related StbHLH proteins on adjacent clades of the phylogenetic tree had the same or similar motif structures. For example, StbHLH33, 99, 98, 75 and 118 each contained two motifs, one and 13. The strong sequence diversity outside of the bHLH domain suggested that the bHLH family experienced extensive domain shuffling after the genome duplication [10]. In our research, 20 different motifs in various arrangements were found among potato bHLH family members. Thus, broad domain shuffling occurred in the protein structures of the bHLH family members.  Table 1. CDS: coding sequence.

Phylogenetic Analysis of the StbHLH Protein Family
To analyze the evolutionary relationships among the StbHLH members, 124 StbHLH proteins were aligned with 135 bHLH proteins from Arabidopsis [28] using ClustalX 1.83 [65]. An unrooted phylogenetic tree was constructed by MEGA6. In total, 259 bHLH protein sequences were classified into 15 distinct groups, which were named from A to O (Figure 2). The result corroborated the previously proposed classification of the bHLH family [27,66]. Group A was the largest subfamily with 34 proteins, while the smallest, Group N, contained only two proteins, both bHLH proteins (AT1G22380 and AT5G50010) were from Arabidopsis. StbHLH proteins were more closely related to those in the same subfamily from Arabidopsis than with the other bHLH proteins from potato. Most StbHLH proteins in the same group shared consensus motifs, and similar exon-intron structures within the corresponding genes (Table S4). For instance, 10 StbHLH genes in Group I contained two or three introns, with four genes having two introns and the others having three. Genes with the same number of introns only varied in lengths of exon and non-coding regions. Additionally, the 10 proteins in Group I shared the same motif arrangement of motifs three, four and six, except for StbHLH39 and StbHLH121, which lacked motif 6, and in StbHLH49, which had motif 1 instead of motif 3. A few of the clades of the phylogenetic tree were supported by low bootstrap values, which may be caused by relatively less informative character positions outside of the short and highly conserved bHLH domains [33].

Phylogenetic Analysis of the StbHLH Protein Family
To analyze the evolutionary relationships among the StbHLH members, 124 StbHLH proteins were aligned with 135 bHLH proteins from Arabidopsis [28] using ClustalX 1.83 [65]. An unrooted phylogenetic tree was constructed by MEGA6. In total, 259 bHLH protein sequences were classified into 15 distinct groups, which were named from A to O (Figure 2). The result corroborated the previously proposed classification of the bHLH family [27,66]. Group A was the largest subfamily with 34 proteins, while the smallest, Group N, contained only two proteins, both bHLH proteins (AT1G22380 and AT5G50010) were from Arabidopsis. StbHLH proteins were more closely related to those in the same subfamily from Arabidopsis than with the other bHLH proteins from potato. Most StbHLH proteins in the same group shared consensus motifs, and similar exon-intron structures within the corresponding genes (Table S4). For instance, 10 StbHLH genes in Group I contained two or three introns, with four genes having two introns and the others having three. Genes with the same number of introns only varied in lengths of exon and non-coding regions. Additionally, the 10 proteins in Group I shared the same motif arrangement of motifs three, four and six, except for StbHLH39 and StbHLH121, which lacked motif 6, and in StbHLH49, which had motif 1 instead of motif 3. A few of the clades of the phylogenetic tree were supported by low bootstrap values, which may be caused by relatively less informative character positions outside of the short and highly conserved bHLH domains [33].

Chromosomal Location and Gene Duplication
The distribution of StbHLH genes was uneven on the chromosomes (Figure 3). The number of StbHLH genes on each chromosome varied from three to 17. Chromosome 1 contained the largest number, 17, of StbHLH genes. Chromosome 11 had only three StbHLH genes. The majority of StbHLH genes were located on the proximate or the distal ends of potato chromosomes. The detailed chromosomal locations of StbHLH genes are shown in Table S1. Additionally, the lengths of chromosomes can be estimated by the scale on the left. The results were consistent with those of tomato [36].

Chromosomal Location and Gene Duplication
The distribution of StbHLH genes was uneven on the chromosomes (Figure 3). The number of StbHLH genes on each chromosome varied from three to 17. Chromosome 1 contained the largest number, 17, of StbHLH genes. Chromosome 11 had only three StbHLH genes. The majority of StbHLH genes were located on the proximate or the distal ends of potato chromosomes. The detailed chromosomal locations of StbHLH genes are shown in Table S1. Additionally, the lengths of chromosomes can be estimated by the scale on the left. The results were consistent with those of tomato [36].  Genome duplication events occurred during the process of plant evolution [67,68]. The expansion of gene families and genomic evolutionary mechanisms mainly depend on gene duplication events [69]. The major duplication patterns are tandem and segmental duplication [70]. Gene duplication events were also identified for StbHLH genes. According to the defined criteria, 20 (16.1%) StbHLH genes were confirmed to be tandem duplicated genes. In addition, four pairs of StbHLH genes were found to be segmental duplicates. The 20 tandem duplicated genes formed eight gene clusters that were indicated by red blocks in Figure 3. Paralogs of segmental duplications were linked to each other by orange lines. A total of 28 (22.6%) StbHLHs were duplicated genes, which indicated that gene duplication played an important role in the expansion of StbHLH family.

GO Annotation of StbHLH Proteins
The highly divergent sequences outside of the conserved bHLH domain indicate that bHLH proteins are involved in multiple biological processes. In Arabidopsis, a number of bHLH proteins were functionally characterized [71][72][73][74][75]. Thus, to understand the biological processes associated with StbHLH genes, we used Arabidopsis as the reference species to perform a GO annotation of potato bHLH proteins. The results are shown in Figure 4 and Table S5. The majority of StbHLH proteins were involved in protein and DNA binding. Meanwhile, four proteins (StbHLH43, StbHLH70, StbHLH88 and StbHLH122) functioned in chromatin binding. In addition, StbHLH119 was involved in both catalytic activity and nucleotide binding. Most of the StbHLH proteins (102, 82.3%) were located on the nucleus. However, there were also StbHLH proteins located on plastids (15) and mitochondrion (10), and in the cytoplasm (8). A small number of StbHLH proteins were located on the nucleoplasm (StbHLH8, 42, 71), cytosol (StbHLH68, 103, 117), vacuoles (StbHLH59 and 119), extracellular regions (StbHLH119) and the endoplasmic reticulum (StbHLH119). Additionally, some StbHLH proteins existed on multiple cellular components. For example, StbHLH119 was located on three cellular components, vacuoles, extracellular regions and the endoplasmic reticulum, which may reflect its multifunction in various biological processes. The biological process analysis showed that StbHLH proteins participated in various biological processes. The biosynthetic process and nucleobase-containing compounds' metabolic processes involved the greatest number of StbHLH proteins. Besides, StbHLH proteins could respond to abiotic stress. Also, there werẽ 25-45 StbHLH proteins involved in post-embryonic development, flower development, anatomical structure morphogenesis and cell differentiation. Thus, based on the biological process analysis, StbHLH proteins could function in multiple biosynthetic and metabolic processes, in response to abiotic and biotic stresses, in the development of various tissues and organs, in signal transduction and in cell communication.

The StbHLH Expression Pattern in Various Tissues
To dissect the expression patterns of StbHLH genes in various tissues, RNA-seq data, represented by fragments per kilobase of transcript per million mapped reads (FPKM , Table S6), was downloaded from PGSC. We processed the FPKM data and generated a heatmap. Twelve different tissues and organs were included in the analysis. The StbHLH genes with FPKM values less than two in all 12 tissues were considered to be barely expressed. Thus, 30 unexpressed genes were excluded from the heatmap ( Figure 5). The remaining 94 StbHLH genes used in the heatmap were expressed

The StbHLH Expression Pattern in Various Tissues
To dissect the expression patterns of StbHLH genes in various tissues, RNA-seq data, represented by fragments per kilobase of transcript per million mapped reads (FPKM , Table S6), was downloaded from PGSC. We processed the FPKM data and generated a heatmap. Twelve different tissues and organs were included in the analysis. The StbHLH genes with FPKM values less than two in all 12 tissues were considered to be barely expressed. Thus, 30 unexpressed genes were excluded from the heatmap ( Figure 5). The remaining 94 StbHLH genes used in the heatmap were expressed in at least one tissue. Two genes (StbHLH85 and StbHLH99) showed high expression levels across all of the analyzed tissues. Some genes have been identified as highly expressed in various tissues [76]. These highly expressed genes may possess specific housekeeping activities. Additionally, some genes were expressed in most of the analyzed tissues, and some gene expressions were tissue specific. StbHLH82, 47, 123 and 72 were expressed only in the callus. StbHLH76 and StbHLH86 had a relatively high expression level in the tuber and stolon compared with in other tissues. This suggests that the two genes may be involved in tuber and stolon development. StbHLH68, 41, 1, 118, 55, 60 and 26 were highly expressed in flower tissues, including the sepal, stamen, flower and petal. These genes may be related to flower development. In addition, StbHLH1, 41 and 60 were also included in flower development by the GO analysis ( Figure 4, Table S5), which further suggested our conclusion.
To further examine the expression patterns of StbHLH in different tissues, we used qRT-PCR to quantify the expression levels of several StbHLH genes in Desiree. Desiree is a tetraploid that is similar to cultivars in its genetic background. Three tissues, flower, stolon and tuber, were used for qRT-PCR analysis. StbHLH117, with low expression in the three tissues, and 14 StbHLHs, with high expression in at least one of the three tissues, were selected for analysis ( Figure 6). Three StbHLHs (StbHLH26, 85 and 99) had relatively higher expression levels in the three tissues than other StbHLHs. Three genes (StbHLH60, 85 and 98) expressed similar abundance levels between flower and tuber. StbHLH19 was observed with a higher expression level than in the other two tissues, while StbHLH41 was mainly expressed in the flower. The expression of StbHLH87 could be barely detected. However, based on qRT-PCR, only three genes (StbHLH26, 41 and 78) had the same expression patterns as determined by the RNA-seq data. The differences in expression patterns between the qRT-PCR and heatmap in various tissues may have been caused by the vastly divergent genetic backgrounds of the two varieties.
Based on the phylogenetic tree, StbHLH22 and AtSACL3 (AT1G29950) were orthologs. AtSACL3 is a member of a negative feedback loop which contributes to the maintaining of root apical meristem size and proper root growth [77]. Thus, StbHLH22 may function in the same way in potato, and highly express in the root. In the same way, StbHLH78 was found to be an ortholog of AtPIF4 (AT2G43010) and AtPIF5 (AT3G59060). The AtPIF genes are the central signaling hub that functions in regulating plant growth and development [78]. AtPIF4 is also found to contribute to hybrid vigor through the auxin pathway [79]. StbHLH78 had a relatively high expression in the leaf, petiole ( Figure 5) and flower ( Figure 6). It may be inferred that StbHLH78 has the same function in regulating potato growth and development.  and 99) had relatively higher expression levels in the three tissues than other StbHLHs. Three genes (StbHLH60, 85 and 98) expressed similar abundance levels between flower and tuber. StbHLH19 was observed with a higher expression level than in the other two tissues, while StbHLH41 was mainly expressed in the flower. The expression of StbHLH87 could be barely detected. However, based on qRT-PCR, only three genes (StbHLH26, 41 and 78) had the same expression patterns as determined by the RNA-seq data. The differences in expression patterns between the qRT-PCR and heatmap in various tissues may have been caused by the vastly divergent genetic backgrounds of the two varieties. Based on the phylogenetic tree, StbHLH22 and AtSACL3 (AT1G29950) were orthologs. AtSACL3 is a member of a negative feedback loop which contributes to the maintaining of root apical meristem size and proper root growth [77]. Thus, StbHLH22 may function in the same way in potato, and highly express in the root. In the same way, StbHLH78 was found to be an ortholog of AtPIF4 (AT2G43010) and AtPIF5 (AT3G59060). The AtPIF genes are the central signaling hub that functions in regulating plant growth and development [78]. AtPIF4 is also found to contribute to hybrid vigor through the auxin pathway [79]. StbHLH78 had a relatively high expression in the leaf, petiole ( Figure 5) and flower ( Figure 6). It may be inferred that StbHLH78 has the same function in regulating potato growth and development.

Expression Analysis of StbHLH under Abiotic Stresses
To analyze the stress responses of StbHLH genes, we compared the FPKM values (Table S7) of StbHLH genes under heat, salt and drought stress with those of the corresponding controls. A total

Expression Analysis of StbHLH under Abiotic Stresses
To analyze the stress responses of StbHLH genes, we compared the FPKM values (Table S7) of StbHLH genes under heat, salt and drought stress with those of the corresponding controls. A total of 38 StbHLH genes had FPKM values lower than two under the three stress treatments and in the corresponding controls, and were, therefore, excluded from the heatmap (Figure 7). Based on the heatmap, we concluded that a number of StbHLH genes respond to abiotic stresses, including salt, drought and heat. Under heat stress, several StbHLH genes were extremely up-regulated, such as genes StbHLH65, 76 and 79, or downregulated, such as genes StbHLH19, 60 and 78. However, the variations in the StbHLH genes' expression levels under salt and drought stress were not as divergent. In addition, a few StbHLH genes responded to all three stresses. For instance, StbHLH51 was sensitive to salt, drought and heat stress, and was up-regulated distinctly under the three stresses. In addition, several StbHLH genes, such as StbHLH69 and StbHLH78, showed contrasting expression patterns among the three stresses. StbHLH78 was not sensitive to salt stress, but it was up-regulated under drought stress and down-regulated under heat stress. One single bHLH protein could interact with one or more bHLH proteins and even non-bHLH proteins [3]. The StbHLH genes with different stress responses may form heterodimers with specific bHLH proteins, resulting in diverse stress responses and expression patterns.
among the three stresses. StbHLH78 was not sensitive to salt stress, but it was up-regulated under drought stress and down-regulated under heat stress. One single bHLH protein could interact with one or more bHLH proteins and even non-bHLH proteins [3]. The StbHLH genes with different stress responses may form heterodimers with specific bHLH proteins, resulting in diverse stress responses and expression patterns.  Moreover, three StbHLHs (StbHLH45, 51 and 81) which were upregulated under three abiotic stresses and three StbHLHs (StbHLH9, 21 and 121) which were in response to drought or heat stress were analyzed for their expression profiles (Figure 8). Of the six StbHLH genes, StbHLH45, StbHLH81 and StbHLH121 were more sensitive to the three abiotic stresses than the other three StbHLH genes and were notably upregulated (>2-fold) under the three abiotic stresses. In comparison, StbHLH9, StbHLH21 and StbHLH51 showed different expression patterns within the three abiotic stresses. Additionally, we compared the results of qRT-PCR with the heatmap. It was confirmed that two genes (StbHLH45 and StbHLH81) had the same expression patterns under the three abiotic stresses. For the other genes, the same expression levels were observed under one or two abiotic stresses. The differences between the two sets of results acquired by qRT-PCR and RNA-seq may be caused by various factors. Although the same potato variety was used, only the aboveground part of the plantlet was sampled in our research, while the whole plant was collected for RNA-seq. Under heat stress, the plantlets were treated for 24-h under a normal photoperiod (16 h light/8 h dark) in our study, but they were treated in darkness all the time for RNA-seq. The potato RNA-seq data downloaded from PGSC was presented as FPKM values. Compared with raw read counts, FPKM values can better reduce sample differences. However, there are also deficiencies in the FPKM data. For example, some highly expressed genes could alter the FPKM values [80]. The bias in FPKM values can lead to different expression patterns compared with qRT-PCR data. The orthologs of six StbHLHs have not been functionally characterized in Arabidopsis except for AtABS5 (AT1G68810), which is an ortholog of StbHLH45. In Arabidopsis, AtABS5 are involved in vascular cell division in root apical meristem [81] and leaf morphogenesis [25]. It may be inferred that StbHLH45 functions in the regulation of potato growth and development. The sensitive response to the abiotic stress of StbHLH45 may reflect its role in stress tolerance, which needs to be further analyzed. Moreover, three StbHLHs (StbHLH45, 51 and 81) which were upregulated under three abiotic stresses and three StbHLHs (StbHLH9, 21 and 121) which were in response to drought or heat stress were analyzed for their expression profiles (Figure 8). Of the six StbHLH genes, StbHLH45, StbHLH81 and StbHLH121 were more sensitive to the three abiotic stresses than the other three StbHLH genes and were notably upregulated (>2-fold) under the three abiotic stresses. In comparison, StbHLH9, StbHLH21 and StbHLH51 showed different expression patterns within the three abiotic stresses. Additionally, we compared the results of qRT-PCR with the heatmap. It was confirmed that two genes (StbHLH45 and StbHLH81) had the same expression patterns under the three abiotic stresses. For the other genes, the same expression levels were observed under one or two abiotic stresses. The differences between the two sets of results acquired by qRT-PCR and RNA-seq may be caused by various factors. Although the same potato variety was used, only the aboveground part of the plantlet was sampled in our research, while the whole plant was collected for RNA-seq. Under heat stress, the plantlets were treated for 24-h under a normal photoperiod (16 h light/8 h dark) in our study, but they were treated in darkness all the time for RNA-seq. The potato RNA-seq data downloaded from PGSC was presented as FPKM values. Compared with raw read counts, FPKM values can better reduce sample differences. However, there are also deficiencies in the FPKM data. For example, some highly expressed genes could alter the FPKM values [80]. The bias in FPKM values can lead to different expression patterns compared with qRT-PCR data. The orthologs of six StbHLHs have not been functionally characterized in Arabidopsis except for AtABS5 (AT1G68810), which is an ortholog of StbHLH45. In Arabidopsis, AtABS5 are involved in vascular cell division in root apical meristem [81] and leaf morphogenesis [25]. It may be inferred that StbHLH45 functions in the regulation of potato growth and development. The sensitive response to the abiotic stress of StbHLH45 may reflect its role in stress tolerance, which needs to be further analyzed.

Conclusions
In summary, we performed a genome-wide analysis of the bHLH family in potato, and 124 StbHLH genes were identified. The divergent biochemical characteristics of StbHLH proteins were analyzed. Based on the phylogenetic tree, StbHLH proteins were classified into 14 subfamilies, named from A to O, except for group N that did not have a StbHLH protein member. The similar exon-intron structures of the genes and motif arrangement of the StbHLH proteins within the subfamilies further supported the classification predicted by the phylogenetic tree. StbHLH genes were distributed on 12 potato chromosomes. A total of 28 StbHLH genes were confirmed to be duplicated genes, which indicated the important role of gene duplication in the expansion of the StbHLH family. The GO analysis revealed multiple functions for the StbHLH proteins. The RNA-seq and qRT-PCR illustrated that StbHLH genes were expressed in various tissues and responded to different abiotic stresses on the transcriptional level. The results provide comprehensive information for further functional analyses of the StbHLH gene family.
Supplementary Materials: The following are available online at http://www.mdpi.com/s1. Table S1: Gene IDs and names of Arabidopsis bHLH proteins. Table S2: Primer sequences used in qRT-PCR.