Genome-wide characterization of the WRKY gene family in cultivated strawberry (Fragaria × ananassa Duch.) and the importance of several group III members in continuous cropping

WRKY transcription factors play important roles in many plant developmental processes and adaptation to the environment. However, little knowledge is available about the WRKY gene family in cultivated strawberry (Fragaria × ananassa Duch.), an important soft fruit worldwide. In this study, a total of 47 WRKY gene members were identified and renamed on the basis of their order on the chromosomes. According to their evolutionary events and conserved structure, the 47 FaWRKYs were divided into three major groups with several subgroups. A cis-element analysis showed that all FaWRKYs possessed at least one stress response-related cis-element. Comprehensive analysis, including phylogenetic analysis and expression profiling, based on real-time qPCR analysis in root, stem, leaf and fruit was performed on group III FaWRKY genes. The phylogenetic tree of the WRKY III genes in cultivated strawberry, wild Strawberry, Arabidopsis, tomato, and rice was divided into five clades. Additionally, the expression profiles of the FaWRKY genes in response to continuous cropping were further investigated based on RNA-seq data. FaWRKY25, FaWRKY32, and FaWRKY45, which are group III FaWRKY genes, were upregulated after continuous cropping. The level of reactive oxygen species (ROS) and the expression levels of PR1 and peroxidase were higher in continuous cropping (CC) than in non-continuous cropping (NCC). The results indicated that group III FaWRKYs might play an important role in continuous cropping. These results provide a foundation for genetic improvements for continuous cropping tolerance in cultivated strawberry.


Phylogenetic analysis and multiple sequence alignment.
To explore the phylogenetic relationship of the FaWRKY proteins, we constructed a phylogenetic tree of 47 FaWRKY proteins using MEGA 7.0. The phylogenetic analysis showed that the cultivated strawberry WRKY proteins could be divided into three major groups (groups I, II and III) with five subgroups (II a, II b, II c, II d, and II e) corresponding to the AtWRKY proteins 9 (Fig. 2a). Among the 47 FaWRKY proteins, 10 belong to group I; 31 belong to group II, including 3 to group II a, 6 to group II b, 10 to group II c, 5 to group II d, and 7 to group II e; and 6 belong to group III. However, FaWRKY34 could not be clustered into any group.
Multiple sequence alignment of the WRKY domains, which span approximately 60 amino acids, revealed that the conserved amino acid sequences were defined by WRKYGQ(K)K at the N-terminal end, together with a zinc finger-like motif at the C-terminal end (Fig. 2b,c). The domain number, conserved heptapeptides and zinc finger types of each group are shown in Table 2. The group I FaWRKY proteins contained two WRKY domains (WRKYGQK) and C2H2-type zinc finger motifs, group IN and group IC. Groups II and III contained one WRKY domain and zinc finger motif. Most members of group II (a, b, c, d, e), except for FaWRKY50, which was defined by WRKYGKK and C2H2-type zinc finger motifs, were defined by WRKYGQK and C2H2-type zinc finger motifs. All six members in group III contain the conserved WRKYGQK and the special C2HC-type zinc fingers. stress-related cis-elements in FaWRKY promoter regions. Plants use complex defense signaling pathways, including stress-related cis-elements, to regulate their adaptation to ever-changing stresses 18 . The WRKY TFs could regulate gene expression by binding to their cis-elements during stress responses. To further determine if WRKY TFs engage in potential regulatory mechanisms during stress responses, the promoter regions, sequences of approximately 1.5-kb upstream from the translation start sites, were submitted into PlantCARE to identify the cis-elements. In this study, ten stress response elements, including ABRE (cis-acting element involved in abscisic acid responsiveness), AuxRR-core (cis-acting regulatory element involved in auxin responsiveness), CGTCA-motif (cis-acting regulatory element involved in MeJA responsiveness), LTR (cis-acting element involved in low-temperature responsiveness), MBS (MYB transcription factor binding site involved in drought inducibility), P-box (gibberellin-responsive element), TCA-element (cis-acting element involved in salicylic acid responsiveness), TC-rich repeats (cis-acting element involved in defense and stress responsiveness), TGA-element (auxin-responsive element) and W-box (WRKY transcription factor binding site in defense responses), were analyzed, and the data are displayed in Fig. 3.
In this study, all FaWRKY TFs had at least 1 stress response-related cis-element. The elements associated with hormone regulation, including ABRE, AuxRR-core, CGTCA motif, P-box, TCA-element, and TGA-element, were identified in many FaWRKY promoter regions. In total, 38 FaWRKYs (80.8%) had one or more ABRE, suggesting a potential abscisic acid response under stress conditions. One or more CGTCA motifs, which are involved in MeJA responsiveness, existed in 33 FaWRKYs (70.2%). TCA-element, TGA-element, P-box and AuxRR-core were located in 14, 14, 10 and 3 FaWRKYs, respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ Cis-elements related to other stresses were found in several FaWRKY promoter regions. For example, 26 FaWRKYs contained one or more W-box elements in their promoter regions. In addition, several LTR, MBS and TC-rich repeats, which are involved in low temperature, drought inducibility and defense responsiveness, were also found in many FaWRKYs. evolutionary analysis of WRKY group III TFs in strawberry and several different species. The group III WRKY TFs are thought to play a key role in plant evolution and adaptation 19 . To further investigate the phylogenetic relationship of the WRKY III genes in cultivated strawberry, wild strawberry, Arabidopsis, tomato, and rice, an unrooted phylogenetic tree of WRKY III complete protein sequences was constructed using MEGA 7.0 (Fig. 4). The phylogenetic tree indicated that the WRKY III proteins were divided into five clades. Clades 5 had the most Group III gene members (19), followed by clade 3 (17), clade 1 (12) and clade 2 (12); clades 4 contained the least WRKY Group III TF members (8) (Fig. 4). Clade 3 and 5 included WRKY Group III TFs in all 5 species, while the members of clades 1 and 2 contained four species. Clade contained other four species except strawberry, while clade 2 only contained all dicots, cultivated strawberry, wild strawberry, Arabidopsis, and tomato. Clade 4 was composed of rice only, which might be a monocot-specific clade. Clade 5 consisted of two sub-branch, one was rice, other belong to dicots. This distribution may be related to the split of monocots and dicots. Among those WRKY Group III TFs, six cultivated strawberry WRKY Group III proteins were classified into clade 2 (FaWRKY31, FaWRKY32 and FaWRKY44), clade 3 (FaWRKY25 and FaWRKY45), and clade 5 (FaWRKY43). Five of the six cultivated strawberry WRKY Group III TFs were clustered together with the wild strawberry members. Based on the phylogenetic analysis, five pairs (FaWRKY25 and FvWRKY27, FaWRKY32 and FvWRKY38, FaWRKY43 and FvWRKY53, FaWRKY44 and SlWRKY80 and FaWRKY45 and FvWRKY56) were identified as orthologous genes.
Expression patterns of cultivated strawberry WRKY III genes in different tissues. To obtain insight into the potential functions of FaWRKY group III genes during development, the expression patterns of six FaWRKY group III genes were analyzed in roots, stems, leaves and fruits using qRT-PCR. The six FaWRKY group III genes revealed significant tissue-specific expression patterns (Fig. 5). All six FaWRKY group III genes were expressed in roots and stems. Among the six genes, three showed the highest expression in roots (, FaWRKY25, FaWRKY31 and FaWRKY45), two in stems (FaWRKY32 and FaWRKY44), and one in leaves  www.nature.com/scientificreports www.nature.com/scientificreports/ cropping (FDR < 0.01 and log2FC ≥ 2). Interestingly, most of the upregulated genes (3/4) belonged to FaWRKY group III. The special expression of group III genes exhibited significant trends in response to continuous cropping. The FPKM of FaWRKY group III genes is shown in Fig. 6b. FaWRKY45 had significantly higher expression (P-value < 0.01) and FaWRKY25 and FaWRKY32 had higher expression (P-value < 0.05) in response to continuous cropping.   www.nature.com/scientificreports www.nature.com/scientificreports/ other changes associated with upregulation of FaWRKY genes. Reactive oxygen species (ROS) play a vital role in plant-environment interactions. Massive ROS generation is toxic to plant cells and damages cellular membranes. In this study, the level of ROS in continuous cropping roots was significantly higher than that in non-continuous cropping roots (Fig. 7a). The expression level of PR1 was higher in CC lines than in NCC plants (Fig. 7b). Similarly, the expression levels of peroxidase genes were also significantly higher in CC roots than in NCC roots (Fig. 7c). www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
The WRKY transcription factor genes are involved in the regulation of a large number of processes in plants.
Previous studies have revealed some information on the WRKY gene family in many species, such as Arabidopsis, rice, tomato, cotton, pineapple and wild strawberry. However, little is known about WRKY gene families in cultivated strawberry.
In the present study, 47 WRKY gene members were identified in cultivated strawberry, and their evolutionary events, conserved structure, stress-related cis-elements, comprehensive analysis of group III WRKY genes and expression patterns in continuous cropping were also examined. Distinct expansion in group III WRKY genes was detected in cultivated strawberry under continuous cropping. These results showed that three group III WRKY gene members (FaWRKY25, FaWRKY32 and FaWRKY45) were upregulated in response to continuous cropping.
The WRKY gene members were classified into three major groups (I, II and III), and group II was divided into five distinct subgroups (II a-e), as previously described for the model herbaceous plant Arabidopsis 9 . The phylogenetic analysis of FaWRKY TFs also indicated that the 47 FaWRKY proteins can be divided into the same groups (I, II a-e, and III). However, FaWRKY34 could not be clustered into any group, similar to AtWRKY38 and AtWRKY52, which were found in Arabidopsis 9 .
The loss of the WRKY domain usually occurs in many monocotyledon species, such as rice and maize 13,[20][21][22] . Group I WRKY TFs in cultivated strawberry all contain two WRKY domains, and no domain loss events were found. The conserved structural domains of their encoded proteins were assessed in this study. Multiple sequence alignments showed that FaWRKY37 in group II c had unique sequences in the WRKY domain (WRKYGKK). Based on previous studies, sequence variation in the WRKY domain might influence the normal interactions and binding specificities with downstream target genes 15,23,24 .
Stress-related cis-elements were identified in the promoter regions of 47 FaWRKYs involved in different functions, such as hormone regulation (ABRE, AuxRR-core, CGTCA motif, P-box, TCA-element, and TGA-element), abiotic stress (LTR and MBS), and disease resistance (TC-rich repeats and W-box elements). The WRKY transcription factors could be regulated by binding different cis-elements in their own promoters [25][26][27] . The WRKY TF promoters could be regulated by autoregulation or cross-regulation by interaction with each other 28,29 . In total, 26 FaWRKYs had one or more W-boxes, suggesting that those WRKY TFs might be regulated by autoregulation or cross-regulation.
The group III WRKY gene members might be the most dynamic group with regard to gene family evolution 15,19 . There are 13 group III WRKY genes in Arabidopsis 30 , 6 in grape 19 , 28 in rice 19 , 10 in Populus 19 , and 10 in wild strawberry 14 . However, the number of group III WRKY gene members was related to the diversity of the WRKY gene family size 19 . In this study, group III FaWRKY gene members totaled six, which was fewer than the corresponding number in most other plants; this is a potential cause of the smaller number of cultivated strawberry WRKY family members.
The group III gene members play an important role in plant evolution, and their evolutionary history would provide more clues to the origin and evolution of the WRKY gene family 19 . In the current study, the plant WRKY III members were clustered into five clades, and the closely related species might tend to be clustered together. www.nature.com/scientificreports www.nature.com/scientificreports/ Differences in the orthologous gene pairs between cultivated strawberry/wild strawberry (5) and cultivated strawberry/tomato (1) showed a closer relationship in different strawberries. FaWRKY44, which was clustered together with SlWRKY80, is not found in wild strawberry.
The expression patterns of WRKY group III genes in different tissues have been evaluated in many species, and there is no uniform gene expression profile for plant WRKY group III genes 15,30 . According to the qRT-PCR expression patterns of all FaWRKY group III gene members in different cultivated strawberry tissues, different FaWRKY group III proteins may have diverse functions. In this study, the expression analysis revealed that FaWRKY25, FaWRKY31, and FaWRKY45 were highly expressed in the roots, suggesting a putative role in the development of roots. Moreover, those three genes showed similar expression patterns in different tissues, suggesting that these three genes might have retained redundant functions in regulating the same functions 19 .
In plants, WRKY gene members play significant roles in regulating defense gene expression in response to adverse conditions 19 , including biotic and abiotic stresses. Among those WRKY members, the Group III WRKY proteins have been considered to play important roles in regulating plant immunity, resistance and development. For example, the majority of group III members in Arabidopsis are involved in pathogen attack and salicylic acid (SA) treatment 30 . The WRKY Group III transcription factors in tomato were identified to participate in the TYLCV defense signaling pathway 25 . Group III GhWRKY genes are involved in fiber development and leaf senescence and can be induced by plant hormones, such as jasmonic acid (JA), abscisic acid (ABA), ethylene, and salicylic acid (SA) 6 . Therefore, certain WRKY Group III proteins have significant functions in defense regulation against abiotic and/or biotic stresses.
Long-term continuous cropping often brings a complex change in the structure of soil, and strawberry is vulnerable to this problem. The complex stresses to which strawberry responds include biotic stresses, such as the accumulation of soil-borne pathogens and plant-feeding nematodes, and abiotic stresses, such as nutrient availability imbalance, soil physicochemical property deterioration, and autotoxic substance accumulation [31][32][33][34][35] . The ROS network plays a vital role in the signal transduction of resistance to environmental stresses. In plants, WRKYs played essential roles in the ROS scavenging system in plant-environment interactions. During continuous cropping, three FaWRKY Group III gene members (FaWRKY25, FaWRKY32 and FaWRKY45) may respond to adverse stress by participating in plant immune ROS bursts by several signaling pathways, such as plant hormone signal transduction and plant-pathogen interactions (Fig. 8). FaWRKY25, FaWRKY32 and FaWRKY45 have high sequence similarity with AtWRKY53, AtWRKY70 and AtWRKY41, respectively. Previous studies showed that AtWRKY41, AtWRKY53 and AtWRKY70 play important roles in plant basal defense 36,37 . The expression of WRKY is activated by MAPKs and induces the expression of a series of defense-related proteins, such as PR1 protein, peroxidase and plant hormone signaling, similar to salicylic acid (SA) and jasmonic acid (JA) 38,39 . AtWRKY70 was demonstrated to be involved in regulating SA-JA-mediated signaling pathways, and AtWRKY41, AtWRKY53 and AtWRKY70 are induced by SA 37,39,40 . The hormone signaling pathway is important in plant immunity and can induce related protein expression to regulate secondary metabolism 25,41 . The extensive cross-regulation mechanism of FaWRKY25, FaWRKY32 and FaWRKY45, which belong to FaWRKY Group III, might play an important role in cultivated strawberry continuous cropping defense.

Materials and Methods
Data collection. The whole Fragaria x ananassa annotated genome sequences were obtained from Strawberry GARDEN (http://strawberry-garden.kazusa.or.jp) 42 . The family assignment rules in PlantTFDB (http://planttfdb.cbi.pku.edu.cn/) 43 were used to identify strawberry WRKY TFs. Finally, the sequences containing WRKY DNA-binding domain (PF03106) were identified as candidates. The annotation of these candidate www.nature.com/scientificreports www.nature.com/scientificreports/ genes was further checked using BLASTP analysis in NCBI (https://www.ncbi.nlm.nih.gov). The Arabidopsis thaliana and Fragaria vesca WRKY transcription factor (TF) Group III members were downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). The Oryza sativa and Solanum lycopersicum WRKY TFs were also taken from PlantTFDB. Phylogenetic analysis and sequence alignment. The FaWRKY genes in cultivated strawberry were classified into different groups based on the AtWRKY classification 9 . Multiple alignments of amino acid sequences   www.nature.com/scientificreports www.nature.com/scientificreports/ Analysis of cis-acting elements in FaWRKY promoter regions. The upstream sequences (1.5 kb) of the FaWRKY-coding sequences were downloaded from Strawberry GARDEN (http://strawberry-garden.kazusa. or.jp) 42 . PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was utilized to identify seven regulatory elements of FaWRKYs 46 . plant materials and stress treatments. Fragaria × ananassa Duch. Benihoppe, a typical cultivated variety, were planted in Beijing Academy of Forestry and Pomology Sciences, Haidian District, Beijing, China (40°1′21″N, 116°16′32″E). All plants were planted in greenhouses at 22 ± 1 °C in a 16 h light/8 h dark photoperiod. The plant materials included two groups: Non-continuous cropping (NCC) and continuous cropping (CC). For NCC strawberry, the plants were cultivated in NCC soil, which cultivated with strawberry for the first time. For CC strawberry, the plants were cultivated in CC soil, which were annually mono-cultivated with strawberry for more than 12 years. The root, stem, leaf and fruit were collected separately for qRT-PCR analysis. Root samples of noncontinuous cropping and continuous cropping treatments were collected at harvest stage and used for further RNA-seq analysis. Each sample contains 3 replicates, and each replicate including 3 plants. All of the collected samples were snap-frozen in liquid nitrogen and kept at −80 °C until further use.
Real-time PCR was performed using a QuantStudio ™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR ® Select Master Mix (Applied Biosystems, Foster City, CA, USA). Each reaction mixture contained 10 μl of 2 × SYBR ® Select Master Mix, 1 μl of diluted cDNA product from reverse-transcription PCR, 0.8 μl of each of two primers, and 7 μl of DNase/RNase-free water, and each reaction was repeated using three independent biological and technical replicates. The housekeeping strawberry DNA binding protein (BDP, EU727547) gene was used as an internal control 48 . Gene expression was presented as relative units after standardization using the 2 −ΔΔCT method 49 . RNA-seq analysis was performed using an Illumina platform at Beijing BioMarker Corporation. The FaWRKY gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped (FPKM). The resulting FDR (false discovery rate) was adjusted by the PPDE (posterior probability of being DE). The FDR < 0.01 & |log2(foldchange)| ≥ 2 was set as the threshold for significant differential expression. The heat maps were created by OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools).