Genome-Wide Identiﬁcation and Characterization of Argonaute , Dicer-like and RNA-Dependent RNA Polymerase Gene Families and Their Expression Analyses in Fragaria spp.

: In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modiﬁcation at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identiﬁed in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO , six DCL , and nine RDR genes were identiﬁed in diploid strawberry Fragaria vesca . We also identiﬁed 33 AGO , 18 DCL , and 28 RDR genes in octoploid strawberry Fragaria × ananassa , studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, ﬁnding that almost all genes respond to the ﬁve hormone stresses. This study is the ﬁrst report of a genome-wide analysis of AGO , DCL , and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.

Fragaria are perennial herbaceous berry fruits, including a diversity of species with wild strawberries ranging from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70), and a unique domestication of octoploid (2n = 8x = 56) cultivated strawberry (Fragaria × ananassa) [79,80]. Among these, wild diploid strawberry Fragaria vesca, with a small and sequenced genome (240Mb), is an excellent model for genetic transformation [81], and cultivated octoploid strawberry F. × ananassa, with an estimated genome size of 813.4 Mb, is an economically important fruit crop due to their alluring appearance, distinctive flavor, and health benefits [82]. Moreover, F. vesca is universally accepted as one of the diploid ancestors of the F. × ananassa, and the F. vesca subgenome has increased by retaining significantly more ancestral genes and a greater number of tandemly duplicated genes than other subgenomes in F. × ananassa [82][83][84][85][86][87][88]. Considering this, we performed a genome-wide analysis so as to identify AGO, DCL, and RDR gene families in diploid and octoploid strawberry. In order to comprehensively understand the function of these genes, we analyzed their protein structure, duplication events, evolutionary selections, and promoter cis-regulatory elements. Furthermore, the expression patterns of these genes were analyzed across various tissues during the development of strawberry. Finally, we examined the expression patterns of AGO, DCL, and RDR gene families in response to hormonal treatments. Above all, our study provides foundational genomic information for the AGO, DCL, and RDR gene families and its probable roles in strawberry growth and development.

Identification and Characterization of AGO, DCL and RDR Genes in Fragaria
The genome information and related annotation files of diploid strawberry F. vesca (v4.0.a2) [89] and octoploid strawberry F. ×ananassa (v1.0.a1) [80] were obtained from the Genome Database for Rosaceae [90]. Additionally, the protein sequences of all the AGO, DCL, and RDR genes of A. thaliana and Oryza sativa were downloaded from the TAIR (https://www.arabidopsis.org/ (accessed on 22 July 2020)) and Phytozome databases (https://phytozome.jgi.doe.gov (accessed on 22 July 2020)) and used to perform a BLAST (E-value 1e-10) search against strawberry genome sequences to obtain Fragaria orthologous genes (Table S1). Meanwhile, hidden Markov models using HMMER 3.0 [91] software were employed for the identification of DCL, AGO, and RDR genes, respectively. Conserved domains of all candidate genes were analyzed and annotated using the Pfam database [92] and Conserved Domain Database in NCBI [93]. For the different transcripts of the same gene, the transcript with the longest open reading frame (ORF) is selected as the representative of the gene, and the transcript with the same length of ORF, the longest untranslated region (UTR) transcript is selected as the candidate gene for analysis. The physical and chemical parameters of the proteins were calculated using the ProtParam tool (https://web.expasy.org/protparam/ (accessed on 12 September 2020)).

Chromosomal Localization, Phylogenetic and Duplication Analysis
The images of all the candidate genes' chromosomal locations were generated using MapGene2ChromosomeV2 (http://mg2c.iask.in/mg2c_v2.0/ (accessed on 20 September 2020)), according to the genome annotations of F. vesca and F. ×ananassa. The respective AGO, DCL, and RDR protein sequences were aligned using MEGA X [94], and phylogenetic trees were constructed using the neighbor-joining (NJ) method with the bootstrap test replicated 1000 times. Motif analysis was performed using MEME Suite [95]. TBtools was used to predict the segmental duplications and tandem duplications [96].

Hormone Treatments and Quantitative Real-Time PCR Analysis
F. × ananassa 'Benihoppe' plants were chosen for the study. The seedlings were sprayed with 0.2 mM naphthylacetic acid (NAA), 0.2 mM abscisic acid (ABA), 0.2 mM gibberellin 4 (GA 4 ), 0.2 mM methyl jasmonate (MeJA), and 1.0 mM salicylic acid (SA). Additionally, the leaves of strawberry seedlings were collected after treating for 12, 24, and 48 h. All samples were rapidly frozen using liquid nitrogen and stored in a −80 • C freezer. Total RNA isolation and quantitative real-time PCR (RT-qPCR) were performed with reference to Zhang et al. [98]. EF-1α (XM_004307362) was used as the reference gene to normalize the expression level of target genes. The relative expression levels of genes were calculated using the 2 −∆∆Ct method. All the statistical analyses were performed using t tests. The primers for the quantitative real-time PCR are listed in Table S2.

Identification and Characterization of AGO, DCL and RDR Genes in Fragaria
Using the BLAST and HMMER search, we identified the putative AGO, DCL and RDR genes of F. vesca and F. × ananassa. The resulting sequences were further confirmed by analyzing conserved domains as putative family members according to the Pfam database [92] and Conserved Domain Database in NCBI [93]. As a result, we identified a total of 13 AGO genes, six DCL genes, and nine RDR genes in F. vesca, as well as 33 AGO genes, 18 DCL genes, and 28 RDR genes in F. × ananassa. Then, the candidate genes were named, and their basic information is listed in Table S3.
Based on HMM research of PAZ (PF02170) and PIWI (PF02171) and BLASTp, 13 AGO genes were identified in diploid strawberry F. vesca, and we found that the lengths of FvAGO coding sequences varied from 2557 bp for FvAGO5a to 3381 bp for FvAGO1b and the code for proteins between 858 and 1126 amino acids. Additionally, most of them contain more than 19 exons, except for FvAGO2, FvAGO7 (three exons), and FvAGO4b (one exon) ( Figure S1A, Table S3). However, the lengths of the 33 identified FaAGOs greatly vary in octoploid strawberry. FaAGO7d has the smallest coding sequence of 1686bp and contains two exon regions, and FaAGO1c encodes the longest coding sequence of 6552bp containing 44 exons ( Figure S1B, Table S3). The pI ranged from 8.6 to 9.7 and molecular weight was 100 kDa in all the AGO proteins, except for FaAGO1a, FaAGO1b and FaAGO1c, where the molecular weight was more than 200 kDa. Similar to the foxtail millet [30], the protein instability index showed that most of the FvAGO/FaAGO proteins are unstable (Table S4) AGO proteins, except in octoploid strawberry, FaAGO4b, and FaAGO7d. Similarly, only CsAGO5b in Citrus sinensis lacks the ArgoL2 domain [23], and an FvAGO5a is also present in strawberry. Interestingly, we found that all AGO7 proteins also lack the ArgoL2 domain in diploid and octoploid strawberry. Moreover, the Gly-rich Ago1 domain was found in all AGO1 proteins, which is conserved in AtAGO1. Furthermore, we found that the ArgoMid domain is present in all the AGO1 and AGO10 proteins in Fragaria, and FaAGO20 is the only domain starting and ending with an F-box domain and containing two FBA domains ( Figure 1A).    The lengths of the 24 newly identified DCL genes coding sequences in F. vesca and F. × ananassa range from 3724 to 5895 bp with the coding potential for 1241~1964 amino acids, and the number of exons was greater than 19 ( Figure S1, Table S3). The molecular weight of FvDCL/FaDCL proteins varies from 147 kDa to 220 kDa and pI varies between 5.89 and 7.22. All the DCL proteins in Fragaria were unstable except FaDCL3e (Table S4). All DCL proteins possessed DEAD, Helicase-C, Dicer_dimer (Duf283), PAZ, and Ribonuclease_3 (RNaseIII) domains, as reported for AtDCLs. In addition, FaDCL2e contains the RCRF and RF-1 domain ( Figure 1B).
All nine FvRDR genes and 28 FaRDR genes found in the F. vesca and F. × ananassa genomes, code for proteins ranging between 587 and 2042 amino acids (Table S3). The pI and molecular weight of these proteins range from 5.92 to 8.75 and 65 to 229 kDa, respectively (Table S4). Compared with other FvRDRs in F. vesca, the FvRDR3 subfamily contains more exons with 22~25 exons ( Figure S1A, Table S3). Interestingly, this is also true in F. × ananassa. FaRDR3c contains 47 exons and has the most exons and the longest DNA sequence with 19,000 bp in the RDR family in strawberry ( Figure S1B, Table S3). Furthermore, a conserved domain analysis shows that FaRDR3c contains two RdRP domains, while all the RDRs in Fragaria share a common RdRP conserved domain ( Figure 1C).

Chromosomal Localization of AGO, DCL, and RDR Genes in Fragaria
In order to comprehensively understand the evolution of multiple AGOs, DCLs, and RDRs, we analyzed their precise physical locations on the F. vesca and F. × ananassa chromosomes ( Figure 2). The chromosomal location map shows that thirteen FvAGOs, six FvDCLs, and nine FvRDRs genes were unevenly distributed in all chromosomes. Five FvAGOs were detected on chromosome 4 (Fvb4), four on Fvb3, two on Fvb5, and one each on Fvb6 and Fvb7. Additionally, they were not localized on chromosomes 1 and 2. Three pairs of diploid strawberry AGOs, namely FvAGO5a-FvAGO5b, FvAGO6a-FvAGO6b, and FvAGO1a-FvAGO1b, were closely localized on Fvb3, Fvb4, and Fvb5, respectively. Therefore, we conjectured that they represent tandem duplications, as well as FvRDR3a-FvRDR3b on chromosome 1, FvRDR6a-FvRDR6b on chromosome 2 and FvRDR1a-FvRDR1c-FvRDR1d on chromosome 4. FvDCLs were unevenly distributed on chromosomes 1, 2, 6, and 7, and FvRDR genes were unevenly distributed on chromosomes 1, 2, 4, and 5. Chromosome 3 contains only members of the AGO family. Chromosome 7 contains nine genes and has the most gene members of the analyzed families ( Figure 2A). The chromosome distribution of FaAGO/FaDCL/FaRDR genes families in octoploid strawberry is similar to that of diploid strawberry. The physical location map reveals the identified FaAGO, FaDCL and FaRDR genes on all the chromosomes of F. × ananassa, except for chromosome 7-4 that comprise no members of the analyzed families. Chromosomes 3-1, 3-2,3-3, 3-4, and 6-4 only contain the AGO genes. Chromosomes 1-1 and 7-3 have only one DCL gene each: FaDCL4c and FaDCL1a, respectively. Additionally, only RDR genes are distributed on 1-2 and 2-3. Importantly, FaAGO6a and FaAGO15 appear very close on Fvb4-3, and the same applies to FaAGO6b and FaAGO16 on Fvb4-1, as well as FaAGO6e and FaAGO17 on Fvb4-4. For FaRDRs, most of the FaRDR1 genes appear very close in the genome, such as FaRDR1b and FaRDR1h on chromosome 4-3 and FaRDR1a, FaRDR1i, and FaRDR1j on chromosome 4-4. Interestingly, all FaRDR6 genes appear close together in the chromosomes except for FaRDR6e. FaRDR6b and FaRDR6d appear on Fvb2-1, FaRDR6a and FaRDR6e on Fvb2-2, and FaRDR6c and FaRDR6g on Fvb2-4 ( Figure 2B).

MEME Analysis of AGO, DCL and RDR Genes in Fragaria
After going through the MEME program, we identified 10 of 20 conserved motifs in the AGOs from Arabidopsis, F. vesca and F. × ananassa ( Figure 3A). Interestingly, half of the AGO proteins start with motifs 20 and 10, while the other half starts with motifs 20, 18, and 10, containing the motif 19, except for FaAGO4b and FaAGO7d. FaAGO1a, FaAGO1b, FaAGO1c, and FaAGO5a have two each of the motifs 5, 6, 8, 10, 11, 13, 17, and 20, which may be due to fragment duplication. In addition, FvAGO6b, FaAGO15, and FaAGO16 contain two of motif 13. FaAGO20 contains three of motif 20. For the DCL protein family, 15 conserved motifs were found in all DCL proteins ( Figure 3B). Motif 19 is a mark that distinguishes the DCL1 subfamily from other subfamilies. In the DCL1 subfamily, motif19 is distributed at the end of the protein, and some DCL1 proteins contain two motif 19. FvDCL2a, FaDCL2a, FaDCL2b, and FaDCL2b contain two motif 14. FaDCL2e contains two motif 9. Compared with the DCL3 protein of Arabidopsis, the DCL3 protein of strawberry has one motif 10 and motif 12. In addition, we found that both the DCL1 subfamily and DCL4 subfamily contain motif 16. In the RDR protein family, the MEME analysis revealed eight distinct motifs, including motifs 8, 12, 6, 9, 3, 2, 1, and 5, which were identified as major motifs among the RDR family members ( Figure 3C). However, the arrangement of these motifs in the RDR family is not the same. Motifs 12 and 9 had distinct diversification in the RDR3s proteins, different from all other RDRs. The above differences may lead to differences in the function of these proteins.

Phylogenetic Tree Analysis and Classification of AGO, DCL and RDR Genes in Fragaria
In order to examine the phylogenetic relationship of AGO, DCL, and RDR families, we constructed unrooted phylogenetic trees of all the identified AGO, DCL, and RDR protein sequences along with their A. thaliana homologs. FvAGO1a could not be included for the phylogenetic tree construction because the results of sequence alignment are unsatisfactory. The 12 FvAGO, 33 FaAGO, and 10 AtAGO proteins were subdivided into four clades, namely AGO1/10, AGO5, AGO2/3/7, and AGO4/6/8/9, following the nomenclature of the ten AGO genes of Arabidopsis ( Figure 4A). The DCL family consists of four clades, including DCL1, DCL2, DCL3, and DCL4 ( Figure 4B). Finally, the tree derived from RDR sequences also consists of four clades (RDR1, RDR2, RDR3, and RDR6) ( Figure 4C).

Synteny and Ka/Ks Analysis of AGO, DCL and RDR Genes in Fragaria
Gene duplications plays an important role in the expansion and evolution of gene families. Gene duplications include whole-genome duplications (WGDs) and single-gene duplications. Single-gene duplications include tandem (TD), proximal (PD), and dispersed duplication (DD) [99]. WGDs are mainly responsible for genome evolution and genetic diversity in auto-polyploids [100], while segmental duplications and TDs are known to play an important role in the generation and maintenance of gene families [101]. PDs arise from ancient tandem duplicates interrupted by other genes or localized transposon activities [102]. DD denotes any DNA sequence non-locally duplicated in a genome, such as transposon element insertions and copy number variations [103]. The collinearity analysis of diploid strawberry and octoploid strawberry shows that, among the 107 identified genes in strawberry, 71 WGD events, 18 DD events, 10 TD events, and eight PD events were involved. Additionally, seven TD gene pairs were found: FvAGO1a-FvAGO1b, FvAGO5a-FvAGO5b, FaAGO6b-FaAGO16, FvRDR3a-FvRDR3b, FvRDR6a-FvRDR6b, FaRDR6a-FaRDR6e, and FaRDR6b-FaRDR6d. Notably, in the DCL family, all FvDCLs derived from DD duplication events, all FaDCL derived from WGD events, and no TD gene pair was found. Furthermore, we identified 77 (AGO 24, DCL 19, RDR 34) gene pairs arising from segment duplications in F. vesca and 55 (AGO 22, DCL 16, RDR 17) gene pairs arising from segment duplications between the F. vesca and F. ×ananassa ( Figure 5). Then, Ka and Ks were calculated via DnaSP [97] using all orthologous and paralogous gene pairs CDs sequences identified via a synteny analysis. The findings show that most of the gene pairs exhibited Ka/Ks < 1, implying that the purifying selection may promote evolution. However, three orthologous gene pairs, FvAGO6c-FaAGO14, FvDCL1-FaDCL1e, FvRDR1b-FaRDR1c, and paralogous gene pairs, FaAGO11-FaAGO14, FaDCL4b-FaDCL4c, FaRDR3b-FaRDR3e, exhibited Ka/Ks > 1, suggesting a positive selection of these gene pairs (Table S5). gene pairs arising from segment duplications in F. vesca and 55 (AGO 22, DCL 16, RDR 17) gene pairs arising from segment duplications between the F. vesca and F. × ananassa ( Figure 5). Then, Ka and Ks were calculated via DnaSP [97] using all orthologous and paralogous gene pairs CDs sequences identified via a synteny analysis. The findings show that most of the gene pairs exhibited Ka/Ks < 1, implying that the purifying selection may promote evolution. However, three orthologous gene pairs, FvAGO6c-FaAGO14, FvDCL1-FaDCL1e, FvRDR1b-FaRDR1c, and paralogous gene pairs, FaAGO11-FaAGO14, FaDCL4b-FaDCL4c, FaRDR3b-FaRDR3e, exhibited Ka/Ks > 1, suggesting a positive selection of these gene pairs (Table S5).

Promoter Cis-acting Element Prediction of AGO, DCL and RDR Genes
In order to further comprehend the cis-acting elements located upstream of identified genes, 2 kb sequences upstream from translations start sites of the three gene families in strawberry were analyzed. More than 16 cis-acting elements were identified in the promoters of the putative the three gene families in F. vesca and F. × ananassa ( Figure 6). These include hormone response elements, stress response elements, light-responsive elements, and tissue-development-related elements. Among the hormone-responsive elements, we found that 63.

Promoter Cis-acting Element Prediction of AGO, DCL and RDR Genes
In order to further comprehend the cis-acting elements located upstream of identified genes, 2 kb sequences upstream from translations start sites of the three gene families in strawberry were analyzed. More than 16 cis-acting elements were identified in the promoters of the putative the three gene families in F. vesca and F. × ananassa ( Figure 6). These include hormone response elements, stress response elements, light-responsive elements, and tissue-development-related elements. Among the hormone-responsive elements, we found that 63.

Promoter Cis-Acting Element Prediction of AGO, DCL and RDR Genes
In order to further comprehend the cis-acting elements located upstream of identified genes, 2 kb sequences upstream from translations start sites of the three gene families in strawberry were analyzed. More than 16 cis-acting elements were identified in the promoters of the putative the three gene families in F. vesca and F. ×ananassa ( Figure 6). These include hormone response elements, stress response elements, light-responsive elements, and tissue-development-related elements. Among the hormone-responsive elements, we found that 63.0% (29 out of 46) AGOs, 45.8% (11 out of 24) DCLs, and 54.0% We also observed the presence of five other very important stress-responsive regulatory elements in our promoter sequences: defense and stress-responsive element, wound-responsive element, drought-responsive element, low-temperature-responsive element, and anaerobic induction element. In total, we identi-fied the defense and stress-responsive element in 45 identified gene promoter sequences, drought-responsive element in 59 sequences, low-temperature-responsive element in 48 sequences, anaerobic induction element in 94 sequences, and wound-responsive element only in the FvAGO6a and FvAGO6b promoter sequences. All identified promoter sequences carry the light-responsive element. Additionally, some tissue-development-related elements were found, such as the meristem (29 sequences) and endosperm (21 sequences) expression elements, seed-specific regulation element (five sequences), zein metabolism regulation element (39 sequences), and circadian control element (11 sequences). Altogether, there are various types and numbers of regulatory elements that can provide vital evidence for the understanding of functions of AGO, DCL, and RDR genes in strawberry.

The Tissue-Specific Expression Patterns of AGO, DCL and RDR Genes in Diploid Strawberry
To analyze the functions of AGO, DCL, and RDR genes, we studied their expression pattern in the development of the flower tissues, seed tissues, and other tissues in diploid strawberry according to annotation of the F. vesca v4.0.a2 genome [89]. FvAGO4a were observed to be highly expressed in all tissues and predominantly expressed in embryo and anther12. FvAGO1a showed a high expression level in the style 2 stage and root. FvAGO1b was more highly expressed in the development of the wall than other genes. FvAGO1b, FvAGO4a, and FvAGO5b greatly accumulate in all developmental stages of cortex and pith. FvAGO4b and FvAGO5c presented very weak expression levels in all tissues except for pollen ( Figure 7A). Additionally, we found that FvAGO5b had a higher expression level than FvAGO5a in the ghost, which had relations with the endosperm expression elements in the promoter of FvAGO5b (Figure 6). For the FvDCL family, all the FvDCL genes were weakly expressed in the pollen and embryo 3 stage. Tissue-specific higher expression of FvDCL1 and FvDCL3b was observed in the anther 9 and 10 stages. Unlike FvDCL3b, FvDCL1 was expressed at all stages of anther development. In style and seeding, all genes were weakly expressed, except for FvDCL1. Compared with the expression level of FvDCL3a in other tissues, it has the highest expression at anther78 stage. FvDCL2a shows a low expression level in all tissues ( Figure 7B). Among FvRDRs, we found that FvRDR1b, FvRDR1c, and FvRDR1d all showed a very weak expression in the FvRDR1 subfamily. In the FvRDR3 subfamily and FvRDR6 subfamily, the expression levels of FvRDR3b and FvRDR6a are very low. FvRDR2 was significantly expressed during carpel development. Additionally, it showed a high expression in the flower meristem (FM), receptacle meristem (REM) and anther 78 stage. FvRDR3a has a higher expression level in the shoot apical meristem (SAM) ( Figure 7C).

RT-qPCR Analysis of AGO, DCL and RDR Genes in Octoploid Strawberry Related to Hormones
Promoter cis-acting element prediction of AGOs, DCLs, and RDRs shows that five types of hormone-responsive elements were identified in their promoter regions, including ABAresponsive elements, and GA, MeJA, IAA, and SA-responsive elements ( Figure 6). Based on this, the expression level of AGO, DCL, and RDR genes under five hormone stresses were analyzed by RT-qPCR. Herein, we found that almost all genes respond to all hormone stresses. Among them, FaAGO6a has a lower expression level in all hormone response ( Figure 8). Moreover, we revealed that  S2 and S3). This finding is mostly consistent with the data of promoter cis-acting element prediction. For instance, three ABA-responsive elements were found in the promoters of FaAGO2 and FaAGO10a, and two MeJA-responsive elements in the FaAGO7a and FaRDR1d. Auxin-responsive element was found in the FaAGO7b, and GA-responsive elements was found in the FaDCL4b, FaRDR2e and FaRDR6d (Figure 6). In addition, homologous genes have a similar expression pattern. For instance, FaAGO15, FaAGO16, and FaAGO17 have a high expression level following 12 h of treatment ( Figure 8). FaDCL1 subfamily genes were also highly expressed after 12 h of treatment, except for FaDCL1a, FaDCL4a, and FaDCL4b, which show a clear response to GAs ( Figure S2).

AGO Proteins in Fragaria
In this study, the members of the AGO4/6/8/9 subfamily are the most common in diploid strawberry and octoploid strawberry, while AGO3 is not found in diploid strawberry, and there is only one AGO3 in octoploid strawberry. The expression profiling of the small RNAs revealed that AGO4, AGO6, and AGO9 bind 24-nt siRNAs that derive from repeat and heterochromatic loci [49]. AGO3 and AGO2 are in the same subfamily, but the study found that the spectra of AGO3-associated sRNAs were different from those bound to AGO2, and AGO3 could not complement the signature function of AGO2 in host antiviral defense. Surprisingly, AGO3 predominantly bound 24-nt sRNAs with 5 -terminal adenines, and the expression of AGO3 driven by the AGO4 promoter partially complemented AGO4 function and rescued a DNA methylation defect in the ago4-1 background, indicating that AGO3, similar to AGO4, is a component in the epigenetic pathway [46]. Therefore, we consider that this may be the reason for the low amount of AGO3 in strawberries. FvAGO4a shows a higher expression level in embryo than ghost tissue, including endosperm and seed coat ( Figure 7A), and our recent research revealed more 24-nt sRNAs present in the embryo than the endosperm [104]. These results show that 24-nt sRNAs recruited by AGO4 may play an indispensable regulatory role in the developing embryo and endosperm of strawberry. Recent studies found that these 24-nt siRNAs play an important regulatory role in the genetic molecular mechanism of endosperm genome imprinting [11]. Compared with AtAGO7, strawberry AGO7 is missing the ArgoL2 domain. FaAGO7d and FaAGO4b are slightly shorter than other AGOs, and their domains are incomplete. We conjecture that they may be the product of octoploid strawberry genome-wide replication events. Next, we analyzed the characteristic DDH/H and DDD/H motifs in the AGO proteins of strawberry. However, FaAGO4b contains a DDH/P motif, FaAGO7a did not present this motif, suggesting that FaAGO7a may lack slicing activity (Table S6). We also found that FvAGO5a, FaAGO13, FaAGO15, FaAGO17, and FaAGO19 lack the slicing activity found among members of the strawberry AGO family. Two tandem duplication events FvAGO1a and FvAGO1b, FvAGO5a and FvAGO5b were found in chromosomes Fvb5 and Fvb3 in F. vesca. While the tissue-specific expression patterns suggested that these two gene pairs showed different expression regulation ( Figure 7A). Two F-box domains and two F-boxassociated domains, FBA_1 and FBA_3, were found in FaAGO21. In plants, many F-box proteins are represented in gene networks broadly regulated by microRNA-mediated gene silencing via the RNA interference [105], and these proteins play an important role in ubiquitin proteolysis machinery [106]. F-box proteins are also involved in many plants vegetative and reproduction growth and development. For example, F-box protein-FOA1 is involved ABA signaling to affects seed germination [107]. Interestingly, we found four ABA-responsive elements in FaAGO21. As a result, we concluded that FaAGO21 may be significant in the development of strawberry.

DCL Proteins in Fragaria
Among the plethora of proteins involved in RNA silence or RNA interference (RNAi) by small RNA molecules, Dicer or DCL proteins are the primary key factors. The role of these RNaseIII-like enzymes is to excise 21-24 nt sRNA duplexes from structural dsRNA precursor molecules. In the A. thaliana, four DCL proteins mediate the production of various classes of sRNA. Additionally, in this study, six FvDCLs and eighteen FaDCL genes clustered into four subgroups were found in diploid and octoploid strawberry ( Figure 4B). All the identified strawberry DCL genes contain the DEAD, Helicase-C, Dicer_dimer, PAZ, and two Ribonuclease_3 domains, whereas the DCL1 and DCL4 subfamilies had an additional dsrm domain, also known as dsRBD (double-stranded RNA-binding motif). The dsrm proteins are mainly involved in the post-transcriptional regulation by preventing the expression of proteins or mediating the localization of RNAs [108]. Interestingly, we found that motif 16 is conserved in the dsrm domain, which may be responsible for post-transcriptional regulation. FvDCL1 and FvDCL3 showed a higher expression level in flowers tissues than other genes, especially in the anther ( Figure 7B). In pepper, CaDCL1 and CaDCL3 also exhibited a higher expression in flowers [21], and previous studies revealed that the double mutant of dcl1 and dcl3 exhibited a delay in the flowing of Arabidopsis [109]. Additionally, in peach, PrupeDCL3 also showed a high level of expression in flowers [63]. These findings suggest that DCL1 and DCL3 play positive roles in the flower development of plants. In addition, DCL2 and DCL4 are key control virus replication levels, even in susceptible plants. Additionally, DCL4 is also involved in the production of some miRNA, including miR822, miR839, and miR869 in A. thaliana [110], and plays important roles in the biogenesis of transposon-derived siRNAs that specifically target transposon (TE) transcripts and endogens, when TEs are epigenetically reactivated [111][112][113]. In this study, FaDCL2e showed two peptide chain release factors related to the RCRF and RF-1 domains, compared with other DCL2 proteins. In addition, FaDCL2e also has a highly significant expression pattern in hormonal treatments ( Figure S2), which may have a special function in octoploid strawberry.

RDR Proteins in Fragaria
RDR is responsible for the synthesis of dsRNA on ssRNA substrates in either a primerdependent or primer-independent manner [64]. The first plant RDR, LeRDR, was isolated in tomato [114]. In the A. thaliana genome, six RDR proteins were identified [64]. We identified nine RDR genes in the diploid strawberry, which is consistent with the number of RDR genes found in Populus trichocarpa [26]. Additionally, 28 RDR proteins were found in octoploid strawberry. The biological function of RDR proteins is usually linked to the subsequent action of specific DCL proteins. Plant RDR1 is an important element of the RNA silencing pathway in plant defenses against viruses. RDR1 expression can be elicited by viral infection and the exogenous application of salicylic acid (SA) and jasmonic acid (JA) [69,115]. Our results show that FaRDR1d and FaRDR1g are not only elicited by SA and MeJA, but also enhanced by GA ( Figure S3). Additionally, FaRDR1k is also induced by ABA ( Figure S3), which is similar to the AcRDR1 [66]. RDR6 also plays an important role in plant antivirus. However, unlike RDR1, the transcription of NgRDR6 is induced by ABA, GA, JA, and CMV, but there is no significant change under the treatment of PVY, TMV, SA, and H 2 O 2 [116]. Among the five members of the rice RDR family, only OsRDR6 is induced by ABA and KINETIN but is not induced by SA, IAA, GA, and ethylene treatment [117]. In strawberry, FaRDR6d is significantly enhanced by GA treatment ( Figure S3). RDR2, DCL3, and AGO4 participate in siRNA-mediated DNA methylation to regulate gene expression. RDR2 is mainly related to the epigenetic modification of plants. It participates in siRNAdirected DNA methylation to regulate gene expression together with DCL3 and DCL4. In addition, AtRDR2 plays a critical role in the development of the female gametophyte [118]. In diploid strawberry, we found one RDR2 gene that was divided in the same clade as AtRDR2. RDR2 is most highly expressed in the ovules of pineapple [66], while in diploid strawberry it has a high expression level in the development of carpel. Although they do not have the same organization, it was also suggested that these proteins may be involved in female reproductive development.

Evolution between Diploid F. vesca and Octoploid F. × ananassa
The genus of Fragaria contains approximately 25 species, diversifying ploidy, ranging from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70) [78]. Among them, F. × ananassa is a modern cultivated species, an allo-octoploid (2n = 8x = 56), was derived from spontaneous hybrids between two wild allo-octoploid species F. virginiana and F. chiloensis in the 18th century in Europe [119]. However, there are still disputes about its diploid ancestors. For example, Tennessen et al. [81] clarified that F. × ananassa originated with four subgenomes, including F. vesca, F. iinumae, and two unknown ancestors, by presenting an approach Phylogenetics of Linkage-Map-Anchored Polyploid Subgenomes (POLiMAPS). However, Yang and Davis [82] reported that octoploid species of genetic signatures from at least five diploid ancestors, including F. vesca, F. iinumae, F. bucharica, F. viridis, and at least one additional allele contributor of unknown identity. With the chromosome-scale genome of F. ×ananassa assembled, Edger et al. [80] and Hardigan et al. [88] proposed that four species, including F. vesca, F. iinumae, F. viridis, and F. nipponica, contributed to the origin of octoploid strawberry. Moreover, Hardigan et al. [88] proposed the ABCD subgenome nomenclatures, and among them, F. vesca was the dominant source of the genic DNA, revealing that the chromosomes of F. × ananassa, Fvb1-4, Fvb2-2, Fvb3-4, Fvb4-3, Fvb5-1, Fvb6-1, and Fvb7-2 were the closest diploid F. vesca chromosomes. We found that these results were consistent with the result of gene location and synteny analysis in our study. For example, the number of AGO, DCL, and RDR in the F. vesca chromosomes was the same as those for the subgenome A of F. × ananassa except for Fvb3 and Fvb7 ( Figure 2). Additionally, collinearity analysis showed that gene duplication events were discovered in the F. vesca and F. ×ananassa subgenome A ( Figure 5).

Conclusions
In this study, a total of 13 AGO, six DCL, and nine RDR, and 33 AGO, 18 DCL, and 28 RDR genes were identified in diploid F. vesca and octoploid F. × ananassa, respectively. The conserved domains, motifs, and gene structures analyses showed the functional characterization of these gene families. Chromosome localization, phylogenetic tree, and collinearity analysis revealed that gene duplication events contributed to the evolution of these genes and provided some evidence of the evolution between diploid and octoploid strawberry. Moreover, their expression patterns in various tissues, developmental stages, and hormone stresses indicated the genes involved in the growth and development of strawberry. Therefore, our study provides a basis for further study on the functions of these genes and provides some evidence for the evolution between diploid and octoploid strawberries.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14010121/s1, Figure S1: The gene structures of AGO, DCL, and RDR genes in F. vesca (A) and F. ×ananassa (B); Figure S2: qRT-PCR analyses of FaDCL genes expression under five hormone treatments; Figure S3: Expression profiles of strawberry RDRs analyzed by qRT-PCR after five hormone treatments; Table S1: The information of the query sequence. Table S2: Primers used to perform RT-qPCR for FaAGO, FaDCL and FaRDR genes; Table S3: Basic information of AGO, DCL, and RDR genes in Fragaria; Table S4: Physical and chemical parameters of AGO, DCL, and RDR proteins in Fragaria; Table S5: Ka/Ks analysis of AGO, DCL and RDR genes in Fragaria; Table S6: The characteristic DDH/H and DDD/H motifs in AGO proteins of strawberry.