Genome-Wide Identification, Expression Pattern and Sequence Variation Analysis of SnRK Family Genes in Barley

Sucrose non-fermenting 1 (SNF1)-related protein kinase (SnRK) is a large family of protein kinases that play a significant role in plant stress responses. Although intensive studies have been conducted on SnRK members in some crops, little is known about the SnRK in barley. Using phylogenetic and conserved motif analyses, we discovered 46 SnRK members scattered across barley’s 7 chromosomes and classified them into 3 sub-families. The gene structures of HvSnRKs showed the divergence among three subfamilies. Gene duplication and synteny analyses on the genomes of barley and rice revealed the evolutionary features of HvSnRKs. The promoter regions of HvSnRK family genes contained many ABRE, MBS and LTR elements responding to abiotic stresses, and their expression patterns varied with different plant tissues and abiotic stresses. HvSnRKs could interact with the components of ABA signaling pathway to respond to abiotic stress. Moreover, the haplotypes of HvSnRK2.5 closely associated with drought tolerance were detected in a barley core collection. The current results could be helpful for further exploration of the HvSnRK genes responding to abiotic stress tolerance in barley.


Introduction
Under natural conditions, plants are constantly exposed to a variety of abiotic stresses, such as drought, salinity and extreme temperatures, resulting in the inhibition of growth and development. In responding to these abiotic stresses, plants have evolved a complex genetically regulated system of tolerance or adaptation [1]. For example, barley (Hordeum vulgare) could rapidly adapt its enzyme system to scavenge reactive oxygen species (ROS) when exposed to salt stress [2,3]. However, the genetic mechanisms of the tolerance to abiotic stresses in plants are still not fully understood.
Protein kinases and phosphatase-mediated signal transduction are important regulators involved in the genetic networks responding to environmental changes or abiotic stresses [4]. Sucrose non-fermenting 1 (SNF1)-related protein kinase (SnRK) is a protein kinase family involved biochemical response to stresses in plants [5]. All SnRK proteins share a conserved serine/threonine protein kinase domain in the N-terminal, while they are variable in the C-terminal. The SnRK proteins could be grouped into three subfamilies according to the structures of the C-terminal [6]. The SnRK1 subfamily consists of three domains, including a kinase domain in the N-terminal, an ubiquitin-associated (UBA) domain and a kinase-associated 1 (KA1) domain in the C-terminal, which is the homologues of SNF1 in yeasts and AMP-activated protein kinases (AMPK) in animals [7]. The SnRK2 subfamily harbors the regulatory C-terminal domain containing acidic amino acids, either Glu or Asp [8]. The SnRK3 subfamily contains two conserved domains in the C-terminal,

Identification and Phylogenetic Analysis of SnRKs in Barley
Based on the query protein sequences from Arabidopsis and rice (Table S1), totally 46 proteins were identified as the SnRK members in barley (Table S2). The 46 HvSnRK proteins consist of 341 to 797 amino acids, with a molecular weight of 38.5 to 89.9 kDa.
To verify the evolutionary relationship of SnRK proteins among diverse plant species, we constructed a phylogenetic tree utilizing 48, 39 and 46 SnRKs from rice, Arabidopsis and barley, respectively ( Figure 1). The 133 SnRK proteins were clustered into 3 groups, as found previously in Arabidopsis [6]. In barley, 4 proteins with Pkinase (PF00069), UBA (PF00627) and KA1(PF02149) domains are clustered into the HvSnRK1 subfamily; 11 proteins belonged to the HvSnRK2 subfamily with high similarity to the AtSnRK2 and OsSnRK2 subfamilies; and another 31 proteins with Pkinase and NAF (PF03822) domains were grouped into the HvSnRK3 subfamily ( Figure 2A). Subcellular localization prediction indicated that HvSnRK1s were mainly located in cytoplasm, HvSnRK2s were mainly located in nuclear and HvSnRK3s were mainly located in the plasma membrane (Table  S2), suggesting that the three subfamilies of HvSnRKs may have the different biological functions.       Table S4.

Gene Structures, Protein Motifs and 3D Structures of HvSnRKs
The gene structures of HvSnRK in the same subfamily are similar. Four genes of the SnRK1 subfamily contain eleven exons. Meanwhile, 10 out of 11 members in the HvSnRK2 subfamily contain 8 or 9 exons, and only HvSnRK2.6 contains 3 exons. The HvSnRK3 subfamily genes have a large variation in exon amount, ranging from 1 to 16 ( Figure 2B). In addition, HvSnRK3s can be divided to two subgroups, while one subgroup member has at least eight exons and the other group members have no more than four exons, except HvSnRK3.26, which contains eight exons ( Figure 2B, Table S3).
The protein sequences of 46 HvSnRKs were analyzed, and 10 conserved motifs were obtained ( Figure 2C). The motifs 1/2/3/5/6 are related to the conserved domains of phosphokinase, while motif 9 is related to the NAF domain (Table S4). The conserved motif analysis reveals that identical conserved motifs are found in the same subfamily. For example, all members in the HvSnRK1s contain the motifs 1/2/3/4/5/7/10, and the motifs 1/2/3/4/5/6/7 exist in the HvSnRK2 subfamily, while the motifs 1/2/3/4/5/7/8/9/10 exist in the HvSnRK3 subfamily. These results indicate that the same subfamily of HvSnRK has high similarity in the gene structure and amino acid sequence. It can be seen from 3D structures of HvSnRK proteins that three subfamily proteins have the similar 3D structures in the N terminal but differ in the C terminal ( Figure 3).

Gene Structures, Protein Motifs and 3D Structures of HvSnRKs
The gene structures of HvSnRK in the same subfamily are similar. Four genes of the SnRK1 subfamily contain eleven exons. Meanwhile, 10 out of 11 members in the HvSnRK2 subfamily contain 8 or 9 exons, and only HvSnRK2.6 contains 3 exons. The HvSnRK3 subfamily genes have a large variation in exon amount, ranging from 1 to 16 ( Figure 2B). In addition, HvSnRK3s can be divided to two subgroups, while one subgroup member has at least eight exons and the other group members have no more than four exons, except HvSnRK3.26, which contains eight exons ( Figure 2B, Table S3).
The protein sequences of 46 HvSnRKs were analyzed, and 10 conserved motifs were obtained ( Figure 2C). The motifs 1/2/3/5/6 are related to the conserved domains of phosphokinase, while motif 9 is related to the NAF domain (Table S4). The conserved motif analysis reveals that identical conserved motifs are found in the same subfamily. For example, all members in the HvSnRK1s contain the motifs 1/2/3/4/5/7/10, and the motifs 1/2/3/4/5/6/7 exist in the HvSnRK2 subfamily, while the motifs 1/2/3/4/5/7/8/9/10 exist in the HvSnRK3 subfamily. These results indicate that the same subfamily of HvSnRK has high similarity in the gene structure and amino acid sequence. It can be seen from 3D structures of HvSnRK proteins that three subfamily proteins have the similar 3D structures in the N terminal but differ in the C terminal ( Figure 3).

Chromosomal Location and Gene Duplication of HvSnRKs
In order to reveal the evolution of the barley SnRK genes, the chromosome location of the SnRK genes was determined ( Figure 4). HvSnRK1 subfamily genes were distributed in chromosomes 1, 3 and 4; HvSnRK2 subfamily genes are found on chromosomes 1, 2, 3, and 4; and HvSnRK3 subfamily genes distributed over all chromosomes. Interestingly, HvSnRK3.12 and HvSnRK3.13 were clustered within 10 kb in chromosome 3.

Chromosomal Location and Gene Duplication of HvSnRKs
In order to reveal the evolution of the barley SnRK genes, the chromosome location of the SnRK genes was determined ( Figure 4). HvSnRK1 subfamily genes were distributed in chromosomes 1, 3 and 4; HvSnRK2 subfamily genes are found on chromosomes 1, 2, 3, and 4; and HvSnRK3 subfamily genes distributed over all chromosomes. Interestingly, HvSnRK3.12 and HvSnRK3.13 were clustered within 10 kb in chromosome 3. In the colinear segment of the barley genome, six HvSnRKs gene pairs were discovered, with the two genes of each pair being located on the distinct chromosomes. Moreover, the collinearity of the SnRK family genes between the genomes of barley and rice was also analyzed, with 33 pairs of SnRK genes being detected ( Figure 5B and Table S6). In the colinear segment of the barley genome, six HvSnRKs gene pairs were discovered, with the two genes of each pair being located on the distinct chromosomes. Moreover, the collinearity of the SnRK family genes between the genomes of barley and rice was also analyzed, with 33 pairs of SnRK genes being detected ( Figure 5B and Table S6).

Stress-Related Cis-Elements in the Promoters of HvSnRK Genes
For determining the expression pattern of HvSnRK genes, 2 kb promoter sequences of all HvSnRK genes were extracted from the barley genome database to analyze the cisacting elements. We analyzed the ABA-signal-related components ABRE (ACGTGG/TC), drought-response components MBS (CAACTG) and low-temperature-related components LTR (CCGAAA) ( Figure 6, Table S2). As a result, we found 43 of all 46 HvSnRKs, except HvSnRK3.12, HvSnRK3.13 and HvSnRK3.28, contained the cis-elements responding to the 3 abiotic stresses. Among the 43 HvSnRKs, 42 genes had ABRE, 19 genes had MBS and 26 genes had LTR. It is indicated that many ABRE, MBS and LTR elements were identified in the promoter of HvSnRK family genes, which may respond to abiotic stresses by modulating gene expression.

Stress-Related Cis-Elements in the Promoters of HvSnRK Genes
For determining the expression pattern of HvSnRK genes, 2 kb promoter sequences of all HvSnRK genes were extracted from the barley genome database to analyze the cisacting elements. We analyzed the ABA-signal-related components ABRE (ACGTGG/TC), drought-response components MBS (CAACTG) and low-temperature-related components LTR (CCGAAA) ( Figure 6, Table S2). As a result, we found 43 of all 46 HvSnRKs, except HvSnRK3.12, HvSnRK3. 13 and HvSnRK3.28, contained the cis-elements responding to the 3 abiotic stresses. Among the 43 HvSnRKs, 42 genes had ABRE, 19 genes had MBS and 26 genes had LTR. It is indicated that many ABRE, MBS and LTR elements were identified in the promoter of HvSnRK family genes, which may respond to abiotic stresses by modulating gene expression.

Expression Profiles of HvSnRKs in Different Tissues and under Different Abiotic Stresses
All HvSnRKs were expressed at various levels in 15 tissues of the barley cultivar Morex, but there was no expression in the developing young inflorescences ( Figure 7A and Table S7). According to the expression levels in different tissues, HvSnRKs can be divided into three groups. Group-1 consists of 18 genes with high expression level, including 3 HvSnRK1s, 6 HvSnRK2s and 8 HvSnRK3s. Group-2 consists of 17 genes with moderate expression level, including 2 HvSnRK1s, 3 HvSnRK2s and 12 HvSnRK3s. Group-3 had 13 genes with the low expression level, including 1 HvSnRK1s, 2 HvSnRK2s and 10

Expression Profiles of HvSnRKs in Different Tissues and under Different Abiotic Stresses
All HvSnRKs were expressed at various levels in 15 tissues of the barley cultivar Morex, but there was no expression in the developing young inflorescences ( Figure 7A and Table S7). According to the expression levels in different tissues, HvSnRKs can be divided into three groups. Group-1 consists of 18 genes with high expression level, including 3 HvSnRK1s, 6 HvSnRK2s and 8 HvSnRK3s. Group-2 consists of 17 genes with moderate expression level, including 2 HvSnRK1s, 3 HvSnRK2s and 12 HvSnRK3s. Group-3 had 13 genes with the low expression level, including 1 HvSnRK1s, 2 HvSnRK2s and 10 HvSnRK3s.
These results indicate the dramatic difference in the expression level among HvSnRK genes.

Functions and Regulatory Networks of HvSnRKs
The protein-protein interaction (PPI) was predicted to further understand the biological roles and regulatory networks of HvSnRKs. A total of 20 functional proteins that interact with HvSnRK proteins were discovered ( Figure 8). Most of the proteins that interacted with HvSnRKs were functionally confirmed ABA signaling components, such as PP2C and PYR/PYL (Figure 8). It was also discovered that the majority of SnRK3 proteins interacted with Calcineurin B-like proteins (CBLs). These SnRKs interaction proteins can be grouped into four categories according to their function annotation, including phosphatase 2C family proteins (ABI5, PP2C6, PP2C9, PP2C30 and PP2C50), ion channel proteins (CBL1, CBL4, CBL6 CBL8 and AKT1), PYR/PYL proteins (PYR1, PYL1, PYL2, PYL4, PYL5, PYL8 and PYL9) and stress-or development-related transcription factors (bZIP27, VP1 and TRAB1). In order to understand the expression profiles of the barley SnRK family genes in response to abiotic stresses, we integrated the transcriptome data of barley SnRK genes responding to abiotic stresses including drought [28], high salt [29], high temperature [30] and waterlogging [31]. On the whole, most HvSnRK genes showed a significant change in expression levels under various abiotic stresses, and a few of them showed little change ( Figure 7B, Table S8). For example, HvSnRK2.5 showed increased expression under drought and salt stresses, while HvSnRK2.10 had little change under the two stresses. From the expression profiles of HvSnRK genes under abiotic stresses, it can be seen that the different HvSnRK subfamilies are not consistent in their functions.

Functions and Regulatory Networks of HvSnRKs
The protein-protein interaction (PPI) was predicted to further understand the biological roles and regulatory networks of HvSnRKs. A total of 20 functional proteins that interact with HvSnRK proteins were discovered (Figure 8). Most of the proteins that interacted with HvSnRKs were functionally confirmed ABA signaling components, such as PP2C and PYR/PYL (Figure 8). It was also discovered that the majority of SnRK3 proteins interacted with Calcineurin B-like proteins (CBLs). These SnRKs interaction proteins can be grouped into four categories according to their function annotation, including phosphatase 2C family proteins (ABI5, PP2C6, PP2C9, PP2C30 and PP2C50), ion channel proteins (CBL1, CBL4, CBL6 CBL8 and AKT1), PYR/PYL proteins (PYR1, PYL1, PYL2, PYL4, PYL5, PYL8 and PYL9) and stress-or development-related transcription factors (bZIP27, VP1 and TRAB1).

Sequence Variation of HvSnRKs in a Barley Core Collection
The SNPs of HvSnRKs from 100 barley core accessions were detected. On average, a SnRK gene contained 10 SNPs (Table S9). However, the SNP density of various subfamilies differed greatly, with the averages of the 3 subfamilies being 7.0, 14.5 and 9.2 SNPs/gene, respectively. Moreover, the SNP density of each HvSnRK gene within the same subfamily also showed the obvious difference. Thus, no SNP was detected for HvSnRK1.4, while there were 15 SNPs for both HvSnRK2.5 and HvSnRK3.24. Here, the detailed SNP distribution of HvSnRK2.5 and HvSnRK3.24 was shown in Figure 9. For HvSnRK2.5, 2, 10 and 3 SNPs were detected in the promoter, exon/intron region and 3′UTR regions, respectively ( Figure 9A). For HvSnRK3.24, 3.10 and 2 SNPs were detected in the promoter, exon/intron and 3′UTR regions, respectively ( Figure 9B).
We analyzed the haplotypes of HvSnRK2.5 in 100 barley core accessions and found that the expression of HvSnRK2.5 was highly induced in the leaves under drought treatment ( Figure 8). Five haplotypes were identified on the basis of the SNPs in the promoter and exon regions (Table S10). It was found that the barley accessions with HvSnRK2.5 Hap3 kept a higher leaf water content, while the accessions with HvSnRK2.5 Hap4 are quite sensitive to drought stress (Figure 9c). Therefore, it may be suggested that the sequence variation of SnRKs affects their expression and response to abiotic stresses.

Sequence Variation of HvSnRKs in a Barley Core Collection
The SNPs of HvSnRKs from 100 barley core accessions were detected. On average, a SnRK gene contained 10 SNPs (Table S9). However, the SNP density of various subfamilies differed greatly, with the averages of the 3 subfamilies being 7.0, 14.5 and 9.2 SNPs/gene, respectively. Moreover, the SNP density of each HvSnRK gene within the same subfamily also showed the obvious difference. Thus, no SNP was detected for HvSnRK1.4, while there were 15 SNPs for both HvSnRK2.5 and HvSnRK3.24. Here, the detailed SNP distribution of HvSnRK2.5 and HvSnRK3.24 was shown in Figure 9. For HvSnRK2.5, 2, 10 and 3 SNPs were detected in the promoter, exon/intron region and 3 UTR regions, respectively ( Figure 9A). For HvSnRK3.24, 3.10 and 2 SNPs were detected in the promoter, exon/intron and 3 UTR regions, respectively ( Figure 9B).
We analyzed the haplotypes of HvSnRK2.5 in 100 barley core accessions and found that the expression of HvSnRK2.5 was highly induced in the leaves under drought treatment ( Figure 8). Five haplotypes were identified on the basis of the SNPs in the promoter and exon regions (Table S10). It was found that the barley accessions with HvSnRK2.5 Hap3 kept a higher leaf water content, while the accessions with HvSnRK2.5 Hap4 are quite sensitive to drought stress (Figure 9c). Therefore, it may be suggested that the sequence variation of SnRKs affects their expression and response to abiotic stresses.

Discussion
In this research, 46 HvSnRK genes classified into three subfamilies were discovered in the barley genome. The comprehensive studies were performed on the HvSnRK family genes, including phylogenetic analysis, gene structures, protein 3D structures, gene distribution on genome, gene duplication and cis-elements identification in the promoters. Moreover, utilizing publicly available data, the analysis of HvSnRKs expression patterns and SNPs distribution was performed. In our understanding, the current study should be helpful for better understanding the functions of HvSnRK genes responding to abiotic stresses.
Previously  [37], respectively. In the barley genome, 46 HvSnRK genes classified into 3 subfamilies were discovered, including 4 HvSnRK1s, 11 HvSnRK2s and 31 HvSnRK3s. Furthermore, the phylogenetic tree of the AtSnRK, OsSnRK and HvSnRK proteins showed that the HvSnRKs were closer to the OsSnRKs than AtSnRKs, as expected.
In AtSnRK, OsSnRK and HvSnRK gene families, various subfamily genes showed the large difference in length and exon-intron structure. In barley, all genes of HvSnRK1 subfamily contain 11 exons, while 3 of 4 OsSnRK1 genes have 11exons. All HvSnRK2 subfamily genes contain eight or nine exons except for HvSnRK2.6, which contains three exons due to intron deletion during the evolution process, being consistent with SnRK2s in Arabidopsis, rice, maize and sorghum [38,39]. The SnRK3 subfamily can be divided into 2 subgroups, SnRK3-1, with more than 10 exons, and SnRK3-2, with fewer than 4 exons. The similarity of exon number in the same subfamily may be attributed to their close evolutionary relationship.
The cis-elements in the promoter can regulate gene expression during plant development and the process in response to environmental changes [40,41]. In this study, the promoter sequence analysis showed that various types of cis-elements were contained in HvSnRKS, such as MBS, ABRE and LTR. The mutations in the MBS element of ZmSOP2 gene cause the attenuated expression under ABA and dehydration condition in Zea Mays [42]. The ABRE element of the HAHB4 gene was identified to be responsible for ABA, NaCl and drought regulation when the site-directed mutagenesis on the promoter of HAHB4 was performed on Arabidopsis and sunflower [43]. Low temperature response element LTR played a key role in the regulation of BLT4.9 gene under low temperature

Discussion
In this research, 46 HvSnRK genes classified into three subfamilies were discovered in the barley genome. The comprehensive studies were performed on the HvSnRK family genes, including phylogenetic analysis, gene structures, protein 3D structures, gene distribution on genome, gene duplication and cis-elements identification in the promoters. Moreover, utilizing publicly available data, the analysis of HvSnRKs expression patterns and SNPs distribution was performed. In our understanding, the current study should be helpful for better understanding the functions of HvSnRK genes responding to abiotic stresses.
Previously  46 HvSnRK genes classified into 3 subfamilies were discovered, including 4 HvSnRK1s, 11 HvSnRK2s and 31 HvSnRK3s. Furthermore, the phylogenetic tree of the AtSnRK, OsSnRK and HvSnRK proteins showed that the HvSnRKs were closer to the OsSnRKs than AtSnRKs, as expected.
In AtSnRK, OsSnRK and HvSnRK gene families, various subfamily genes showed the large difference in length and exon-intron structure. In barley, all genes of HvSnRK1 subfamily contain 11 exons, while 3 of 4 OsSnRK1 genes have 11exons. All HvSnRK2 subfamily genes contain eight or nine exons except for HvSnRK2.6, which contains three exons due to intron deletion during the evolution process, being consistent with SnRK2s in Arabidopsis, rice, maize and sorghum [38,39]. The SnRK3 subfamily can be divided into 2 subgroups, SnRK3-1, with more than 10 exons, and SnRK3-2, with fewer than 4 exons. The similarity of exon number in the same subfamily may be attributed to their close evolutionary relationship.
The cis-elements in the promoter can regulate gene expression during plant development and the process in response to environmental changes [40,41]. In this study, the promoter sequence analysis showed that various types of cis-elements were contained in HvSnRKS, such as MBS, ABRE and LTR. The mutations in the MBS element of ZmSOP2 gene cause the attenuated expression under ABA and dehydration condition in Zea Mays [42]. The ABRE element of the HAHB4 gene was identified to be responsible for ABA, NaCl and drought regulation when the site-directed mutagenesis on the promoter of HAHB4 was performed on Arabidopsis and sunflower [43]. Low temperature response element LTR played a key role in the regulation of BLT4.9 gene under low temperature stress in barley [44]. We found that most HvSnRK genes have at least one of the above cis-elements in the promoters.
In addition, 3D structure modeling was constructed to characterize the potential functions of different HvSnRK subfamilies. A noteworthy characteristic of HvSnRK2 is the well-ordered SnRK2 box, which forms a single α-helix and is packed parallel against the αC-helix in the N-terminal lobe. The SnRK2 box-αC interaction has been reported to be crucial for kinase activity [45]. SnRK3 has a junction region in the C terminal, which packs against αB and αC helices, and the central β-sheet was reported to connect the catalytic domain and the regulatory domain [46]. These modeled 3D structures of HvSnRK proteins lay the foundation for us to understand their biological functions.
Gene expression profiles may provide valuable clues for characterizing gene functions. Here, we analyzed the expression levels of HvSnRKs in different barley tissues using published transcriptome data. The findings revealed that the expression patterns of 46 genes could be classified into three groups. (Table S7). Interestingly, we found that the HvSnRKs in group-3 contained fewer cis-elements in their promoters than the other two groups. On average, groups-1, -2 and -3 contain 3.94, 4.29 and 2.69 ABRE, respectively. This indicates that HvSnRKs activities may be correlated with the disparities of cis-elements in the promoter regions.
In this study, expression patterns of HvSnRKs in response to drought, salinity and temperature stresses were also investigated. HvSnRK3.12 is an orthologous gene of Os-CIPK12 ( Figure 5B, Table S6), which regulates the synthesis of proline and soluble sugar in rice exposed to drought stress [47], indicating the identical functions in response to drought stress. However, HvSnRK2.5, which was also significantly induced under drought stress, does not have the orthologous gene in rice. According to the predicted PPI networks (Figure 8), HvSnRK2.5 may respond to drought stress through regulating ABA signaling. Moreover, we determined the SNPs in each HvSnRK gene using the re-sequenced data of a barley core collection. As a result, both potential drought-tolerant and sensitive haplotypes were identified in HvSnRK2.5. For example, barley accessions with HvSnRK2.5 Hap4 are tolerant to drought stress, while those accessions with HvSnRK2.5 Hap4 are sensitive (Fig9C). Obviously, these drought-tolerant haplotypes could be used as the elite alleles in drought-tolerant breeding.
In conclusion, the current study provides a comprehensive understanding of the SnRK family genes in barley. Although phylogenetic relation, structure, expression pattern and sequence variation in HvSnRK genes were analyzed, further studies are required to reveal the roles of HvSnRK family genes in responding to various stresses.

Analysis of Protein Motifs and Gene Structures
The amino acid sequences of the SnRKs in barley were uploaded to the MEME online tool (http://meme.sdsc.edu/meme/itro.html (accessed on 29 August 2021)) [51] to analyze the conserved motifs. The settings were selected based on numerous analyses' experience value: the motif length was set to 6-100 bp, and the maximum number of motifs was set to 10. InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/ (accessed on 29 August 2021)) was used to annotate the motifs. The sequences of barley SnRK genes were extracted from EnsemblePlants database (http://plants.ensembl.org/index.html (accessed on 17 August 2021)), and the gene structure map was created using the Tbtools software [52]. The upstream (−2 kb) sequences of the promoters of HvSnRK genes were submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 29 August 2021)) [53] to analyze the cis-acting elements related to abiotic stress such as drought response components MBS (CAACTG) and low temperature response components LTR (CCGAAA).

Phylogenetic Analysis of SnRK Family Proteins in Barley
The full-length SnRK protein sequences of Arabidopsis, rice and barley were aligned with Clustal X for multiple sequence alignment (MSA) [54], and 1000 bootstrap tests were performed with MEGA-X software to construct the phylogenetic tree by the Neighbor-Joining (NJ) method [55].

Modeling of 3D Structures of HvSnRKs
The 3D structures of HvSnRK proteins were predicted by homology modeling method. Firstly, the most similar homology of each HvSnRK protein were found in the PDB database (http://www.rcsb.org/ (accessed on 30 August 2021)) by the position-specific iterated BLAST algorithm (PSI-BLAST) [56], then the 3D structures of HvSnRK proteins were predicted by the Swiss-Model interactive tool (https://swissmodel.expasy.org/interactive/ (accessed on 30 August 2021)) [57]. In addition, the 3D structure quality of the HvSnRK proteins was tested by SAVES server (http://nihserver.mbi.ucla.edu/SAVES/ (accessed on 30 August 2021)). Finally, the 3D structures were displayed by the Pymol software.

Chromosomal Location and Gene Duplication of HvSnRKs
The chromosomal location data of HvSnRKs was retrieved from the EnsemblePlants, and their distribution was mapped to 7 barley chromosomes using the MapChart v. 2.3 software [58]. SnRK duplication patterns, including segmental and tandem duplications, were classified using the Multiple Collinearity Scan toolkit (MCScanX) software [59]. A tandem duplication was defined as a chromosomal area of less than 200 kb containing two or more genes with more than 70% sequence identity between them [60]. Moreover, the synteny relationships of SnRK genes between H. vulgare and O. sativa were performed with python. Non-synonymous (Ka) and synonymous (Ks) substitutions of each duplicated SnRK gene pair were calculated using the KaKs _Calculator 2.0 [61].

Expression Patterns and Interaction Networks Analysis of HvSnRKs
The transcriptome data of 16 different tissues of barley cultivar Morex were obtained from the barley transcriptome database (https://apex.ipk-gatersleben.de/apex/f?p=284 (accessed on 3 October 2021)), and the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values of HvSnRKs were normalized by log2 for further analysis. In addition, barley transcriptome data of drought [28], salt [29], heat [30] and waterlogging [31] treatments were obtained from published references. The heatmaps were performed by the TBtools software. Moreover, the interaction networks of barley SnRK genes were identified based on the database STRING (http://string-db.org/cgi (accessed on 3 October 2021)) [62] and the predicted interaction networks were created with the Cytoscape software [63].

Sequence Variation and Haplotypes of SnRKs in a Barley Core Collection
SNPs in the coding regions of the HvSnRK genes were identified in 100 barley accessions from the International Barley Core Selected Collection according to previously re-sequenced data (Table S11) [64]. For further analysis, SNPs with Minor Allele Frequencies (MAF) more than 5% and a missing rate of less than 20% were employed. Moreover, we performed a soil-culture experiment using the core collections to investigate the relationship between the haplotypes of SnRK genes and their drought tolerance. Barley seeds were sowed and grown in the mixture of peat soil and vermiculite with the ratio of 2:1, and watering was stopped for 2 weeks when seedlings were at the 3-leaf stage. The drought tolerance of barley accessions was evaluated based on water content of shoots. Then, we compared shoot water content in each haplotype of the HvSnRK genes.

Conclusions
In this study, 46 SnRK family genes were found and classified into 3 subfamilies in barley. Analysis on phylogenetic tree, conserved motifs, gene structure and protein 3D structure of these SnRK family genes were performed. The expression profiles showed that the HvSnRK genes differed greatly in their expression patterns in 15 barley tissues and in response to various abiotic stresses. The expression of HvSnRK2.5 and HvSnRK3.24 were significantly upregulated under drought stress. A protein-protein interaction analysis showed that HvSnRK proteins probably responded to abiotic stresses through ABA signaling. Moreover, the SNPs in each HvSnRK gene were detected, and the haplotypes showing drought tolerance and sensitivity were identified in some genes. The current results could be helpful for further exploration of the HvSnRK genes associated with abiotic stress tolerance in barley.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11070975/s1,  Table  S9. List of SNPs in HvSnRKs identified in this study. Table S10. List of haplotypes of HvSnRK2.5 identified in this study. Table S11. The information of 100 barley core collection accessions.