Genome-Wide Identification of DUF668 Gene Family and Expression Analysis under F. solani, Chilling, and Waterlogging Stresses in Zingiber officinale

The domains of unknown function (DUF) superfamilies contain proteins with conserved amino acid sequences without known functions. Among them, DUF668 was indicated widely involving the stress response of plants. However, understanding ZoDUF668 is still lacking. Here, 12 ZoDUF668 genes were identified in ginger by the bioinformatics method and unevenly distributed on six chromosomes. Conserved domain analysis showed that members of the same subfamily had similar conserved motifs and gene structures. The promoter region of ZoDUF668s contained the light, plant hormone and stress-responsive elements. The prediction of miRNA targeting relationship showed that nine ginger miRNAs targeted four ZoDUF668 genes through cleavage. The expression patterns of 12 ZoDUF668 genes under biotic and abiotic stress were analyzed using RT-qPCR. The results showed that the expression of seven ZoDUF668 genes was significantly downregulated under Fusarium solani infection, six ZoDUF668 genes were upregulated under cold stress, and five ZoDUF668 genes were upregulated under waterlogging stress. These results indicate that the ZoDUF668 gene has different expression patterns under different stress conditions. This study provides excellent candidate genes and provides a reference for stress-resistance research in ginger.


Introduction
Zingiber officinale Roscoe, a perennial herb belonging to Zingiberaceae, is widely cultivated for its distinctive edible and medicinal values [1].With the increase in market demand, the planting scale of ginger has gradually expanded, and the problems restricting the yield and quality of ginger have become increasingly prominent.Among them, the factors that affect the yield and quality of ginger mainly include various biotic and abiotic stresses, including ginger blast, stem rot, ginger borer, drought, and waterlogging [2].Cultivating stress-tolerant varieties is currently the most economical and effective way to reduce losses.Therefore, identifying and excavating genes related to stress resistance and screening resistant ginger germplasm resources are of great significance for ginger stress-resistance breeding.
The domains of unknown function protein families (DUFs) refer to a class of protein families with unknown functional domains [3,4].At present, the Pfam database has included 6565 DUFs or unspecified protein families (UPFs), accounting for approximately 34% of the entire protein family in the database [5].The DUFs not only participate in plant growth and development, but also play a positive regulatory role in plant response to biotic/abiotic stresses [3,6].For example, Yuan et al. [7] found that a protein STS1 containing the DUF726 domain plays a crucial role in the development of rice tapetum and pollen wall formation.AtDUF569 positively regulates stem growth and negatively regulates root growth in Arabidopsis thaliana under the stress of nitric oxide [8].In Gossypium hirsutum, the DUF1117 protein encoded by GhRDUF4D increased its expression level after inoculation with Verticillium dahlia [6].In Triticum aestivum, after TaDUF966-9B was silenced, its leaves exhibited severe leaf curling symptoms under salt stress, indicating that TaDUF966s played a positive regulatory role in wheat's response to salt stress [9].
DUF668 is one of the highly conserved plant-specific transcription factors, characterized by a conserved domain containing 29 amino acids, which are widely present in monocotyledonous and dicotyledonous plants [10].This domain was initially discovered in Arabidopsis thaliana and is located at the C-terminus of the A. thaliana PSI protein, which has a promoting effect on plant growth [11].DUF668 genes have shown the potential importance of participating in stress resistance in plants [3].In Glycine max, GmDUF668-8, GmDUF668-20, GmDUF668-30 respond to salt stress by upregulating their expression levels [12].In T. aestivum, TaDUF668-26 and TaDUF668-27 participate in the infection process of Fusarium graminearum and Puccinia striiformis [10].In Ipomoea batatas, IbDUF668-6, IbDUF668-7, IbDUF668-11 and IbDUF668-13 could respond to salt and drought stresses [13].
So far, DUF668 family members have been identified and analyzed in rice [3], cotton [6], sweet potato [13], soybean [12] and wheat [10], and their stress response patterns have been studied.However, there is still a lack of systematic understanding of DUF688 family members in ginger.Therefore, this study systematically identified the members of the DUF668 family in ginger and analyzed the expression characteristics of this gene family under various stress conditions, aiming to preliminarily understand the function of the DUF668 gene in ginger and to provide a theoretical basis for resistance breeding of ginger.

Identification of ZoDUF668s
Through BLASTp alignment and domain analysis using Pfam database, 12 ginger ZoDUF668 members were identified.According to the evolutionary relationship of the phylogenetic tree, they were designated as ZoDUF668-1~ZoDUF668-12.To determine the evolutionary relationship between ZoDUF668s and other plants, a phylogenetic tree was constructed using 143 DUF668 members, including Z. officinale, A. thaliana, O. sativa, Z. mays, and Gossypium.The results showed that the DUF668 protein of five species can be divided into three groups (Group I, II, and III).Group III has the highest number of DUF668, containing 53 genes, including six ZoDUF668s, followed by Group II and Group I, containing 46 and 44 genes, respectively, including zero and six ZoDUF68s, respectively (Figure 1).

Chromosomal Distribution of ZoDUF668s
As can be seen in Figure 2, 12 ZoDUF668 genes are unevenly distributed on six chromosomes.Among them, Chr12 (Chromosome 12) has the highest number of genes, including five ZoDUF668 genes, followed by Chr8 and Chr20, both containing two ZoDUF668 genes, and the remaining chromosomes only contain one ZoDUF668 gene.

Characterization Analysis of ZoDUF668 Proteins Sequence
As shown in Table 1, the average length of the ZoDUF668 protein is 674.75 aa, with a distribution range of 471-1428 aa.The average molecular weight of the protein is 75.27 kDa, and the distribution range is 52.91-156.83kDa.Among them, ZoDUF668-7 has the largest molecular weight of 156.83 kDa, and the remaining proteins are all less than 100 kDa.The ZoDUF668 isoelectric point ranges from 6.64 to 9.68, and 10 ZoDUF668 proteins are predicted to be alkaline proteins (pI > 8).The average protein instability index is 53.57, with a distribution range of 36.44-65.43.Among them, ZoDUF668-7 is a stable protein, while the rest are unstable proteins (instability index > 40).The hydrophilicity (GRAVY) of ZoDUF668 proteins varied from −0.598 to −0.12, suggesting that they are hydrophilic proteins (hydropathicity < 0).The subcellular localization prediction results show that 50% of ZoDUF668 proteins are located in chloroplasts, 42% of ZoDUF668 proteins are located in the nucleus, and only ZoDUF668-7 is located in the cell membrane.

Chromosomal Distribution of ZoDUF668s
As can be seen in Figure 2, 12 ZoDUF668 genes are unevenly distributed on six chromosomes.Among them, Chr12 (Chromosome 12) has the highest number of genes, including five ZoDUF668 genes, followed by Chr8 and Chr20, both containing two Zo-DUF668 genes, and the remaining chromosomes only contain one ZoDUF668 gene.

Chromosomal Distribution of ZoDUF668s
As can be seen in Figure 2, 12 ZoDUF668 genes are unevenly distributed on six chromosomes.Among them, Chr12 (Chromosome 12) has the highest number of genes, including five ZoDUF668 genes, followed by Chr8 and Chr20, both containing two Zo-DUF668 genes, and the remaining chromosomes only contain one ZoDUF668 gene.The three-dimensional structure prediction results indicate that the ZoDUF668 protein is mainly composed of α-helix, random coil, extended strand, and β-turn (Figure 3), with α-helix accounting for the largest proportion (41.53-64.54%),with an average value of 52.33%; Next is the random coil (23.99-39.14%),with an average value of 30.48%, followed by extended strand (6.79-16.46%),with an average of 10.91%;The proportion of β-turn is the smallest (3.85-9.66%),with an average of 6.27%.
proteins (hydropathicity < 0).The subcellular localization prediction results show that 50% of ZoDUF668 proteins are located in chloroplasts, 42% of ZoDUF668 proteins are located in the nucleus, and only ZoDUF668-7 is located in the cell membrane.The three-dimensional structure prediction results indicate that the ZoDUF668 protein is mainly composed of α-helix, random coil, extended strand, and β-turn (Figure 3), with α-helix accounting for the largest proportion (41.53-64.54%),with an average value of 52.33%; Next is the random coil (23.99-39.14%),with an average value of 30.48%, followed by extended strand (6.79-16.46%),with an average of 10.91%;The proportion of βturn is the smallest (3.85-9.66%),with an average of 6.27%.

Gene Structure and Conservative Motif Analysis
Based on the annotation information provided in the GFF3 file, the ZoDUF668 exon/intron structures in the ginger genome were visualized by TBtools.As shown in Figure 4, except for ZoDUF668-2, ZoDUF668-3, ZoDUF668-5, and ZoDUF668-6, and all ZoDUF668s contain 1-13 introns.ZoDUF668-8 contains both 5 ′ and 3 ′ UTR regions, and the remaining Zo-DUF668s contain no UTR regions.Conservative motif analysis showed that the distribution of the five motifs of the ZoDUF668 protein varies among different subfamilies, with each ZoDUF668 containing 4-6 conserved motifs (Figure 4C).Among them, Motif 4 and Motif 5 both constitute the key functional domains DUF347; Motif 3 constitutes DUF668 functional domain; and the other motifs do not match any known functional domain.Furthermore, all 12 ZoDUF668 genes contain Motif 1, Motif 2, and Motif 4, indicating that Motif 1, Motif 2, and Motif 4 exhibit high conservatism during evolution.

Promoter Analysis of ZoDUF668s
Plant CARE was used to predict the 1500 bp upstream promoter sequence of the ZoDUF668 genes.The results showed that 42 types of cis-acting regulatory elements were identified from the promoter regions of ZoDUF668 genes (Figure 5).These cis-elements can be divided into four groups.Among them, 19 cis-acting elements belong to "light-responsive elements", 11 belong to "growth and development", eight belong to "plant hormone response", and four belong to "abiotic stress".A large number of basic cis-acting elements, including TATA-box and CAAT-box, were commonly present in each ZoDUF668 promoter, implying that the ZoDUF668 gene plays an important role in the regulation of ginger growth and development.In addition, there are 19 kinds of light-responsive elements, including GT1-motif, AE-box, Box4, GATA-motif, I-box, G-box, and Sp1.Plant hormone response, including methyl jasmonate (TGACG-motif, CGTCA-motif), auxin (TGA-element, AuxRR-core), gibberellins (P-box, GARE-motif), abscisic acid (ABRE), and salicylic acid (TCA-element), were also detected in the ZoDUF668 promoters.In addition, abiotic response elements, low-temperature response elements (LTR), oxidative stress response elements (ARE), drought-induced response elements (MBS), and defense and stress response elements (TC-rich repeats) were also present in the ZoDUF668 promoter region, suggesting that ZoDUF668s may play a key regulatory role in stress response.
ZoDUF668s participate in the growth and development, with expression in roots and aerial stem and leaves higher than in rhizomes.ZoDUF668-2, ZoDUF668-8, ZoDUF668-10, ZoDUF668-11, and ZoDUF668-12 show increased expression levels as the aerial stem leaves grow, while ZoDUF668-3, ZoDUF668-7, and ZoDUF668-11 show decreased expression levels as the aerial stem leaves grow.

Post-Transcription Regulation of miRNA to ZoDUF668s
As can be seen from Figure 7, all nine miRNAs target four ZoDUF66s genes through the cleavage effect.Notably, ZoDUF668-8 was potentially targeted by two miRNAs, including miR159 and miR319e_1; miR529a regulates the expression of ZoDUF668-5; and miR396a_5p, miR396b, miR396b_5p and miR396e were predicted to target to ZoDUF668-5 and ZoDUF668-12 simultaneously.These results indicate that multiple miRNAs target ZoDUF668s and form a miRNA-mRNA regulatory network, participating in the post-transcriptional expression regulation of ZoDUF668s.cluding miR159 and miR319e_1; miR529a regulates the expression of ZoDUF668-5 miR396a_5p, miR396b, miR396b_5p and miR396e were predicted to target to ZoDUF 5 and ZoDUF668-12 simultaneously.These results indicate that multiple miRNAs t ZoDUF668s and form a miRNA-mRNA regulatory network, participating in the postscriptional expression regulation of ZoDUF668s.

GO Function Enrichment Analysis
GO (Gene Ontology) analysis of the ZoDUF668 protein (Figure 8) showed tha biological pathways that the ZoDUF668 protein mainly participates in include cel processes (3 ZoDUF668s), molecular functions (ZoDUF668-7), and single biological cesses (12 ZoDUF668s).These ZoDUF668 proteins constitute cellular components, su cells, organelles, membrane and so on.It is worth noting only ZoDUF668-7 exerts m ular function, including nucleotide binding (GO:0000166, GO:0001882), ATP bin (GO:0005524), and transferase activity (GO:0016740).In addition, the molecular func of "response to stimulus (GO:0050896)", "positive regulation of growth (GO:00459 and "biological regulation (GO:0065007)" provide evidence of the involvement o DUF668 in ginger growth and development, as well as its response to environm stressors.

GO Function Enrichment Analysis
GO (Gene Ontology) analysis of the ZoDUF668 protein (Figure 8) showed that the biological pathways that the ZoDUF668 protein mainly participates in include cellular processes (3 ZoDUF668s), molecular functions (ZoDUF668-7), and single biological processes (12 ZoDUF668s).These ZoDUF668 proteins constitute cellular components, such as cells, organelles, membrane and so on.It is worth noting only ZoDUF668-7 exerts molecular function, including nucleotide binding (GO:0000166, GO:0001882), ATP binding (GO:0005524), and transferase activity (GO:0016740).In addition, the molecular functions of "response to stimulus (GO:0050896)", "positive regulation of growth (GO:0045927)", and "biological regulation (GO:0065007)" provide evidence of the involvement of ZoDUF668 in ginger growth and development, as well as its response to environmental stressors.

ZoDUF668s Respond to Biotic Stress
To obtain a comprehensive comprehension of the functional significance of ZoDUF668s in response to biotic stress, the expression patterns of 12 ZoDUF668s were analyzed using quantitative RT-qPCR in response to F. solani stress (Figure 9).The results showed that the expression levels of ZoDUF668-5, -6, and -12 continuously decreased within 3 days after treatment with F. solani.Among them, ZoDUF668-5 exhibited an expression level of only 4% compared to the CK at 3 days post-treatment.In contrast, the expression level of ZoDUF668-10 increased continuously over time, reaching 3.2-fold higher than the CK at 3 days post-treatment.

ZoDUF668s Respond to Biotic Stress
To obtain a comprehensive comprehension of the functional significance of Zo-DUF668s in response to biotic stress, the expression patterns of 12 ZoDUF668s were analyzed using quantitative RT-qPCR in response to F. solani stress (Figure 9).The results showed that the expression levels of ZoDUF668-5, -6, and -12 continuously decreased within 3 days after treatment with F. solani.Among them, ZoDUF668-5 exhibited an expression level of only 4% compared to the CK at 3 days post-treatment.In contrast, the expression level of ZoDUF668-10 increased continuously over time, reaching 3.2-fold higher than the CK at 3 days post-treatment.

ZoDUF668s Respond to Biotic Stress
To obtain a comprehensive comprehension of the functional significance of Zo-DUF668s in response to biotic stress, the expression patterns of 12 ZoDUF668s were analyzed using quantitative RT-qPCR in response to F. solani stress (Figure 9).The results showed that the expression levels of ZoDUF668-5, -6, and -12 continuously decreased within 3 days after treatment with F. solani.Among them, ZoDUF668-5 exhibited an expression level of only 4% compared to the CK at 3 days post-treatment.In contrast, the expression level of ZoDUF668-10 increased continuously over time, reaching 3.2-fold higher than the CK at 3 days post-treatment.

ZoDUF668s Respond to Abiotic Stress
As illustrated in Figure 10, ZoDUF668s play an implant role in plants respond to abiotic stress.Under chilling injury, the expression levels of ZoDUF668-2, -3, and -11 significantly increased after 4 h of treatment, among them, ZoDUF668-3 showed the most significant increase, reaching 13.8-fold higher than the CK; subsequently, the expression levels decreased after 12 h but remained higher than the CK.The expression levels of ZoDUF668-7 and -8 continued to increase within 12 h of treatment, and were 2.3-and 2.1-fold higher than CK at 12 h of treatment, respectively.otic stress.Under chilling injury, the expression levels of ZoDUF668-2, -3, and -11 significantly increased after 4 h of treatment, among them, ZoDUF668-3 showed the most significant increase, reaching 13.8-fold higher than the CK; subsequently, the expression levels decreased after 12 h but remained higher than the CK.The expression levels of ZoDUF668-7 and -8 continued to increase within 12 h of treatment, and were 2.3-and 2.1-fold higher than CK at 12 h of treatment, respectively.Under waterlogging stress, the expression levels of 7 ZoDUF668s showed an initial increase followed by a decrease.However, the expression level of ZoDUF668-12 continued to increase within 6 days of treatment and was 1.7-fold higher than the CK at 6 days.Additionally, ZoDUF668-9 showed an initial increase in expression level, reaching 1.5 times higher than the control at 3 days and remained stable thereafter.

Discussion
Ginger is an important spice crop, and its cultivation process is susceptible to various biotic and abiotic stresses [14].Relevant research shows that that the DUF668 gene plays an important role in plant growth and development and stress responses [6].Despite this, the classification, evolution, and function of this gene family in ginger have not been systematically studied.In this study, 12 ZoDUF668s were identified from the ginger genome and separated into two subfamilies according to the phylogenetic tree branches and designated as ZoDUF668-1 to ZoDUF668-12.
Structure determines function, and the structural diversity of exons/introns often plays a key role in the evolution of gene families [15].The gene structure result shows that there are more introns of ZoDUF668 in Group II than in Group I.This phenomenon, with one set of DUF668 introns significantly higher than the other, is consistent across various plants such as wheat [10], rice [3], and cotton [6].The difference in the number of introns may be one of the reasons for the functional differentiation of ZoDUF668s [16].
The cis-elements in the promoter region participate in plant growth and development and respond to environmental changes by regulating the expression of related genes [17].There was a significant positive correlation between upstream promoter response genes and their cis-elements [18].Previous studies have shown that cis-elements, such as AE-box, ATCT-motif, G-box, GT1-motif, GATA-motif, Box-4, AT-rich, and I-box, are important for the regulation of light-mediated transcriptional activity [19].In this study, we found the G-box, Box-4, and GT1 motifs are the most abundant elements in ginger ZoDUF668s; simultaneously, these cis-elements have been identified in the study of Ipomoea batatas [13] and Triticum aestivum [10], strongly implying that, through these light-responsive ciselements, ZoDUF668s play a role in photosynthesis and therefore regulate gene expression.Furthermore, cis-elements in response to abiotic stress, such as LTR in response to low temperature, ARE related to hypoxia stress, MBS related to drought stress and TC-rich in response to defense and stress, were also detected in the promoter region of ZoDUF668s.RT-qPCR analysis revealed that the expression levels of 6 ZoDUF668s increased under cold stress, while the expression levels of 10 ZoDUF668s increased within 3 days of waterlogging stress.Therefore, we speculate that when plants are subjected to chilling and waterlogging stresses, the cis-elements in the promoter region upregulate the expression of ZoDUF668s in response to these stresses.
Research has indicated that DUF668 plays a significant role in plant responses to biotic stresses.In G. hirsutum, GhDUF668-05/08/11/23/28 were significantly upregulated under drought and Verticillium dahliae, thus participating in the stress response process of cotton [6].In O. sativa, the expression levels of OsDUF668-1/4/5 are significantly upregulated under the pathogen stress of rice blast fungus, while the expression levels of OsDUF668-6/7/11/12 are significantly decreased [3].Similarly, the expression levels of ZoDUF668s are also regulated by the pathogen stress of R. solanacearum and F. solani.Meanwhile RT-qPCR analysis revealed that three genes (ZoDUF688-2, -10, and -11) were upregulated under F. solani stress.Among them, ZoDUF668-10 showed significantly higher expression than the control (CK) within 3 days of treatment, implying ZoDUF668-10 may be a resistance gene.Therefore, in the subsequent study, we will construct an overexpression vector for ZoDUF668-10 to further validate its role as a disease-resistant gene and elucidate its biological function.
There are two main mechanisms by which miRNAs exert their effects.One class of miRNAs exhibits complementarity to their target mRNA and interferes with gene expression through a cleavage activity, leading to degradation of the target mRNA; the other class of miRNA functions by inhibiting the translation process, thereby regulating gene expression at the post-transcriptional level [20].Previous studies have shown that miR159 can enhance plant tolerance to abiotic stress [21], such as in G. max, where the expression of miR159 decreases under cold stress [22].In this study, we observed that the expression level of ZoDUF668-8, which is targeted by miR159, specifically increased under chilling stress conditions.Based on these findings, we speculate that in ginger, chilling stress leads to a downregulation of miR159 expression, consequently resulting in an upregulation of ZoDUF668-8 expression.In addition, as most members of the miR396 family showed downregulation in expression levels under waterlogging stress in Cucumis sativus [23], and combined with RT-qPCR analysis, we speculate that waterlogging stress in ginger would also lead to a decrease in the expression levels of miR396, resulting in an upregulation of the expression levels of ZoDUF668-5 and ZoDUF668-12 to respond to the waterlogging stress.

Chromosome Location Analysis
The chromosome locations of ZoDUF668s are extracted from the ginger genome GFF file, according to the start and end position information of ZoDUF668s on the chromosome, and the chromosome distribution map is drawn with "MapInspect" [9].

Gene Structure and Conserved Motifs Analysis of the ZoDUF668 Gene Family
According to GFF3 gene structure annotation information, the gene exon/intron structure map of ZoDUF668s was drawn using TBtools [9].Conservative motifs were analyzed using MEME (https://meme-suite.org/meme/, accessed on 15 October 2023), parameter setting: the width of motifs ranged from 6 to 50 aa; any number of repetitions; the maximum fundamental number was 5.Then, the conserved motifs were visualized by TBtools [27].

Cis-Element Analysis
For cis-acting element analysis, the upstream promoter sequence (1-1500 bp) of Zo-DUF668 was manually extracted from the genome sequence and uploaded into the Plant-CARE database to identify cis-elements in the promoter region [31].The R software package "pheatmap" (R-4.2.2) was used to organize and display the analysis results.

Transcriptomic Analysis of ZoDUF668s
Ginger RNA-seq data were downloaded from the NCBI database and mapped to ginger genome using Hisat2 [32].The expression level of ZoDUF668s (Fragments Per Kilobase of exon model per million mapped fragments) was calculated by Cufflinks.Log 2 (FPKM + 1) values and R package "pheatmap" (R-4.2.2) were used to generate the heatmap to illustrate the expression profiling of ZoDUF668s under various growth stages and stress conditions [33].

Prediction of miRNAs Targeting Relationship
The mature coding sequences of ginger miRNAs were collected from the previous studies, the coding sequence and miRNA sequences were uploaded to psRNATarget (https://www.zhaolab.org/psRNATarget/,accessed on 22 October 2023) database [34].Then, the mapping of the targeting relationship between miRNAs and ZoDUF668s was performed using the "ggalluvia" package in R software (R-4.2.2) [30].

Gene Ontology (GO) Data Analysis and Prediction of ZoDUF668s
The GO (Gene Ontology) database was used for functional annotation of the Zo-DUF668 gene.GO (Gene ontology) analysis is performed in python.The full-length amino acid sequence of ZoDUF668 proteins was uploaded to the original program, and then drawn and annotated [35].The analysis results provide a three-layer structure definition method for ZoDUF668 protein, which are biological process (BP), cellular component (CC), and molecular function (molecular function, MF), used to describe the related functions of ZoDUF668 protein.

Plant Materials
Ginger ("Fengtoujiang", Jingzhou, Hubei Province, China) is cultivated in a glass greenhouse at the college of Horticulture and Gardening, Yangtze University.Healthy and uniformly sized ginger seeds were selected for disinfection and germination, and then planted directly (the temperature was set to 26 • C, relative humidity of 70~80% with 12 h/12 h day/night period).When the plant reached a height of about 90 cm, seedlings with the same growth were selected for stress treatment.The different treatments were as follows: (a) inoculation with Fusarium solani, all tested strains were provided by the Xiangxin Crop Research Institute of Yangtze University.It was cultured on a PDA plate for 3-4 d under dark conditions of 28 • C. The spores were collected by rinsing the culture medium with sterile water, and the concentration of spore suspension was adjusted to 1 × 10 4 sporangia ml −1 .The ginger seedling basal part of stems were uniformly wounded using a needle prick method and then injected with 30 µL of spore suspension, with sterile water treatment as the CK control.The infection sites were collected 1 and 3 d after inoculation with F. solani and immediately frozen in liquid nitrogen.After (c) chilling (10 • C) for 0, 4 and 12 h of treatment, the leaves were collected and immediately frozen in liquid nitrogen.The samples treated for 0 h were used as CK control.After (d) waterlogging (water submerged 1 to 2 cm above the rhizome of ginger) for 0, 3 and 6 d of treatment, the roots were collected.The samples treated for 0 h were used as CK control [36].The collected tissue samples will be stored at temperature of −80 • C until further use.For each treatment, there were three biological replications and three technical replications, and at least ten plants were planted.

RNA Extraction and Quantitative Real-Time PCR Analysis
Total RNA extracted from collected ginger tissue using TRIzol reagent (GenStar, Beijing, China), and then reverse transcribe the total RNA into cDNA using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China).Each treated cDNA sample was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on the CFX 96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA).The reaction system (20 µL) is as follows: 10 µL 2 × ChamQ SYBR qPCR Master Mix, forward and reverse primers each 0.4 µL, cDNA 2 µL, add ddH 2 O to 20 µL.The reaction procedure was as follows: 95 • C for 3 min, 95 • C for 30 s, 55 • C for 30 s, 72 • C for 20 s, a total of 35 cycles.The relative expression level of ZoDUF668s was calculated using the 2 −∆∆Ct method [37].Each treatment includes three biological replications, and three technical duplications were performed to each cDNA.

Statistical Analysis
Excel 2021 was used to sort out and analyze the data, and student's t-test was used for difference significance analysis.

Conclusions
In this study, a total of 12 DUF668 family members were identified from the ginger genome, which were located on six chromosomes, and divided into two subfamilies according to evolutionary relationships.All of them were hydrophobic proteins and unstable proteins.The ZoDUF668 genes may be involved in the regulation of biotic and abiotic stress responses of ginger, including F. solani, waterlogging, and chilling stresses.This study laid a foundation for elucidating the function of ZoDUF668 genes in ginger.

Figure 2 .
Figure 2. Chromosomal distribution of ZoDUF668s.Chr stands for chromosome.The ruler on the left represents the length of chromosomes (Mb).

Figure 2 .
Figure 2. Chromosomal distribution of ZoDUF668s.Chr stands for chromosome.The ruler on the left represents the length of chromosomes (Mb).

Figure 2 .
Figure 2. Chromosomal distribution of ZoDUF668s.Chr stands for chromosome.The ruler on the left represents the length of chromosomes (Mb).

Figure 4 .
Figure 4. Gene structure and conservative motif analysis.(A) Phylogenetic tree of ZoDUF668s, different colored represent different species.(B) Gene structure analysis of ZoDUF668s, the green boxes, yellow boxes, and blacklines represent the exon, untranslated regions, and intron, respectively.(C) Motifs identification of ZoDUF668s.(D) Conserved amino acid sequences and functional domain of 5 motifs in ZoDUF668s.The seqlogo diagram shows that the conservatism of motifs at each position, with higher letters indicating better conservatism at that position.Different amino acids in the same position will be scaled according to their frequency.

Figure 5 .
Figure 5. Analysis of cis-elements in promoter regions of ZoDUF668 genes.

Figure 6 .
Figure 6.Expression profiles of the ZoDUF668 gene under multiple growth stages, and Fusarium solani and Ralstonia solanacearum infection.St, aerial stem and leaves; Ro, roots; Rh1 to Rh5, rhizomes collected at five developmental stages based on their growth segments.Rs, Ralstonia solanacearum; Fs, Fusarium solani; WFPS, water-filled pore space; Ch, chitosan.

Figure 5 .
Figure 5. Analysis of cis-elements in promoter regions of ZoDUF668 genes.

Figure 5 .
Figure 5. Analysis of cis-elements in promoter regions of ZoDUF668 genes.

Figure 6 .
Figure 6.Expression profiles of the ZoDUF668 gene under multiple growth stages, and solani and Ralstonia solanacearum infection.St, aerial stem and leaves; Ro, roots; Rh1 to Rh5, r collected at five developmental stages based on their growth segments.Rs, Ralstonia solan Fs, Fusarium solani; WFPS, water-filled pore space; Ch, chitosan.

Figure 6 .
Figure 6.Expression profiles of the ZoDUF668 gene under multiple growth stages, and Fusarium solani and Ralstonia solanacearum infection.St, aerial stem and leaves; Ro, roots; Rh1 to Rh5, rhizomes collected at five developmental stages based on their growth segments.Rs, Ralstonia solanacearum; Fs, Fusarium solani; WFPS, water-filled pore space; Ch, chitosan.

Figure 7 .
Figure 7. Sankey diagram of miRNA targeting relationship with ZoDUF668s.The three col represent miRNA, mRNA, and inhibition effect.

Figure 7 .
Figure 7. Sankey diagram of miRNA targeting relationship with ZoDUF668s.The three columns represent miRNA, mRNA, and inhibition effect.

Figure 8 .
Figure 8. Gene Ontology (GO) classification based on ZoDUF668 annotation.GO terms are divided into three main categories, orange represents cellular components, blue represents molecular functions, and orange represents biological processes.

Figure 9 .
Figure 9. Analysis of ZoDUF668 expression under F. solani stress.The y axis represents the relative expression level, and the x axis represents the time point after stress treatment.Each bar represents the average of nine replicates, and error bar represents standard deviation (SD), n = 9.Different letters in columns indicate significant differences between the treatments at p < 0.05 level.

Figure 8 .
Figure 8. Gene Ontology (GO) classification based on ZoDUF668 annotation.GO terms are divided into three main categories, orange represents cellular components, blue represents molecular functions, and orange represents biological processes.

Figure 8 .
Figure 8. Gene Ontology (GO) classification based on ZoDUF668 annotation.GO terms are divided into three main categories, orange represents cellular components, blue represents molecular functions, and orange represents biological processes.

Figure 9 .
Figure 9. Analysis of ZoDUF668 expression under F. solani stress.The y axis represents the relative expression level, and the x axis represents the time point after stress treatment.Each bar represents the average of nine replicates, and error bar represents standard deviation (SD), n = 9.Different letters in columns indicate significant differences between the treatments at p < 0.05 level.

Figure 9 .
Figure 9. Analysis of ZoDUF668 expression under F. solani stress.The y axis represents the relative expression level, and the x axis represents the time point after stress treatment.Each bar represents the average of nine replicates, and error bar represents standard deviation (SD), n = 9.Different letters in columns indicate significant differences between the treatments at p < 0.05 level.

Figure 10 .
Figure 10.Analysis of ZoDUF668 expression under chilling and waterlogging stresses.(A) Chilling and (B) waterlogging stress.The y axis represents the relative expression level, and the x axis represents the time point after stress treatment.Each bar represents the average of nine replicates, and error bar represents standard deviation (SD), n = 9.Different letters in columns indicate significant differences between the treatments at p < 0.05 level.

Figure 10 .
Figure 10.Analysis of ZoDUF668 expression under chilling and waterlogging stresses.(A) Chilling and (B) waterlogging stress.The y axis represents the relative expression level, and the x axis represents the time point after stress treatment.Each bar represents the average of nine and error bar represents standard deviation (SD), n = 9.Different letters in columns indicate significant differences between the treatments at p < 0.05 level.