Genomic Analysis of Sugar Transporter Gene Family in Cultivated Peanut (Arachis Hypogaea): Evolution, Expression Proles and Gene Variation

transporter gene family, information genome-wide investigation into and

genome sequences indicated multiple variations occurred in gene sequence or promoter region, and provided valuable clues for the different expression pro les of the four genes during seed development in subsp. hypogaea and subsp. fastigiate.

Conclusion
Thirty-six STP genes were identi ed in cultivated peanut and their protein character,structure,evolution characteristics, expression patterns and gene variation were analyzed.This study provided a foundation for further functional characterization of STP genes with an aim of cultivated peanut crop improvement.
Substantial evidence indicates that the STPs are involved in the uptake of hexose hydrolyzed from sucrose in the apoplast and sugar unloading which contribute to carbon allocation, yield, and environmental adaptation in crops [1]. In Arabidopsis, for example, after wounding, AtSTP3 and AtSTP4 transcript levels transiently rise [11,12]; pathogen attack can induce the expression of AtSTP4 [12]; AtSTP1 and AtSTP14 show circadian oscillations and are strongly dark-inducible [13,14].
Previous studies have shown that STP genes have different expression patterns during plant growth and respond to different stress conditions. In Arabidopsis, most of the AtSTPs show sink-speci c expression [2]. For example, AtSTP1 is expressed in germinating seeds, roots and guard cells [13,15,16]. AtSTP2, AtSTP4, AtSTP6, AtSTP9 and AtSTP11, are expressed in pollen during different developmental stages [17][18][19][20]. AtSTP3 is expressed in the source tissues (green leaves) under normal condition [12]. AtSTP4 show expression in root tips [11], AtSTP5 in silique and whole seedling [21], AtSTP13 in the vascular tissue of petals [22]. AtSTP14 is expressed most prominently in leaves and siliques [23]. As the newly report, AtSTP7 and AtSTP12 can be detected at whole open owers, leaves, stems, roots, whole seedlings, and siliques; while AtSTP8 is expressed exclusively in oral tissues [21]. Although previous studies have been explored in the expression patterns and functional analysis of STP genes, the extensive expression pro les of the STP genes in subterranean pod crops, such as peanut, remain poorly understood.
Cultivated peanut is an important edible and oil crop planted in the tropics and semi-arid tropics of the world, providing high-quality vegetable oil and protein. Genetic, cytogenetic, phylogeographic and molecular evidences have suggested that cultivated peanut is an allotetraploid (AABB, 2n = 4x = 40) formed through hybridization between A. duranensis (AA, 2n = 2x = 20) and A. ipaensis (BB, 2n = 2x = 20) [24][25][26][27][28][29]. It's speci c in ower stalks (peg) elongation and developing pods under the ground, which contains sugar transportation, accumulation and transition processes [30]. But until now, little information on STP gene family expression in peg elongation, pod development and other tissues of peanut is available for us. In this study, we took advantage of the peanut genomes and transcriptome data to identify the complete set of STP gene family in peanut. Their sequence characteristics and gene structures were detailly analyzed. Segmental and tandem duplication of peanut STPs were investigated by synteny analysis. The expression pro les of AhSTP genes in different tissues, including peg and pod, were analyzed using the publicly available transcriptome data, in an attempt to understand their possible roles in peg and pod development. In addition, the cause for speci c AhSTP genes was explored through gene variation analysis, possessing different expression pro les during seed development in peanut subspecies.

Results
Identi cation and protein character analysis of STPs in A. hypogaea, A. duranensis and A. ipaënsis A total of 36 STP genes was identi ed in peanut genome, while, 15 and 16 STP genes were identi ed in the haploid ancestors of cultivated peanut, A. duranensis and A. ipaënsis, respectively (Table S1 and  Table S2). These STP genes were named AhSTP1 to AhSTP36 in A. hypogaea, AdSTP1 to AdSTP15 in A. duranensis, and AiSTP1 to AiSTP16 in A. ipaënsis, respectively, according to their order on the chromosomes. The STP genes were unevenly located on the three genomes, and no STP gene was found to distribute on chromosome A07 of A. duranensis, B08 of A. ipaënsis or A. hypogaea (Table S1).

Gene structure and motif composition of peanut STPs
The exon-intron organizations of all the identi ed STP genes were analyzed to get more insight into the evolution of the STP gene family in peanut. As shown in Fig. 3B, in A. hypogaea, the exon of AhSTPs uctuated from 1 to 11, of which 20 AhSTP genes contained 3 exons and 7 with 4 exons. In A. duranensis, all the STP genes had only 1 exon to 8 exons. In A.ipaënsis, all the STP genes possessed 1 exon to 6 exons. The majority of the AhSTP had a similar structure with its ancestor STP (AdSTP or AiSTP) at the bottom branch of phylogenetic tree (Fig. 3). Furthermore, in A. hypogaea, only 3 AhSTP genes had no untranslated sequence (UTR), while, A. duranensis and A. ipaënsis both had 6 STP genes with no UTR.
Conserved motif analysis of all the peanut STP proteins was performed using the MEME program. The results showed that 12 motifs were identi ed with their length ranging from 21 to 49 aa, 9 motifs were distributed on all STP proteins except motif 3, 4 and 9 (Fig. 3C). Most STP proteins had similar motif architecture at the bottom branch of phylogenetic tree. For example, AhSTP20 and AhSTP24, with 12 motifs, had the same motifs distribution in the same branch of group I, as well as AdSTP6, AdSTP7, AhSTP25 and AiSTP7 in the same branch of group III. Comparing to the other STPs, the motifs distribution on AiSTP16 was unique, since that motif 1/7/9 distributed 3 times, motif 2/3/4/5/8/10/11 distributed 2 times, and motif 6/12 distributed once (Fig. 3B). The unique motifs distribution on AiSTP16 might indicate its unique evolution and function. To further understand the character of the motifs, the motif sequence was predicted by Pfam program, and motifs 1, 2, 3, 5, 6, 7, 8 were part of the putative sugar_tr domain (Table 1). Overall, the similar gene structures and conserved motif compositions of the STPs in the same branch, together with the phylogenetic analysis results, could strongly support the reliability of the group classi cations.

Synteny analysis of peanut STP genes
According to the study of Yu [31], segmental duplications were characterized as multiple genes of one family occurring through polyploidy followed by chromosome rearrangements. In A. hypogaea, 24 AhSTP genes were clustered into 18 segmental duplication events in 18 chromosomes except A02 and B08 (Fig. 4A). Intriguingly, AhSTP9, AhSTP15, AhSTP28, and AhSTP33 exhibited segmental duplication with each other on chromosome A05, A09, B05 and B09. The same phenomenon was also observed in AhSTP13, AhSTP14 and AhSTP32 on chromosome A07, A08 and B07 (Fig. 4A). Moreover, AhSTP13 and AhSTP31 were identi ed as segmental duplication genes. Besides the segmental duplication events, a tandem duplication event region harboring 2 genes (AhSTP35/36) was identi ed on chromosome B10. The results indicated that gene duplication might generate some AhSTP genes and segmental duplication played a key role in driving the AhSTP evolution.
Orthologous pairs between peanut and the four species (A. duranensis, A. ipaënsis, Arabidopsis and soybean) were 28, 28, 7, 44. Some AhSTP genes exhibited to associate with more than one gene pair, especially between peanut and soybean STP genes. For instance, both AhSTP14 and AhSTP32 were associated with four GmSTP genes. These genes might play an important role in the evolution of STP gene family.
Six (AhSTP5/16/18/20/26/36) of the 36 AhSTP genes were not found collinear STP genes in its ancestors A. duranensis and A. ipaënsis, which may indicate that these genes diverged after the formation of cultivated peanut. And some STP genes in A. duranensis and A. ipaënsis, such as AdSTP7/14 and AiSTP9, were not found collinear STP genes in A. hypogaea, implied that gene loss may happen during A. hypogaea evolution. In addition, 6 AhSTP genes (AhSTP3/15/19/32/33/35) were identi ed collinear genes among all the four species, indicating that these orthologous pairs may exist before the species divergence.

Ka and Ks calculation of orthologous pairs
To better understand the evolution of peanut STP gene family, the Ka/Ks ratios of the STP gene pairs were calculated. All segmental and tandem duplicated AhSTP gene pairs, and most of the orthologous STP pairs between peanut and its ancestors, had Ka/Ks < 1, indicating that the peanut STP gene family might have experienced strong purifying selective pressure during evolution ( Table 2, Table 3 and Table  S3).  Further, the divergence time of the STP gene pairs was also estimated to characterize the formation of the peanut STP genes. The divergence time of the orthologous STP pairs between peanut and its ancestors showed that most of the genes were diverged after the formation of A and B genome, according to the divergence time of A and B genome by Bertioli [32] ( Table 3 and Table S3). All the Ks values of 5 orthologous STP pairs in A genome and 10 orthologous STP pairs in B genome were zero (Table 3), which revealed these genes kept conservation during the evolution of allotetraploid peanut formed from its wild diploid ancestors. These also suggested that the evolution of AhSTP genes was asymmetrically in A and B sub-genome and AhSTP genes in B sub-genome were possible to be more conservative than in A sub-genome. Moreover, AhSTP25 (B04) and AhSTP33 (B09) in B sub-genome showed nearest divergence time with orthologous STP pairs AdSTP6/AhSTP7 (A04) and AdSTP13/AhSTP15 (A09) in A genome, respectively (  (Table 3, Fig. 5). This result implied the location conversion of these AhSTP genes might happen through genetic exchange (or homologous chromosome rearrangements) between A and B sub-genome in A. hypogaea after the polyploidy event. The divergence time of tandem duplicated gene pair AhSTP35/36 was much earlier than that of the A and B genome as estimated in previous studies [33,34]. And the divergence time of orthologous pairs AiSTP16/ AhSTP35 was also much earlier than that of the other nearest orthologous pairs showing the complicated formation of these genes.

Gene expression Pro le Analysis
The transcriptome data, derived from different developmental stages of 22 peanut tissues [35] (Table S4), were used to investigate the expression patterns of all 36 AhSTP genes. All the 36 AhSTP genes were expressed with different expression patterns among development stage of peanut (Fig. 6). Eleven AhSTP genes were expressed in all the 22 tissues (FPKM > 0) and 4 genes (AhSTP1/3/19/28) showed constitutive expression with FPKM > 1 in all the tissues. Based on the FPKM value, the transcripts of AhSTP genes were exhibited a most abundance in the tissue 13 (Peg tip to fruit Pattee 1). AhSTP3 showed the highest total transcript abundance of all the 22 tissues, and also showed the highest transcript abundance in tissue 1-4, 7, 11, 13-17 and 22. AhSTP4 was the highest expressed gene in tissue 5; while AhSTP 19 was the highest expressed gene in tissue 6, 8, 10, 12; AhSTP 28 was the highest expressed gene in tissue 9, AhSTP 9 was the highest expressed gene in tissue 18-19; AhSTP 18 was the most expressed gene in tissue 20-21 (Fig. 6). Furthermore, some genes also exhibited preferential expression in the detected tissues. 13 genes in pistil (tissue 10), 3 genes (AhSTP2/4/21) in repr shoot (tissue 5), 2 genes (AhSTP6/22) in nodule (tissue 7) and 2 genes (AhSTP13/31) in seed pat 5 and 6 (tissue 18 and 19) showed relatively highest transcript abundances (Fig. 6).
The transcriptional level of 36 AhSTP genes was further analyzed to understand the gene expression in response to peg elongation and pod development. Four genes (AhSTP 3/9/19/28), were found signi cantly high expression in the stage of peg tip to fruit Pattee (tissue 13) (Fig. 7A), indicating these genes may participate the process of fertilized egg development in Tifrunner. The three constitutive expression genes (AhSTP3/19/28), had relative lower expression from development tissue 13 to 14, and kept up-regulated expression from tissue 14 to 16, while AhSTP9, an induced expression gene, kept relatively high expression in the stage of fruit pat 1(tissue 14) to pericarp Pat 5 (tissue 16). However, no signi cant expression change of the genes was observed in seed developmental stage of Tifrunner.
Moreover, using the gene expression data of cultivated peanut subsp. fastigiata ICGV 91114 [35], the four gene expression were analyzed in seed developmental stage of 5, 15 and 25 days after pegging. The four genes were found signi cantly high expression in seed_25, a seed developing phase of seed coat epidermal cell differentiation, fatty acid and defense proteins synthesis [36] (Fig. 7B). Overall, these results suggested that some AhSTP genes may participate in early pod formulation and seed development, and have different expression pro le in subspecies of cultivated peanut.

Gene variation analysis
To explore the cause of different expression pro les of AhSTP3/9/19/28 during seed development in peanut subspecies, genic-SSR in the four genes were identi ed with no SSR in AhSTP3, three SSR in both AhSTP9 and AhSTP19 and four SSR in AhSTP28 (Table S5). SSR markers were successfully developed for all the SSR loci and used for genotyping the 37 diverse cultivars (Table S6). Unfortunately, no allelic variation or substantial structure variation in the three genes was detected in the different cultivars of subsp. hypogaea or subsp. fastigiata.
Secondly, gene sequence of the four genes was extracted from the subsp. hypogaea and subsp. fastigiata genomes. Sequence analysis showed that AhSTP19 kept consistent in gene level of the genomes, eight-base deletion and single-base insertion were detected in the rst intron of AhSTP9 and AhSTP28 respectively, and three single-base substitution were detected in the coding region sequence (CDS) of AhSTP3 ( Fig.S1A and Fig.S1B). Two of the three substitutions in AhSTP3 were non-synonymous substitutions ( Fig.S1B and Fig.S1C), occurring in 804 and 993 bp of the CDS, resulted in glutamic acid replaced by lysine and aspartic acid transformed to glycine in 268 and 328 aa of the protein from subsp. hypogaea to subsp. fastigiata. Protein secondary structure prediction of AhSTP3 indicated that the extended strand increased while the beta turn and random coil decreased (Fig.S2), and the protein binding site increased two sites in subsp. fastigiata (Fig.S3).
Thirdly, the promoter region of the genes, the initiator codon upstream 2000 bp sequences, was extracted from the genomes. Sequence alignment analysis indicated that no variation was detected in the promoter region of AhSTP9 and AhSTP28, while, single-base substitution or insertion/deletion was observed in AhSTP3 and AhSTP19 respectively (Fig.S1E). The single-base substitution in AhSTP3 promoter region caused the AT1-motif deletion in subsp. fastigiata, which is an important part of light responsive module (Fig.S1D, Table S7). The single-base substitution, four-base insertion and deletion in AhSTP19 promoter region resulted in changes of cis-acting regulatory elements (CREs) in the subspecies, such as deletion of CAAT-box, TATA-box and AT ~ TATA-box (Table S7).
The above results indicated that multiple variations were discovered in the four genes or promoter region of the two subspecies, and were potential causes for the different expression pro les during seed development.

Discussion
The release of peanut genomes [32,34,37] provided an availability to explore the peanut STP gene family and discover its potential function in pod formulation and development. In this study, we rst analyzed protein properties, gene phylogenetic, structure, evolution, expression pro les and gene variation of STP gene family in cultivated peanut. A total of 36 AhSTPs was identi ed in cultivated peanut genome through HMMER and BLAST search, and named AhSTP1 to AhSTP36, with 17 AhSTPs in A sub-genome and 19 AhSTPs in B sub-genome. To better understand the evolution of AhSTP genes, STP gene family in the two diploid wild peanut genomes was identi ed and named in the same way as cultivated peanut, with AdSTP1 to AdSTP15 in A genome and AiSTP1 to AiSTP16 in B genome.
STP proteins contain a common structure with 12 transmembrane domains in plants [1]. As observed in tomato [6], Cabbage [10] and cassava [8], the loss of TMD, ranging from one to four TMDs, were also occurring in AhSTPs, AdSTPs and AiSTPs. Unlike other species, 3 STP genes (AdSTP1 and AiSTP1/16) in the diploid genomes contained excessive TMDs. AiSTP16 contained double sugar_tr domain, and all of the conserved motifs were doubled at least as well, while AhSTP35, the orthologous gene of AiSTP16, contained only 1 sugar_tr domain. These results indicated that the domain gain and loss may have happened during evolution from A.ipaënsis to A.hypogaea as happened in other gene families [38].
In this study, comparison of STP genes in cultivated peanut with other dicot plants had revealed that cultivated peanut contained more STP genes and the STP gene number in sub-genome remained comparatively stable through evolution from its ancestors. Gene duplication is a primary driving force leading to functional speciation and diversi cation in evolution [39,40]. Tandem and segmental duplication events play a critical role in the expansion of STP gene family [8][9][10]. Here, segmental duplication events were the major force driving the expansion of AhSTP genes. Evidence of STP Gene loss and new STP gene gain were also observed during the polyploid of cultivated peanut as in cabbage [10] and cassava [8]. In cultivated peanut, no AhSTP gene was identi ed collinear with AdSTP7/14 and AiSTP9 in its ancestor genomes, while, 6 AhSTP were not found collinear STP genes from its ancestor genomes. These results indicated that AhSTP gene family was generated by different patterns of evolution events, and the 6 genes may have new features.
Moreover, the Ks analysis of orthologous STP genes pairs provided more clues about the evolution of STP gene family in cultivated peanut. Previous studies have showed that after polyploid formation of allotetraploid peanut, A and B sub-genomes were subjected to asymmetric homoeologous sequence exchanges (or homeologous chromosomes rearrangement), gene family expansion and contraction, homoeolog expression divergence, and selection [33,34,37]. In this study, Ks value or divergence time of orthologous STP gene pairs between peanut and its ancestors', indicated that STP genes in cultivated peanut B sub-genome were more conservation than in A sub-genome. This may due to the human domestication, which has larger effects on homoeologous structural variation genes of A sub-genome in cultivated peanut, according to the study of Yin [33]. In addition, genetic exchange (or homeologous chromosomes rearrangement) between the sub-genomes was also observed in this study, the divergence time of AhSTP25 (B04), AhSTP33 (B09) and AhSTP13 (A07) implied these genes were likely to come from homeologous chromosome A04, A09, and B07. All these results suggested that the AhSTP genes were more advanced in terms of evolution in A sub-genome, seemed to have played important roles in plant adaption, and explained why A sub-genome was more different to its ancestors A. duranensis from the perspective of a gene family.
Phylogenetic analysis showed 36 AhSTPs were clustered into four groups with the other STP proteins and AhSTPs grouped with AtSTP, GmSTP, AdSTP and AiSTP in each group. A close relationship was observed between AhSTPs and its ancestor STPs (AdSTP and AiSTP), then the GmSTPs, and the relationship between STPs are consistent with the species divergence [34,37]. Homologous proteins clustered with the characterized proteins in the same group possibly possessing similar biochemical properties [8]. Group I contained AtSTP1/12, group II contained AtSTP4/9/10/11, group III contained AtSTP2/6/7/8/13/14, and group IV contained AtSTP3/5. Previous studies about AtSTP genes provide valuable clues about the functional role of AhSTP genes that involved in the speci c peanut physiological process. For example, in group I, AtSTP1 is a guard cell-speci c localization gene involved in carbon acquisition and plays a possible role in osmoregulation [16,41], while, AtSTP12 is highly expressed in reproductive organs, and its protein product might contribute to sugar uptake into the pollen tube and the embryo sac [41].
The functional roles of some AhSTP genes involved in speci c peanut physiological process were inferred through valuable clues. For example, AhSTP3 and AhSTP19, a segmental gene pair, exhibited high expression in the stage peg tip developed to form a pod, which is a stage embryo cell division facilitating geocarpic pod [30]. While their orthologs in Arabidopsis, AtSTP12, located in the inner integument, could uptake sugar to supply the embryo development [41], indicating that AhSTP3/19 may share the similar function in cultivated peanut. AhSTP35 was preferentially expressed in the pistil, and with no expression in other detected tissues. Interestingly, its orthologs in Arabidopsis, AtSTP11 was found to be the most prominent AtSTP in germinating pollen and in the growing pollen tube [42,43], participating in the allocation of sugars to growing pollen tubes. AhSTP15 and AhSTP33 were orthologs of AtSTP6, a pollen-speci c H + -monosaccharide symporter, and their function should be further studied. AhSTP32 exhibited expression in all the tissues detected, while its ortholog, AtSTP5 might be a pseudogene with a nonfunctional protein product [21], indicating AhSTP32 may have no function in cultivated peanut.
Peg is an important yield related trait, with the capacity for embryo positive gravitropism transportation and penetrating the soil to form subterranean pods [30]. The development stage from peg to pod is a biological process participated by phytohormone, signals, energy, et al. [30], while monosaccharides (e.g., glucose, fructose, and mannose) are important to generate energy and synthesize cellulose and starch [4,21]. Therefore, it is important to investigate the expression of AhSTP genes in the peg and pod of cultivated peanut and dissect the potential roles of these genes in sugar distribution. Previous studies in Arabidopsis have showed that STP genes are always expressed coupling with cell wall invertase genes [44], and after the sugar cleavage by cell wall invertases, the resulting monosaccharides could be imported into the inner integument by AtSTP12 to supply the early development of embryo [21]. Using the transcriptome data of subsp. hypogaea Tifrunner [35], four AhSTP genes were found high expressed in the stage of pod formulation, and two genes were orthologs of AtSTP12, indicating these genes may have important function in early development of embryo. While, the embryo of cultivated peanut is unique in positive gravitropism transportation under soil through the tube tissue peg, then the embryo development to a pod, suggesting that the tissue these genes performing function perhaps had altered.
Meaningful, using the transcriptome data of ICGV 91114 [36], a subsp. fastigiata cultivated peanut, we found these genes also high expressed in the developing seed, while in the subsp. hypogaea no signi cant expression change were observed in these genes during seed developing, indicating these genes may associate with seed development in subsp. fastigiate and the gene function need to study further. Therefore, AhSTP genes exhibiting high expression in the pod at the early growth stage or in the seed at the mature stage may be involved in pod and seed development.
Gene expression pro le is a complex biological process regulated by a variety of factors, such as alternative splicing, epigenetic modi cations, etc. [45], and gene variation is probably to be the most direct cause for altering gene expression pattern. The expansion or contraction of SSR, the transition or transversion of SNP and the insertion or deletion of InDel in gene sequence or promoter region, all can lead to changes in gene expression pattern and function via mutation in CDS, UTR, intron or promoter region [46][47][48]. Gene sequence of AhSTP19 was found conservation in the two subspecies, but SNP and InDel in the promoter region caused altering of CREs. None of genic-SSR, CDS or promoter variation was observed in AhSTP9/28, but small altering was found in the rst intron of the genes. For these genes, more evidences are needed to verify the effect on gene expression patterns and functions. While, for AhSTP3, farther study is essential to evaluate the effect of two non-synonymous substitutions in the CDS on protein structure and function. Overall, the above ndings provide valuable clues for the different expression pro les of AhSTP3/9/19/28 during seed development in peanut subspecies.

Conclusions
This study presents the rst genome-wide analysis of STP gene family in cultivated peanut genome.
Thirty-six AhSTP genes were characterized and classi ed into four groups, with high similar exon-intron structures and motif compositions at the bottom branch. Synteny analysis and phylogenetic comparison of STP genes from its ancestor and two other dicots provided valuable clues for the evolutionary characteristics of peanut STP genes. Segmental duplication events were the major force driving the expansion of AhSTP genes, and homeologous chromosomes rearrangement may lead the exchange of STP genes between the A and B sub-genome. The transcriptome data exhibited distinct expression patterns of AhSTP genes in various tissues, and four AhSTP genes may involve in the development of pod and seed. Gene variation was speculated to be potential causes of the four genes with different expression pro les during seed developing phase in peanut subspecies. This study provided a foundation for further understanding the biological roles of STP genes in cultivated peanut. Gene structure and conserved motif analysis Peanut STP gene structures were analyzed using TBtools software [33] via comparing the cDNA to the gene sequence. Conserved motifs in peanut STP proteins were analyzed using MEME programs (http://meme-suite.org/tools/meme) with the following parameters: optimum width, 20-60; number of repetitions, any; maximum number of motifs, 12.

Synteny analysis
Multiple Collinearity Scan toolkit (MCScanX) was adopted to analyze the STP gene duplication events, with the default parameters. The circos map of peanut STP genes was constructed to reveal the synteny relationship between cultivated peanut STP genes by TBtool software [49]. The syntenic maps were constructed using Tbtool software to reveal the synteny relationship of STP genes between cultivated peanut and selected plants. Non-synonymous (Ka) and synonymous (Ks) substitution of each duplicated STP gene pairs were calculated using DnaSP 6.0 [50]. The divergence time was estimated according to the published method: T = Ks/2*m, in which m means molecular clock [32,33]. The average mutation rate for Arachis was considered as 8.12 × 10 − 9 mutations per base per year, according to the previous work [32,33].

Gene expression Pro le Analysis
To explore the expression pro les of AhSTPs in 22 different peanut tissues of Tifrunner, the FPKM (fragments per kilobase per million fragments mapped) values of the AhSTP genes were download from the peanut developmental transcriptome map dataset in Peanutbase (https://peanutbase.org/) (BioProject ID: PRJNA291488) submitted by Clevenger [35]. The 22 peanut tissues, including leaf, root, oral organ, peg, pod, were described in Table S4. The heatmap of AhSTPs expression pro les was performed using the Tbtools software [49] based on the normalization method of Z-score standardization. The gene expression atlas for fastigiata subspecies of cultivated groundnut were download from National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (BioProject ID: PRJNA484860) submitted by Sinha [36]. And the genes expression data of the seeds_5, seeds_15 and seeds_25 were used to analyze AhSTPs expression pro les in seed developmental stage.

Plant materials and gene variation analysis
Thirty-seven diverse peanut cultivars were used as plant material to detect the gene sequence variation, with 14 cultivars belonging to subsp. hypogaea and 23 cultivars belonging to subsp. fastigiata respectively. Detailed information for the 37 peanut cultivars was listed in Table S6. Genomic DNA was extracted from the young leaves of these cultivars using a modi ed cetyltrimethyl ammonium bromide (CTAB) method. Gene associated simple sequence repeats (genic-SSR) were identi ed using MISA with the default parameters and primers of the identi ed SSRs were designed using Primer 3 software described by Zhao [51]. PCR reactions were performed as previously described by Huang [52].

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