Genome-wide identification of peanut IGT family genes and their potential roles in the development of plant architecture

IGT family genes play essential roles in shaping plant architecture. However, limited amount of information is available about IGT family genes in peanuts (Arachis hypogaea). In the current study, 13 AhIGT genes were identified and classified into three groups based on their phylogenetic relationship. Gene structure, conserved domain analyses indicated all AhIGTs were observed to share a similar exon–intron distribution pattern. AhIGTs within the same subfamily maintained a consistent motif composition. Chromosomal localization and synteny analyses showed that AhIGTs were unevenly localized on 9 chromosomes and that segmental duplication and purifying selection may have played important roles in the evolution of AhIGT genes. The analysis of conserved motifs, GO annotation, and transcript profile suggested that AhLAZY1-3 may play roles in gravity sensing and shaping peanut plant architecture. Transcript profile analysis suggested that AhTAC1 could potentially be involved gynophore (‘peg’) penetration into the soil. The cis-element analysis revealed that the light-responsive elements accounted for most of all cis-acting elements. Furthermore, qRT-PCR analysis showed that the expression of several AhIGT genes, like AhTAC1-2/4, was light-dependent, indicating that these genes may regulate plant architecture in response to light signals. This study may facilitate functional studies of the IGT genes in peanut.

was light dependent 24 .The expression of AtIGT genes was also found to respond differentially to alterations in light signaling, and the loss of several LAZY and DRO genes in Arabidopsis resulted in a lack of branch angle reaction to light stimuli 25 .These studies have demonstrated that IGT family genes play a role in regulating plant architecture in response to both light and gravity stimuli.
The peanut (Arachis hypogaea L.) is one of the world's most important economic oilseed crops.The peanut seeds are rich in vegetable oils, proteins, vitamins and minerals.Peanuts produce aerial flowers but subterranean fruits (pods).Embryo development remains arrested until the fertilized ovary is buried in the soil with the help of a specialized organ called the peg or gynophore.Positive gravitropism is the most typical features of pegs as it enables them to grow downwards and penetrate in the soil 26 .Then the height of flowers directly influences pegs to penetrate the soil and affect peanut yield.As a result, optimizing plant architecture improves planting density and increases pod number per plant in peanut.Although the critical importance of IGT genes involved in regulating gravitropism and shaping the structure of plants [27][28][29] , little is known about this gene family in A. hypogaea.This study is the first genome-wide study to identify the IGT genes in the genome of peanuts.To gain a better understanding of IGT genes in peanuts, their phylogenetic relationships, chromosome locations, synteny study, gene structures, conserved motifs, and cis-element were examined.Additionally, their expression profiles in numerous tissues and their response to light and dark signals were evaluated.The results may facilitate functional studies of the IGT genes in peanut.

Identification and characterization of IGT genes in peanut
The protein and genomic sequences of A. hypogaea var.Shitoqi were downloaded from the Peanut Genome Resource (PGR) database (http:// peanu tgr.fafu.edu.cn/ Downl oad.php) 30 .The sequences of seven AtIGTs (protein sequences are listed in supplementary Table S1) were downloaded from the Arabidopsis Information Resource (TAIR) database (https:// www.arabi dopsis.org/).These sequences were then subjected to the blastp program to search for the peanut protein sequence database with an E-value ≤ 1.0e−6.Further, 7 AtIGT protein sequences were subjected to MAFFT version 7.0 software 31 for multiple sequence alignment.The alignment results were used to build HMM profile using hmmbuild program 32 .Then, the HMM profile was used to screen protein sequences of peanuts on HMMER software 32 with an E-value ≤ 1.0e−6.The amino acid sequences of candidate AhIGTs from blastp and HMMER search were aligned with those of the corresponding AtIGTs using DNAMAN version 5.0 software (Lynnon Biosoft, San Ramon, CA, USA) to confirm whether they were the orthologs of the target AtIGTs or not.The protein sequences of Arachis duranensis and Arachis ipaensis were obtained from the PeanutBase database (https:// data.legum einfo.org/ Arach is/) 33 , and the IGT proteins in these two species were identified using the blastp method.The sequences of the confirmed proteins were submitted to the ExPASy tool 34 to predict protein molecular weight (MW) and isoelectric point (PI).Subcellular localization of the proteins was predicted by WoLF PSORT 35 .
The structure of AhIGT genes was analyzed by the Gene Structure Display Server 2.0 36 , and conserve protein motifs were predicted by the MEME software 37 with a maximum number of motifs (10) and motif length ranging from 6 to 20 amino acids.The conserved motifs were visualized using the TBtools software 38 .The final figures prepared for gene structures and conserved motifs were refined using Adobe Illustrator software (Adobe Systems Incorporated, San Jose, CA, USA).

Chromosome location, phylogenetic tree, and synteny analysis of AhIGT genes
The chromosomal location of AhIGTs was obtained from the PGR database 30 .The distribution of AhIGTs was visualized using MapChart software 39 , and the final figure was refined with Adobe Illustrator software (Adobe Systems Incorporated, San Jose, CA, USA).
The full-length amino acid sequences of IGT proteins from A. thaliana, A. hypogaea, A. duranensis and A. ipaensis were submitted to ClustalW software 40 to perform sequence alignment.Sequence alignment results were used as the basis to construct a phylogenetic tree by maximum likelihood method using MEGA 7.0 software 41 .The phylogenetic tree was visualized by the Evolview website 42 .
The synteny analysis of AhIGTs in A. hypogaea was performed with McScanX software and the results were plotted by Circos software 43 .The collinear relationships among IGT proteins in A. hypogaea, A. duranensis and A. ipaensis were determined using the Python version of McScanX 44 .The Ka/Ks values of IGT proteins were calculated using the KaKs calculator 2.0 program 45 .

Analysis of cis-regulatory element distribution in AhIGT gene promoters and GO annotation
The 1500-bp sequences occurring upstream of the start codon of AhIGTs were extracted from the PGR database 30 .The putative cis-elements of each promoter were analyzed using the PlantCARE database 46 , and the results were visualized using the Gene Structure Display Server 2.0 online tools 36 .The full-length amino acid sequences of AhIGTs were submited to the eggNOG website 47 .GO enrichment analysis was performed using the TBtools software 38 .
A peanut variety PX05 provided by the Crops Research Institute, Anhui Academy of Agricultural Science, Hefei, China, was used as the plant material and exposed to continuous light and dark conditions.The use of plant parts and seeds in the present study complies with relevant institutional, national, and international guidelines and legislation.The seedlings were grown in a growth chamber at 25-30 ℃ under 14:10 h light/dark conditions.The 5-or 6-week-old seedlings were transferred to chambers in continuous darkness for 48 h, and thereafter, leaves were sampled at 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 h after the treatment.The plants were then transferred to chambers under continuous light, and leaves were sampled at 0.5, 1, 2, 4, 8, 12, 24 and 48 h after the treatment.We combined equal amounts of leaves from 5 plants as a biological replicate, and three biological replicates were considered in our experiment.Total RNA was extracted using the SDS-LiCl method which was described by Vennapusa 49 .We used the PrimeScript™ RT reagent Kit (TaKaRa, Japan) to synthesize cDNA according to developer instructions.qRT-PCR assays were performed using SYBR Premix Ex Taq™ (TaKaRa, Japan) with three technical replicates on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA).The conditions for the qRT-PCR reaction were set as follows: initial denaturation at 95 ℃ for 5 min, then 40 cycles of denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 1 min.The quantified data were calculated using the 2 −ΔΔCt method.The Actin gene from peanut (A.hypogaea) 50 was used as an internal control and all AhIGT primers are listed in Supplementary Table S2.

Identification and characterization of IGT family genes in peanut
A total of 13 IGT genes unevenly distributed along 9 out of the 20 A. hypogaea chromosomes, were identified in peanut genome, with 7 genes located on AA subgenome and 6 located on BB subgenome.Chromosome A06 contained the maximum number of IGT genes (3); while chromosomes A02 and B02 each contained 2 IGT genes, and chromosomes A03, A09, B03, B04, B06, and B09 each contained 1 IGT gene (Table 1, Fig. 1).Most homologous IGT gene pairs that had strong collinear relationships were observed to be located on the homologous chromosomes between the AA and BB subgenomes.For example, the IGT genes AhTAC1-1 and AhLAZY5-1 present on chromosome A02 were found to have strong collinear relationships with the IGT genes AhTAC1-2 and AhLAZY5-2 present on chromosome B02.The lengths of AhIGT proteins varied from 241 (AhTAC1-2) to 417 (AhLAZY1-2) amino acids, whereas their predicted molecular weight ranged from 27.67 (AhTAC1-2) to 46.50 kDa (AhLAZY1-2), and the isoelectric points ranged from 5.03 (AhLAZY3-1) to 9.73 (AhLAZY5-2) (Table 1).After conducting in silico prediction of subcellular localization, AhTAC1-1 and AhTAC1-3 were shown to be located in the cytoplasm, while the others were located in the nuclear (Table 1).Additional information such as gene position and coding sequence lengths can be found in Table 1.

Phylogenetic relationships among IGT genes
To investigate the evolutionary relationships among IGT genes, a phylogenetic tree was constructed using the sequences of 33 IGT proteins from A. thaliana, A. hypogaea, A. duranensis, and A. ipaensis (protein sequences are shown in Table S1).As shown in Fig. 2, the tree was divided into three groups (LAZY-like, DRO-like, and TAC).The LAZY-like group included LAZY1, LAZY5, and LAZY6, while the DRO-like group consisted of LAZY2,

Gene structures and conserved motifs of IGT genes in peanut
To have a better understanding of the structure of AhIGT genes, the exon-intron arrangements were generated (Fig. 3).The results revealed that most AhIGT genes shared similar structural patterns.For example, all 13 AhIGT genes consisted of two short exons followed by a long exon.In addition, a short exon was observed to appear at the end of AhLAZY1-3 and all members of the AhLAZY2/4, AhLAZY3, AhLAZY5 clades.Expect for LAZY1-1, which had 3 exons, all LAZY-like genes and DRO-like genes contained 5 exons, while all AhTAC1 clade members contained 4 exons.Furthermore, the full-length amino acid sequences of all AhIGTs were examined to identify their conserved motifs (Fig. 3).As shown in Fig. 3, motif 1 contained the family-specific GφL (A/T) IGT sequence, which was identified in all AhIGT proteins except for the proteins of the AhLAZY5 clade.All AhIGTs contained motif 2 at the N terminus.Motif 3, which contained the EAR motif (Figure S1), was found in all members of AhLAZY3,

Duplication and synteny analysis of IGT genes in peanut
Synteny analysis was performed to explore the evolutionary relationships among IGT genes in A. hypogaea (Fig. 4A).A total of 9 segmentally duplicated gene pairs were detected, and no tandem duplication was found.
After calculating the Ka/Ks ratio, all duplicated IGT gene pairs in peanut were shown to have a Ka/Ks ratio less than 1 (Table 2), indicating that purifying selective pressure acted on evolutionary process of the AhIGT genes.Further, the collinearity analysis of genes among A. hypogaea, A. ipaensis and A. duranensis was performed (Fig. 4B).A total of 10 AhIGTs in A. ipaensis and A. duranensis showed the correspondent relationships.Four AhIGTs in the AA subgenome exhibited a one-to-one homologous relationship with their orthologs in A. duranensis, and 6 AhIGTs in the BB subgenome had a one-to-one homology relationship with their orthologs in A. ipaensis.No IGT gene pair was found between the AA subgenome and the A. ipaensis genome or between the BB subgenome and the A. ipaensis genome.The results indicated that gene duplication played a crucial role in the evolution of the IGT gene family in the peanut genome.The Ka/Ks ratio was also determined for A. hypogaea and its two diploid ancestors (Table S3).All duplicated gene pairs had a Ka/Ks ratio < 1, indicating the role of strong purifying selective pressure in the evolutionary process of IGT genes in these species.

Cis-element analysis and functional annotation of IGT genes in peanut
The 1500-bp upstream sequences of AhIGT genes were extracted to perform the cis-element analysis.Apart from the general transcriptional regulators and elements with unknown functions, most cis-elements could group into three categories, which involved light-responsive elements, hormone-responsive elements, and transcription   www.nature.com/scientificreports/factor (TF)-binding elements (Fig. 5).Light-responsive elements included AE-box, Box4, G-box, GA-motif, GATA-motif, GT1-motif, TCT-motif, chs-CMA1a, chs-CMA2a and Sp1.Hormone-responsive elements included CGTCA-motif and TGACG-motif (Methyl jasmonate, MeJA), P-box (gibberellic acid, GA), TCA-element (salicylic acid, SA), ABRE (abscisic acid, ABA) and TGA-element (auxin).TF-binding elements, however, included Myb, MYB, Myc, MYC.Interestingly, the light-responsive elements accounted for the majority of all cis-acting elements, which indicated that AhIGT genes may play roles in the regulation of photoactivity.GO annotation and GO enrichment analysis of AhIGTs were performed to gain an understanding of their functions (Table S4).Only three members of AhLAZY1 were annotated, which were found to be involved in the biological process (BP) and the cellular component (CC).In the biological process, AhLAZY1-3 was annotated in gene ontology (GO) terms of hormone transport and regulation of polar auxin transport, whereas AhLAZY1-1 and AhLAZY1-2 were annotated in gravitropism.In the cellular component, three annotated genes were located in the nucleus, which was consistent with the subcellular location prediction of these genes.

Expression patterns of AhIGT genes in various tissues
To explore the expression patterns of AhIGT genes in different tissues, we downloaded the transcriptome profiles of 13 AhIGT genes across 9 different tissues from the Peanut Genome Resource (PGR) database.As shown in Fig. 6 and Table S5, some AhIGT genes displayed relatively lower expression.For example, AhLAZY3-1, AhLAZY3-2, AhLAZY5-1, AhLAZY5-2, and AhAZY1-1 had FPKM values less than 1 in all 9 tissues.In addition, most AhIGT genes were also observed to have relatively lower FPKM values in cotyledon, root, testa, pericarp, and embryo.High expression of AhIGTs was usually detected in the stem, leaves, and stem tip.For example, AhLAZY1-3 had the highest FPKM values in the stem, while AhLAZY2/4-1 and AhLAZY2/4-2 had the highest FPKM values in the stem tip.These genes also exhibited relatively higher expression in leaves or the stem.The highest expression of AhTAC1s was detected in the gynophore, and their relatively higher expression was observed in the stem, leaves and some other tissues.

Expression patterns of AhIGT genes in continuous darkness and light
The cis-elements analysis showed that the light-responsive elements accounted for the majority of all cis-acting elements in the promoter region of AhIGT genes.Next, we explored the dynamic expression profiles of these genes under continuous dark and light conditions (Fig. 7).A total of 9 genes including AhLAZY1-1, AhLAZY1-3, AhLAZY3-1, four paralogs of AhTAC1 and two paralogs of AhLAZY5 were evaluated, out of which three (AhL-AZY3-1 and two paralogs of AhLAZY5) exhibited no expression in leaves.
In continuous darkness, the expression of AhLAZY1-1 increased after 4 h of the treatment and peaked at 12 h, followed by a decrease thereafter and an increase after 24 h of darkness (Fig. 7A).Similarly, the expression of AhLAZY1-3 increased after 1 h, peaked at 4 h, and then declined, with a final increase after 12 h of dark treatment (Fig. 7B).The expressions of both AhTAC1-1 and AhTAC1-3 were variable (Fig. 7C, D).However, AhTAC1-2 and AhTAC1-4 showed different expression patterns.Although their expression showed a relatively lower level at 1 and 12 h, it started increasing in continuous darkness and peaked at 48 h (Fig. 7E, F).
In continuous light, the expressions of both AhLAZY1-1 and AhLAZY1-3 were variable (Fig. 7A, B).AhTAC1-1 and AhTAC1-3 showed similar expression patterns, i.e., their expression was first downregulated gradually and then upregulated in a stepwise manner in the late stage.In addition, both these two genes demonstrated sharply increased expression at 8 h (Fig. 7C, D).Similar expression patterns with little difference were also observed in AhTAC1-2 and AhTAC1-4, with a sharp decrease after 30 min, followed by a gradual decline after 24 h (Fig. 7E, F).

Discussion
The IGT family genes are known as key players in gravity sensing, which is critical to the optimization of crop architecture 1 .Currently, 4, 2, 27 and 4 members of the IGT gene family have been identified in rice 27 , maize 1 , rapeseeds 28 and apple 29 .In this study, 13 AhIGT genes were identified, unevenly distributed on 9 chromosomes.The 13 AhIGT genes could be classified into three group, consistent with previous studies' results 1,25 .WGD is recognized as a major driving force of plant evolution, resulting in gene duplication and subsequent silencing and removal of duplicate genes 51 .Phylogenetic, chromosomal locations and synteny analysis revealed strong collinear relationships among most IGT genes in A. hypogaea between the AA and BB subgenome.Additionally, most AhIGT genes in the AA and BB subgenomes showed corresponding relationship with those in A. duranensis and A. ipaensis, respectively.These indicated the crucial role of segmental duplications in the expansion of the IGT gene family.Although tetraploid peanut contains the same number of IGT genes in A. duranensis and A. ipaensis combined, the number of IGT homologous genes between their subgenome in A. hypogaea and its two wild progenitors was not equivalent.This suggests that the chromosome doubling and then sequence arrangement would cause the loss of IGT genes during the polyploidization process.
In Arabidopsis, 5 short conserved regions and a unique intro-exon arrangement were presented in IGT family genes 1,52 .These conserved regions and intro-exon arrangement are also identified in IGT family genes in rice 27 , rapeseeds 28 and apple 29 .In this study, motif 1, 2 and 3 corresponded exactly to the conserved regions II, I and V, and a similar intro-exon arrangement was also presented in AhIGT family.The exon-intron arrangement could also explain the evolutionary history of the gene family 53 .It is wide accepted that homologous proteins with similar gene structures and conserved motifs across different species may possess identical or similar functions 1,52,54 .So, it is conjectured that AhIGTs might participated gravity sensing and shaping plant architecture in peanuts.
Using public databases 30 , the expression patterns of AhIGTs across 9 different tissues were obtained.Interestingly, the expression level of AhLAZY1-3 in the stem tip and stem was much greater than that of the other two paralogs AhLAZY1-1 and AhLAZY1-2.GO annotation analysis indicated that AhLAZY1-3, not AhLAZY1-1/2, were annotated in the regulation of auxin polar transport.It is known that IGT genes regulate the gravitropic responses through asymmetrical auxin distribution 1,15,52 .In the meantime, only AhLAZY1-3 contained motif 3 which is a key domain of AtLAZY1 in Arabidopsis 6 .Hence, it is reasonable to conjecture that AhLAZY1-3 might participate in gravity sensing and shaping peanut architecture.However, this prediction needs to be tested by more experimental evidence.A much higher expression level of all AhTAC1 genes was found in the gynophore.Gynophore in peanuts is specialized to develop peg which is essential for burying the fertilized ovary into the soil.Positive gravitropism is the most typical features of pegs 26 .Whether AhTAC1s participate in peg penetration into the soil is another interesting project supported by further experimental evidence in the future.
Cis-element analysis showed that light-responsive elements accounted for most cis-acting elements in the promoter of AhIGTs.Several recent studies have reported that the signals of light and gravity function together in shaping plant architecture [21][22][23] .In Arabidopsis, the expression of TAC1 gradually declined once plants were transferred into the dark and returned to their normal levels 48 h after it was transferred back into the light 24 .The expression of TAC1, which plays an important role in regulating plant architecture in response to light signals, was light dependent 24 .qRT-PCR revealed that the expression of AhTAC1-2/4 was also light dependent but with a different pattern.AhTAC1-2/4 expression increased in the dark and then decreased sharply once transferred back in the light.Hence, it is speculated that AhTAC1-2/4 may have regulated plant architecture in response to light signals.

Conclusions
In this study, a genome-wide analysis of IGT family genes was performed in the genome of cultivated.A total of 13 AhIGT genes, which were found to be unevenly distributed on 9 chromosomes and classified into three groups, were identified.Gene structure, conserved motifs, and cis-element were examined to gain insights into IGT genes in peanuts.Their expression profiles across different tissues, and under different light and dark conditions were also evaluated.Results revealed that AhLAZY1-3 may play roles in shaping peanut architecture, AhTAC1 could potentially be involved in the process of peg penetration into the soil, and several AhIGT genes, like AhTAC1-2/4 may regulate plant architecture in response to gravity signals.The results provide a theoretical basis for further characterization of the biological functions of the AhIGT genes in shaping plant architecture.

Figure 1 .
Figure 1.The physical map of AhIGT genes located on chromosomes; The diagram was drawn by the Mapchart software, and the physical location of AhIGTs was obtained from the Peanut Genome Resource (PGR) database (http:// peanu tgr.fafu.edu.cn/ Downl oad.php).

Figure 2 .Figure 3 .
Figure 2. A phylogenetic tree of IGT family genes in A. thaliana, A. hypogaea, A. duranensis, and A. ipaensis constructed using the maximum likelihood method; Overall, 13 IGTs from A. hypogaea (red stars), 7 IGTs from A. thaliana (light blue triangles), 7 IGTs from A. ipaensis (orange boxes), and 6 IGTs from A. duranensis (light green boxes) were clustered into three major classes, denoted by different colors.The bootstrap is displayed in orange circles.

Figure 4 .
Figure 4. Duplications and synteny analysis of IGT genes in peanut; (a) Collinearity analysis of AhIGTs in A. hypogaea; Grey lines in the background represent all collinear blocks among different chromosomes, while red lines indicate segmentally duplicated gene pairs.(b) Collinearity analysis of AhIGTs in A. hypogaea, A. ipaensis and A. duranensis; All collinear blocks among A. hypogaea, A. ipaensis and A. duranensis are represented by grey lines in the background and syntenic IGT gene pairs are represented by red lines.

Figure 5 .
Figure 5. Cis-element analysis of IGT genes in A. hypogaea.Different color boxes represent different ciselements.

Figure 6 .
Figure 6.Expression profiles of AhIGT genes in various tissues; The transcriptome data were obtained from the PGR database.The heat map was created using the log2 based FPKM expression values.

Figure 7 .
Figure 7. Expression patterns of AhIGTs under continuous darkness and light; Dark and light conditions represented by black and white columns, respectively.Error bars represent the standard deviation (SD).

Table 1 .
Information on the identified IGT genes in A. hypogaea.CDS coding sequence, MW molecular weight, PI isoelectric point.

Table 2 .
The Ka and Ks values of duplicated AhIGT gene pairs in peanut.