Genome-wide identification, structural characterization and expression profiling of AP2/ERF gene family in bayberry (Myrica rubra)

doi:10.21203/rs.3.rs-4318206/v1

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Genome-wide identification, structural characterization and expression profiling of AP2/ERF gene family in bayberry (Myrica rubra)

https://doi.org/10.21203/rs.3.rs-4318206/v1

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Background: Bayberry is the most economically significant fruit within the Myricaceae family, having high nutritional and medicinal value. The AP2/ERF family is a class of transcription factors found mainly in plants. However, the bayberry AP2/ERF gene family has not previously been studied.

Results: In this study, 113 members of the bayberry AP2/ERF gene family were identified. According to the phylogenetic tree, the members of this group are divided into three subfamilies. The gene structure and conserved motifs were analyzed. Chromosome localization showed that 95 genes were unevenly distributed on 8 chromosomes and 18 genes were located on the skeleton. In addition, we further investigated the collinearity between these genes and related genes in six other species. Transcriptomic data showed that the expression pattern of AP2/ERFgene was different in bayberry space electric field treatment and at different stages of development.

Conclusions: The AP2/ERFgene was identified in the genome of bayberry, and its structure, conserved motif, and phylogenetic relationship were analyzed. These findings of this study serve as a reference for the genome-wide identification of the AP2/ERFgene family in other species and groundwork for future research on the function of AP2/ERF genes in bayberry.

Bayberry

AP2/ERF gene family

Expression pattern

Phylogenetic analysis

Transcription factor

Transcription factors (TFs) are critical regulatory proteins which can modulate the initiation of transcription of other genes [1]. They exert control over the transcription and expression of downstream genes, thus playing an indispensable role in plant growth and development, and the response to environmental stimuli [2–4]. As research in this field expands, various transcription factor families, including MYB [5], bHLH [6], NAC [7], WRKY [8] and AP2/ERF, have been identified across different species. These families are important because of their involvement in regulating diverse biological processes in plants.

The APETALA2/Ethylene-Response factor (AP2/ERF) transcription factors family is one of the largest plant-specific TFs families [9], attracting considerable attention over the past decade. Extensive investigations have led to the successful identification of numerous AP2/ERF genes in various plant species, e.g., 172 in oil palm [10], 134 in California poppy [11], 125 in longan [12], 155 in pear [13], 218 in sugarcane [14], 64 in strawberry [15], 158 in Actinidia eriantha [16], 134 in Tartary buckwheat [17] and 88 in Masson pine [18].

The AP2/ERF family is a class of transcription factors found mainly in plants. At least one AP2 domain is typically contained within members of the AP2/ERF family [19]; this encodes a domain consisting of 60–70 amino acids [20]. Direct interaction with cis-acting elements, such as the dehydration response element (DRE)/C-repeat element (CRT) and/or the GCC box located within the promoter region of target genes, can be facilitated by these AP2/ERF proteins [21, 22]. Furthermore, based on the type and number of conserved structural domains, the AP2/ERF family can be divided into five subfamilies, namely the AP2 (APETALA2), DREB (Dehydration reaction element binding factor), ERF (Ethylene Reaction factor), RAV (Related to Abscisic Acid Insensitive 3/Viviparous 1) and Soloist subfamilies [23–25]. The AP2 subfamily contains two AP2/ERF structural domains, whereas only one AP2 structural domain is contained within the ERF subfamily. The RAV subfamily contains one AP2 domain and a specific B3 motif [26]. In addition, the DREB and ERF subfamilies can be further subdivided into six groups known as evolutionary clades B1–B6 and evolutionary clades A1–A6 [27].

AP2/ERF, a large family of transcription factors with multiple functions, holds a pivotal role in plant growth, hormone regulation and response to environmental stresses [28]. In recent years, there has been increasing evidence that these TFs are also key regulators of fruit quality attributes such as ripening, color, texture, and flavor [29, 30]. Studies have shown that the transcriptome and expression profile analysis of ERF gene family showed that it plays the role of transcriptional activator and suppressor in durian fruit ripening process [31]. PpERF is a candidate gene for ethylene biosynthesis in peach and plays an important role in peach fruit ripening [32]. Li et al. [33] found that CitERF71, a member of the AP2/ERF family, can activate the CitTPS16 promoter, thereby promoting the production of E-geraniol in citrus fruits, which plays an important role in the flavor of sweet oranges. Wang et al. [34] found that PpERF5 and PpERF7 can combine to form a protein complex that enhances peach aroma by upregulating PpLOX4 expression. AP2/ERF transcription factor PavRAV2 negatively regulates sweet cherry fruit size by directly inhibiting the expression of PavKLUH[35]. These studies provide a new target for exploring the biological function and role of AP2/ERF gene family in fruit and provide a reference for further understanding and studying the role of AP2/ERF in regulating fruit quality.

The bayberry (Myrica rubra, family Myriaceae) is a tropical or subtropical fruit tree. Bayberries are esteemed for their high economic and nutritional value and are favored by consumers for their distinctive flavor and texture [36, 37]. Originating from China, a staggering 305 cultivars of bayberries have been documented, with introductions to countries such as Japan, Australia, and the United States [38]. Bayberry cultivation boasts a history spanning over 2,000 years. The fruit are commonly reddish-purple owing to the presence of cyanidin-3-O-glucoside (C3G) [36].

A variety of transcription factor families have been identified in bayberry, e.g., 174 of the MYB gene family members[39], 152 of the UDP-glycosyltransferase (UGT) gene family [40], 15 of the β-Galactosidase gene family [41], 54 of the MADS-box gene family [42], and 42 of the glutathione S-transferases (GSTs) gene family [43]. Nevertheless, despite the extensive genetic exploration in bayberry, the AP2/ERF gene family remains unreported in this species.

In the present comprehensive study, 113 AP2/ERF family candidates were identified in bayberry, including 22 AP2 genes, 56 ERF genes, and 133 DREB genes. Sequence conservation, gene structures, chromosomal locations, gene duplications, and cis-regulatory elements in bayberry were systematically investigated. These results will provide new clues for the future exploration of AP2/ERF gene functions in bayberry.

Identification of AP2/ERF genes in bayberry

By combining Hidden Markov Model (HMM) and BLAST searches, all putative members of the AP2/ERF gene family were identified within the bayberry genome. After removing redundancies, a total of 113 AP2/ERF members was identified. The 113 genes were analyzed for amino acid (aa) sequence length, protein isoelectric point (pI), molecular weight (MW) and subcellular localization (Additional file 1: Table S1). The protein lengths in bayberry ranged from 70 aa (KAB1199489.1) to 724 aa (KAB1203436.1) with an average of 282 amino acids. The smallest molecular weight is 7830.21Da (KAB1199489.1), the largest is 77776.76Da (KAB1203436.1), and the average is 31069.83Da. The theoretical pI of these proteins ranged from 4.74 (KAB1222311.1) to 10.22 (KAB1199489.1) with an average of 6.88. Most AP2/ERF proteins (94 of 113) were identified as being in the nucleus, while 10 AP2/ERF proteins were located in the chloroplast, 5 AP2/ERF proteins were located in the mitochondrion, and the other four were in the cytoplasm.

Phylogenetic analysis and classification of the bayberry AP2/ERF

To determine the evolutionary relationships of the identified bayberry AP2/ERF, a neighbor-joining (NJ) phylogenetic tree was constructed through multiple sequence alignment of 249 AP2/ERF protein sequences. Among them, 113 AP2/ERF members from bayberry and 136 from Arabidopsis thaliana (Fig. 1). According to the A. thaliana classification, all the AP2/ERF genes from these two species were grouped into three subfamilies, AP2, ERF and DREB, of which the ERF was the largest group, comprising 56 AP2/ERF family members, further subdivided into six distinct subgroups (designated as B1 to B6). Among these, subgroup B3 was the largest comprising 25 members, while subgroups B2 and B5 were the smallest, each containing only 4 members. The DREB subfamily has 35 AP2/ERF family members and could be further divided into six subgroups (A1 to A6). Subgroups A2 and A4 were the largest with 9 members each, while subgroup A3 was the smallest with only 1 member. Finally, the AP2 subfamily comprised 22 genes.

Gene structure and conserved motif analysis of the bayberry AP2/ERF

To elucidate further the phylogenetic evolutionary relationships and structural characteristics of bayberry AP2/ERF genes, we conducted an analysis of their exon-intron gene structure and conserved motifs [44]. The structural domains show that each gene has at least one AP2 structural domain. Notably, the number of exons in AP2/ERF genes varied significantly, ranging from 1 to 10 (Fig. 2). In particular, the DREB subfamily had more exons than the other two subfamilies. Most genes, 82.3% of the total, contained one or two exons. However, three genes, namely KAB1214741.1, KAB1226102.1, and KAB1223904.1, each had 10 exons. Furthermore, 63.7% of AP2/ERF gene family members did not have introns. These findings showed a relatively high degree of conservation in the AP2/ERF gene family. A total of 10 conserved motifs was identified and named motif 1 to motif 10 (Fig .2, Additional file 2: Fig. 1). Intriguingly, no member of the bayberry AP2/ERF family had a complete set of all 10 conserved motifs, but instead had one to six conserved motifs. Nine genes in the DREB subfamily had six conserved motifs. Eight out of 113 AP2/ERF members contained one motif. Motif 1, motif 2, motif 3 were observed in the majority of members. The finding that closely related members of the phylogenetic tree tend to have the same motif suggests that the AP2/ERF family subfamily genes are highly conserved and similar and may have the same biological function.

Chromosomal location of the bayberry AP2/ERF

The location of genes on chromosomes is often one of the important factors of gene exercise function. Each bayberry chromosome contains more than 7 AP2/ERFs (Fig. 3). Of the 113 identified AP2/ERF genes, 95 are unevenly distributed across the eight chromosomes, while the remaining 18 are localized to the chromosomal skeleton. The highest number of AP2/ERF family members were found on Chr 8, Chr 3 and Chr 5, with 18, 16 and 15 family members, respectively. In contrast, the family on Chr 1 has the fewest, only 7 members. Intriguingly, although Chr 7 is the longest chromosome, it does not have the most AP2/ERF members, suggesting a lack of significant correlation between chromosome length and AP2/ERF distribution.

Synteny analysis of the bayberry AP2/ERF

To investigate whether these genes have duplication events in the bayberry genome, segmental duplications and tandem duplications in the bayberry genome were analyzed. Gene collinearity analysis of the AP2/ERF gene family showed 12 segmental duplication events, involving 21 AP2/ERFs, accounting for 18% of the whole gene family (Fig. 4). In the 12 pairs of collinear relationships, KAB1218028.1, KAB1204620.1, and KAB1199840.1 were paired with 2 genes each, and all others were one-to-one paired (Additional file 1: Table S2). The Ka/Ks ratios of homologous gene pairs can be used as an indicator of selection pressure during gene evolution, and Ka/Ks ratios less than 1.0 indicate low selection pressure [45]. Remarkably, in this study, the Ka/Ks ratios of 6 homologous gene pairs were less than 1.0 (Additional file 1: Table S3), indicating that these genes underwent purification selection pressure.

To enhance our comprehension of genetic differentiation, gene duplication, and evolutionary patterns within the AP2/ERF gene families of Arabidopsis, grape, pomegranate, peach, Populus trichocarpa, apple, and bayberry, we conducted an analysis of homologous AP2/ERF genes across these species (Fig. 5, Additional file 1: Table S4). The results showed that 153 homologous gene pairs existed in bayberry and Populus trichocarpa. This was followed by 141 homologous gene pairs in bayberry and apples. There were 88, 87, 85 and 65 homologous gene pairs between the bayberry AP2/ERF gene and peach, grape, pomegranate, and Arabidopsis AP2/ERF genes, respectively.

Cis-element analysis of the bayberry AP2/ERF

Transcription factors play a crucial role in orchestrating plant responses to both biotic and abiotic stresses. Within the promoter sequences of transcription factors, various cis-regulatory elements act as key regulators of gene expression. In this study, we extracted the 2000 bp promoter region upstream of the AP2/ERF gene and analyzed the cis-elements present using the PlantCARE online tool. It is well-established that AP2/ERF genes are pivotal in governing essential processes of plant growth and development. Moreover, they exhibit remarkable versatility in their ability to respond to many different phytohormones and environmental stresses [1]. The results indicate that there are many cis-acting elements in the AP2/ERF promoter, such as phytohormone response elements, light response elements, stress response elements, as well as abiotic and biotic stresses (Fig. 6). A total of 113 AP2/ERF promoters exhibited light-responsive elements, 98 had MeJA-responsive elements, 61 had gibberellin-responsive elements, 61 had low-temperature responsive elements, 57 had auxin-responsive elements, and 44 had defense and stress responsive elements.

Expression analysis of AP2/ERF genes in response to space electric field treatment

The expression patterns of bayberry AP2/ERF family members during space electric field storage were analyzed (Fig. 7). Out of the total AP2/ERF genes examined, only 89 were expressed during space electric field storage. Among these, 60 AP2/ERF genes exhibited significantly higher expression levels compared to the control group after 30 days of storage in the space electric field. Furthermore, KAB1221974.1, KAB1214033.1, KAB1213936.1, KAB1200465.1, and KAB1212199.1 demonstrated a gradual decrease in expression levels in both the space-field storage and control groups. Conversely, KAB1202427.1, KAB1222005.1, and KAB1203769.1 genes showed a progressive increase in expression levels during the space electric field storage process, whereas they showed a gradual decrease in the control group, indicating that these three genes play an important role in the post-harvest space electric field storage process of bayberry.

Expression analysis of the AP2/ERF genes at different developmental stages

To understand specific spatiotemporal expression patterns of AP2/ERF genes, we analyzed the identified genes’ expression profiles in different cultivars at different developmental stages (Fig. 8). Notably, 81 genes exhibited expression across various developmental stages. KAB1214033.1, KAB1204136.1, and KAB1199840.1 were highly expressed at all developmental stages of ‘ShuiJing’ and ‘DongKui’, indicating that these genes were likely essential functional genes. Additionally, KAB1213936.1 was highly expressed at S5 in both cultivars. The expression of KAB1204136.1, KAB1204975.1, KAB1203948.1, KAB1212863.1, KAB1214485.1, and KAB1205694.1 gradually increased during the maturation of ‘DongKui’ bayberry fruit. Meanwhile, the expression of KAB1217174.1 increased progressively at different developmental stages in both cultivars, implying their potential significant roles in bayberry development.

GO and KEGG analysis of the bayberry AP2/ERF genes

GO and KEGG analysis of the bayberry AP2/ERF gene family members was performed using transcriptome data (Fig. 9). The results of GO annotation revealed that the predominant biological processes were associated with cellular processes, single-organism processes, and metabolic processes. Within the cellular component classification, the primary subgroups encompass cell, cell parts, and organelle. The key subcategories of molecular function include catalytic activity and binding. Moreover, the KEGG enrichment analysis indicated that these genes predominantly cluster within the pathways plant hormone signal transduction and ubiquitin-mediated proteolysis.

The APETALA2/ethylene response factor (AP2/ERF) proteins represent a significant group of transcription factors (TFs) within plants, potentially implicated in various aspects of plant growth and stress responses [46], including drought stress [47], organ development [48, 49], hormone synthesis [50], cold stress [51], and disease resistance [52].

The bayberry, indigenous to China, has considerable medicinal and economic importance [53]. Despite extensive research on the AP2/ERF family across diverse plant species, the structural characteristics, and functional roles of the bayberry AP2/ERF gene family was largely unexplored. Therefore, we identified AP2/ERF transcription factors in bayberry, which is valuable for the analysis of the AP2/ERF family in bayberry.

In this study, 113 members of the bayberry AP2/ERF gene family were identified. By phylogenetic analysis, these 113 AP2/ERF gene families were categorized into three distinct subfamilies, namely AP2, ERF, and DREB. The ERF subfamily is the largest subfamily with 56 family members. The number of AP2/ERF gene family members depends on the number of ERF subfamily members [54]. While the AP2/ERF gene family has been documented in various species, significant variations exist in the number of its members. For instance, there are 104 AP2/ERF family members in Liriodendron chinense [55], including 43 members of the ERF subfamily. There are 198 AP2/ERF family members in sweet potato [56], with 172 members of the ERF subfamily. 149 AP2/ERF family members were identified in Vitis vinifera [57], with 122 members of the ERF subfamily. 119 AP2/ERF family members were identified in Chinese jujube [58], including 57 members of the ERF subfamily. 364 members of the AP2/ERF family were identified in Chinese willow [59], with 301 members of the ERF subfamily.

Gene structure analysis plays a crucial role in revealing gene functions [60]. Structural analysis showed that 63.7% of AP2/ERF gene family members do not have introns. Of those AP2/ERF gene family members that do have introns, 82.3% have only one or two exons. This is consistent with that observed in other plants [13, 16]. Studies have indicated a relationship between the number and distribution of introns and plant evolution, suggesting that introns may have been lost from ERF and RAV family genes during the evolution of higher plants [14, 61]. Most AP2/ERF gene family members have motif 1, motif 2, and motif 3, whereas motif 1, motif 2, motif 3, and motif 7 are prevalent in the ERF subfamily and motif 4, motif 5, and motif 10 are prevalent in the DREB subfamily. Similar results have been reported in both Rosa chinensis [46] and almond [62]. Combined analyses of intron-exon structure and conserved motifs indicate that members of the same taxon share similar features, suggesting a high degree of conservation among most AP2/ERF genes during evolution.

Gene duplication events that occur during genome evolution are a major factor leading to gene amplification and evolution and are also important in helping plants cope with environmental changes during growth and development [63, 64]. To understand better the amplification mechanism of the AP2 gene, we analysed the replication events of the gene in bayberry. We identified at least 12 pairs of AP2/ERFs. In addition, Arabidopsis, grape, pomegranate, peach, Populus trichocarpa, and apple shared 65, 87, 85, 88, 153, and 141 pairs of collinear genes respectively with members of the AP2/ERF family of bayberry. 95 of the 113 bayberry AP2/ERF gene family members are unevenly distributed across 8 chromosomes, there are only 7 genes on Chr 1, while there are up to 18 genes on Chr 8. These results suggest that gene duplication events promote the evolution and expansion of the AP2/ERF gene family in bayberry.

The yield and quality of bayberry fruits are susceptible to a wide range of biotic and abiotic stresses. Methyl jasmonate (MeJA) treatment induces the initiation of a defense response in bayberry to Penicillium citrinum [65]. Gibberellin and abscisic acid can break the dormancy of bayberry seeds and promote germination [66]. Cis-acting elements are recognized by transcription factors and are involved in the expression of tissue-specific and stress-responsive genes [67]. In this study, several cis-elements mainly involved in stress response, phytohormones and plant growth and development were identified from the promoter region of the AP2/ERF family genes of bayberry. The presence of a diverse array of cis-regulatory elements within the promoter region of bayberry AP2/ERF genes suggests their potential involvement in responding to various stresses and phytohormones. This indicates a likely role for these genes in regulating bayberry growth and development.

In addition, transcriptome analysis showed that 89 genes were expressed under space electric field storage, of which 60 genes were significantly expressed after 30 days of space electric field storage, and expression of 3 genes gradually increased during space electric field storage. The fruit of bayberry is very perishable but was preserved in the space electric field for a long time, indicating that these genes play an important role in the storage process of the space electric field. 81 genes were expressed in different developmental stages of bayberry. Expression of 6 genes gradually increased in different developmental stages of ‘DongKui’ bayberry fruit, and expression of only one gene gradually increased in different developmental stages of the two cultivars, indicating that these genes played an important role in different developmental stages of the bayberry fruit.

In conclusion, our results showed that a total of 113 members of the bayberry AP2/ERF family had been identified; these were distributed in three different subfamilies and unevenly distributed on eight chromosomes. Collinearity showed that gene replication events occurred in the AP2/ERF gene family of bayberry, Arabidopsis, pomegranate, peach, grape and other species. Transcriptome data showed that the expression level of KAB1217174.1 increased gradually in the development stages of different cultivars, indicating that KAB1217174.1 played an important role in the ripening process of bayberry fruit. These results not only help to screen valuable candidate AP2/ERF genes for further functional studies, but also provide valuable resources for further understanding of the biological role of AP2/ERF genes in bayberry.

In summary, genome-wide detection and analyses of the AP2/ERF gene family in bayberry identified a total of 113 AP2/ERF members. According to phylogenetic analysis, the AP2/ERF genes were classified into 3 subfamilies. Subsequently, the domains, conserved motifs, gene structures, collinearity, cis-elements, and different developmental stages expression profiles of 113 AP2/ERF transcription factors were comprehensively identified. Comprehensive insights into the molecular underpinnings of the AP2/ERF family in bayberry were provided by the findings of this study, offering valuable information for further research endeavors.

Plant materials and treatment

Different cultivars (‘DongKui’ and ‘ShuiJing’) of bayberry fruits were purchased from Wenrong Yangmei Professional Cooperative in Lin’an District, Hangzhou City, Zhejiang Province. Fruits were harvested at five developmental stages, including 51, 58, 65, 72 and 80 days after full blossom (DAB). Three biological replicates, each with five fruits were harvested, frozen in liquid nitrogen, and stored at -80^o C until further analysis.

The mature fruits of ‘DongKui’ (80 days after flowering) were divided into two groups for postharvest storage and preservation experiments. One group was exposed to a -2°C cold storage equipped with space electric field (electric field strength of 3000V, frequency of 50Hz) for 10, 20 and 30 days. A control group was placed in -2°C cold storage without space electric field.

Identification of AP2/ERF genes

The genome sequence and annotation information of bayberry (Myrica rubra) were downloaded from the Chinese bayberry genome (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_003952965.2/), the protein sequences of AP2/ERF genes in Arabidopsis thaliana were obtained from The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/). Hidden Markov Models (HMMs) of the AP2/ERF conserved domain (PF00847) were acquired from the Pfam Protein Family Database (http://pfam-legacy.xfam.org/). Subsequently, HMMER and BLASTP tools were employed to screen candidate AP2/ERF genes of bayberry using the AP2 domain model retrieved from Pfam database and A. thaliana AP2/ERF protein sequences. First, the A. thaliana sequences were used as query sequences to carry out BLASTP program against the whole protein sequences of bayberry (E-value < 1e-10 and identity > = 40). AP2/ERF proteins in the bayberry genome were identified using the HMM search program in TBtools. Redundant sequences were removed from the above results. Subsequently, the conserved domains of the candidate AP2/ERF genes were searched for batch comparison to verify whether they contained AP2 conserved domains using the NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Molecular weight (Mw) and isoelectric point (pI) of each AP2/ERF protein were calculated by the online program ExPASy (https://web.expasy.org/compute_pi/). The subcellular location of AP2/ERF proteins in bayberry was predicted by WoLF PSORT online Sites (https://wolfpsort.hgc.jp/).

Phylogenetic analysis and Chromosomal locations of AP2/ERF genes

To generate a phylogenetic tree, multiple sequence alignments of the complete AP2/ERF protein sequences in Arabidopsis and bayberry were carried out using ClustalX software. The Neighbor-joining (NJ) method was used to establish the phylogenetic tree in FigTree v1.4.4 program. The positions of all genes were extracted from the bayberry GFF3 profile and displayed on the 8 chromosomes via TBtools software.

Gene Structure and Conserved Motif of AP2/ERF Genes

Exon-intron structures of AP2/ERF genes were visualized using TBtools. Conserved motifs were detected in bayberry AP2/ERF family members using the motif analysis tool MEME (https://meme-suite.org/meme/doc/meme.html) with the selected number of motifs was set to 10. Then, TBtools software was used to generate conserved motif diagrams.

Gene duplication and cis-regulatory elements of AP2/ERF

Multiple collinear scanning tool (MCScanX) was used to evaluate interspecies replication events of bayberry. The genomic data of Arabidopsis thaliana, Malus pumila, Vitis vinifera, Punica granatum, Prunus persica and Populus trichocarpa were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/), and the collinear gene relationships between bayberry and these six species were analyzed using Multiple Synteny Plot function in TBtools and visualized by TBtools. To analyze the cis-acting elements on the promoter sequence, the genomic DNA sequence of 2000 bp upstream of the start codon of each AP2/ERF gene was extracted from the bayberry genome, and the number of cis-acting elements of the promoter was predicted on the Plant CARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Gene expression analysis

About 1 g of bayberry fruit was used for total RNA extraction. The quality and concentration of RNA were determined by NanoDrop One spectrophotometer at A260/A280 absorbance ratio. Libraries for high-throughput Illumina strand-specific RNA-seq were prepared for gene expression analysis. All clean reads were mapped to the Chinese bayberry genome, which was used as the reference genome. Raw sequencing data files were uploaded to the NCBI Sequence Read Archive database (project number: PRJNA1105392). The data was calculated by reads per kilobase per million mapped read (RPKM) values as transcript abundance. Three biological replicates for various fruit development and ripening stages and treatment were performed for gene expression analysis. The heat maps of gene expression were visualized using the Heatmap illustrator program in TBtools.

Go and KEGG analysis of AP2/ERF genes

Utilizing transcriptome data from various developmental stages of bayberry, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on the members of the bayberry AP2/ERF gene family.

TFs

Transcription factors

AP2/ERF

APETALA2/Ethylene-Response factor

DREB

Dehydration reaction element binding factor

ERF

Ethylene Reaction factor

RAV

Related to Abscisic Acid Insensitive 3/Viviparous 1

Acknowledgements

We thank Prof. Ross Ferguson (Plant & Food Research, New Zealand) for the critical revisions to the manuscript, and Dr. Shaojia Li (Zhejiang University, China) for his valuable assistance with modification.

Authors’ contributions

YL and BW designed the experiments. YL, LC and JZ performed the experiments. YL, MC, HZ and XF participated in sample collection and data analyses. YL wrote the manuscript. BW and KX modified the manuscript. All authors have read and approved the final manuscript.

Funding

This work was funded by the Natural Science Foundation of Zhejiang province (Q21C150002), the National Natural Science Foundation of China (30871718) and the Scientific Research and Development Foundation of Zhejiang A & F University (2019FR046).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary files. The datasets analysed during the current study are available in the NCBI Sequence Read Archive database (project number: PRJNA1105392). Further inquiries can be directed to the corresponding author.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

Additionalfile1TableS1.xlsx
Additional file 1: Table S1. The AP2/ERFgene family in bayberry.
Additionalfile2Fig.1.tif
Additional file 2: Fig.1. Sequence information for each motif (Motif 1–Motif 10).
Additionalfile3TableS2..xlsx
Additional file 3: Table S2. Gene duplication pairs of the AP2/ERF genes within the bayberry genome.
Additionalfile4TableS3.xlsx
Additional file 4: Table S3. Ka/Ks ratios of the duplicated AP2/ERF genes in bayberry.
Additionalfile5TableS4.xlsx
Additional file 5: Table S4. Syntenic gene pairs between AP2/ERF genes in bayberry and other species.

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Genome-wide identification, structural characterization and expression profiling of AP2/ERF gene family in bayberry (Myrica rubra)

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Abstract

Figures

Background

Results

Phylogenetic analysis and classification of the bayberry AP2/ERF

Gene structure and conserved motif analysis of the bayberry AP2/ERF

Chromosomal location of the bayberry AP2/ERF

Synteny analysis of the bayberry AP2/ERF

Cis-element analysis of the bayberry AP2/ERF

Discussion

Conclusions

Materials and methods

Plant materials and treatment

Gene expression analysis

Go and KEGG analysis of AP2/ERF genes

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1