Identification of Peanut Aux/IAA Genes and Functional Prediction during Seed Development and Maturation

Auxin-responsive genes AUX/IAA are important during plant growth and development, but there are few relevant reports in peanut. In this study, 44 AhIAA genes were identified from cultivated peanut, of which 31 genes were expressed in seed at varying degrees. AhIAA-3A, AhIAA-16A and AhIAA-15B were up-regulated, while AhIAA-11A, AhIAA-5B and AhIAA-14B were down-regulated with seed development and maturation. The expression patterns of seven genes, AhIAA-1A, AhIAA-4A, AhIAA-10A, AhIAA-20A, AhIAA-1B, AhIAA-4B and AhIAA-19B, were consistent with the change trend of auxin, and expression in late-maturing variety LM was significantly higher than that in early-maturing EM. Furthermore, allelic polymorphism analysis of AhIAA-1A and AhIAA-1B, which were specifically expressed in seeds, showed that three SNP loci in 3′UTR of AhIAA-1A could effectively distinguish the EM- and LM- type germplasm, providing a basis for breeding markers development. Our results offered a comprehensive understanding of Aux/IAA genes in peanut and provided valuable clues for further investigation of the auxin signal transduction pathway and auxin regulation mechanism in peanut.


Introduction
As one of the important plant hormones, auxin can not only participate in all aspects of plant growth and development, such as embryo development, apical dominance, organogenesis and morphogenesis, cell division and tissue differentiation, tropism, fruit ripening and organs aging, etc., but also be involved in the response to various abiotic stresses, such as salt and drought stress and exogenous hormone treatment [1][2][3][4][5][6][7]. Studies have also shown that auxin is the most important hormone that controls the ovary developing into fruit and causes parthenocarpy [8]. The regulated network of auxin involves complex processes such as regulation of gene transcription and protein expression, and according to the response degree, the regulated genes can be divided into primary response genes and secondary induced genes to auxin. The primary auxin-responsive genes mainly include Auxin/indole-3-acetic acid (Aux/IAA), Small Auxin-up RNA (SAUR) and Gretchen Hagen 3 (GH3), which are always represented by a large multigene family in plants [5,7,9,10]. Among them, Aux/IAA is in relatively more research studies so far, and it is also a gene family with expression specificity in different tissues and developmental stages [7,11].
The Aux/IAA genes were isolated from soybean for the first time [12]. In Arabidopsis thaliana, 29 Aux/IAA genes were identified, which were found to distribute on five chromosomes [13]. In addition, studies on Aux/IAA genes and their function analysis have been reported in many other plants, such as cucumber and tomato, as well as in crops including wheat, rice and maize [1,3,11,[14][15][16]. The Aux/IAA gene family encodes nuclear localization proteins with a short half-life period, and the Aux/IAA proteins are characterized by the presence of four highly conserved domains (domain I to IV) [17]. Domain I and domain II are located at the N-terminus, and their functions are inhibiting transcription and maintaining protein stability, respectively; domain III and domain IV are located at the C-terminus, which showed a very high homology with the auxin response factors (ARFs) [18][19][20]. With the development and wide application of molecular genetics technology, we are moving towards understanding the clear signal transduction pathway and feedback regulation mechanism of plant auxin.
With about 50% oil and 26% protein, peanut (Arachis hypogaea L.) is an important oil and economic crop that is grown worldwide with great nutritional value [21]. However, cultivated peanut is an allotetraploid (AABB, 2n = 4x = 40) with a total genome size of about 2.7 Gb, which is very large and complicated [22]. For a long time, research studies related to peanut genomes and functional genes have been relatively scarce due to the lack of genome information. In recent years, the released cultivated peanut genome data have provided strong support for the in-depth genetic research at the molecular and genetic level in peanut [22][23][24][25][26]. Previous studies on auxin in peanut mainly focused on dynamic changes in endogenous IAA content and distribution, and its influence on embryogenesis, but even such reports are limited [27][28][29]. In other plants, auxin has a strong effect on attracting and transporting nutrients, promoting fruit setting, assimilating transportation and dry matter accumulation in fruit and seed development. For example, in the grain filling stage of wheat, kernel growth and dry matter accumulation depend mainly on the concentration level of IAA and cytokinin (CTK) [30]. The exogenous auxin can also promote nutrient transportation, and auxin spraying to grain has an obvious effect on increasing yield in rice, and grain weight of cultivar with application of auxin under stress conditions also increased significantly [31]. Moreover, exogenous auxin treatment has a positive effect on increasing seed oil content in rapeseed [32]. Seed development and maturity are closely related to dry matter accumulation rate. In Zoysia japonica, IAA and gibberellin (GA) content slowly reduced with the seed becoming mature, while abscisic acid (ABA) content increased gradually, indicating that auxin plays an important role in the regulatory process of seed maturation [33]. In peanut, there are significant differences in growth period and seed development and maturation among varieties. Generally speaking, the early-maturing varieties usually have a faster seed development process, with a shorter growth period and earlier senescence of up-ground plants. On the contrary, the seed development process of late-maturing peanut varieties was relatively slow, with later senescence of above-ground plants. One of our previous studies also showed that seed fullness and dry matter accumulation rate were closely related to IAA content, and the peak value of IAA was significantly higher in late-maturing variety [29]. However, few studies were conducted at the molecular and genetic level in peanut. Only four possible Aux/IAA genes in peanut, named PNIAA1, PNIAA2, PNIAA3 and PNIAA4, were identified, and the expression of PNIAA3 was relatively higher in seeds [34], but no complete sequences and further research was reported.
In this study, we provide insight into a comprehensive understanding of Aux/IAA genes in peanut, and a total of 44 AhIAA genes were identified. The bioinformatics characters of these genes were analyzed, and expression differences analysis between earlymaturing and late-maturing peanut varieties were further performed during the seed development process. Detailed sequence polymorphisms of the two genes specifically expressed in seeds were also analyzed among different germplasms, and the key SNP regulatory sites were predicted. The results will provide basic knowledge of Aux/IAA genes in peanut and provide valuable clues for further investigation of the auxin signal transduction pathway and auxin regulation mechanism in peanut.

Identification of AhIAA Genes in Peanut
The hypothetical Aux/IAA protein sequences obtained by the Hidden Markov Model (HMM) analysis in peanut genome were submitted to websites NCBI-CDD, Pfam and SMART to confirm the Aux/IAA domain. Finally, a total of 44 sequences were confirmed to be peanut Aux/IAA genes and named as AhIAA-1A~AhIAA-22A and AhIAA-1B~AhIAA-22B based on their chromosomal locations. Detailed characteristics of AhIAA genes identified are listed in Table 1. The validated AhIAA genes and protein sequences are available in Supplementary Tables S1 and S2. Among the 44 AhIAA proteins, AhIAA-9A and AhIAA-9B were smallest with 133 aa, whereas AhIAA-15A was the largest with 455 aa. The molecular weight (MW) of protein ranged from 14.9 to 50.5 kDa, and the isoelectric point (pI) ranged from 4.86 (AhIAA-3B) to 9.13 (AhIAA-11A). The instability index (II) classified the proteins AhIAA-21A (36.24), AhIAA-8B (39.80), AhIAA-13B (37.92) and AhIAA-20B (36.98) as stable, which may be difficult to be degraded. Subcellular localization prediction showed peanut AhIAA proteins were located in nucleus and contained no transmembrane structure.

Bioinformatics Analysis of AhIAA Genes
The sequences and gene function were well known in Arabidopsis thaliana, so 29 IAA protein sequences from Arabidopsis thaliana, 44 sequences from our study and one sequence of PNIAA3 from the previous report [34] were used to construct an unrooted phylogenetic tree ( Figure 2a). The 74 AUX/IAA proteins were classified into eight groups (from I to VIII), and most of them were clustered in group VIII. Remarkably, AhIAA proteins were more closely related to those AUX/IAAs in the same group than to the other AhIAAs from peanut, indicating that IAA proteins shared a relatively high conservation between the same group across species. Genes in the same cluster may have a similar function. PNIAA3, identified to be expressed in peanut seeds at high levels, was clustered with AhIAA-1A and AhIAA-1B, speculating that they may function during seed development. The protein sequences of 44 peanut AhIAA genes could be classified into five major clusters, namely cluster 1 to 5 (  (Figure 2c). Among all genes, AhIAA-21B and AhIAA-22B had the longest 5 UTR. We also used the MEME web server to search the conserved motifs which were shared with the AhIAA proteins. A total of 10 distinct conserved motifs were detected, and motif 1 and motif 2 were common in all AhIAA proteins, while motif 3 and motif 4 were not detected in most proteins in cluster 5, and genes AhIAA-4A and AhIAA-4B lacked motif 4 only (Figure 2d). The motif 5, motif 8, motif 9 and motif 10 were unique to genes in cluster 1. Motif 10 was detected only in three genes, AhIAA-22A, AhIAA-21B and AhIAA-22B. Obviously, genes and proteins in the same cluster usually share a similar intron-exon formation and motif distribution, especially within the pairwise homologous genes, with some exceptions.
Four conserved domains (domain I to IV) were also identified in most AhIAA proteins through sequences alignment (Figure 3), and motif 4, motif 3, motif 2 and motif 1 in Figure 2d were found to encode domains I to IV, respectively. These domains classified proteins to be Aux/IAAs [17]. Domain I was located at the N-terminus and showed a relatively higher variation, while six genes AhIAA-1A, AhIAA-1B, AhIAA-9A, AhIAA-9B, AhIAA-18A and AhIAA-17B lacked domain I. Aux/IAA proteins degradation was closely related to domain II [35], but AhIAA-3B lacked domain II, and may affect its stability and degradation. Domain III and IV, similar to ARF proteins [4,6,7], were located at the C-terminus, and both of them showed very high conservation among all AhIAA proteins (except AhIAA-3B). Based on the genomic sequence, we obtained the potential promoter sequences, which are 2000 bp in length, within the upstream of 5 UTR of 44 AhIAA genes. Aux/IAA belongs to the early auxin responsive genes, in addition to typical eukaryotic promoter elements including CAAT-box and TATA-box, and many auxin-responsive elements were found in their promoter region from cis-acting elements prediction (Supplementary Figure S1). In addition, most genes contained cis elements in response to GA, ABA, salicylic acid (SA) and methyl jasmonate (MeJA), strongly indicating that AhIAA must play a key role in the intersection and interaction of different hormonal signaling pathways.

Tissue-Specific Expression of Peanut AhIAA Genes
Based on a published transcriptome dataset of 22 different tissue types in cultivated peanut [36], 44 AhIAA genes can be classified into four categories ( Figure 4). Class 1 contained four genes, AhIAA-13A, AhIAA-13B, AhIAA-19A and AhIAA-18B, which had a relatively high gene expression level in most tissues except seeds. Class 2 consisted of 15 genes, and most of them were expressed at relatively higher levels in other tissues than leaves and seeds, while genes AhIAA-14A and AhIAA-12B were expressed at relatively lower level in shoot tips and root tissues, like that of AhIAA-6A and AhIAA-6B in the roots. Class 3 included 14 genes, and all of them had high gene expression in flowers; moreover, genes AhIAA-4A and AhIAA-4B were also expressed highly in leaves. The fourth class contained 11 genes, most of which had obvious gene expression only in shoot tips, while genes AhIAA-1A and AhIAA-1B were expressed in seeds at relatively higher level and AhIAA-18A and AhIAA-17B in the young pods with high expression. The above results indicated that AhIAA genes had tissue-and organ-expression specificity. Compared with other genes, AhIAA-1A and AhIAA-1B was specifically highly expressed in seeds, suggesting that they may play a more important role in seed development. AhIAA genes were classified into four classes (class 1 to 4). The 22 tissues were renamed as S_1 to S_22. S_1, Seedling leaves; S_2, Main stem leaves; S_3, Lateral stem leaves; S_4, Vegetative shoot tip; S_5, Reproductive shoot tip; S_6, Roots; S_7, Nodule roots; S_8, Perianth; S_9, Gynoecium; S_10, Androecium; S_11, Aerial gynophore tip; S_12, Subterranean gynophore tip; S_13, Pattee 1 stalk; S_14, Pattee 1 pod; S_15, Pattee 3 pod; S_16, Pattee 5 pericarp; S_17, Pattee 6 pericarp; S_18, Pattee 5 seed; S_19, Pattee 6 seed; S_20, Pattee 7 seed; S_21, Pattee 8 seed; S_22, Pattee 10 seed. All FPKM values were normalized by log 2 and scaled to 0-1 within each row.

Differential Analysis of AhIAA Genes in Peanut Seeds between Early and Late-Maturing Varieties
The AUX/IAA genes were found to play important roles in fruit development and maturation [37,38], but there is no relevant research in peanut. In our study, an earlymaturing variety Fenghua2 (EM) and a late-maturing variety D666 (LM) were used as materials (Figure 5a) for exploring the relationships of AhIAA genes and seed development and maturation. According to a previous report on pod development in peanut [39], we divided the pod developing period into nine stages, from R1 to R9. The R1 and R2 were the early stages of pod development, and seed development was nearly unable to be observed, while R9 was the late stage of pods over-maturing, and various contents in seeds had been stabilized accompanied by developmental cessation. Therefore, we selected six stages (R3 to R8) of the developing seed from two varieties (Figure 5b) for IAA content and AhIAA genes expression analysis. The IAA content increased rapidly in the early stage of seed development, reached the maximum at the R4 stage, and then decreased sharply and maintained at a low level. The peak value in the early-maturing variety EM was significantly lower than that of the latematuring variety LM (Figure 5c). The expression data of AhIAA genes were extracted from our transcriptomic dataset for differential analysis between the two varieties. Data analysis showed that 13 of the 44 peanut AhIAA genes were not expressed or were rarely expressed with FPKM <0.5 in seed, including AhIAA-2A, AhIAA-7A, AhIAA-8A, AhIAA-14A, AhIAA-18A and AhIAA-22A, and their homologous B subgenome genes AhIAA-2B, AhIAA-7B, AhIAA-8B, AhIAA-12B, AhIAA-17B, AhIAA-21B and AhIAA -22B (Supplementary Table S4). These 13 genes may not play major roles in seed development and maturation, and they were not involved in the subsequent analysis. From the expression patterns of 31 AhIAA genes in the developing seed (Figure 5d), seven genes' expression was completely consistent with the IAA content changes, showing a trend of increasing at the early stage and then decreasing to a low level, including AhIAA-1A, AhIAA-4A, AhIAA-10A, AhIAA-20A, AhIAA-1B, AhIAA-4B and AhIAA-19B. It is speculated that these seven genes are more important in response to auxin during seed development. The expression of these seven genes reached the peak value at R5 stage, and expression in the LM variety was significantly higher than that in the EM variety, which was also similar to the IAA content difference. In addition, the expression of genes including AhIAA-3A, AhIAA-16A and AhIAA-15B showed an increasing trend with seeds becoming mature, while expression of another three genes including AhIAA-11A, AhIAA-5B and AhIAA-14B showed a decreasing trend with seed maturity, suggesting that these genes were also closely related to the seed developing process.
Combined with the tissue-specific results of gene expression (Figure 4), allelic polymorphism of AhIAA-1A and AhIAA-1B was further analyzed in LM and EM. Based on the results of simplified genome sequencing, three SNP loci were detected within 3 UTR of Furthermore, we randomly selected 30 early-maturing and 30 late-maturing peanut germplasms, including landraces and breeding varieties (lines) (Supplementary Figure S2), based on the sequencing results, and SNP with a frequency of more than 5% was used for sequence diversity analysis (Supplementary Table S5). Three SNP loci detected within 3 UTR of AhIAA-1A formed two haplotypes Hap-1A1 and Hap-1A2 (Figure 6a), of which 34 germplasm were Hap-1A1, including 30 late-maturing germplasm and 4 early-maturing germplasm, while 26 Hap-1A2 germplasm were the early-maturing type (Table 2, Figure S2). As to AhIAA-1B gene, only one SNP locus was detected, forming two haplotypes Hap-1B1 and Hap-1B2 (Figure 6b), including 56 and 4 germplasms, respectively, and 4 Hap-1B2 germplasms were the late-maturing type (Table 2, Figure S2). All the germplasms can be divided into three haplotype-combination genotypes, namely Hap-1A1/1B1, Hap-1A1/1B2 and Hap-1A2/1B1, including 30, 4 and 26 germplasms, respectively (Table 2, Figure S2). From the above results, it can be seen that haplotype Hap-1A1 and Hap-1A2 can effectively distinguish the germplasm maturity of the LM and EM types.

Discussion
Auxin is an important plant hormone, which is widely distributed and plays a vital role in plant growth and development. Aux/IAA is one of the most important primary auxin-response gene families, and its function should not be underestimated. A total of 29 Aux/IAA genes were isolated from Arabidopsis, which distributed on five chromosomes [13], and 31 Aux/IAA genes were isolated from rice [16], and 25 Aux/IAA genes were isolated from sorghum which was located on 9 chromosomes [40]. In this study, 44 AhIAA genes were identified from the peanut genome by bioinformatics methods, and they were located on 16 chromosomes (Table 1, Figure 1).
Cultivated peanut is an allotetraploid (AABB), and it is derived by the natural hybridization of the diploid wild species between A and B genomes [22]. Most of the homologous genes from the A and B subgenome usually have high sequence similarity [22,41]. Through sequence alignment analysis of peanut AhIAA genes, 21 pairs of homologous genes were identified between the A and B subgenome (Table S3). In phylogenetic evolutionary analysis, the paired homologous genes were preferentially clustered together, and sequence similarity of their coding and amino acid sequence were also very high; for example, the sequence similarity between pairwise AhIAA-7A and AhIAA-7B, AhIAA-9A and AhIAA-9B and AhIAA-14A and AhIAA-12B reached 100%, and similar gene structure and motif patterns were found among them, respectively (Table S3, Figure 2). The results also confirmed high conservation of this gene family during the evolution process [16,40]. However, gene sequences and expression levels of pairwise homologous genes were not always consistent (Figure 3, Figure 5, Figure S1). The results suggested that there may be differences in the regulation of homologous genes, and such differences probably caused a different contribution to the gene function. Further analysis of AhIAA-1A and AhIAA-1B showed AhIAA-1B is more conservative among the germplasm, while the variation of AhIAA-1A has a stronger correlation with maturity traits in peanut. Similar results had been reported on two homoeologous genes, FAD2A and FAD2B, encoding for the desaturase located on the A and B subgenome, respectively [41]. Some of our previous studies on the chloroplast Cu/Zn-SOD gene also showed consistent research findings [42].
Most of the isolated members of the Aux/IAA family are involved in the growth and development of roots [43,44], and a few reports showed that Aux/IAA genes influence fruiting; for example, silencing of the SlIAA9 caused parthenocarpy, while silencing the SlIAA27 gene not only caused parthenocarpy, but also changed the tomato size and shape [45]. Among the 44 AhIAA genes identified in our study, 13 genes were not expressed or were rarely expressed in seed from transcriptomic data analysis (Table S3), and the same result was obtained from gene expression in 22 different peanut tissue types (Figure 4), not excluding that these genes may play major roles in other tissues [7,11]. For example, AhIAA-22A, AhIAA-21B and AhIAA-22B were specifically expressed highly in shoot tips, while AhIAA-14A and AhIAA-12B were expressed relatively higher in flowers, young pods and pod pericarps (Figure 4). Among the remaining 31 AhIAA genes, seven of them, AhIAA-1A, AhIAA-4A, AhIAA-10A, AhIAA-20A, AhIAA-1B, AhIAA-4B and AhIAA-19B, had the highest expression in the middle stage of seed development, and the change trend was completely consistent with the auxin changes ( Figure 5), indicating that they are key genes responding to auxin during seed development.
The maturing time varies greatly in peanut germplasm resources, and the longer growth period may provide conditions for seeds to accumulate more nutrients. The seed development process of early-maturing varieties are likely to be faster than that of latematuring varieties. Therefore, peanut seed development and maturation is closely related with growth period, and maturing time is also a very important breeding character in peanut. Due to its underground fruiting characteristics, the key to judging seed maturity in peanut lies in the maturity of above-ground plants. The seed development process of late-maturing peanut varieties was relatively slow, with later senescence of above-ground plants. On the contrary, the up-ground plants aged earlier in early-maturing varieties, with a shorter growth period and relatively faster seed development process. Previous studies have shown that auxin plays a very important role in the regulation of fruit and seed development and has a close relationship with the dry matter accumulation ability, seed fullness and maturation [29,30], and exogenous auxin can promote nutrient transportation, showing a positive effect on increasing yield and seed oil content [31,32]. In our study, late-maturing variety LM and early-maturing EM were used for exploring the relationships between AhIAA genes and seed development. The middle stages (R4-R6) are the rapid accumulation period for nutrients and dry matter during peanut seed development, and seven AhIAA genes showed significantly higher expression in LM than that of EM ( Figure 5). Among them, AhIAA-1A and AhIAA-1B were specifically expressed in seeds, and we further analyzed their sequence polymorphism. Thirty early-maturing germplasms and 30 late-maturing germplasms were randomly selected. After sequence analysis, we found that three SNP within AhIAA-1A could effectively distinguish the two types ( Figure 6, Table 2, Figure S2, Table S5). The results showed that AhIAA-1A was closely related to peanut seed development and maturation. Based on the results, we are now developing corresponding PCR markers, which are expected to be used in breeding research. However, seed development and maturation is a complex biological process, which is certainly not determined by a single gene. In addition to AhIAA-3A, AhIAA-16A and AhIAA-15B showed an increasing trend with seeds becoming mature, while AhIAA-11A, AhIAA-5B and AhIAA-14B showed a decreasing trend, which are also closely related to the process of seed development. In addition, a lot of regulatory genes must be involved ( Figure S1).
As an important regulator of the auxin signal transduction pathway, the Aux/IAA gene family is involved in many processes of plant growth and development through auxin-mediated transcription regulation, and it is a key protein in the transduction of auxin signaling [6]. In recent years, more and more studies are focused on the functional research of Aux/IAA family members and great progress has been made in some plants. In this study, we established a preliminary understanding of peanut AhIAA genes, analyzed the expression pattern in developing seed, discussed the differences between the EM-and LM-type germplasm, and predicted the key genes related to seed development. The results laid a foundation for the follow-up mechanism analysis of AhIAA regulatory network in peanut growth and development.

Identification of Aux/IAA Genes in Peanut Genome
The HMM file corresponding to the Aux/IAA domain (PF02309) was downloaded from the Pfam database (https://pfam.xfam.org/, accessed on 10 March 2020) [46] and used it as the query (p <0.001) to search the peanut protein sequence data. All candidate genes that may contain Aux/IAA domain based on hmmsearch [47] results were submitted to websites NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd, accessed on 15 March 2020), Pfam and SMART (http://smart.embl-heidelberg.de/, accessed on 15 March 2020) to confirm the Aux/IAA domain. The assumed genes with bitscore lower than 80 and genes with more conserved domains other than Aux/IAA were manually excluded. All the nonredundant and high-confidence genes were assigned as peanut Aux/IAA genes (AhIAA).
Finally, a total of 44 sequences were confirmed to be peanut AhIAA genes and they were renamed based on position on chromosomes (Table 1).

Plant Materials and Sampling
An early-maturing variety Fenghua2 and a late-maturing variety D666 both developed by our lab were used as experimental materials, renamed as EM and LM, respectively ( Figure 5a). Moreover, landraces or breeding varieties (lines) including 30 EM-type (EM_1 to EM_30) and 30 LM-type (LM_1 to LM_30) were also used for maturing phenotype and genotype identification ( Figure S2). All the materials were grown in the test field of Agricultural Experiment Station of Shandong Agricultural University (36.15 • N, 117.15 • E), Tai'an, China. Developing seeds without seed coat at different stages (R3 to R8) of EM and LM were sampled for IAA content and transcriptome RNA-sequencing (RNA-seq) analysis. The fresh leaves were sampled for DNA extraction and genotype identification. All the collected samples were frozen in liquid nitrogen rapidly and stored in an −80 • C refrigerator before DNA or RNA extraction. After 110 days of sowing, the growth performance of the germplasm was photographed.

IAA Content Determination
According to\previous report [29], a high-performance liquid chromatography (HPLC) method was used to determine the IAA content. The chromatographic conditions were as follows: symmetry C18 column (150 mm × 4.6 mm, 5 µm), mobile phase of methanol and acetic acid aqueous solution (5‰ acetic acid), a flow rate of 0.9 mL/min, a column temperature of 25 • C, an injection volume of 15 µL, and a detection wavelength of 254 nm.

Data Analysis
Transcriptome RNA sequencing of developing seeds (R3 to R8) of EM and LM and simplified genomic resequencing of peanut germplasm were carried out in Biomarker Technologies Co., Ltd. The transcript abundance of peanut AhIAA genes was calculated as FPKM (fragments per kilobase of exon model per million mapped reads), and the average of three replicates was taken in analysis. The genes with FPKM values <0.5 were not selected for the following analysis [58]. Moreover, a published transcriptome dataset (BioProject PRJNA291488) of 22 different tissue types that represent the full development of cultivated peanut was obtained from the website PeanutBase (https://www.peanutbase.org/, accessed on 10 January 2022) [23]. The gene expression heatmap was created by TBtools [56] with standardized FPKM values by log2 and scaled to 0-1 within each row.

Conclusions
Auxin-responsive gene AUX/IAA has been widely studied in plants, but there are few relevant reports in peanut. In this study, 44 peanut AhIAA genes were identified by bioinformatics methods, of which 31 genes were expressed in seed at varying degrees. During seed development, the up-regulated genes AhIAA-3A, AhIAA-16A and AhIAA-15B, and down-regulated genes AhIAA-11A, AhIAA-5B and AhIAA-14B may be closely related to peanut seed development and maturation. Expression patterns of seven genes, AhIAA-1A, AhIAA-4A, AhIAA-10A, AhIAA-20A, AhIAA-1B, AhIAA-4B and AhIAA-19B, were consistent with the change trend of auxin, and gene expression level in LM was significantly higher than that in EM, so it was speculated that they were the key genes which responded to auxin and affected the seed maturation process. Furthermore, allelic polymorphisms of AhIAA-1A and AhIAA-1B, which were specifically expressed in peanut seeds, were analyzed among germplasms with different maturing times. The three SNP detected in 3 UTR of AhIAA-1A could effectively distinguish the EM-and LM-type germplasms, which may be the key regulatory sites for peanut maturity differences. We speculated that AhIAA-1A was closely related to peanut seed development and maturation. Furthermore, we are developing breeding markers based on the SNP loci of the gene AhIAA-1A. All the results provided a comprehensive understanding of Aux/IAA genes in peanut and provide valuable clues for further investigation of the auxin signal transduction pathway and the auxin regulation mechanism in peanut.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/plants11040472/s1, Figure S1. Some of the predicted cis-acting elements in potential promoter sequence of AhIAA genes in peanut. Figure S2. Genotypes and growth performance of germplasm after 110 days of sowing. Table S1. The cDNA sequences of validated AhIAA genes in peanut. Table S2. The protein sequences of validated AhIAA genes in peanut. Table S3. The similarity of coding and protein sequences of pairwise homologous AhIAA genes. Table S4. The FPKM value of 44 AhIAA genes from transcriptome data in peanut. Table S5. Allele polymorphism analysis of AhIAA-1A and AhIAA-1B among early maturing and late-maturing peanut germplasms.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.