Identification, Phylogeny, Divergence, Structure, and Expression Analysis of A20/AN1 Zinc Finger Domain Containing Stress-Associated Proteins (SAPs) Genes in Jatropha curcas L.

Jatropha is a small woody perennial biofuel-producing shrub. Stress-associated proteins (SAPs) are novel stress regulatory zinc-finger proteins and are mainly associated with tolerance against various environmental abiotic stresses in Jatropha. In the present study, the JcSAP gene family were analyzed comprehensively in Jatropha curcas and 11 JcSAP genes were identified. Phylogenetic analysis classified the JcSAP genes into four groups based on sequence similarity, similar gene structure features, conserved A20 and/or AN1 domains, and their responsive motifs. Moreover, the divergence analysis further evaluated the evolutionary aspects of the JcSAP genes with the predicted time of divergence from 9.1 to 40 MYA. Furthermore, a diverse range of cis-elements including light-responsive elements, hormone-responsive elements, and stress-responsive elements were detected in the promoter region of JcSAP genes while the miRNA target sites predicted the regulation of JcSAP genes via a candid miRNA mediated post-transcriptional regulatory network. In addition, the expression profiles of JcSAP genes in different tissues under stress treatment indicated that many JcSAP genes play functional developmental roles in different tissues, and exhibit significant differential expression under stress treatment. These results collectively laid a foundation for the functional diversification of JcSAP genes.


Introduction
Plant growth and productivity is severely affected by various biotic and abiotic stresses because of their sessile nature. These environmental stresses down-or upregulate a large pool of genes. To eliminate or reduce the harsh environmental effect, plants have evolved complex internal molecular mechanisms to modulate stress-responsive or regulatory genes [1,2]. Regulatory genes code for sensors that perceive stress signals, kinases that transmit the signals, and transcription factors that are down-or upregulated as a result of perceived stress. Thus, defence mechanisms start with the perception of stress, followed by signal transduction, synthesis of transcription factors, and finally down-or upregulation of genes that produce protective proteins and metabolites [2]. Hitherto, a large number of genes have been characterized that play an important role during different stresses and are involved at different levels of regulation, such as perception, signalling, transcription, and production of protective biomolecules [3][4][5][6][7]. The characterisation of genes belonging to the signal transduction and transcription factor categories is of great importance because of their effect on a wide range of stress-related genes. Zinc finger proteins are associated

Gene Duplication Events, Homology and Synteny Analysis of JcSAP Genes
For Gene conservation, duplication events, homology, and Synteny analysis, a comparative Synteny analysis was performed by using circoletto Tool (https://www.tools.bat. infspire.org/circoletto/) (accessed on 2 September 2022) to visualize genome conservation. Protein sequences of Arabidopsis SAP genes were used against the identified JcSAP sequences and were finally exhibited by circus by running the Circoletto tool [27].

Conserved Domains, Motifs, and Gene Structure Organization of JcSAP Genes
The identified JcSAP protein sequences were subjected to NCBI CDD online software (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 2 September 2022) for domain analysis, and the obtained results were visualized via the TBtools software (https://github.com/CJ-Chen/TBtools) (accessed on 2 September 2022). Similarly, to analyze the JcSAP proteins for the conserved motifs, the protein sequences of JcSAP were submitted to MEME suite software 5.4.1 (https://meme-suite.org/meme/tools/meme) (accessed on 2 September 2022). Consistently, to display the gene structure organization of JcSAP genes, the gene structure display server (http://gsds.gao-lab.org/) (accessed on 2 September 2022) was used by submitting the CDS and genomic sequences of JcSAP genes [22,24].

Divergence Analysis
For divergence analysis of JcSAP genes, the server Ka/Ks calculation tool (http:// services.cbu.uib.no/tools/kaks) (accessed on 2 September 2022) was used and the nonsynonymous substitution per nonsynonymous site (Ka) and synonymous substitution per synonymous site (Ks) was determined by inputting the DNA sequences of JcSAP genes using default parameters. The divergence time was calculated by the given formula [24,28].

Protein Structure Analysis of JcSAP Genes
To identify the structural composition of 11 JcSAP genes, an online tool for the prediction of secondary structure SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/secpredsopma.pl) (accessed on 2 September 2022) were used. The tertiary structure of JcSAP proteins were visualized via uniprot (https://www.uniprot.org) (accessed on 2 September 2022) to further support the secondary structure of JcSAP proteins [29].

Cis-Elements Analysis and Predicted miRNA Target Sites
To analyse the cis-regulatory element, the upstream region of 1500 bp of each genomic sequence of the JcSAP genes was submitted to PlantCARE server (https://bioinformatics. psb.ugent.be/webtools/plantcare/html/) (accessed on 5 September 2022) and was searched for the presence of cis-regulatory elements. The results were then visualized using TBtools software (https://github.com/CJ-Chen/TBtools) (accessed on 6 September 2022) [24].

Gene Expression Profiling of JcSAP Genes
For JcSAP gene expression patterns in different plant tissues under abiotic stress, the raw expression data of leaf and root tissues subjected to drought stress was retrieved from the public database of NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE61109) (accessed on 11 September 2022) under the GEO accession number GSE61109. The expression profiles of all JcSAP genes were exhibited as reads per kilobase per million (RPKM) values and illustrated via heat map using TBtools [30,31].

Identification and Phylogenetic Analysis of JcSAP Genes
To gain insight into the identification and evolution of JcSAP genes, the Hidden Markov model profiles of the A20 domain and AN1 domain were used as a probe to screen all the candidate proteins and were then used with the protein sequences of Arabidopsis thaliana and Oryza sativa to construct a phylogenetic tree (see materials and methods). Results demonstrated that A. thaliana, having a genome size of 135 mb and 2n chromosome number of 10, had a total of 14 SAP genes following both the A20 and AN1 domain. Oryza sativa, having a genome size of 372 mb and 2n chromosome number of 24, had a total of 18 SAP genes in which 12 members were following both the A20 and AN1 domain, 5 members were following only the AN1 domain, while 1 member was following only the A20 domain. Jatropha curcas, having a genome size of 416 mb and 2n chromosome number of 22, had a total of 11 SAP genes in which 8 members were following both the A20 and AN1 domain, 3 members were following only the AN1 domain, while there were no single gene members following the A20 domain only ( Figure 1B, Table S1). To further investigate the classification and the evolutionary characteristics of the JcSAP proteins, an unrooted phylogenetic tree was constructed based on the SAP protein sequences of Arabidopsis thaliana, Oryza sativa, and Jatropha ( Figure 1A). All available 34 sequences, including 14 AtSAP, 18 OsSAP, and 11 JcSAP, were mainly clustered into four groups. Group A contained four JcSAP members (JcSAP810a, JcSAP810b, JcSAP111314, and JcSAP12), clustered to AtSAP8, AtSAP10, AtSAP11, AtSAP13, and AtSAP14. Group B consisted of three JcSAP members (JcSAP179, JcSAP3, JcSAP46), clustered to AtSAP1, AtSA3, AtSAP4, At-SAP6, AtSAP7, and AtSAP9. Group C consisted of three JcSAP members (JcSAP2a, JcSAP2b, and JcSAP2c), clustered to AtSAP2. Group D included only one JcSAP member (JcSAP5) clustered to AtSAP5. These results of the comparative phylogenetic relationship predicted that the SAP members clustered together or with other species may share similar biological functions against stresses.

Physicochemical Characteristics of JcSAP Genes
To further get into the physicochemical characteristic of the total 11 identified SAP genes in Jatropha in a comprehensive manner (Figure 1), the physicochemical parameters of JsSAP genes including gene code, location on chromosome, amino acids and CDS length, molecular weight (MW/kDa), isoelectric point (PI), GRAVY, Formula, and Predicted subcellular localization were investigated insilico and exhibited in Tables 1 and S1. Results demonstrated that the amino acid length JcSAP genes were varied from 133 (JcSAP2a) to 288 (JcSAP111314). CDS was ranged from 402 (JcSAP2a) to (867) (JcSAP111314). Molecular weight (MW/kDa) JcSAP genes varied from 14686.65 kDA (JcSAP2a) to 31998.28 (JcSAP111314), while the isoelectric point (PI) ranged from 8 PI (JcSAP179) to 9.4 PI (JcSAP5). Moreover, the predicted subcellular localization revealed that all JcSAP genes are cytoplasmic expect JcSAP111314 and JcSAP12, which appeared to be extracellular. The presence of each JcSAP gene member on a specific chromosome was not predicted in silico, however the GRAVY and formula were given in Table 1. These results provide information about the basic known parameters of the JcSAP genes.

Physicochemical Characteristics of JcSAP Genes
To further get into the physicochemical characteristic of the total 11 identified SAP genes in Jatropha in a comprehensive manner (Figure 1), the physicochemical parameters of JsSAP genes including gene code, location on chromosome, amino acids and CDS length, molecular weight (MW/kDa), isoelectric point (PI), GRAVY, Formula, and Predicted subcellular localization were investigated insilico and exhibited in Tables 1 and S1. Results demonstrated that the amino acid length JcSAP genes were varied from 133 (JcSAP2a) to 288 (JcSAP111314). CDS was ranged from 402 (JcSAP2a) to (867) (JcSAP111314). Molecular weight (MW/kDa) JcSAP genes varied from 14686.65 kDA (JcSAP2a) to 31998.28 (JcSAP111314), while the isoelectric point (PI) ranged from 8 PI (JcSAP179) to 9.4 PI (JcSAP5). Moreover, the predicted subcellular localization revealed that all JcSAP genes are cytoplasmic expect JcSAP111314 and JcSAP12, which appeared to be extracellular. The presence of each JcSAP gene member on a specific chromosome was not predicted in silico, however the GRAVY and formula were given in Table 1. These results provide information about the basic known parameters of the JcSAP genes.

Synteny Analysis of JcSAP Genes
To further support the identification and homologous relationship among SAP genes, Synteny analysis of all the identified SAP genes of Jatropha and Arabidopsis was subjected to the circoletto tool to make a map of comparative synteny circos (see materials and methods). The Synteny map illustrated the relationship among the SAP genes of Jatropha and Arabidopsis species regarding their function, expression, duplication events, and evolution ( Figure 2). The sequences were placed clockwise around a circle, starting at 12 o'clock, and the ideograms were placed in order to maximally untangle the ribbons; the queries and database entries were intertwined. Ribbon colors of the map diagram represent the alignment length, visualizing the sequence similarity and identity level, i.e., blue ≤ 0.25, green ≤ 0.50, orange ≤ 0.75, red > 0.75, providing an essential first glimpse at sequence relationships. The obtained results revealed that Arabidopsis AtSAP1, AtSAP7, and AtSAP9 showed Synteny with JcSAP179 of Jatropha. Similarly, AtSAP2 showed Synteny with JcSAP2a, JcSAP2b, and JcSAP2c. AtSAP3 showed Synteny with JcSAP3. AtSAP4 and AtSAP6 showed Synteny with JcSAP46. AtSAP5 showed Synteny with JcSAP5. AtSAP10 showed Synteny with JcSAP810a, JcSAP810b. AtSAP11, AtSAP13, and AtSAP14 showed Synteny with JcSAP111314. AtSAP12 showed Synteny with JcSAP12. Moreover, based on the color intensity, the inward and outward tangling of ribbons showed conservation and duplication events, respectively, suggesting that SAP genes were conserved in Jatropha during evolution.

Determination of Non-Synonymous (Ka) and Synonymous (Ks) Substitution Rate
The divergence analysis was performed to gain insight into the evolutionary aspects of JcSAP genes by determining the non-synonymous substitution per non-synonymous site (Ka) and synonymous substitution per synonymous site (Ks) for each pair of paralogous JcSAP genes according to the phylogenetic tree ( Figure S1) generated by Ka/Ks calculation server, in order to indicate the evolutionary discretion among JcSAP genes ( Table  2). Results with a Ka/Ks value of < 1 each pair of paralogous genes indicated the purifying selection pressure during the evolution. The divergence time for each pair of JcSAP genes ranged from 9.1 to 40 million years ago (MYA) ( Table 3).

Determination of Non-Synonymous (Ka) and Synonymous (Ks) Substitution Rate
The divergence analysis was performed to gain insight into the evolutionary aspects of JcSAP genes by determining the non-synonymous substitution per non-synonymous site (Ka) and synonymous substitution per synonymous site (Ks) for each pair of paralogous JcSAP genes according to the phylogenetic tree ( Figure S1) generated by Ka/Ks calculation server, in order to indicate the evolutionary discretion among JcSAP genes ( Table 2). Results with a Ka/Ks value of <1 each pair of paralogous genes indicated the purifying selection pressure during the evolution. The divergence time for each pair of JcSAP genes ranged from 9.1 to 40 million years ago (MYA) ( Table 3).

Gene Structure, Domain and Motif Analysis of JcSAP Genes
To further explore the critical fundamental function of the identified JcSAP genes, the JcSAP were further investigated for conserved domains, gene structure organization, and motifs. Eight JcSAP genes like JcSAP179, JcSAP2a, JcSAP2b, JcSAP2c, JcSAP3, JcSAP46, JcSAP5, and JcSAP810b followed both the A20 domain at the N terminal and AN1 domain at C terminal, except JcSAP111314, JcSAP12, and JcSAP810a, which followed only the AN1 domain and had no A20 domain ( Figure 3A). Similarly, the motif analysis revealed that all the JcSAP genes followed five conserved motifs corresponding to the A20 and AN1 domains ( Figure 4A). The logos of the five identified motifs are present in Figure 4B. The demonstration of the gene structure organization further revealed the coding region (CDS) distribution ( Figure 3B), indicating that JcSAP179, JcSAP2a, JcSAP5, JcSAP810a, and JcSAP810b have one exon, 2 JcSAP3, JcSAP46, JcSAP111314, and JcSAP12 have two exons, while JcSAP2b and JcSAP2c have three exons. All these results collectively further support the conserved domain, motif, and structure and thus suggested to share similar biological functions in response to environmental abiotic stresses.

Protein Structure Analysis of JcSAP Genes
To delve further into the structure of the JcSAP genes, the secondary and tertiary structures of the JcSAP proteins were visualized (Table 2, Figure 5). The secondary structure of the JcSAP proteins explored the way these proteins fold and coil. The secondary structure of the JcSAP proteins consisted of four main elements including the α-helix (H%), β-turn (T%), extended chain (E%), and random coil (RC%). In the secondary structure of JcSAP proteins, the random coil (RC%) had the highest value ranging from 49.19% (JcSAP5) to 68.89% (JcSAP8/10a), followed by the α-helix (H%) ranging from 13.2% (JcSAP12) to 37.84% (JcSAP5), again followed by the extended chain (E%) ranging from 10.27% (JcSAP5) to 13.71% (JcSAP12), and β-turn (T%) ranging from 1.04% (JcSAP11/13/14) to 4.68% (JcSAP2b). Similarly, the tertiary structures of the JcSAP proteins visualized via uniprot (https://www.uniprot.org) (accessed on 2 September 2022) further supported the secondary structure of JcSAP proteins. domains ( Figure 4A). The logos of the five identified motifs are present in Figure 4B. The demonstration of the gene structure organization further revealed the coding region (CDS) distribution ( Figure 3B), indicating that JcSAP179, JcSAP2a, JcSAP5, JcSAP810a, and JcSAP810b have one exon, 2 JcSAP3, JcSAP46, JcSAP111314, and JcSAP12 have two exons, while JcSAP2b and JcSAP2c have three exons. All these results collectively further support the conserved domain, motif, and structure and thus suggested to share similar biological functions in response to environmental abiotic stresses.

Protein Structure Analysis of JcSAP Genes
To delve further into the structure of the JcSAP genes, the secondary and tertiary structures of the JcSAP proteins were visualized (Table 2, Figure 5). The secondary structure of the JcSAP proteins explored the way these proteins fold and coil. The secondary structure of the JcSAP proteins consisted of four main elements including the α-helix
Similarly, to predict the miRNA target sites of the JcSAP genes, the coding sequences of the identified JcSAP genes were used against the miRNAs of Jatropha (see material and methods). The results revealed that only three miRNAs (Jcu-miR393, Jcu-miR1628, and Jcu-miR2111) showed interaction with only four JcSAP genes (JcSAP2a, JcSAP2b, JcSAP810a, and JcSAP12) ( Figure 6C). Moreover, miRNA Jcu-miR393 showed interaction with two JcSAP genes, JcSAP2a and JcSAP2b, miRNA Jcu-miR1628 showed interaction with JcSAP12,
Similarly, to predict the miRNA target sites of the JcSAP genes, the coding sequences of the identified JcSAP genes were used against the miRNAs of Jatropha (see material and methods). The results revealed that only three miRNAs (Jcu-miR393, Jcu-miR1628, and Jcu-miR2111) showed interaction with only four JcSAP genes (JcSAP2a, JcSAP2b, JcSAP810a, and JcSAP12) ( Figure 6C). Moreover, miRNA Jcu-miR393 showed interaction with two JcSAP genes, JcSAP2a and JcSAP2b, miRNA Jcu-miR1628 showed interaction with JcSAP12, while miRNA Jcu-miR2111 showed interaction with JcSAP810a. The Excel spreadsheet containing targeting sites predicted miRNAs ID, and the alignment with JcSAP gens are given in the Supplementary Table S2. Genes 2022, 13, x FOR PEER REVIEW 11 of 15 while miRNA Jcu-miR2111 showed interaction with JcSAP810a. The Excel spreadsheet containing targeting sites predicted miRNAs ID, and the alignment with JcSAP gens are given in the supplementary Table S2.

Gene Expression Profiling of JcSAP Genes
To investigate the possible function of JcSAP genes during environmental abiotic stress in Jatropha, the expression data were analysed from leaf and root tissues of Jatropha plants subjected to drought stress [32] ( Figure 7A). The heat map showed the expression level of 11 JcSAP genes in different tissues of leaf and root during drought stress and after recovery ( Figure 7B). The results further revealed that JcSAP179, JcSAP3, and JcSAP2b were highly expressed in leaf and in root, followed by JcSAP111314, JcSAP810a, and JcSAP810b as compared with the other JcSAP genes. Contrarily, JcSAP2a, JcSAP5, JcSAP2c were highly expressed in roots only. These results provide the possible involvement of JcSAP genes in abiotic stress tolerance in Jatropha.

Gene Expression Profiling of JcSAP Genes
To investigate the possible function of JcSAP genes during environmental abiotic stress in Jatropha, the expression data were analysed from leaf and root tissues of Jatropha plants subjected to drought stress [32] ( Figure 7A). The heat map showed the expression level of 11 JcSAP genes in different tissues of leaf and root during drought stress and after recovery ( Figure 7B). The results further revealed that JcSAP179, JcSAP3, and JcSAP2b were highly expressed in leaf and in root, followed by JcSAP111314, JcSAP810a, and JcSAP810b as compared with the other JcSAP genes. Contrarily, JcSAP2a, JcSAP5, JcSAP2c were highly expressed in roots only. These results provide the possible involvement of JcSAP genes in abiotic stress tolerance in Jatropha.

Discussion
Stress-associated proteins (SAP) are novel stress regulatory zinc-finger proteins and are strongly associated with tolerance against various abiotic stresses [1,[12][13][14][15]. SAP gene family has previously identified and comprehensively studied in many plant species including Oryza sativa [5], Arabidopsis thaliana [5], Hordeum vulgare [13], Glycine max [12], Gossypium hirsutum [16], Solanum lycopersicum [2], Cucumis sativus [17], Solanum melongena [1], and Prunus dulcis [18], etc. However, there is no systematic study of SAP genes in the most important biofuel-producing shrub, Jatropha. In present study, 11 SAP genes were identified genome-wide in Jetropha and the phylogenetic divided the JcSAP genes into four groups ( Figure 1); Synteny analysis showed that Jatropha SAP genes had a high homology with the Arabidopsis SAP genes (Figure 2). These results are highly in consistence with the previously reported results of [1,2], suggesting very little variation in SAP gene family members.
To obtain further insight into the similar features and biological functions of JcSAP genes, a search for the conserved domain, motif, and structure was conducted (Figure 3,  4), resulting in the presence of A20 or AN1 domains and their respective motifs, thus suggesting that they share a similar biological function in response to stresses. Zinc-finger A20 or AN1 domains are highly conserved in all plant species and these results are in

Discussion
Stress-associated proteins (SAP) are novel stress regulatory zinc-finger proteins and are strongly associated with tolerance against various abiotic stresses [1,[12][13][14][15]. SAP gene family has previously identified and comprehensively studied in many plant species including Oryza sativa [5], Arabidopsis thaliana [5], Hordeum vulgare [13], Glycine max [12], Gossypium hirsutum [16], Solanum lycopersicum [2], Cucumis sativus [17], Solanum melongena [1], and Prunus dulcis [18], etc. However, there is no systematic study of SAP genes in the most important biofuel-producing shrub, Jatropha. In present study, 11 SAP genes were identified genome-wide in Jetropha and the phylogenetic divided the JcSAP genes into four groups ( Figure 1); Synteny analysis showed that Jatropha SAP genes had a high homology with the Arabidopsis SAP genes (Figure 2). These results are highly in consistence with the previously reported results of [1,2], suggesting very little variation in SAP gene family members.
To obtain further insight into the similar features and biological functions of JcSAP genes, a search for the conserved domain, motif, and structure was conducted (Figures 3 and 4), resulting in the presence of A20 or AN1 domains and their respective motifs, thus suggesting that they share a similar biological function in response to stresses. Zinc-finger A20 or AN1 domains are highly conserved in all plant species and these results are in agreement with the previous reports [15,33]. Moreover, the domain organization revealed some domainwise grouping, illustrating the presence of solely the AN1 domain in three JcSAP genes, with the others having both the A20 and AN1 domains (Figure 3). Except for Arabidopsis and tomato, similar reports were also exhibited in some other plant species like Amborella trichopoda, soybean, rice, and eggplant [1]. Such results may be due to the existence of a homology structure beyond the domain sequences [2]. These cases may also indicate the ancient origin of the specific AN1 domain with respect to its characteristics as compared with the A20 domain, or may also be due to the loss of one domain during evolution, as such cases occur in prologue genes ( Table 2) and were also in line with the previous report on Brassica napus [14]. Moreover, the presence of corresponding motifs of the A20 and AN1 domain and the diversity of exon in the gene structure organization further strengthen the understanding of the evolutionary mechanisms in the JcSAP gene members [34,35]. In the present study, no intron has been found in JcSAP genes ( Figure 3B), and this intron-free characteristic of SAP genes is usually found in other plant species like eggplant, rice, and apple [1,5,12,36]. It may be attributed to the fact that intron-free gene families can reduce posttranscriptional processing and rapidly adjust transcript expression [37].
The differential expression pattern of JcSAP genes in leaf and root tissues against drought stress revealed a potential role of these genes in stress response (Figure 7). Various studies have declared the role of SAP genes in different biotic and abiotic stresses [12,13]. Previous studies indicated that SAP genes play a great role in mediating abiotic stresses including cold, salt, and drought [1,2]. Our results are also in line with the previous studies on other species [12,36]. Altogether, the present study provides a baseline for understanding the molecular role of JcSAP genes and for further study of these genes against different abiotic stresses.

Conclusions
In conclusion, a total 11 SAP genes were identified during this study of Jatropha and were divided into four groups based on the phylogenetic analysis and amino acid sequence similarity; they may share similar biological functions against stresses. The physicochemical properties of JcSAP genes uncovered the basic gene parameters like amino acids and CDS length, molecular weight (MW/kDa), isoelectric point (PI), GRAVY, and molecular formula, and further revealed that most of the JcSAP genes are cytoplasmic, however, no detailed information was found regarding the chromosomal localization of JcSAP genes. The Synteny analysis showed that most of the JcSAP proteins were highly homologous to the Arabidopsis SAP proteins, indicating that SAP genes are conserved in Jatropha during evolution. Further domains and motifs analysis revealed that the A20 and AN1 domains are conserved in JcSAP genes and their similar gene structure features may be due to the duplication events during evolution. The divergence analysis further provided insight into the evolutionary aspects of JcSAP genes revealing the time of divergence from 9.1 to 40 MYA. The promoter region analysis of JcSAP genes resulted in a diverse range of cis-elements including light-responsive elements, hormone-responsive elements, and stressresponsive elements. The predicted miRNA target sites revealed that JcSAP genes may be regulated by a complicated miRNA mediated posttranscriptional regulatory network. In addition, the expression profiles of JcSAP genes in different tissues and stress treatments indicated that many JcSAP genes play functional developmental roles in different tissues, and exhibit significant differential expression under different stress treatments. All these results collectively suggested that JcSAP genes share similar biological functions in response to stresses and provide valuable clues for further investigation of JcSAP genes' function and diversity.