1. Introduction
Transcription factors (TFs), one of the master regulatory proteins types, perform a key role in the plant response to a range of abiotic and biotic stresses [1]. The APETALA2/Ethylene Responsive Factor (AP2/ERF) superfamily is one of the largest groups which regulates plant physiological processes, such as growth, development, and stress response [2,3]. The AP2/ERF transcription factors contain one or two highly conserved AP2 DNA binding domains with 60–70 amino acid residues that consist of three-stranded anti-parallel β-sheets, followed by a parallel α-helix [4]. The AP2 domain contains two conserved elements (YRG and RAYD). The YRG element is located at the N-terminal of the AP2 domain consisting of about 19–22 amino acid residues, and the RAYD element is located at the C-terminal consisting of about 43 amino acid residues [5]. The AP2/ERF TFs have been classified into the AP2, ERF, RAV, and Soloist subfamilies [6]. The AP2 subfamily contains either single or double AP2 domains and plays an important role in the plant developmental processes [7]. The ERF subfamily was subdivided into the dehydration-responsive element-binding protein (DREB), and ERF subfamilies based on the amino acid sequences in the AP2 domain. The ERF subfamilies were further categorized into 12 groups: I–X, VI-L, and Xb-L [8]. The RAV subfamily consists of AP2 and B3 domains [9]. Additionally, a small group of TFs containing one AP2 domain, but a different structure, was classified as being in the Soloist subfamily [6].
With more extensive genome sequences, the AP2/ERF superfamily has been identified in various plant species, such as Arabidopsis [6,8], rice [8], poplar [10], oil palm [11], dark jute [12], cucumber [13], wheat [14], and sunflower [15]. These studies have shown that AP2/ERF superfamily members are involved in abiotic and biotic stress responses in plants. The AP2 subfamily binds to the GCAC(A/G)N(A/T)TCCC(A/G)ANG(C/T) element [16] and has been shown to be involved in organ development, including spikelet meristem differentiation, leaf epidermal cell designation, floral organ patterning, and seed yield [17,18,19]. Members of the RAV subfamily also have important functions in plant hormone signal transduction and the regulation of responses to biotic and abiotic stress by interacting with CAACA and CACCCTG in the promoter region [20,21]. The ERF subfamily members are generally involved in hypoxic stress response, pathogen stress response, injury response, and ethylene signaling pathway by binding a specific cis-acting component GCC-Box (sequence AGCCGCC) [22]. Moreover, DREB subfamily members generally bind to CRT/DRE elements (A/GCCGAC) to regulate stress response, including drought, salinity, and heat [23,24].
The pecan (Carya illinoinensis (Wangenh.) K. Koch) belongs to the Juglandaceae family and is an extremely important nut crop for daily diet in the world [25]. With the continuous expansion of planting area, the pecan is facing more and more adverse conditions, such as waterlogging, drought, and saline–alkali stress, so it is urgent to cultivate the resistant varieties. In the past three decades, traditional genetic methods, such as hybrid breeding, have contributed considerably to the production of new pecan varieties that increased productivity and stress resistance. However, the classical breeding technology is limited due to cross-incompatibility and its being time-consuming [26]. Current genetic-engineering methods, such as CRISPR/Cas9 [27] and TAILEN [28], have great potential for molecular improvement in short periods and high efficiency, but that requires the knowledge of the specific role of gene families.
To the best of our knowledge, the mechanisms underlying the regulation of pecan growth, development, and response to different environmental stresses remain poorly understood. Numerous reports have documented that the AP2/ERF-type TFs were important regulators involved in plant growth and development, as well as in hormonal regulation. For example, ERF-VII members play important roles in waterlogging tolerance in case of Arabidopsis and rice [29]. Overexpression of RAP2.2, RAP2.12, HRE1, and HRE2 increased hypoxia tolerance in Arabidopsis, and SUB1A-1 improved waterlogging tolerance in rice [30,31].
In the present study, the AP2/ERF TFs of pecan were investigated. The AP2/ERF genes structure, chromosome localization, conserved motif, phylogenetic analysis, duplication events, and cis-acting elements analysis were studied. Meanwhile, transcriptome data were used to analyze the expression of family members in various degrees of development and stress. Finally, five ERF-VII genes were selected to explore expression patterns under waterlogging stress, using real-time-PCR analysis. This study hopefully provides an important resource for further functional characterization of CiAP2/ERF genes and their utilization for the genetic improvement of the pecan.
2. Results
2.1. Identification, Classification, and Physical Properties of CiAP2/ERF TFs
A total of 202 AP2/ERF genes from the pecan genome were identified (Table 1 and Supplementary Table S1), and identified AP2/ERF genes were categorized into four families (ERF, AP2, RAV, and Soloist) depending on the occurrence of conserved AP2 domains.
Following the classification method in the AP2/ERF factors of Arabidopsis [8], these genes were further analyzed and classified into two subfamilies (ERF and DREB) according to their specific amino acid variation in the conserved domain region. The ERF subfamily contained 107 members with alanine (A) in the 14th and aspartic acid (D) in the 19th positions, while the DREB subfamily had 61 members having valine (V) and glutamine (E) residues at respective positions. Twenty-six genes were classified into the AP2 family, including 23 genes containing two AP2 domains and 3 genes with only one AP2 domain. The RAV family had 6 genes with the AP2 and B3 domain. Moreover, 2 specific genes contained a single domain and showed the highest homology with the At4g13040 gen; they were in the Soloist family (Table 1 and Supplementary Table S1).
The length of CiAP2/ERF genes ranged from 387 to 2283 bp, and the proteins contained 128–760 of amino acids length. The molecular weight (MW) and Isoelectric point (PI) varied widely, which was 13906.60–82684.48 and 4.47–10.73, respectively. Predicted subcellular localization revealed that a majority of AP2/ERF genes were located in the nucleus and cytoplasm (Supplementary Table S2).
2.2. Phylogenetic, Gene Structure, and Conserved Motif Analysis of CiAP2/ERF TFs
A phylogenetic tree was constructed by using the deduced protein sequences of AP2/ERF genes from pecan and Arabidopsis that were clustered into distinct ERF, AP2, RAV, and Soloist clades (Figure 1). Most of the members were classified into the ERF family. In-depth analysis subdivided the ERF clades into twelve sub-clades (I–X, Xb-L, and VI-L), similar to gene classification in Arabidopsis. The first four sub-clades (I–IV) were grouped as DREB subfamily, and the remaining eight sub-clades (V–X, Xb-L, and VI-L) were in the ERF subfamily. Interestingly, CiAP2-26 was assigned to group ERF-IV (Figure 1).
The number of introns varied in different subfamilies of the AP2/ERF superfamily. Except CiAP2-26, which had 1 intron, the AP2 family members contained more introns (4–10). Most of ERF family members did not have any introns, and a few of them had 1 or 2 introns. CiRAV-05 had 3 introns, and the other RAV family members had no introns. The remaining two Soloist family members (CiSoloist1 and CiSoloist2) contained 6 and 7 introns, respectively (Figure 2D). In general, closely related members from the same subfamily had similar numbers and intron lengths. Furthermore, the conserved motifs of the AP2/ERF family members were studied. In total, 10 conserved motifs were presented and designated Motifs 1 to 10 (Figure 2B and Supplementary Figure S2). Motifs 1 and 2 were presented in almost all sequences except CiAP2-26, which contained Motifs 3, 5, 7, and 9. Most ERF family members had Motifs 1, 2, and 3; most DREB family members contained Motifs 1, 2, 3, 4, and 10; the majority of AP2 family members and all RAV family members had Motifs 1, 2, 3, 4, and 5; all Soloist family members contained Motifs 1, 2, and 4 (Figure 2B).
Based on the presence, position, and phase of introns, the intron distribution within the AP2 domain of all of the reported AP2/ERF genes was analyzed. Eleven different distribution patterns (designated A to K), ranging from zero to seven introns within the domain, were observed. (Figure 3). The results showed that 81.7% identified members of the family did not have introns in their AP2 domain, thus indicating that the members were conserved. All DREB and RAV, and most ERF subfamily members belonged to Pattern A, and the remaining ERF members belonged to Pattern B. Soloist subfamily members had Pattern C. Except for CiAP2-26, other AP2 subfamily members covered from Pattern D to Pattern K (Figure 3 and Supplementary Table S4).
2.3. Chromosomal Distribution and Genes Duplication Analysis of 202 CiAP2/ERF TFs
All 202 identified CiAP2/ERF genes were mapped to 16 chromosomes of pecan, and the distribution of CiAP2/ERFs varied through 16 chromosomes (Figure 4). Chromosome 1 anchored the highest number of 22 AP2/ERF genes, followed by Chromosome 3 (20) and Chromosome 4 (20). Chromosome 16 had the lowest number (6) of CiAP2/ERFs. ERF and DREB subfamily members were distributed on all 16 chromosomes of pecan. However, none of the AP2 subfamily members were mapped to Chromosome 3, 4, 5, 11, or 12. Moreover, RAV subfamily members were only mapped to Chromosomes 4, 13, 14, 15, and 16. At the same time, Soloist subfamily members were located only on Chromosomes 1 and 2 (Figure 4).
The calculated Ka/Ks ration values of all AP2/ERF genes were found to be less than 1 (Supplementary Table S5). These results indicated that the evolution of pecan AP2/ERF genes happened via strong purifying selection. Moreover, a total of 165 pairs of gene duplications were identified and divided into tandem duplications and segmental duplications. Among them, 19 pairs were identified as tandem duplication, and 146 pairs were classified as segmental duplication (Figure 4 and Figure 5). All 16 chromosomes of pecan genome contained segmental duplicated AP2/ERF family members. Ten chromosomes (1, 3, 4, 7, 8, 9, 10, 11, 12, and 13) had tandem duplication, whereas Chromosome 8 contained more (Figure 4 and Figure 5). The tandem and segmental duplication is the driving force for gene-family expansion in plants [32]. These results demonstrated the prominent role of tandem and segmental duplication in the expansion of AP2/ERF gene family members in the pecan genome.
2.4. Analysis of Cis-Acting Elements in Promoter Regions of CiAP2/ERFs
To investigate the cis-elements of the 202 CiAP2/ERF genes, 2000 bp of sequence upstream from the start codon was analyzed. The cis-elements of the CiAP2/ERF genes contained 26 categories, which were related to plant growth and development, stress response, and plant hormone response (Figure 6 and Supplementary Figure S1). Light response elements were found in the largest number in all AP2/ERF genes’ promoter sequences, followed by abscisic acid responsive elements and MeJA-responsive elements, which were also identified in most members (Figure 6 and Supplementary Figure S1).
2.5. RNA-Seq Expression Analysis of CiAP2/ERF Genes in the Response of Stress and Development
RNA-Seq analysis was used to identify the function of AP2/ERF genes in different development and abiotic stress conditions. For the drought condition, 0 (control), 3, 6, 9, 12, and 15 days were considered where 19 genes were not expressed (Figure 7A). Among the 26 AP2 subfamily members, 10 had the highest expression at 3 days, and 8 showed the highest expression at 15 days. This indicated that these genes might be induced at 3 and 15 days. Most ERF members were upregulated at 15 days, while DREB members showed a similar trend. Three RAV members had the h ighest expression at 6 days, while CiRAV-05 had the highest expression at 0 days. The Soloist subfamily was mainly expressed at 12 and 15 days (Figure 7A).
In the pistillate flowering development, FB1 indicated the initial stage of flower-bud differentiation, FB2 indicated the inflorescence formation stage, FB3 indicated that the female flower primordium continues, FL1 indicated the early blooming stage, and FL2 indicated the full blooming stage (Figure 7B). There were 20 genes that were not expressed. Most AP2 subfamily genes were highly expressed at the FL stage, while the majority of ERF and DREB genes was upregulated at the FB stage. Particularly, most members were highly expressed in the FB3 stage. Except for CiRAV-02 and CiRAV-03, the other members of RAV subfamily were also highly expressed at the FB3 stage.
In the graft union development, nine genes were not expressed. Most AP2 subfamily genes were mainly upregulated at 0 and 8 days, while the other subfamily members were mainly upregulated at 15 and 30 days (Figure 7C). Similarly, in the kernel development (Figure 7D), thirty-five genes were not expressed. AP2 subfamily genes were mainly upregulated at the later water stage and gel stage. Most ERF genes were upregulated at the later water stage, while DREB genes were upregulated at the later water and mature stage. RAV and Soloist subfamily members showed similar expression patterns which were upregulated at the late water stage and mature stage.
2.6. ERF-VII Members in Pecan and Arabidopsis
There were five group ERF-VII genes in the Arabidopsis genome, namely AtRAP2.2, AtRAP2.3, AtRAP2.12, AtHRE1, and AtHRE2. Similarly, five members of ERF-VII were identified in the pecan genome: CiERF-005, CiERF-010, CiERF-020, CiERF-052, and CiERF-064. The amino acid sequences of these ten genes were used for multi-sequence alignment and tree construction. The resultant showed that these ERFs are characterized by a conserved DNA-binding domain by the extremely conserved N-terminal motif MCGGAI/VI (Figure 8). In order to estimate the evolutionary relatedness among the members of this group, a neighbor-joining method was used to build a phylogenetic tree of the ERF-VII proteins from Arabidopsis and pecan. CiERF-010, CiERF-020, AtRAP2.12, and AtRAP2.2 cluster together. In the same way, CiERF-005 and AtRAP2.3 cluster together. CiERF-052, CiERF-064, and AtHRE2 cluster together.
2.7. ERF-VII Genes’ Expression Analysis of Different Pecan Cultivars under Waterlogging Stress
To further explore the response of CiAP2/ERF genes under waterlogging stress, the expression patterns of 5 CiAP2/ERF genes (CiERF-064, CiERF-052, CiERF-020, CiERF-010, and CiERF-005) from ERF-VII group were examined via RT-qPCR analysis (Figure 9). In the case of waterlogging stress, CiERF-064 was significantly upregulated before 12 h in all varieties except C10, which was downregulated at 4 h. CiERF-052 followed downregulation trends but moved up at 12 h. In addition to J23, CiERF-020 showed a trend of first rising and then decreasing. In comparison to other varieties, it showed a trend of first decreasing and then increasing. CiERF-010 was downregulated at 24 h. CiERF-005 was downregulated before 24 h and upregulated at 48 h. (Figure 9). Thus, these experiments indicated that ERF-VII genes might have a strong correlation with waterlogging stresses.
3. Discussion
The AP2/ERF is one of the major transcription factor families in plants. This gene family plays a key role in complex growth processes, including seed germination, flower growth, leaf senescence, response to biotic, and abiotic stress [6,33,34,35]. Genome-wide analysis based on genome sequence can provide a basis for the comprehensive identification of the AP2/ERF family and an understanding of the function and evolution of AP2/ERF factors [36]. To date, the AP2 subfamily of pecan has been studied [37]. However, genome-wide studies have not been conducted to explore the structure and role of AP2/ERF superfamily genes in the pecan. Therefore, we report the AP2/ERF transcription factors in pecans from the whole genome. In addition, we also explored their role in different pecan cultivars under waterlogging stress based on real-time PCR analysis and the function of development and stress (drought, pistillate flowering development, graft union development, and kernel development), using transcriptome data.
In our study, 202 AP2/ERF family genes of pecan were identified. Compared with other plants, the total number of AP2/ERF genes in the pecan is greater than that in many plants, but less than the sunflower (288) [15], Brassica rapa (281) [38], and sugarcane (218) [39]. Previous studies reported that the AP2/ERF number was increased with the plant evolution or whole-genome duplication [40], which may be in close relationship with functional specificity in plants. The numbers of AP2/ERF genes varied significantly in these plants that suggested their expansion during evolution. The wide variation of molecular weight (13906.60–82684.48) and isoelectric point (4.47–10.73) indicated the putative novel variants in CiAP2/ERF genes. Predicted subcellular localization suggested that all AP2/ERF members were located in the nucleus and cytoplasm. This suggested that these genes might function in transcription and post-transcriptional modification.
The number and position of introns give clues on evolutionary relationships of proteins. The pecan AP2/ERF gene structural analysis revealed that a higher number of AP2 subfamily members contained more introns (4–10) compared to ERF, RAV, or Soloist subfamily members. This was consistent with the results observed in other plants [11,15,41,42]. It suggested that the AP2/ERF superfamily members of pecan were functionally diverse, but the functions of members of the same subfamily were similar, which further proved the accuracy of AP2/ERF genes evolution and classification. Introns can be divided into three types based on phases: Phase 0, Phase 1, and Phase 2 [43]. An exon flanked by two introns of the same phase was called a symmetrical exon, and exons that were subjected to alternative splicing were always symmetrical exons [43]. Intron distribution of AP2 domain suggested that ERF, RAV, DREB, and Soloist subfamily members were more conserved than AP2 subfamily members in pecan.
In addition, conserved motifs analysis was used to determine transcriptional activity, protein–protein interactions, and DNA-binding activity of TFs [44]. The conserved motifs analysis of pecan AP2/ERF TFs indicated that these motifs might be related to specific functions. Gene-duplication events are one of the main evolutionary mechanisms for the generation of new genes and an important way to cope with the environmental changes during growth and development stages [45]. Our study demonstrated the occurrence of tandem and segmental duplications in pecan for the expansion of all AP2/ERF superfamily members during evolution. The Ka and Ks measure the course of divergence, and the ratio of Ka/Ks was a checkpoint for positive selection pressure after duplication. The Ka/Ks > 1 means accelerated evolution with positive selection, Ka/Ks = 1 stands for neutral selection, and Ka/Ks < 1 represents purifying selection [46]. The mean value of Ka/Ks (0.239) of pecan suggested a purifying selection of the AP2/ERF genes that underwent great selective constraint and substitutions elimination by natural selection.
Gene functions can be preliminarily predicted by analysis of the gene-expression profiles [47]. DREB2A, DREB2B [48], and CBF4 (DREB1D) [49] were induced by drought stress in Arabidopsis. Similarly, drought transcriptome data suggested that AP2/ERF genes were upregulated at 3 and 15 days in pecan. Three AP2/ERF family genes, PhAp2A, PhAp2B, and PhAp2C, were isolated from petunia and were verified to perform function in flower development [50]. In pecan, transcriptomic data of flower development also suggested that the AP2/ERF genes may play a key role in early and middle stages. Previous research showed that ERF subfamily members, specifically the ERF-VII members, were involved in the submergence stress response. For example, water-tolerance gene, Sub1A, restricts rice elongation at the seedling stage during flash floods [31]. In addition, there were five genes in the ERF-VII of Arabidopsis, which were RAP2.2 (At3g14230), RAP2.12(At1g53910), RAP2.3 (At3g16770), HER1 (At1g72360), and HER2 (At2g47520). RAP2.2, RAP2.12, HRE1, and HRE2 were proved to play vital roles in hypoxia response [30,51]. Similarly, five ERF-VII members in pecan were identified. In Arabidopsis, the stability of most ERF-VII proteins was regulated by the N-end rule pathway [52]. Conserved motif MCGGAI/VI in N-terminal of ERF-VII genes in pecan were also found. This suggested that members of ERF-VII in pecan and Arabidopsis may perform similar functions. Then the quantitative analysis for ERF-VII genes in pecan by qRT-PCR suggested that their expression levels also changed significantly with the time of flooding in pecan.
Overall, the abovementioned findings provided insights into the potential functional roles of pecan AP2/ERF genes. The comprehensive analyses were beneficial to screening candidate AP2/ERF genes for further functional characterization, as well as for the genetic improvement in the agronomic characters of pecan.
4. Materials and Methods
4.1. Identification of the CiAP2/ERF TFs in Pecan Genome
The whole-genome sequences and deduced protein sequences of pecan (Carya illinoinensis) were downloaded from the NCBI (
4.2. CiAP2/ERF Gene Structure, Chromosome Localization, and Conserved Motif Analysis
The conserved motifs of AP2/ERF genes were searched by using MEME (
4.3. Phylogenetic Analysis and Duplication Events Analysis of CiAP2/ERF Genes
The evolutionary relationships among AP2/ERF protein sequences of pecan (202 AP2/ERFs) and Arabidopsis thaliana were established by constructing a phylogenetic tree via the maximum likelihood method, using IQ-TREE version 1.6.12 software. Best-fit model according to BIC (VT + F + R10) was used to construct the phylogenetic tree. Finally, iTOL (
The evolutionary pattern and the divergence of homologous genes were estimated by computing the synonymous (Ks) and nonsynonymous (Ka) substitution. The TBtools [53] software was used to calculate Ka and Ks values of pecan.
4.4. Cis-Acting Elements Analysis of CiAP2/ERFs
The cis-acting regulatory elements in the 2000 bp genomic sequence upstream of the coding CiAP2/ERF gene sequences were investigated by using the online PlantCARE databases (
4.5. Transcriptome Data Sources and Expression Analysis of the AP2/ERF Genes
The transcriptome data of pecan (drought, pistillate flowering development, graft union development, and kernel development) were downloaded from the SRA (Sequence Read Archive) database of the NCBI website. Kernel development data were downloaded from accession number PRJNA504494 that were previously generated by our group [25]. Transcriptome data of graft union development were retrieved from SRA accession number PRJNA411951 [54]. Pistillate flowering development transcriptome data were considered, and the raw data were retrieved under the accession number PRJNA533506 [55]. Moreover, drought transcriptome data were downloaded from the SRA database under accession number PRJNA743302 [56]. All the transcriptome sequencing data obtained from Illumina sequencing were quality checked by FastQC v.0.11.9 (
4.6. Pecan Plant Materials and Stress Exposure
The pecan (Carya illinoinensis) plantlets were raised at the test site at the Research Institute of Subtropical Forestry, Hangzhou, China. Healthy pecan grafting plantlets of the same age (13 months old) were chosen to undergo waterlogging. Before stress exposure, all plants were shifted to a growth chamber at 27 °C for one week. Waterlogging treatment was conducted after 0 (control), 4, 12, 24, and 48 h, using different plantlets (3 for each point in time). At different time intervals (0, 4, 12, 24, and 48 h) of the flood-water-stress exposures, the mature leaves were collected and immediately frozen in liquid nitrogen for further qRT-PCR analysis.
4.7. Quantitative Real-Time PCR (qRT-PCR) Analysis of CiAP2/ERFs
Five genes were selected for expression analysis of four pecan varieties (Mahan, C10, C20, and J23). The primers of these genes were designed by PrimerExpress software v3.0.1 (Applied Biosystems, Foster City, CA, USA). Electrophoresis with 1% agarose and melting-curve analysis was used to calculate specificity during quantitative real-time PCR analysis.
For RNA extraction, samples (2 g) were crushed into a fine powder with liquid nitrogen and mortar–pestle. Then total RNA was isolated from the leaf samples of the above waterlogging treatment by using a Tiangen RNAprep plant kit (Tiangen, Beijing, China), and gel electrophoresis was used to check RNA integrity. First-strand complementary DNA (cDNA) was prepared with PrimeScript™ RT Master Mix (TaKaRa) as per the manufacturer’s instructions. A 20 μL final reaction was prepared in a 96-well plate containing 10 μL of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X), 0.4 μL of ROX Reference Dye, 2 μL cDNA, 6.8 µL ddH2O, and 0.4 μL of each primer. The quantitative RT-PCR expression was carried out with a QuantStudio 7 Flex Real-time PCR system (Applied Biosystems) with the following steps: (1) denaturation stage, 95 °C/30 s; and (2) PCR stage, 95 °C/3 s, 60 °C/30 s for 40 cycles. All the amplifications were performed with three repeats. The expression analysis was calculated by using the comparative CT method (2−ΔΔCt) [59]. The list of qPCR primers used in this study is provided in Supplementary Table S3. One-way analysis of variance (ANOVA) was used to determine the statistical significance at the p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001 levels.
5. Conclusions
A total of 202 AP2/ERF genes were identified and analyzed through conserved domain, motif distribution, chromosomal distribution, genes duplication, and cis-acting elements. These results help to classify this gene family and provide insight into AP2/ERF genes’ evolution. Furthermore, the role of AP2/ERF genes in various development and abiotic stress conditions, using transcriptome data and quantitative experiments, was analyzed. The results suggest that members of ERF-VII in pecan may perform similar functions as members of Arabidopsis. In short, these results provide a valuable resource for better understanding the biological roles of AP2/ERF genes in the pecan.
B.Y., and X.Y. designed the study; B.Y. prepared samples and generated the experiments; B.Y. collected data and wrote the manuscript. Y.Z. and C.Z. provided suggestions and revised the paper. All authors read the final manuscript and approved submission. All authors have read and agreed to the published version of the manuscript.
This project was supported by the National Key Research and Development Program Project “characteristic economic forest ecological economic varieties screening and supporting cultivation technology”, grant number 2019YFD1001600; and Jiangxi Forestry Bureau forestry Science and technology innovation special project “Introduction test and Screening of pecan clones” innovation special project, grant number (2020) 04.
Not applicable.
Not applicable.
Not applicable.
The authors declare no conflict of interest.
| MeJA | methyl jasmonate |
| Ka | nonsynonymous substitution rates |
| Ks | synonymous substitution rates |
| SMART | simple modular architecture research tool |
| PFAM | database of protein families |
| CDD | conserved domain database |
| MEME | multiple em for motif elicitation |
| qRT-PCR | quantitative real-time polymerase chain reaction |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 1. An unrooted phylogenetic tree constructed by the maximum likelihood method, using AP2/ERF proteins of pecan and Arabidopsis. All of the families from pecan and Arabidopsis are grouped and represented in various colors.
Enlarge this image.Figure 2. Gene structure, conserved domain, and motifs of AP2/ERF genes according to the phylogenetic relationship. (A) The phylogenetic tree was conducted based on the full-length sequences of pecan 202 AP2/ERF proteins, using IQ-TREE software. (B) The motif composition of pecan AP2/ERF proteins. The motifs, numbers 1–10, are displayed in different colored boxes. (C) The AP2 domains are highlighted by green boxes and B3 domain by yellow boxes. (D) Exon–intron structure of pecan AP2/ERF genes. Blue boxes indicate untranslated 5′- and 3′-regions; pink boxes indicate exons and black lines indicate introns.
Enlarge this image.Figure 3. Intron distribution within the AP2 domains of the CiAP2/ERF genes. Scheme of the intron distribution patterns (color-coded and designated A–K) within the AP2 domains of the CiAP2/ERF proteins. White triangles and numbers within them indicate the locations of introns and splicing phases (0, Phase 0; 1, Phase 1; and 2, Phase 2), respectively. The percentage of CiAP2/ERF proteins in each pattern is shown on the right.
Enlarge this image.Figure 4. Chromosomal mapping of 202 pecan AP2/ERFs. The length of the pecan chromosomes is indicated on the vertical grayscale. The chromosome numbers (1–16) are indicated on the bottom of each chromosome. Red lines indicate tandem duplication.
Enlarge this image.Figure 5. Schematic representations of the inter-chromosomal relationships of pecan AP2/ERF genes. The red lines indicate duplicated AP2/ERF gene pairs in pecan. The chromosome number is indicated in the middle of each chromosome.
Enlarge this image.Figure 6. Cis-elements of AP2/ERF genes in pecan: (A) AP2 subfamily, (B) DREB subfamily, (C) ERF subfamily, and (D) RAV–Soloist subfamily. The numbers of different cis-elements in the CiAP2/ERF genes are indicated in different colors in the grid.
Enlarge this image.Figure 7. Expression profiling of CiAP2/ERF genes: (A) drought, (B) pistillate flowering development, (C) graft union development, and (D) kernel development. Gene expression was measured by quantified transcription levels (fragments per kilobase of exon model per million mapped reads, FPKM) derived from RNA-Seq analysis. Green indicates AP2 subfamily, brown indicates ERF subfamily, blue indicates DREB subfamily, violet indicates RAV subfamily, and pink indicates Soloist subfamily.
Enlarge this image.Figure 8. Group VII ethylene responsive factors (ERFs) in pecan and Arabidopsis. (A) Alignment of the protein sequences of the pecan and Arabidopsis group VII ERFs. Amino acidic sequences were aligned by using ClustalX2 software. (B) Phylogenetic tree illustrating the relatedness of group VII ERFs among pecan and Arabidopsis. An unrooted neighbor-joining tree was created by using MEGA11.
Enlarge this image.Figure 9. Real-time PCR analysis of 5 AP2/ERF genes in different pecan cultivars under waterlogging stress (0, 4, 12, 24, and 48 h) conditions. X-axis represents the hours of stress exposure. Y-axis represents the relative expression of a transcription factor. Data represent the mean ± SE of three replicates. Asterisks represent significant difference at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001(***), and p ≤ 0.0001 (****).
Summary of the AP2/ERF superfamily in pecan genome.
| Classification | Group | No. |
|---|---|---|
| AP2 family | Double AP2/ERF domain | 23 |
| Single AP2/ERF domain | 3 | |
| DREB family | I | 10 |
| II | 9 | |
| III | 32 | |
| IV | 10 | |
| ERF family | V | 19 |
| VI | 10 | |
| VI-L | 3 | |
| VII | 5 | |
| VIII | 21 | |
| IX | 39 | |
| X | 7 | |
| Xb-L | 3 | |
| RAV family | 6 | |
| Soloist family | 2 | |
| Total | 202 |
Supplementary Materials
The following are available online at
References
1. Singh, K.B.; Foley, R.C.; Oñate-Sánchez, L. Transcription factors in plant defense and stress responses. Curr. Opin. Plant Biol.; 2002; 5, pp. 430-436. [DOI: https://dx.doi.org/10.1016/S1369-5266(02)00289-3]
2. Li, M.; Xu, Z.; Huang, Y.; Tian, C.; Wang, F.; Xiong, A. Genome-wide analysis of AP2/ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress. Mol. Genet. Genom.; 2015; 290, pp. 2049-2061. [DOI: https://dx.doi.org/10.1007/s00438-015-1061-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25971861]
3. Licausi, F.; Ohme-Takagi, M.; Perata, P. APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: Mediators of stress responses and developmental programs. New Phytol.; 2013; 199, pp. 639-649. [DOI: https://dx.doi.org/10.1111/nph.12291] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24010138]
4. Allen, M.D.; Yamasaki, K.; Ohme-Takagi, M.; Tateno, M.; Suzuki, M. A novel mode of DNA recognition by a β-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. EMBO J.; 1998; 17, pp. 5484-5496. [DOI: https://dx.doi.org/10.1093/emboj/17.18.5484]
5. Okamuro, J.K.; Caster, B.; Villarroel, R.; Van Montagu, M.; Jofuku, K.D. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl. Acad. Sci. USA; 1997; 94, pp. 7076-7081. [DOI: https://dx.doi.org/10.1073/pnas.94.13.7076]
6. Sakuma, Y.; Liu, Q.; Dubouzet, J.G.; Abe, H.; Shinozaki, K.; Yamaguchi-Shinozaki, K. DNA-Binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem. Biophys. Res. Commun.; 2002; 290, pp. 998-1009. [DOI: https://dx.doi.org/10.1006/bbrc.2001.6299]
7. El Ouakfaoui, S.; Schnell, J.; Abdeen, A.; Colville, A.; Labbé, H.; Han, S.; Baum, B.; Laberge, S.; Miki, B. Control of somatic embryogenesis and embryo development by AP2 transcription factors. Plant Mol. Biol.; 2010; 74, pp. 313-326. [DOI: https://dx.doi.org/10.1007/s11103-010-9674-8]
8. Nakano, T.; Suzuki, K.; Fujimura, T.; Shinshi, H. Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice. Plant Physiol.; 2006; 140, pp. 411-432. [DOI: https://dx.doi.org/10.1104/pp.105.073783]
9. Li, C.; Su, R.; Cheng, C.; Sanjaya,; You, S.; Hsieh, T.; Chao, T.; Chan, M. Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway1. Plant Physiol.; 2011; 156, pp. 213-227. [DOI: https://dx.doi.org/10.1104/pp.111.174268]
10. Zhuang, J.; Cai, B.; Peng, R.; Zhu, B.; Jin, X.; Xue, Y.; Gao, F.; Fu, X.; Tian, Y.; Zhao, W. et al. Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem. Bioph. Res. Co.; 2008; 371, pp. 468-474. [DOI: https://dx.doi.org/10.1016/j.bbrc.2008.04.087]
11. Zhou, L.; Yarra, R. Genome-Wide identification and characterization of AP2/ERF transcription factor family genes in oil palm under abiotic stress conditions. Int. J. Mol. Sci.; 2021; 22, 2821. [DOI: https://dx.doi.org/10.3390/ijms22062821] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33802225]
12. Kabir, S.M.T.; Hossain, M.S.; Bashar, K.K.; Honi, U.; Ahmed, B.; Emdad, E.M.; Alam, M.M.; Haque, M.S.; Islam, M.S. Ge-nome-wide identification and expression profiling of AP2/ERF superfamily genes under stress conditions in dark jute (Corchorus olitorius L.). Ind. Crop. Prod.; 2021; 166, 113469. [DOI: https://dx.doi.org/10.1016/j.indcrop.2021.113469]
13. Hu, L.; Liu, S. Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers. Genet. Mol. Biol.; 2011; 34, pp. 624-634. [DOI: https://dx.doi.org/10.1590/S1415-47572011005000054]
14. Zhuang, J.; Chen, J.; Yao, Q.; Xiong, F.; Sun, C.; Zhou, X.; Zhang, J.; Xiong, A. Discovery and expression profile analysis of AP2/ERF family genes from Triticum aestivum. Mol. Biol. Rep.; 2011; 38, pp. 745-753. [DOI: https://dx.doi.org/10.1007/s11033-010-0162-7]
15. Najafi, S.; Sorkheh, K.; Nasernakhaei, F. Characterization of the APETALA2/Ethylene-responsive factor (AP2/ERF) tran-scription factor family in sunflower. Sci. Rep.; 2018; 8, pp. 1-16. [DOI: https://dx.doi.org/10.1038/s41598-018-29526-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30068961]
16. Nole-Wilson, S.; Krizek, B.A. DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA. Nucleic Acids Res.; 2000; 28, pp. 4076-4082. [DOI: https://dx.doi.org/10.1093/nar/28.21.4076] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11058102]
17. Taketa, S.; Amano, S.; Tsujino, Y.; Sato, T.; Saisho, D.; Kakeda, K.; Nomura, M.; Suzuki, T.; Matsumoto, T.; Sato, K. et al. Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway. Proc. Natl. Acad. Sci. USA; 2008; 105, pp. 4062-4067. [DOI: https://dx.doi.org/10.1073/pnas.0711034105]
18. Jofuku, K.D.; Omidyar, P.K.; Gee, Z.; Okamuro, J.K. Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proc. Natl. Acad. Sci. USA; 2005; 102, pp. 3117-3122. [DOI: https://dx.doi.org/10.1073/pnas.0409893102]
19. Aukerman, M.J.; Sakai, H. Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. The Plant Cell; 2003; 15, pp. 2730-2741. [DOI: https://dx.doi.org/10.1105/tpc.016238]
20. Hu, Y.X.; Wang, Y.H.; Liu, X.F.; Li, J.Y. Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development. Cell Res.; 2004; 14, pp. 8-15. [DOI: https://dx.doi.org/10.1038/sj.cr.7290197]
21. Sohn, K.H.; Lee, S.C.; Jung, H.W.; Hong, J.K.; Hwang, B.K. Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance. Plant Mol. Biol.; 2006; 61, pp. 897-915. [DOI: https://dx.doi.org/10.1007/s11103-006-0057-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16927203]
22. Mantiri, F.; Kurdyukov, S.; Lohar, D.P.; Sharopova, N.; Saeed, N.A.; Wang, X.-D.; VandenBosch, K.A.; Rose, R.J. The Transcription Factor MtSERF1 of the ERF Subfamily Identified by Transcriptional Profiling Is Required for Somatic Embryogenesis Induced by Auxin Plus Cytokinin in Medicago truncatula. Plant Physiol.; 2008; 146, pp. 1622-1636. [DOI: https://dx.doi.org/10.1104/pp.107.110379] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18235037]
23. Bouaziz, D.; Pirrello, J.; Charfeddine, M.; Hammami, A.; Jbir, R.; Dhieb, A.; Bouzayen, M.; Gargouri-Bouzid, R. Overexpression of StDREB1 Transcription Factor Increases Tolerance to Salt in Transgenic Potato Plants. Mol. Biotechnol.; 2012; 54, pp. 803-817. [DOI: https://dx.doi.org/10.1007/s12033-012-9628-2] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23250722]
24. Fujimoto, S.Y.; Ohta, M.; Usui, A.; Shinshi, H.; Ohme-Takagi, M. Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box–mediated gene expression. Plant Cell; 2000; 12, pp. 393-404. [DOI: https://dx.doi.org/10.1105/tpc.12.3.393] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10715325]
25. Zhang, C.; Yao, X.; Ren, H.; Chang, J.; Wang, K. RNA-seq reveals flavonoid biosynthesis-related genes in pecan (Carya illinoinensis) kernels. J. Agric. Food Chem.; 2019; 67, pp. 148-158. [DOI: https://dx.doi.org/10.1021/acs.jafc.8b05239]
26. Islam, S.; Saito, J.A.; Emdad, E.M.; Ahmed, B.; Islam, M.M.; Halim, A.; Hossen, Q.M.M.; Hossain, Z.; Ahmed, R.; Hossain, S. et al. Comparative genomics of two jute species and insight into fibre biogenesis. Nat. Plants; 2017; 3, 16223. [DOI: https://dx.doi.org/10.1038/nplants.2016.223]
27. Shan, Q.; Wang, Y.; Li, J.; Gao, C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat. Protoc.; 2014; 9, pp. 2395-2410. [DOI: https://dx.doi.org/10.1038/nprot.2014.157]
28. Joung, J.K.; Sander, J.D. TALENs: A widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol.; 2013; 14, pp. 49-55. [DOI: https://dx.doi.org/10.1038/nrm3486]
29. Gasch, P.; Fundinger, M.; Müller, J.T.; Lee, T.; Bailey-Serres, J.; Mustroph, A. Redundant ERF-VII transcription factors bind to an evolutionarily conservedcis-motif to regulate hypoxia-responsive gene expression in Arabidopsis. Plant Cell; 2016; 28, pp. 160-180. [DOI: https://dx.doi.org/10.1105/tpc.15.00866]
30. Licausi, F.; Van Dongen, J.T.; Giuntoli, B.; Novi, G.; Santaniello, A.; Geigenberger, P.; Perata, P. HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana. Plant J.; 2010; 62, pp. 302-315. [DOI: https://dx.doi.org/10.1111/j.1365-313X.2010.04149.x]
31. Fukao, T.; Xu, K.; Ronald, P.C.; Bailey-Serres, J. A Variable Cluster of Ethylene Response Factor–Like Genes Regulates Metabolic and Developmental Acclimation Responses to Submergence in Rice. Plant Cell; 2006; 18, pp. 2021-2034. [DOI: https://dx.doi.org/10.1105/tpc.106.043000] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16816135]
32. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol.; 2004; 4, 10. [DOI: https://dx.doi.org/10.1186/1471-2229-4-10] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15171794]
33. Li, A.; Yu, X.; Cao, B.B.; Peng, L.X.; Gao, Y.; Feng, T.; Li, H.; Ren, Z.Y. LkAP2L2, an AP2/ERF transcription factor gene of Larix kaempferi, with pleiotropic roles in plant branch and seed development. Russ. J. Genet.; 2017; 53, pp. 1335-1342. [DOI: https://dx.doi.org/10.1134/S1022795417120079]
34. Woo, H.R.; Kim, J.H.; Kim, J.; Kim, J.; Lee, U.; Song, I.; Kim, J.; Lee, H.; Nam, H.G.; Lim, P.O. The RAV1 transcription factor positively regulates leaf senescence in Arabidopsis. J. Exp. Bot.; 2010; 61, pp. 3947-3957. [DOI: https://dx.doi.org/10.1093/jxb/erq206]
35. Yao, Y.; He, R.J.; Xie, Q.L.; Zhao, X.H.; Deng, X.M.; He, J.B.; Song, L.; He, J.; Marchant, A.; Chen, X.Y. et al. ETHYLENE RESPONSE FACTOR 74 (ERF74) plays an essential role in controlling a respiratory burst oxidase homolog D (RbohD)-dependent mechanism in response to different stresses in Arabidopsis. New Phytol.; 2017; 213, pp. 1667-1681. [DOI: https://dx.doi.org/10.1111/nph.14278]
36. Feng, K.; Hou, X.; Xing, G.; Liu, J.; Duan, A.; Xu, Z.; Li, M.; Zhuang, J.; Xiong, A. Advances in AP2/ERF super-family transcription factors in plant. Crit. Rev. Biotechnol.; 2020; 40, pp. 750-776. [DOI: https://dx.doi.org/10.1080/07388551.2020.1768509]
37. Yang, Z.; Jin, H.; Chen, J.; Li, C.; Wang, J.; Luo, J.; Wang, Z. Identification and Analysis of the AP2 Subfamily Transcription Factors in the Pecan (Carya illinoinensis). Int. J. Mol. Sci.; 2021; 22, 13568. [DOI: https://dx.doi.org/10.3390/ijms222413568]
38. Liu, S.; Wang, X.; Wang, H.; Xin, H.; Yang, X.; Yan, J.; Li, J.; Tran, L.P.; Shinozaki, K.; Yamaguchi-Shinozaki, K. et al. Ge-nome-wide analysis of ZmDREB genes and their association with natural variation in drought tolerance at seedling stage of Zea mays L. PLoS Genet; 2013; 9, e1003790. [DOI: https://dx.doi.org/10.1371/journal.pgen.1003790]
39. Li, P.; Chai, Z.; Lin, P.; Huang, C.; Huang, G.; Xu, L.; Deng, Z.; Zhang, M.; Zhang, Y.; Zhao, X. Genome-wide identification and expression analysis of AP2/ERF transcription factors in sugarcane (Saccharum spontaneum L.). BMC Genom.; 2020; 21, pp. 1-17. [DOI: https://dx.doi.org/10.1186/s12864-020-07076-x]
40. Li, M.; Liu, J.; Hao, J.; Feng, K.; Duan, A.; Yang, Q.; Xu, Z.; Xiong, A. Genomic identification of AP2/ERF transcription factors and functional characterization of two cold resistance-related AP2/ERF genes in celery (Apium graveolens L.). Plant; 2019; 250, pp. 1265-1280. [DOI: https://dx.doi.org/10.1007/s00425-019-03222-2]
41. Xu, W.; Li, F.; Ling, L.; Liu, A. Genome-wide survey and expression profiles of the AP2/ERF family in castor bean (Ricinus communis L.). BMC Genom.; 2013; 14, 785. [DOI: https://dx.doi.org/10.1186/1471-2164-14-785] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24225250]
42. Cui, L.; Feng, K.; Wang, M.; Wang, M.; Deng, P.; Song, W.; Nie, X. Genome-wide identification, phylogeny and expression analysis of AP2/ERF transcription factors family in Brachypodium distachyon. BMC Genom.; 2016; 17, pp. 1-19. [DOI: https://dx.doi.org/10.1186/s12864-016-2968-8] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27527343]
43. Choudhuri, S. Bioinformatics for Beginners: Genes, Genomes, Molecular Evolution, Databases and Analytical Tools; Academic Press: Cambridge, MA, USA, 2014; pp. 1-25.
44. Boeva, V. Analysis of Genomic Sequence Motifs for Deciphering Transcription Factor Binding and Transcriptional Regulation in Eukaryotic Cells. Front. Genet.; 2016; 7, 24. [DOI: https://dx.doi.org/10.3389/fgene.2016.00024] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26941778]
45. Ren, R.; Wang, H.; Guo, C.; Zhang, N.; Zeng, L.; Chen, Y.; Ma, H.; Qi, J. Widespread Whole Genome Duplications Contribute to Genome Complexity and Species Diversity in Angiosperms. Mol. Plant; 2018; 11, pp. 414-428. [DOI: https://dx.doi.org/10.1016/j.molp.2018.01.002] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29317285]
46. Lynch, M.; Conery, J.S. The Evolutionary Fate and Consequences of Duplicate Genes. Science; 2000; 290, pp. 1151-1155. [DOI: https://dx.doi.org/10.1126/science.290.5494.1151]
47. Liu, L.; White, M.J.; Macrae, T.H. Transcription factors and their genes in higher plants. Functional domains, evolution and regulation. JBIC J. Biol. Inorg. Chem.; 1999; 262, pp. 247-257. [DOI: https://dx.doi.org/10.1046/j.1432-1327.1999.00349.x]
48. Nakashima, K.; Shinwari, Z.K.; Sakuma, Y.; Seki, M.; Miura, S.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression. Plant Mol. Biol.; 2000; 42, pp. 657-665. [DOI: https://dx.doi.org/10.1023/A:1006321900483]
49. Haake, V.; Cook, D.; Riechmann, J.L.; Pineda, O.; Thomashow, M.F.; Zhang, J.Z. Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis. Plant Physiol.; 2002; 130, pp. 639-648. [DOI: https://dx.doi.org/10.1104/pp.006478]
50. Maes, T.; Van de Steene, N.; Zethof, J.; Karimi, M.; D’Hauw, M.; Mares, G.; Van Montagu, M.; Gerats, T. Petunia AP2-like genes and their role in flower and seed development. Plant Cell; 2001; 13, pp. 229-244. [DOI: https://dx.doi.org/10.1105/tpc.13.2.229]
51. Licausi, F.; Kosmacz, M.; Weits, D.A.; Giuntoli, B.; Giorgi, F.M.; Voesenek, L.A.C.J.; Perata, P.; van Dongen, J.T. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature; 2011; 479, pp. 419-422. [DOI: https://dx.doi.org/10.1038/nature10536]
52. Varshavsky, A. N-degron and C-degron pathways of protein degradation. Proc. Natl. Acad. Sci. USA; 2019; 116, pp. 358-366. [DOI: https://dx.doi.org/10.1073/pnas.1816596116] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30622213]
53. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for in-teractive analyses of big biological data. Mol. Plant; 2020; 13, pp. 1194-1202. [DOI: https://dx.doi.org/10.1016/j.molp.2020.06.009] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32585190]
54. Mo, Z.; Feng, G.; Su, W.; Liu, Z.; Peng, F. Transcriptomic Analysis Provides Insights into Grafting Union Development in Pecan (Carya illinoinensis). Genes; 2018; 9, 71. [DOI: https://dx.doi.org/10.3390/genes9020071] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29401757]
55. Wang, M.; Xi, D.; Chen, Y.; Zhu, C.; Zhao, Y.; Geng, G. Morphological characterization and transcriptome analysis of pistillate flowering in pecan (Carya illinoinensis). Sci. Hortic.; 2019; 257, 108674. [DOI: https://dx.doi.org/10.1016/j.scienta.2019.108674]
56. Zhu, K.; Fan, P.; Liu, H.; Zhao, J.; Tan, P.; Mo, Z.; Peng, F. Pecan kinome: Classification and expression analysis of all protein kinases in Carya illinoinensis. For. Res.; 2021; 1, pp. 1-12. [DOI: https://dx.doi.org/10.48130/FR-2021-0014]
57. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics; 2014; 30, pp. 2114-2120. [DOI: https://dx.doi.org/10.1093/bioinformatics/btu170]
58. Kim, D.; Paggi, J.M.; Park, C.; Bennett, C.; Salzberg, S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol.; 2019; 37, pp. 907-915. [DOI: https://dx.doi.org/10.1038/s41587-019-0201-4]
59. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods; 2001; 25, pp. 402-408. [DOI: https://dx.doi.org/10.1006/meth.2001.1262]
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Details
; Yao, Xiaohua 1 ; Zeng, Yanru 2 ; Zhang, Chengcai 1
1 The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China;
2 State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China