Identification of AP2/EREBP in Fragaria vesca
A total of 119 AP2/EREBP genes were identified through HMM searches, local BLAST analyses, and domain confirmations. These genes contained at least one AP2 domain (supplementary information). A previous study has identified 115 AP2/EREBP genes in F. vesca (Wang et al., 2019). The difference between that and this study is that the previous study used an older version of the genome, while our study used the latest version (v4.0.a2). The specific differences in numbers and gene IDs are shown in Table S1. According to the classification of AP2/EREBP in Arabidopsis and rice (Sakuma et al., 2002; Nakano et al., 2006), the 119 AP2/EREBP genes in F. vesca were divided into five groups. The phylogenetic tree was constructed based on the alignment of 350 AP2/EREBP proteins from Arabidopsis, rice, and F. vesca (Fig. 1). The phylogenetic tree clearly classified the AP2/EREBP proteins from F. vesca into a soloist as well as the four typical subfamilies, namely, the AP2, RAV, ERF, and DREB clades, which were comprised of 1, 18, 7, 61, and 32 proteins, respectively. Generally, the RAV subfamily has one AP2 domain and one B3 domain, and the FvRAV subfamily contains two members (FvH4_5g19881 and FvH4_6g29430), which had one AP2 domain and no B3 domain.
Identification and Phylogenetic Analysis of FvDREBs
Based on the conserved 14th valine (V14) of the AP2 domain (Fig S1), 32 DREB genes were identified from AP2/ERF in F. vesca and named according to the chromosomal position (Table 1). The identified FvDREBs proteins ranged from 150 to 579 amino acids in length, with theoretical isoelectric points (pI) ranging from 4.63 to 9.48 and molecular weights (MW) ranging from 16447.59 to 65304.13. Subcellular localization analysis predicted that most (26/32, 81.25%) FvDREBs localized to the nucleus, whereas other (6/32, 18.75%) members were localized to the cytoplasm (Table 1).
To investigate the phylogenetic relationships between DREBs in strawberry and other plants, a neighbor-joining phylogenetic tree was generated using the whole-protein sequences of the DREB subfamily between F. vesca and A. thaliana. As shown in Fig. 2, the phylogenetic tree was further divided into six subgroups (A-1 to A-6) as in Arabidopsis, in which the A-4 subgroup was the largest (13 members) and the A-3 subgroup was the smallest (one member). According to the similarities between AtDREB1/CBF and AtDREB2, the A-1 subgroup and A-2 subgroup included two and six members, respectively. Meanwhile, seven orthologous pairs were identified in F. vesca and A. thaliana, and one paralogous pair was identified in F. vesca based on a bootstrap value greater than 90 (supplementary information).
Gene Structure and Conserved Motif Analysis of FvDREBs
The exon-intron structures were analyzed to gain a better understanding of the structural characteristics of FvDREBs genes. Almost all of FvDREBs (30/32, 93.75%) were intronless, except for FvH4_2g38880 (FvDREB3) and FvH4_5g34550 (FvDREB28), which contained only one intron (Fig. 3).
The conserved motifs of all FvDREBs were further examined using MEME. A total of 15 motifs were predicted and named as motifs 1 to 15. Motifs 1 and 2 were found in all FvDREB protein sequences and were related to the AP2 domain. The protein sequences of two members belonging to the A-1 subgroup both contained motifs 5 and 11. Motif 8 was only found in members of the A-2 subgroup, whereas motif 10 was only found in members of the A-6 subgroup. Some other motifs, such as motif 15, were distributed among various subgroups.
Chromosomal Location and Tandem Duplication of FvDREBs
In order to explore the functional differentiation of FvDREB members, their positions on chromosomes were further investigated with the latest annotated genome (v4.0.a2). As shown in Fig. 4, 32 FvDREB members were distributed unevenly on five of the seven chromosomes, and there were no members on chromosomes 3 and 4. Chromosome 5 had the largest number (10, 31.25%) of FvDREB genes, containing one A-1 subgroup member, five A-4 subgroup members, two A-5 subgroup members, and two A-6 subgroup members. Chromosomes 6 and 7 had 25% (8/32) and 18.75% (6/32) FvDREB genes, respectively. The remaining 25% (8/32) members were evenly located on chromosomes 1 and 2. Moreover, five tandem duplication events involving eleven FvDREB genes were observed, namely, FvDREB1 and FvDREB12, FvDREB13 and FvDREB14, FvDREB17 and FvDREB18, FvDREB2 and FvDREB22, FvDREB27, FvDREB15, and FvDREB16. Four of the five tandem duplication events distributed on chromosome 5, including the three members with tandem duplications.
Synteny Analysis of FvDREBs
To provide insights on the genome duplications and evolution of DREB, intergenomic synteny analysis was conducted between F. vesca and F. × ananassa. There were 143 syntenic gene pairs that were syntenic between F. vesca and F. × ananassa, and high levels of collinearity were observed in all FvDREBs between F. vesca and their corresponding F. × ananassa, except FvDREB24 (Fig 5). Moreover, the F. vesca chromosomes where the syntenic FvDREBs were located corresponded to several syntenic genes in F. × ananassa chromosomes. For example, the FvDREB10 gene was in the F. vesca chromosome 1 and its syntenic corresponding genes were in the F. × ananassa chromosome Fvb1-1, chromosome Fvb1-2, chromosome Fvb1-3, and chromosome Fvb1-4, respectively.
Expression Profiles of FvDREBs in Response to Drought Stress in Different Strawberry Leaves
Transcriptome sequencing data from old leaves and young leaves exposed to different drought stress conditions were previously generated by our group to investigate the expression profiles of FvDREBs. In general, different subgroups from different tissues showed different expression patterns (Figs. 6 and 7), suggesting the functional divergence of different subgroups of FvDREB members. In old leaves, D5 and D7 showed a similar clustering relationship (Fig. 6), whereas D3 and D5 displayed a similar clustering relationship in young leaves (Fig. 7). Two genes (FvDREB1 and FvDREB2) of the A-1 subgroup were highly expressed in the later period of drought stress, whereas the expression of FvDREB6, which belonged to the A-2 subgroup, was significantly up-regulated at the initial stage of drought stress. The expression of FvDREB30 of the A-6 subgroup was lower in the early stages of drought stress and that of FvDREB18 of the A-4 subgroup was lower in the middle stages of drought stress. And their expression level was similar in old and young leaves (Figs. 6 and 7), indicating that they may play a negative regulatory role in response to drought stress.
To further verify the expression of these identified FvDREB genes, two genes were randomly selected from each subgroup of the FvDREB gene family (the A-3 subgroup had only one member, so only one gene was selected) to detect their expression levels under different drought stress conditions by qRT-PCR analysis (Fig. 8). The results showed the expression of FvDREB8 of the A-2 subgroup was significantly up-regulated, with the highest expression on the second day under drought stress. The expression level gradually decreased, but all the expression levels were ten times higher than those in the control group. The expression level of FvDREB1 of the A-1 subgroup reached the highest level when it was subjected to drought stress for 4 days, which was more than eight times that of the control group. The expression level of FvDREB20 of the A-4 subgroup reached the highest level when subjected to drought stress for 6 days, which was more than five times that of the control group. The longer the time of exposure to drought stress, the greater the down-regulation of FvDREB30 of the A-6 subgroup. The trend of the expression of FvDREB was consistent with the RNA-Seq data. It could be seen that the expression of FvDREB genes in different subgroups was variable and unstable under drought stress.