Genome-wide characterization and expression analysis of WRKY family genes during development and resistance to Colletotrichum fructicola in cultivated strawberry (Fragaria×ananassa Duch.)

ZOU Xiao-hua,DONG Chao,LIU Hai-ting,GAO Qing-hua

Shanghai Key Laboratory of Protected Horticultural Technology,Forestry and Fruit Tree Research Institute,Shanghai Academy of Agricultural Sciences (SAAS),Shanghai 201403,P.R.China

AbstractBased on the recently published whole-genome sequence of cultivated strawberry ‘Camarosa’,in this study,222 FaWRKY genes were identified in the ‘Camarosa’ genome. Phylogenetic analysis showed that the 222 FaWRKY candidate genes were classified into three groups,of which 41 were in group I,142 were in group II,and 39 were in group III. The 222 FaWRKY genes were evenly distributed among the seven chromosomes. The exon-intron structures and motifs of the WRKY genes had evolutionary diversity in different cultivated strawberry genomes. Regarding differential expression,the expression of FaWRKY133 was relatively high in leaves,while FaWRKY63 was specifically expressed in roots. FaWRKY207,59,46,182,156,58,39,62 and 115 were up-regulated during achene development from the green to red fruit transition. FaWRK181,166 and 211 were highly expressed in receptacles at the ripe fruit stage. One interesting finding was that FaWRKY179 and 205 were significantly repressed after Colletotrichum fructicola inoculation in both ‘Benihoppe’ and ‘Sweet Charlie’ compared with Mock. The data reported here provide a foundation for further comparative genomics and analyses of the distinct expression patterns of FaWRKY genes in various tissues and in response to C.fructicola inoculation.

Keywords:Fragaria×ananassa,FaWRKY,Colletotrichum fructicola,structure,evolution

1.Introduction

The WRKY family genes are among the most important transcription factors in plants,and play a very important role in plant growth and development,as well as responses to various biotic and abiotic stresses (ülker and Somssich 2004;Pandey and Somssich 2009;Valérieet al.2009;Huanget al.2015;Jianget al.2016;Klothet al.2016). Many studies have reported the WRKY domain structural characteristics in many plants (Eulgemet al.2000;Zhang and Wang 2005;Rosset al.2007;Rushtonet al.2011;Brandet al.2013),which provide a strong basis for identifying new WRKY genes.

Since the first WRKY geneSPF1was cloned from sweet potato (Ishiguro and Nakamura 1994),a large number of WRKY proteins have been studied in different plants.AtWRKY6positively affects PR1 promoter activity related to senescence and pathogen defense,and it is likely to be involved in NPR1 function (Singhet al.2002). The ScWRKY1 protein is detectable in potato ovules,and it contains two WRKY domains and is only expressed strongly in fertilized ovules (Lagacé and Matton2004). The AtWRKY70 protein acts as a common regulator of salicylic acid (SA)-and jasmonic acid (JA)-dependent defense pathways (Wanget al.2006;Knothet al.2007). TheAtWRKY57gene inhibits the transcription of senescence-related genes. The function of theAtWRKY57mutant is also related to leaf senescence (Jianget al.2014).VvWRKY11is involved in the response to dehydration stress in grapes (Banerjee and Roychoudhury 2015).AtWRKY12andAtWRKY13regulate flowering through negatively correlated expression profiles,and the interruption ofAtWRKY12results in delayed flowering while the mutation ofWRKY13promotes flowering (Liet al.2016). In addition,WRKY genes also play an important role in many other aspects,such as plant evolution (Zhang and Wang 2005),dormancy,germination (Huanget al.2016),fruit flavour (Zhouet al.2016;Yeet al.2017),biosynthesis of secondary metabolites (Gonzalezet al.2016),development and stress (Xieet al.2018),and resistance(Rinersonet al.2015).

In theFragariavescagenome,59FvWRKYgenes have been identified,and the expression ofFvWRKYfamily genes have been analysed in different developmental stages (Zhouet al.2016). In the cultivated strawberry,FaWRKY1is related to the mediation of defence responses toColletotrichumacutatum(Mackenzieet al.2006;Jianget al.2014). In the ‘Reikou’ genome,49FaWRKYgenes were identified,and responded to continuous cropping based on RNA-seq data (Chenet al.2019).

Cultivated strawberry is an allo-octoploid,and the genomic information of different varieties is quite variable. Although the WRKY gene has been identified in wild strawberry (Zhouet al.2016) and the cultivated strawberry ‘Reikou’ genome (Chenet al.2019),the genome of ‘Camarosa’ has not been analysed thus far.In this study,we identified 222FaWRKYgenes in the‘Camarosa’ genome,and also analysed the classification,structure,conserved motifs and phylogenetic evolution of these genes. Then,we assessed the expression ofFaWRKYin different tissues at various developmental stages. More importantly,the expression patterns ofFaWRKYgenes afterColletotrichumfructicolainfection(Namet al.2013;Hirayamaet al.2016;Fuet al.2018)were also profiled. Our results provide some valuable clues for the characterization and functions of WRKY genes in the ‘Camarosa’ genome.

2.Materials and methods

2.1.ldentification of FaWRKY genes

‘Camarosa’ is a cultivated strawberry from the United States. We used the ‘Camarosa’ genome to analyseFaWRKYfamily genes. Based on the conserved sequence of the WRKY domain (PF03106),we used HMMER 3.0 Software to identify theFaWRKYfamily genes in the ‘Camarosa’ genome (Fragaria×ananassaCamarosa Genome Assembly v1.0.a1). For the HMMER 3.0 Software,the default parameters were adopted,and the cut-off value was set to 0.01. Ultimately,222FaWRKYgenes were finally identified inF.×ananassaCamarosa Genome Assembly v1.0.a1.

2.2.Sequence analysis for WRKY

Multiple sequences of WRKY proteins were aligned using Clustal-X Software with the default parameters. The phylogenetic tree was established by MEGA (7.0) using the neighbour-joining (NJ) method with replicated 100-fold bootstrap tests (Sudhiret al.2016). By using the online program TBtool Software (Chenet al.2020),the exon-intron structure of theFaWRKYgene in ‘Camarosa’genome was analysed. The conserved motifs of the FaWRKY proteins in the ‘Camarosa’ genome were identified using the MEME online programme (http://meme.nbcr.net/meme/intro.html) (Baileyet al.2009). The optimized parameters include:the number of repetitions(anr),the maximum number of motifs (20),and the optimal width of each motif (6 to 50 residues).

2.3.Chromosomal localization

The chromosomal locations of all of theFaWRKYswere analyzed by TBtool Software,based on the genome assembly information on the strawberry genome database website (https://www.rosaceae.org/species/fragaria_x_ananassa/genome_v1.0.a1).

2.4.Colletotrichum fructicola culture and infection

TheC.fructicolaisolate (cgmcc3.17371) used in this study was from the Shanghai Academy of Agricultural Sciences (SAAS),China,and the culture,inoculation and statistical method of the disease index ofC.fructicolawere described in our previous paper (Zouet al.2018).

2.5.Plant materials and sampling for C.fructicola infection

For theC.fructicolainfection experiment,plant material from strawberry cultivars ‘Sweet Charlie’ and ‘Benihoppe’were grown in the greenhouse at SAAS. In order to avoid the influence of photoperiod on the gene expression analysis,we selected a dark treatment during the sampling. The specific culture conditions were set as follows:0-h-light/24-h-dark cycle,96 h post inoculation(Hpi),70% relative humidity (RH),and a constant temperature of 25°C. The 3th,4th and 5th compound leaves were collected at 0,2,12,24,72 and 96 Hpi for qRT-PCR.

2.6.Sampling from different development tissues and organs in ‘Benihoppe’

The method of sampling for strawberry fruit and different developmental stages in this study referred to Liuet al.(2019). The sampled materials were immediately frozen in liquid nitrogen and transferred to a freezer at -80°C until use.

2.7.Total RNA extraction and complementary DNA library construction

The methods of strawberry RNA extraction and cDNA reverse transcription in this study followed those of our group’s previous paper (Zouet al.2018).

2.8.qRT-PCR

The quantitative measurement ofFaWRKYgene expression was performed using the cDNA samples with a LightCycler 480 System (Roche Diagnostics GmbH,Mannheim,Germany) and SYBR Green (TaKaRa,Otsu,Japan). The results were normalized with the internal reference genesFaCHP1andFaGAPDH(Estradajohnsonet al.2017),which are stably expressed in all stages of strawberry growth and development.Normalized expression levels ofFaWRKYgenes were calculated using the geNorm Software with a method derived from the algorithms outlined by Zhaet al.(2016). The primer sequences used for the qRT-PCR are given in Appendix A. To evaluate the differences in the transcript levels of genes between ‘Benihoppe’and ‘Sweet Charlie’,the slopes of standard curves(maintained at approximately -3.4) and the efficiencies of amplification (＞70% and ＜120%) with the same pair of primers for the genes were performed and they were found to be very similar (Appendix B). The statistical analysis method for the relative expression of theFaWRKYtarget gene was as follows:The minimum Ctvalue is found from the targetFaWRKYsat different time points after theC.fructicolainoculation and different tissues of strawberry;(2) The ΔCtvalues are obtained by subtracting the other Ctvalues from the minimum Ctvalue,and then 2-ΔCtvalues are calculated;(3) The relative expression is calculated by dividing 2-ΔCtvalues by the normalization factors.

3.Results

3.1.Identification and chromosome locations of FaWRKY genes in the ‘Camarosa’ genome

Previous research showed that there are 59FvWRKYsin theF.vescagenome (Zhouet al.2016),and there are 62FvWRKYsin the wild diploid woodland strawberry‘Heilongjiang-3’ (Weiet al.2016). Based on the amino acid sequence of the Pfam WRKY domain (PF03106),we identified the WRKY coding genes in the ‘Camarosa’genome. The candidate genes with incomplete open reading frames (ORFs) and redundant sequences of the WRKY gene were excluded. Furthermore,SMART (http://smart.emblheidelberg.de/) and Pfam (http://pfam.xfam.org/) searches were subsequently performed to verify the presence of WRKY domains. Ultimately,a total of 222 non-redundant FaWRKY proteins were identified in the‘Camarosa’ genome (Fig.1). The characteristics of each predicted FaWRKY protein in the ‘Camarosa’ genome are listed in Appendix C. According to the ‘Camarosa’genome information provided by GDR (https://www.rosaceae.org/),we analysed the chromosomal locations of theFaWRKYsin this study. The 222FaWRKYgenes are evenly located on chromosomes,with 23,18,33,21,24,61 and 42FaWRKYgenes located on chromosomes 1,2,3,4,5,6 and 7,respectively (Fig.2). The relatively high densities ofFaWRKYgenes were detected on chromosomes 6 and 7.

Fig.1 Phylogenetic tree of FaWRKY proteins from the ‘Camarosa’ genome. The protein sequences were aligned using Clustal X.The tree was constructed using MEGA 7.0 with the neighbour-joining method. The areas with different colors indicate different groups (or subgroups) of WRKY proteins.

3.2.Phylogenetic analysis of FaWRKY genes in‘Camarosa’

The phylogenetic relationships of the WRKY proteins in cultivated strawberry were displayed by constructing a neighbour-joining phylogenetic tree. In the ‘Camarosa’genome,41 FaWRKY proteins were classified into the group I WRKY subfamily,which contains two WRKY domains with zinc-finger motifs of C-X4-C-X22-H-N-H and C-X4-C-X18-H-N-H. A total of 142 FaWRKY proteins containing C-X4-C-X23-H-N-H or C-X5-C-X23-H-N-H WRKY domains with a C2H2 zinc-finger motif were assigned to the group II WRKY subfamily,which was subdivided into five additional subgroups (IIa-e). A total of 39 FaWRKY proteins,which contain only one WRKY domain C-X7-C-X22-H-N-C and a C2HC zinc-finger motif belong to group III (Fig.1;Appendix C). Similar WRKY domain structures are shared by the same WRKY group members from different species (Rushtonet al.2011).Also,46 FaWRKY proteins were classified into the seven main phylogenetic groups (I,IIa-e,and III) according to the structural features of the full-length FaWRKY proteins in the ‘Reikou’ genome (Appendix D).

Compared with WRKY genes in the wild strawberry and ‘Reikou’ genomes (Zhouet al.2016;Garridoet al.2019),our data showed that theFaWRKYsexpanded and diversified rapidly in the evolutionary process of the‘Camarosa’ genomes. This diversity might have optimized the adaptability of the cultivated strawberry in times of adversity. Similar results have also been reported for other plant species in previous studies (Ayadiet al.2016;Jianget al.2016).

3.3.Gene structure and motif composition of the FaWRKY family genes in the ‘Camarosa’ genome

We further analysed the exon-intron structures and motif compositions ofFaWRKYgenes in the ‘Camarosa’genome. As shown in Fig.3,the number of introns in theFaWRKYgenes varied between 1 to 18. Also,compared with theFvWRKYexon-intron structures,we found that mostFaWRKYgenes had more than two exons,and some genes had up to eight exons,which is different than in the wild strawberry genome (Zhouet al.2016). NoFaWRKYgene with only one exon was found (Fig.3).The lengths of 134 of theFaWRKYgenes (not including introns) were less than 2 000 bp,while the lengths of 81FaWRKYgenes were between 2 000-4 000 bp,and only sevenFaWRKYgenes had lengths longer than 4 000 bp.These results show that theFaWRKYgenes have evolutionary diversity in terms of gene structures and gene lengths (Appendix C).

Fig.3 The exon-intron structure of FaWRKY genes in the ‘Camarosa’ genome. The exon-intron structure was visualized using the TBtool Software.

Further,we analysed the 10 most conserved domains from theFaWRKYgenes in the ‘Camarosa’ genome using the MEME program. The amino acid lengths of the 10 conserved domains ranged from 8 to 50 amino acids,of which motif 1 and motif 3 were WRKY domains. Motif 2 was present in most of theFaWRKYgenes (Figs.4 and 5).We found that theFaWRKYgenes classified in the same category had similar conserved domains. For example,category IFaWRKYs(FaWRKY1-28) all contained two WRKY domains (one motif 1 and one motif 3) (Fig.5),and they also shared motif 5 and motif 10. Most of the genes with motifs 6,7 and 9 appeared in category II of theFaWRKYs.

Fig.4 The architecture of conserved motifs in FaWRKY proteins from the ‘Camarosa’ genome. The motifs,numbers 1-10,are displayed in boxes of different colors,which were anayzed by the MEME program (http://meme-suite.org/),and visualized using TBtool Software.

Fig.5 The sequence logos of 10 conserved motifs in FaWRKY genes were analyzed with the MEME program (http://meme-suite.org/). Each letter represents an amino acid,and the height of letters in the y-axis indicate the degree of conservation of the amino acid. The x-axis represents the number of amino acids. Motif 1 and motif 3 are WRKY domains.

3.4.Expression analysis of FaWRKY family genes in different developing tissues and organs

To further understand the potential role of theFaWRKYgenes,qRT-PCR analysis was employed to detect the expression of selected candidateFaWRKYgenes in different strawberry organs. The results revealed that the expression levels ofFaWRKY63andFaWRKY179,which both belong to category II,were higher in the root tissue than other selected organs. In addition,FaWRKY133was highly expressed in leaves,whileFaWRKY205was more highly expressed in stolon than in flower,leaf,or root. The expression ofFaWRKY181was detected at a very low level in root,stem,leaf,and flower organs simultaneously(Fig.6). The data also proved that the members of a given group ofFaWRKYgenes generally have a similar expression pattern and participate in the same developmental metabolic process (Miaoet al.2004). In diploid strawberry,a large number ofFvWRKYgenes are mainly expressed in roots,followed by fruits and stems(Zhouet al.2016). A previous article only analyzed the expression of sixFaWRKYgenes,and found that they are mainly expressed in roots and stems (Chenet al.2020).Our data showed that differentFaWRKYgenes were specifically expressed in different tissues and organs,including leaves and flowers,which might be related to the performance of different functions.

3.5.Expression analysis of FaWRKY family genes in achenes and receptacles during strawberry fruit development

Strawberry fruit is mainly composed of the receptacle and numerous achenes (Aragüezet al.2013),and we evaluated the expression patterns of theFaWRKYfamily genes in achenes and receptacles during strawberry fruit development. Our analysis revealed that someFaWRKYgenes,especiallyFaWRKY207,59,46,156,58,39and62,were highly expressed in achenes at the fruit transition stage. Also,the expression patterns ofFaWRKY207,59,46,182,156,58,39,62and115were up-regulated during achene development from the green to red fruit transition. Interestingly,the expression ofFaWRKY62was simultaneously up-regulated during achene and receptacle development from the green to red fruit transition (Fig.6).Published data have shown thatFaWRKYgenes were differential expressed in diploid strawberry fruits after flowering at 18-42 days (Zhouet al.2016).

Fig.6 qRT-PCR validation of FaWRKY expression in different tissues:flower (full-bloom flower),stolon (the whole stolon),root(all the roots),leaves (the third fully expanded leaf blades),and four stages of achenes and receptacles (G,green fruits;W,white fruits;T,transition fruits;R,ripe fruits). All the samples were collected at the same time. For qRT-PCR analysis,the relative mRNA levels of all of the FaWRKY genes were normalized with respect to two housekeeping genes,FaCHP1 and FaGAPDH1.Bars are SD (n=3).

Receptacles are the main component of strawberry fruits,and play a very important role in regulating sugars and their direct derivatives (Faitet al.2008). Our data showed thatFaWRKY67,205and145were relatively highly expressed in receptacles at the green and white fruit stage;whileFaWRK143andFaWRKY140were relatively highly expressed in receptacles at the transition fruit stage;andFaWRK181,166and211were highly expressed in receptacles at the ripe fruit stage.FaWRKYsregulate strawberry fruit ripening by participating in anthocyanin synthesis (Wangetal.2020).Many studies have suggested that WRKY genes play an important role in developmental processes,including seed and trichome development,dormancy and germination,and senescence (Silke and Somssichet al.2002;Miaoet al.2004). A transcription factor ofFaMADS9regulates fruit receptacle development in strawberry (Vallarinoet al.2019),while transcription factors of MYB,SPL,NAC,TCP,and ARF participate in strawberry fruit ripening(Wanget al.2020). Our data suggest that theFaWRKYgenes could be differently expressed in fruits at different developmental stages (achenes and receptacles),which might reflect their potential functions in the developmental processes of receptacles and achenes.

3.6.Expression patterns of FaWRKY genes in response to C.fructicola in ‘Sweet Charlie’ and ‘Benihoppe’ leaves

We investigated the interactions betweenFaWRKYgene expression patterns and resistance toC.fructicolain strawberry. In China,‘Benihoppe’ and ‘Sweet Charlie’ are the main cultivated varieties (Zhaoet al.2012;Zouet al.2020). The resistance of ‘Camarosa’ toC.fructicolawas similar to that of ‘Sweet Charlie’ (Appendix E). Here,theC.fructicolainoculations were carried out in these two very important varieties. One variety is ‘Benihoppe’ which is susceptible toC.fructicola(Zouet al.2018),while the other is ‘Sweet Charlie’ which is resistant toC.fructicola(Zhanget al.2014). qRT-PCR analyses were used to assess the expression levels ofFaWRKYgenes at Mock/0,2,12,24,72 and 96 Hpi afterC.fructicolainfection in resistant ‘Sweet Charlie’ and susceptible‘Benihoppe’ leaves. As shown in Fig.7,the expression ofFaWRKY179andFaWRKY205was significantly downregulated afterC.fructicolainoculation (from 2 to 96 Hpi)compared with Mock in both susceptible ‘Benihoppe’and resistant ‘Sweet Charlie’. The expression ofFaWRKY207was repressed at 24,72 and 96 Hpi in susceptible ‘Benihoppe’. The expression ofFaWRKY46,155,156and115was up-regulated with a peak at 2 Hpi in susceptible ‘Benihoppe’ (Figs.7 and 8).FaWRKY181was up-regulated in susceptible ‘Benihoppe’ at 2Hpi,but down-regulated in resistant ‘Sweet Charlie’ at 2 Hpi.These data revealed the different expression profiles between ‘Benihoppe’ and ‘Sweet Charlie’,which might suggest a correlation with resistance toC.fructicola. Our analysis suggested thatFaWRKYgenes might operate in the interaction between strawberry andC.fructicolaby differential expression afterC.fructicolainoculation.

Fig.7 Expression profiles of FaWRKY genes after Colletotrichum fructicola infection by qRT-PCR analysis. The 3rd,4th and 5th compound leaves of strawberry ‘Benihoppe’ and ‘Sweet Charlie’ were sampled at 0 (Mock),2,12,24,72 and 96 hours post inoculation (Hpi). For qRT-PCR analysis,the relative mRNA levels of each FaWRKY gene were normalized with respect to two housekeeping genes,FaCHP1 and FaGAPDH1,at the different stages. Bars are SD (n=3).

4.Discussion

In a recent study,a search for WRKY genes in the Asia‘Reikou’ genome resulted in the identification of 47 members,which were classified into different sub-groups(Chenet al.2019). Here,the FaWRKY proteins in the‘Camarosa’ genome were identified.

Through multiple sequence alignment analysis,we found that the WRKY domain is different in these two cultivated strawberry genomes. In the ‘Camarosa’genome,one WRKY domain was composed of 22 amino acids,with a conserved sequence of‘DILDDGYRWRKYGQKVVKGSPY’;while the other WRKY domain was composed of 50 amino acids,with a conserved sequence of ‘DGYNWRKYGQKQVKGSEYPRSYYKCTHPNCPVKKKVERSLDGQITEIIYK’. In the ‘Reikou’ genome,the WRKY domain was found to have the highly conserved domain ‘WRKYGQK’ as in the ‘Camarosa’ genome,while FaWRKY44 varied by a single amino acid as WRKYGKK in the ‘Reikou’genome (Appendix F). Through the overall analysis of 46 FaWRKYs from the ‘Reikou’ genome,we found that the conserved sequences of the WRKY domains were‘JLDDGYRWRKYGQKPIKGSP’ with 20 amino acids(Appendix G). Meanwhile,in the ‘Reikou’ genome,we found that the conserved sequences of WRKY domains in different groups were different (Appendix F).

Furthermore,the zinc-finger motif also varied among the different classes of FaWRKY proteins in the ‘Reikou’genome. The zinc-finger structures were C-X7-C-X23-H-T-C/C-X7-C-X23-H-N-C in group III and C-X4-C-X23-H-N-H in group I,while the zinc-finger motif varied in the group II FaWRKY subfamily (Appendix D). Compared with the ‘Reikou’ genome,the mutations of the WRKY domain zinc finger motif were less in the ‘Camarosa’genome (Appendices C and D). The stable mutations of amino acids in the WRKY domain are likely to enhance the binding specificity of DNA targets (Zhouet al.2016).The 10 FaWRKY III proteins in the ‘Reikou’ genome with complete amino acid sequences were divided into seven clades. Clades 3,4 and 6 were mainly comprised of monocots. Interestingly,WRKY genes from very different species were included in clade 5 (Appendix G). The group III WRKY proteins are beneficial in plant evolution and adaptation (Guet al.2015;Aslamet al.2019).Therefore,there were some differences in the WRKY domain and motif between the two different octoploid strawberry genomes,which might be related to the evolution of different regional cultivars.

Some valuable clues were obtained regarding the functional role of transcription factors that are involved in resistance toC.fructicola(Zhanget al.2018).Interestingly,the expression patterns ofFaWRKY179andFaWRKY205were down-regulated with a similar expression pattern afterC.fructicolainoculation in both‘Benihoppe’ and ‘Sweet Charlie’ (Fig.8). They also have the closest phylogenetic relationship within Group II(Fig.1),which indicated that both FaWRKYs might share similar functions in strawberry (Wang 2010). TheFaWRKYgenes were differentially expressed afterC.fructicolainfection,highlighting the extensive involvement ofFaWRKYgenes in environmental adaptation and the immune response toC.fructicolainfection.

Fig.8 Pattern diagram of the expression of FaWRKY genes. The expression pattern map is based on FaWRKYs qRT-PCR data,summarizing the preferential expression of FaWRKYs in different tissues (A) and after Colletotrichum fructicola infection (B). For different tissues,the preferentially expressed genes are indicated with bold letters. For C.fructicola infection,an arrow indicates that a FaWRKY is up-regulated,otherwise it indicates down-regulated expression. Hpi,hours post inoculation.

Overall,our data provide some insight into the new functions of cultivated strawberry WRKY genes. The comprehensive analyses were helpful in selecting candidate WRKY genes for further functional characterization,and for the genetic improvement of the agronomic characters and resistance toC.fructicolain strawberry.

5.Conclusion

A comprehensive analysis of WRKY family genes in cultivated strawberry was carried out in the present study. A total of 222 full-lengthFaWRKYgenes were identified and classified into three main groups in the octoploid ‘Camarosa’ genome. We analysed the similar motif compositions,exon-intron organizations and multiple sequence alignment of FaWRKY,which provided significant clues about the evolutionary characteristics ofFaWRKYgenes in different octoploid genomes.Furthermore,FaWRKYswere differentially expressed in different tissues and during fruit development (in receptacles and achenes). More importantly,FaWRKYgenes might play an important role in the response toC.fructicolainoculation,as indicated by their expression patterns afterC.fructicolatreatment in different varieties.All of our results provide valuable information for better understanding the evolutionary characteristics and biological roles of WRKY genes in cultivated strawberry.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31601731).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm