Identifying strawberry DOF family transcription factors and their expressions in response to crown rot

Crown rot is one of the most destructive diseases of cultivated strawberry. The DOF family transcription factors, which involved in biotic stress, has not been studied in responding to strawberry crown rot. In this study, the DOFs of Fragaria × ananassa, F. iinumae, F. nilgerrensis, F. viridis, and F. vesca were characterized. One hundred and eighteen FaDOFs, twenty-two FiDOFs, twenty-three FnDOFs, twenty-five FviDOFs and thirty-seven FvDOFs were identified. Gene cluster analysis showed nearly seventy segmental duplication and seventeen tandem duplications for DOF family expansion in octaploid strawberry. In addition, 59 FaDOFs showed syntenic relationships with 32 AtDOFs, which were located on all F.×ananassa chromosomes except Fvb4-1 and Fvb4-2. Except for five DOFs of diploid strawberries had syntenic relationships to one FaDOF, most of them corresponded to multiple FaDOFs. Gene expression analysis revealed that 107 FaDOFs were expressed in crown, and most of them were downregulated by crown rot, while some FaDOFs such as FaDOF107, 12, 82, 91, 90 and 101 were upregulated, whose regulation was not always consistent with the cis-elements in their promoters. Together, these results provided a basis for further functional studies of the FaDOFs.


Introduction
Cultivated strawberry (Fragaria × ananassa), the important fruit crop species whose fruits with distinctive flavour and rich nutritious value, is widely grown all over the world, but its productivity and quality are seriously limited by crown rot (Han et al., 2016). Crown rot occurs in the root neck, which is manifested as a short plant. After infection, the root neck produces red streaks, and then rapidly expands to dark, sunken spots, and finally the whole plant wilts and withers, which is a destructive disease of strawberry. Under suitable conditions, crown rot can reduce the yield of strawberries by up to 80% (Han et al., 2016).
DOF proteins are a family of plant-specific transcription factors that contain a particular class of zincfinger DNA-binding domain, which has been reported to be involved in biotic stresses. These DOF transcription factors can interact with other related transcription factors and regulate the transcription process by activating or inhibiting target genes, and then regulate their expression to participate in plant resistance to stress. For instance, in barley, two DOFs, BPBF and SAD, involved in gene regulation during seed development

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Notulae Botanicae Horti Cluj-Napoca Agrobotanici 2 have been characterized, as part of the transcriptional regulation of the barley Cystatin Hv-CPI gene upon barley seed germination which has the plant defence function (Martínez et al., 2005). Pepper DOFs CaDOF10 and 11 showed relatively high expression levels after infection of PepMoV and Phytophthora capsici, suggesting that they could play a role in defense response against pathogen (Kang et al., 2016). In Arabidopsis, motif DOF are proven functional in the regulation of ACBP3 confers resistance to Pseudomonas syringae (Zheng et al., 2012).
The DOF family has been characterized in several plants, including A. thaliana (Lijavetzky et al., 2003), Oryza sativa (Lijavetzky et al., 2003), Hordeum vulgare , Glycine max (Guo and Qiu, 2013), Populus trichocarpa (Yang et al., 2006), Triticum aestivum (Shaw et al., 2009), Sorghum bicolor (Kushwaha et al., 2011), Zea mays (Jiang et al., 2012), Solanum lycopersicum (Cai et al., 2013), and Chinese cabbage (Ma et al., 2015). However, F. ananassa specific DOF studies are lacking. In the present study, the strawberry DOF family transcription factors members were identified via bioinformatics tools, and their expression patterns in response to biotic stress were characterized. This study provides basic information on the protein structures, subfamily divisions, chromosome localization in the strawberry genome, and expression patterns of the DOF proteins in response to crown rot.
Sequence alignment and phylogenetic analysis The full-length DOF protein sequences from A. thaliana and strawberries were aligned via muscle in MEGA version 7.0, with default parameters (Edgar, 2004;Kumar et al., 2016). A neighbour-joining (NJ) tree was also generated with bootstrapping (1000 replicates). The phylogenetic relationships among the five kinds of strawberries DOFs and A. thaliana DOFs were estimated.

Conserved motifs and gene structure analysis
Motif analysis was conducted on the MEME website (http://meme-suite.org/tools/meme) to identify conserved motifs with the following optimized parameters: zero or one occurrence per sequence, a maximum of 10 motifs and an optimum motif width between 6 and 50 residues. The default settings were used for all other parameters. The structure of FaDOFs was determined with TBtools by comparing the coding sequences and the corresponding genomic sequences (Chen et al., 2020).
The Strawberries Generic Feature Format (GFF) files were downloaded from the strawberry Genome Database and used to elucidate the structure information of the DOF gene. An illustration of the FaDOFs protein motifs, conserved domain, gene structures and a phylogenetic tree was also constructed in TBtools (Chen et al., 2020).
Chromosomal distribution, gene duplication and collinearity The chromosome locations of the candidate strawberry DOF genes were analyzed from the GFF information and visualized by TBtools (Chen et al., 2020). Gene duplication events of the FaDOFs and collinearity between the A. thaliana DOF protein sequences and five kinds of strawberries DOF protein sequences were investigated by MCScanX (Wang et al., 2012). The results were visualized in TBtools (Chen et al., 2020).
FaDOFs expression in response to biotic stress A single factor experiment was performed using different treatments causing inoculation with Colletotrichum siamense SCR-7, which caused crown rot in Hubei province, China. The healthy and consistent one year old strawberry (F.×ananassa Duch 'Benihoppe') seedlings were divided into SCR-7 inoculation treatment group and control group with 12 pots each. The SCR-7 inoculation treatment group was inoculated with C. siamense SCR-7, while the control group was inoculated with non-toxic medium using the same method. The seedlings were grown with or without C. siamense SCR-7 and 0 or 6 days after vaccination, resulting in four treatment groups: 0-day post inoculation with medium (0DPM), 6-day post inoculation with medium (6DPM), 0-day post inoculation with C. siamense SCR-7 (0DPI) and 6 days post inoculation with C. siamense SCR-7 (6DPI). All the seedlings were grown in light incubator, with 16h photoperiod, 900 μmol m -2 s -1 , 25/15 ℃ day/night temperatures, and a 68% relative humidity. The crowns of seedlings were sampled for transcrptome and qRT-PCR analysis, each treatment had three biological replicates.
Transcriptomic data of seedling crowns from the four treatments were analyzed as described by Shu et al. (2016). Twelve libraries of seedling crowns were sequenced using the Illumina HiSeq 2000 system. Reads that contained adapters, more than 10% unknown nucleotides, and more than 50% bases with a quality value ≤5 were removed to obtain uncontaminated sequences based on the raw data. Uncontaminated sequences were mapped to the genome of F. × ananassa 'Camarosa' (v1.0.a2) for annotation (Liu et al., 2020). The transcriptomic data were uploaded to the NCBI Sequence Read Archive as PRJNA715088. Gene expression was analyzed based on the transcriptomic data, where the transcriptional abundance of FaDOF was calculated as fragments per kilobase of exon model per million mapped reads (FPKM) using the Cufflinks package cuffdiff version 2.2.1. The FPKM value of 0DCK was considered the relevant control. Heat maps were created using TBtools software based on the transformed data of log2 (FPKM+1) values (Chen et al., 2020).
qRT-PCR was performed as in Luo et al. (2020) on three independent biological samples having three technical replications each. Eight genes were selected for RNA-seq verification and the primers used for qRT-PCR were shown in Table S1. The relative gene expression was calculated using the 2 − △△ Ct method, where house-keeping gene (gene11892) was taken as the reference gene. The measured transcripts were normalized to the relative expression value in 0-day post inoculation with medium. Significant differences between treatments were determined by Duncan's Multiple Range Tests at p = 0.05 with SAS 8.1 (SAS Institute, Inc., Cary, NC, USA). Different letters indicate statistically significant differences.

Cis-acting elements of the FaDOFs
The 2000 bp sequences upstream of the transcription initiation site of the candidate genes were extracted from the strawberry genome sequences. The PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) was used to search for cis-acting elements (Rombauts et al., 1999), and the results were visualized in TBtools (Chen et al., 2020).
The FaDOFs in different groups were characterized according to their DOF domain numbers and exon-intron structures. Motifs 1 composed the DOF domain, and all 118 FaDOFs had DOF characteristic domain. The number of introns varied from 0 (42 FaDOFs) to 7 (FaDOF12 and FaDOF17). Most FaDOFs contained motif 1, 5, 7 and 4 or motif 3, 4, 1, 9, 7, 6 and 4 in the group I. Most of the group II had motif 2, 1, 5 and 4 or motif 10 as the end of 3'UTR side. Most of the group III contained one or more codons, and it was also the only group with motif 3 and 10 as the end of 3'UTR side. Most members of the fourth group IV only contained two or three motifs (Figure 4). The phylogenetic tree was constructed using the neighbour-joining method implemented in MEGA 7.0. Reliability of the predicted tree was tested using bootstrapping with 1,000 replicates. 7 Figure 4 Phylogenetic analysis of deduced FaDOF proteins associated with the motif composition and exon-intron composition of FaDOF genes The phylogenetic tree was constructed using the neighbour-joining method (left-hand side of the figure). Reliability of the predicted tree was tested using bootstrapping with 1,000 replicates. The motif composition related to each FaDOF protein is displayed in the middle of the figure. The motifs were numbered 1-10. The information for each motif is provided in Figure S2.
All of the FaDOFs clustered into three major groups, with distinct protein domains, motifs and sequences ( Figure 3). DOF domain was the most conserved region in DOF protein. Motifs 1 composed the DOF domain, and all 118 FaDOFs had one characteristic domain ( Figure S2). The phylogenetic tree which was generated based on the protein sequence alignment of strawberries and A. thaliana segregated the 118 FaDOFs into four large groups. Group members shared similar protein sequence lengths, motif compositions and exon-intron structures, suggesting a close function. Thus, FaDOF5 with their homolog AtDOF18 (OBP1) in the same branch may play similar roles in biotic stress responses (Figure 3). AtDOF18 clustering with FaDOF5 which was induced by crown rot ( Figure 5) speculated that they may interaction with cystatin gene and octopine synthase gene respectively in biotic stress tolerance to enhance its anti-adversity (Zhang et al., 1995;Martínez et al., 2005). From the phylogenetic tree it was predicted that the FaDOFs were involved in pathogen infection interactions, but this hypothesis required verification in future studies. While AtDOF4;2 affects A. thaliana resistance by negative influence on flavonoid biosynthesis and positively influences the production of hydroxycinnamic acids in phenylpropanoid metabolism (Skirycz et al., 2007), the most similar genes FaDOF24 which was repressed by crown rot ( Figure 5) might regulated phenylpropanoid metabolism in octoploid strawberry. In addition, 'defence and stress responsive' cis-elements were identified in the promoters of 43 FaDOFs but some of them such as FaDOF 4, 14 were down-regulated by crown rot. The results suggested FaDOFs expression regulations was not consistent with the cis-elements present in their promoters ( Figure 5, 7), which might be due to the integration of other gene regulating elements, such as transacting factors (Chow et al., 2018;Xie et al., 2018).

Conclusions
DOF family transcription factors are one class of resistance genes involved in biological stress, and the family members in strawberry and its expression pattern in responding to crown rot still unclear. In our current study, we identified 118 FaDOFs, 22 FiDOFs, 23 FnDOFs, 25 FviDOFs and 37 FvDOFs in the F. × ananassa, F. iinumae, F. nilgerrensis, F. viridis, and F. vesca genome, respectively. In the syntenic relationship analysis with A. thaliana, it was found that F. ×ananassa produced 22 new genes during the evolution process. Except for a few DOFs of diploid strawberries (FnDOF10,FviDOF4,FvDOF7,FiDOF4 and FiDOF5) showing syntenic relationships to one FaDOF, most of them corresponded to multiple FaDOFs, with the most DOFs (FnDOF12,18 and 21,FviDOF30,FiDOF11,15,17 and 20) had syntenic relationships to 14 different FaDOFs. It showed that F. ×ananassa may have chromosomal variation during the evolution process, which also proved it is highly conserved during DOF evolution. In addition, 107 FaDOFs were expressed in crown, most of them downregulated by crown rot, while some FaDOFs such as FaDOF107, 12, 82, 91, 90 and 101 were upregulated. The analysis of phylogenetic tree and cis-elements in promoters indicated that the genes may have the ability to regulate the pressure of pathogen infection. However, the study of DOF's mechanism of action is still not thorough, so it is necessary to further study the signal pathways involved in order to further study the specific mechanism of action. Collectively, the results of this study provided a basis for future functional studies of the strawberry DOF and their responses to crown rot.

Authors' Contributions
BS conceived and designed the experiments, supervised and revised the manuscript. CL conducted part of the experiments and wrote the original manuscript. YH conducted part of the experiments.
All authors read and approved the final manuscript Ethical approval (for researches involving animals or humans) Not applicable.