Identification and characterization of transposable element AhMITE1 in the genomes of cultivated and two wild peanuts

Background The cultivated peanut (Arachis hypogaea L., AABB) is an allotetraploid hybrid between two diploid peanuts, A. duranensis (AA genome) and A. ipaensis (BB genome). Miniature inverted-repeat transposable elements (MITEs), some of which are known as active nonautonomous DNA transposons with high copy numbers, play important roles in genome evolution and diversification. AhMITE1, a member of the MITE family of transposons, but information on the peanut genomes is still limited. Here, we analyzed AhMITE1, AuMITE1 and ApMITE1 in the cultivated (A. hypogaea) and two wild peanut (A. duranensis and A. ipaensis) genomes. Results The cultivated and the two wild peanut genomes harbored 142, 14 and 21 AhMITE1, AuMITE1 and ApMITE1 family members, respectively. These three family members exhibited highly conserved TIR sequences, and insertions preferentially occurred within 2 kb upstream and downstream of gene-coding and AT-rich regions. Phylogenetic and pairwise nucleotide diversity analysis showed that AhMITE1 and ApMITE1 family members have undergone one round of amplification bursts during the evolution of the peanut genome. PCR analyses were performed in 23 peanut varieties and demonstrated that AhMITE1 is an active transposon and that hybridization or chemical mutagenesis can promote the mobilization of AhMITE1. Conclusions AhMITE1, AuMITE1 and ApMITE1 family members were identified based on local BLAST search with MAK between the cultivated and the two wild peanut genomes. The phylogenetic, nucleotide diversity and variation copy numbers of AhMITE1, AuMITE1 and ApMITE1 members provides opportunities for investigating their roles during peanut evolution. These findings will contribute to knowledge on diversity of AhMITE1, provide information about the potential impact on the gene expression and promote the development of DNA markers in peanut. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-08732-0.


Introduction
The cultivated peanut (Arachis hypogaea L.), also known as groundnut, is an allotetraploid (2n = 4x = 40) resulting from the hybridization of two wild diploids A. duranensis (AA genome) and A. ipaensis (BB genome). Peanut is an important oil crop with global production of 53.64 Mt (with shells) from an area of 31.57 Mha [1]. Mainland China boasts the largest peanut production of 17.99 Mt (with shells), compared with 9.95 Mt (with shells) in India [1]. Molecular breeding has played an important role in improving peanut varieties, which demands the development of genomic resources like linked markers for various traits. In peanut, genetic diversity is affected by polyploidization and is the source of lower levels of polymorphism than in diploid species [2,3]. This limits the use of molecular marker-assisted breeding to enhance peanut production. Different types of markers should be developed and employed for diversity analysis with the completion of the assembly of peanut genome sequences [4,5]. Among these, transposable elements (TEs) are the most abundant components of the genome and are used as genetic markers in molecular breeding.
TEs are major components of many plant and animal genomes and have been found in virtually all species investigated to date [6,7]. Ds (Dissociation) was the first transposable element discovered by McClintock in maize [8]. TEs were once regarded as 'selfish DNA' , but increasing evidence suggests that TEs are important in the generation of structure, evolution of genomes [9], and the regulation of gene function [10,11]. In animals, Lynch et al. (2015) indicated that ancient TEs transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy [12]. Chuong et al. (2016) revealed that TEs, including endogenous retroviruses (ERVs), were involved in the regulation of essential immune functions [13]. TEs can be classified into class I and class II elements. The replication of class I elements, or retrotransposons and class II elements, DNA transposons, occurs through a 'copy-and-paste' mechanism and 'cut-and-paste' mechanism, respectively [14]. Class II elements are divided into two subclasses autonomous and nonautonomous [15], the difference is whether they contain transposases that mobilize TEs.
Miniature inverted-repeat terminal elements (MITEs) are short nonautonomous class II transposons that do not encode transposase [16][17][18]. MITEs possess a pair of conserved terminal inverted repeats (TIRs) and short target site duplications (TSDs) that are located outside of each TIR [19]. In many plant species including Arabidopsis, rice, maize, wheat, sorghum and peanut, MITEs have been reported and characterized [20][21][22][23]. MITE insertion has generated numerous polymorphisms, which have been exploited to develop molecular markers [24]. MITEs play an important role in gene regulation [25], as they preferentially accelerate the evolutionary process. In rice, the first active MITE mPing was identified in a slender glume mutant and also identified through genomic/computational analysis [18,26,27]. Tang et al. (2019) identified the active MITE mJing by analyzing a rice high-tilling dwarf (htd) rice mutant [28]. In maize, MITE insertion in the promotor of the gene ZmNAC111 is associated with seedling drought tolerance [29]. Previous studies have improved that TEs can influence nearby gene expression. Such as, an 82 bp MITE insertion in the ZmNAC111 promoter region was correlates with lower ZmNAC111 expression in maize [29]. A stowaway-like MITE embedded in the 3'-UTR of the agronomically important gene Ghd2 directly represses its protein synthesis, affecting grain number, plant height, and heading date in rice [30]. In peanut, an insertion of MITE in the ahFAD2B gene caused a frameshift, resulting in the high-oleate phenotype in Mycogen-Flavo and M2-225 mutants [22].  found the AhMITE1 in a gene for fatty-acid desaturase possessed excision activity [31]. Subsequently, 1039 AhMITE1 markers were developed and used for mapping [32][33][34]. With the completion of peanut genome sequencing, TEs have been found to account for approximately 74% of the assembled genome sequence [4,5]. Thus, the complete genome sequence offers excellent opportunities to study TEs and will contribute to our understanding of peanut diversity.
In this study, we used peanut sequencing data to determine the characteristics of AhMITE1. We identified the numbers of AhMITE1 copies in cultivated peanut and its wild ancestors A. duranensis (AA genome) and A. ipaensis (BB genome). Following the genomic sequence, phylogenetic tree analyses of AhMITE1 indicated that these elements clustered into five subfamilies. The genomic distribution and preferences of insertional loci were also investigated. The results improve understanding of the potential impact of AhMITE1 on differentiation in cultivated and wild peanut species.

Identification of AhMITE1s elements in peanut genomes
To investigate the AhMITE1 family in the genomes of peanut, we performed a BLASTN analysis of genome to identify AhMITE1 in the reference genome of A. hypogaea cv. Tifrunner and the two wild genomes of A. duranensis (AA genome) and A. ipaensis (BB genome). A previous study showed that the 205 bp AhMITE1 includes a 9 bp target duplication (TSD) and 25 bp terminal inverted repeats (TIRs) [22,31]. Finally, 142 copies of AhMITE1 harboring the TSD and TIR sequences in the cultivated peanut genome were identified, and these copies shared over 90% similarity (Supplementary Table 1). According to the order of chromosomes 1-20 and the degree of similarity on the same chromosome, we named these AhMITE1 elements AhMITE1_1 to AhMITE1_142 (Supplementary Table 1). We used TBtools to investigate the positions of AhMITE1 insertions in the peanut genome, and all AhMITE1 elements were randomly distributed among the 20 chromosomes ( Fig. 1 and  Table 1). However, these 142 AhMITE1 elements were unevenly distributed and were preferentially located at the ends of chromosome arms rather than in the central portions of chromosome (Fig. 1).
Similarly, AhMITE1 elements were also identified in the two wild genomes. Fourteen and 21 AuMITE1 (AuMITE1_1 to AuMITE1_14) and ApMITE1 (ApMITE1_1 to ApMITE1_21) elements were obtained from the A. duranensis and A. ipaensis genomes, respectively (Supplementary Table 2). The nomenclature for AuMITE1 and ApMITE1 was the same as in the cultivated peanut genome. In the A. duranensis genome, all chromosomes contained AuMITE1 elements except for Aradu.A08 ( Supplementary Fig. 1A). In the A. ipaensis genome, all chromosomes contained ApMITE1 elements, and the chromosome Araip.B01 exhibited the highest number, with 4 ApMITE1 elements ( Supplementary  Fig. 1B).

Analysis of the TIR and TSD sequences of AhMITE1 in the peanut genome
Through alignment of the TIR and TSD sequences of AhMITE1 in the A. hypogaea genome, we found that most of the AhMITE1 elements contained a 25 bp conserved TIR with the sequence 5'-GGT GGA TAC TAC AAT GAA GAT GGC A-3' ( Fig. 2A and B). Almost all of the 9 bp TSD sequences in the 142 copies of AhMITE1 were different, whereas most of them preferred the TTA TTT TAA target site sequences (Fig. 2C).
In the two wild genomes, the TIR and TSD sequences of 14 AuMITE1 and 21 ApMITE1 elements were also analyzed. We found that the TIR and TSD sequences were similar to those in the A. hypogaea genome (Supplementary Fig. 2A, B, D and E). However, in the A. duranensis and A. ipaensis genomes, the TSD preferred AAA AAA TAA/TAA AAA TAA and TTA ATA AAA/TTA TAA AAA (Supplementary Fig. 2C and F).

Analysis of AhMITE1 sequence insertion sites in the peanut genome
Previous studies showed that approximately 45.83% (33 of 72) and 52.0% (133 of 256) of mJing and mPing insertions were within 2 kb and 3 kb of a coding regions, respectively [28,35]. Investigation of the locations of 142 AhMITE1 insertions in the A. hypogaea genome showed that 51 insertion events (35.92%) occurred 2 kb upstream and downstream of gene-coding regions, and 25 (17.61%) and 29 events (20.42%) were located in intergenic and intron regions, respectively (Fig. 3). In addition, 4 (2.82%) and 2 (1.41%) insertion events occurred in the 5' and 3'-UTRs, respectively, no insertion events occurred in exonic regions (Fig. 3). Perhaps these insertion events that occurred in introns do not affect gene function. The location preference of AhMITE1 insertions is consistent with the characteristics of mJing and mPing insertions in rice. The results suggested that the insertion of AhMITE1 elements also occurred preferentially in promoter, intergenic and intron regions. As a control, 142 randomly selected sequences were preferentially inserted into intergenic, exonic and intronic regions rather than the promoter regions ( Supplementary Fig. 3, Supplementary Table 5).
In the A. duranensis and A. ipaensis genomes, we found that the locations of insertion events were similar to those in A. hypogaea. In the A. duranensis genome, 2 (14.29%) and 3 (21.43%) insertion events occurred in introns and 2 kb upstream or downstream of genecoding regions, respectively (Fig. 3B). In the A. ipaensis genome, 7 (33.33%) and 6 (28.57%) insertion events were located in introns and 2 kb upstream or downstream of gene-coding regions, respectively (Fig. 3C). Whether in the cultivated or the two wild peanut genomes, no insertion events occurred in exonic regions (Fig. 3).

Analysis of GC content of AhMITE1 sequences in the peanut genome
Previous studies have demonstrated that GC content averages 36.3589%, 36.0214% and 37.1722% in the A. hypogaea, A. duranensis and A. ipaensis genomes, respectively [4,36,37]. To determine whether the insertion of AhMITE1 affects upstream and downstream sequences, sliding-window analysis of GC content at the AhMITE1 insertions was conducted. One hundred upstream and downstream genome sequences close to the position of each AhMITE1 insertion were examined, while 142 sequences 200 bp in length were randomly selected as a control (Supplementary Table 5). The results showed that the flanking regions near the AhMITE1 insertion sites contained GC contents than those randomly selected 200 bp genome sequences in A. hypogaea (Fig. 4A). In addition, the GC contents of AuMITE1 and ApMITE1 were also lower than in the randomly selected sequences in the two wild peanut genomes (Fig. 4B, C, Supplementary Tables 6 and 7). Taken together, these results suggest that AhMITE1, AuMITE1 and ApMITE1 are all preferentially inserted into T/A rich regions in the peanut genome, which is consistent with the characteristics of other MITE family members [35].

Sequence comparison of the AhMITE1 family in the peanut genome
AhMITE1 elements exhibited significant similarity in their internal sequences (Fig. 5A, Supplementary Fig. 4 and Supplementary Table 8). As with AhMITE1 family, AuMITE1 and ApMITE1 members also exhibited high similarity in the two wild genomes (Supplementary Figs. 5 and 6). Comparing the similarities of AhMITE1, AuMITE1 and ApMITE1 sequences among the cultivated and two wild peanut genomes, we found that the AhMITE1 family exhibited higher similarity in the A. hypogaea genome (Fig. 5A-C, Supplementary Table 9). We suspect that a one-time burst of AhMITE1 family expansion occurred in the cultivated genome, similar to the expansion of the mPing family in rice.

Phylogenetic analysis of the AhMITE1 family in the peanut genome
Phylogenetic analysis of AhMITE1 family members separated these sequences into four clades (I, II, III, IV). Clade I was the largest branch with 120 members. Clade I and Clade II comprised 120 AhMITE1 members and only one member, respectively, and were two subgroups from one branch (Fig. 6). Clade III and Clade IV contained 4 and 7 members, respectively (Fig. 6). In addition, three phylogenetic trees of 14 AuMITE1 and 21 ApMITE1 members from the A. duranensis and A. ipaensis genomes were also constructed, respectively. 14 AuMITE1 members were separated into three clades ( Supplementary Fig. 7A), and 21 ApMITE1 members were divided into five clades ( Supplementary Fig. 7B). However, in Supplementary  Fig. 7C, 14 AuMITE1 and 21 ApMITE1 members were clustered into two Clades, 14 AuMITE1s were clustered into one Clade and 20 ApMITE1s were clustered into another Clade (Supplementary Fig. 7C), implying that the amplification of AuMITE1 and ApMITE1 members might occurred after the differentiation between AA and BB genomes.
To investigate the amplification of AhMITE1, AuMITE1 and ApMITE1 members in the peanut genome, we calculated the pairwise nucleotide diversity. AhMITE1, AuMITE1 and ApMITE1 members exhibited similar peak distributions (Fig. 7). The histogram for the AhMITE1, AhMITE1 and AhMITE1 family has only the front face of a wave, centered at diversity = 0, the low nucleotide diversity indicated that these families are still under rapid amplification, like mPing family in rice [25,35]. The AhMITE1, AhMITE1 and AhMITE1 families with unimodal distribution of pairwise nucleotide diversity have phylogenetic trees of a star shape (Supplementary Fig. 8).

Validation of AhMITE1 insertions in PCR amplification
There are differences in the numbers of AhMITE1 members copies among cultivated peanut varieties. Based on the polymorphism of 142 AhMITE1 members in cultivated peanut, 12 AhMITE1 insertions on the 20 chromosomes were selected and verified by PCR using 23 varieties (Supplementary Table 10).
These different accessions of peanut may be polymorphic in their presence or absence of AhMITE1 ( Fig. 8 and Supplementary Fig. 9). For example, there are no AhMITE1 insertions in the peanut varieties LH11 and YH1, and a homozygous insertion occurred in YH18, which is the hybrid progeny of LH11 and YH1 at the location Arahy.18:48,326,860.0.48327510 (Supplementary Fig. 9). In the mutant lines S19-2, S24-1, S24-6 and S24-11 derived from parental HTY22 by chemical mutagenesis, a homozygous AhMITE1 insertion were present at Arahy. In this study, the effect of AhMITEs on the expression of genes was assessed and compared it between cultivated species. We found that AhMITE1 insertions occurred in some cultivated species, and some did not. We compared the expression levels of adjacent genes (Arahy.7UQ7HQ and Arahy.8H8JUA), and found that AhMITEs insertion into the promoter of  Our findings suggest that the insertion of AhMITEs can influence nearby gene expression.

Discussion
In the present study, 124, 14 and 21 AhMITE1, AuMITE1 and ApMITE1 members were identified from the A. hypogaea, A. duranensis and A. ipaensis genomes, respectively. The representative 205 bp AhMITE1 sequence was AT-rich, with 9 bp TSDs and 25 bp TIRs, similar to those previously reported for peanut [22,31]. Comparison of TIR and TSD sequences revealed complete conservation of TIRs, but not TSDs. AhMITE1 preferentially inserted into the 2 kb upstream and downstream sequences of coding regions, especially T/A-rich regions, in the peanut genome. Pairwise nucleotide difference analyses demonstrated that AhMITE1 members underwent one round of amplification during peanut evolution.
Genome-wide analysis revealed that different copy numbers of AhMITE1, AhMITE1 and ApMITE1 were present in the genomes of cultivated and the two wild peanut genomes. The copy number for AhMITE1 was far greater than those for AuMITE1 and ApMITE1. Differences in the copy numbers of AhMITE1, AhMITE1 and ApMITE1 between cultivated and wild peanut might also affect amplification and selection during peanut evolution. In the A. hypogaea genome, the copy number of AhMITE1 identified was 142 members, which is significantly higher than those in the A. duranensis (14) and A. ipaensis (21) genomes. DNA methylation, generally considered to be a heritable epigenetic modification that functions in silencing TEs, plays roles in the maintenance of genome stability, genetic imprinting and the regulation of gene expression [38][39][40][41]. Methylation was lower in the A subgenome than in the B subgenome [5]. Thus, there may be other reasons, such as chromosome structure, chromosome inversions and small RNAs, that caused the copy number differences in the AA and BB genomes. These differences in copy number suggest that the AhMITE1, AuMITE1 and ApMITE1 members maintained transpositional activation after the differentiation of cultivated and wild peanut.
The AhMITE1 members preferentially inserted within 2 kb upstream or downstream of the gene-coding and genetic regions, and no insertions occurred in exons of the gene regions. Previous studies have also demonstrated that the insertion of both mJing and mPing elements were preferentially in the flanking region of the gene [28]. In addition, 142 AhMITE1 elements were preferentially located at the ends of chromosome arms rather than in the central regions of chromosomes. Bertioli [4]. Taken together, we suspect that this may be the reason why AhMITE1 elements preferentially inserted into the promoters of gene-coding regions and were located in the distal chromosome regions. As for MITEs, the density of genes is higher in distal regions of chromosomes.
The nucleotide diversity among AhMITE1 elements may reflect the amplification characteristics during peanut evolution. The pairwise nucleotide diversity of AhMITE1, AuMITE1 and ApMITE1 members in cultivated and wild peanut revealed the occurrence of different amplification pattern bursts during peanut evolutionary history, which is similar to patterns observed in the MITE families in rice [25]. The pairwise nucleotide diversity of AuMITE1 is lower than that of AhMITE1 and ApMITE1 members indicating that AuMITE1 amplification occurred before AhMITE1 arose during peanut genome evolution. Therefore, both phylogenetic and pairwise nucleotide difference analyses demonstrated that AhMITE1, AhMITE1 and AhMITE1 families have experienced one-time burst expansion during peanut evolution. In Supplementary Fig. 7C, 14 AuMITE1 and 21 ApMITE1 members were clustered into two Clades, 14 AuMITE1s were clustered into one Clade and 20 ApMITE1s were clustered into another Clade (Supplementary Fig. 7C). These results suggested that the amplification of AuMITE1 and ApMITE1 members possibly occurred later than the differentiation of the two wild peanuts.
In our study, common and high-oleic-acid peanut varieties, such as HY20 and YH117, differed in AhMITE1 insertions ( Fig. 8 and Supplementary Fig. 9). This phenomenon also occurs in peanut varieties from different countries (Fig. 8 and Supplementary Fig. 9), such as AT215 and other varieties ( Fig. 8 and Supplementary  Fig. 9). In addition, there were no AhMITE1 insertions in the peanut varieties LH11 and YH1, and a homozygous insertion occurred in YH18, which is the hybrid progeny of LH11 and YH1, at the location Arahy.18: 48,326,860.0.48327510 (Supplementary Fig. 9). The detected de novo insertions in the hybrid progeny and mutant lines suggested that the mobilization of AhMITE1 might be induced by hybridization. Therefore, this study provides evidence to support the hypothesis that transposons are activated by "genome shock" due to plant-wide hybridization [41].
In peanut, DNA polymorphism is very low due to the narrow genetic diversity, which limits the development of markers [42][43][44]. The very limited genotypic polymorphism despite enormous phenotypic differences among peanut genotypes signifies the requirement for a large number of markers [45]. In many studies, have showed that different patterns of MITE insertions in germplasms or individuals have been used as DNA markers in plants [28,[46][47][48]. Thus, the development of AhMITE1 members as DNA markers is worth considering. Targeted high-throughput sequencing is an efficient method for identifying the insertion positions of specific MITE members in the genomes of different peanut varieties [28]. Finally, these polymorphisms involving the presence or absence of AhMITE1 at loci could be used to develop DNA markers. The variation in AhMITE1 copy numbers contributes to our understanding of peanut diversity. Therefore, our identification of AhMITE1, AuMITE1 and ApMITE1 members provides opportunities for investigating their roles during peanut evolution.

Identification of peanut AhMITE1, AuMITE1 and ApMITE1 members in the peanut genome
Both the cultivated (A. hypogaea cv. Tifrunner) and two wild peanut (A. duranensis and A. ipaensis) reference genome sequences were downloaded from the peanut genome database (PeanutBase, https:// peanu tbase. org/ data/ public/). A local BLAST search with MAK was performed using the 205 bp AhMITE1 as a query [49]. As a result, many elements were aligned. Then, the following settings were used length = 205 bp, E-value < 10 -71 and the similarity > 90.00%. The target sequences were discarded if they did not satisfythese conditions. In

Analyses of the chromosomal distribution of AhMITE1, AuMITE1 and ApMITE1 members
All AhMITE1, AuMITE1 and ApMITE1 elements were mapped to chromosomes based on physical location information from the peanut genome database (A. hypogaea, A. duranensis and A. ipaensis genome sequences) using TBtools (Graphics/Show Genes on Chromosome/Gene Location Visualize (advanced)) [50].

Multiple sequence alignment of AhMITE1, AuMITE1 and ApMITE1 members
A multiple sequence alignment of AhMITE1, AuMITE1 and ApMITE1 members was performed using CLUSTALW with default parameters [51]. TSD and TIR sequences was truncated from the 142, 14 and 21 members of AhMITE1, AuMITE1 and ApMITE1. Nine bp and 21 bp TSD and TIR sequences of AhMITE1, AuMITE1 and ApMITE1 were depicted using WebLogo 3.0, respectively [52].

Insertion preferences of AhMITE1, AuMITE1 and ApMITE1 members
To analyze the insertion preferences of AhMITE1, AuMITE1 and ApMITE1 members, 142 AhMITE1, 14 AuMITE1 and 21 ApMITE1 insertion sites were aligned with the A. hypogaea, A. duranensis and A. ipaensis genomes (https:// peanu tbase. org/ gbrow se_ peanu t1.0), respectively. Based on each distance between an insertion site and the annotated gene, the position of insertion was categorized as being in the 3'-UTR, 5'-UTR, exon or intron, upstream or downstream of the annotated gene coding region. If the distance between the insertion site and the annotated gene exceeded 8 kb (> 8 kb), the insertion site was categorized as being in an intergenic region.

Analysis of GC content
Sample 100 bp upstream and downstream sequences with TSDs of 142 AhMITE1, 14 AuMITE1 and 21 ApMITE1 members were extracted from the genomes of A. hypogaea, A. duranensis and A. ipaensis, respectively. In addition, 142, 12 and 21 of sequences 205 bp in length were randomly selected with TBtools (Sequence Toolkit/Fasta Tools/Fasta Extract (Basic)) from the A. hypogaea, A. duranensis and A. ipaensis genomes, respectively [50]. A PERL script (Supplementary Table 11) was used to calculate the GC content in each 5-bp sliding-window at a 1 bp increment.

Estimation of pairwise diversity
To investigate the amplification of AhMITE1, AuMITE1 and ApMITE1 members in the peanut genome, we calculated the pairwise nucleotide diversity for each species. Pairwise nucleotide diversity among AhMITE1, AuMITE1 and ApMITE1 elements in the A. hypogaea, A. duranensis and A. ipaensis genomes was calculated using a PERL script and MEGA [53]. If there was a gap in the sequence alignment, each gap was considered to be a single mismatch. The obtained pairwise nucleotide diversity values were plotted with OriginPro 2018 software. The frequency distribution of pairwise nucleotide diversity was used to describe the amplification patterns during peanut genome evolution.

Phylogenetic analysis
In total, 142 AhMITE1, 14 AuMITE1 and 21 ApMITE1 members from A. hypogaea, A. duranensis and A. ipaensis genomes were aligned using MUSCLE, respectively [54]. The alignment results were used to construct a phylogenetic tree in MEGA 6.0 by the neighbor-joining (NJ) method with a bootstrap test of 1000 replications [38], and visually enhanced by Evolview [55].

PCR amplification of AhMITE1 insertion sites
To validate the AhMITE1 insertions in the cultivated peanut genomes, PCR primers were designed based on the flanking sequences of the AhMITE1 insertion. PCR amplifications were performed using 10 ~ 50 ng peanut genomic DNA in a 5 μL reaction volumes containing 0.2 µL of LA Taq ® DNA polymerase (1 U), 0.2 µL of dNTP (200 mM), 1 µL of 10 × PCR buffer and 1 µL each of forward and reverse primers (30 μM). The PCR protocol was performed with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 45 s, and a final extension at 72 °C for 10 min. The products were separated using polyacrylamide gel electrophoresis (PAGE) in 1% (w/v) agarose gels to distinguish the presence or absence polymorphisms of AhMITE1 insertions in the cultivated peanut genomes.

RNA extraction and RT-PCR
Total RNA from the leaves was extracted using TRIzol reagent (Thermo Fisher Scientific) and purified using a Qiagen RNeasy Kit (Qiagen, Hilden, Nordrhein-Westfalen, Germany) according to the manufacturer's instructions. First-strand cDNAs were synthesized using SuperScript RT Kit (Thermo Fisher Scientific) with an oligo(dT) [12][13][14][15][16][17][18] primer. RT-PCR was performed using a CFX96 real-time system (Bio-Rad, Hercules, CA) and the peanut expressing gene U6 was used as the internal control to normalize the gene expression data. Relative expression levels were calculated from three biological replicates. qRT-PCR was performed with a CFX96 realtime system (Bio-Rad, Hercules, CA). All reactions were performed with three biological replicates. Statistical analysis and sample comparisons were performed using the relative quantification method (2 −ΔΔCT ).

Primers
The primers used in this study are listed in Supplementary Table 12.