Identification and expression analysis of the small auxin-up RNA (SAUR) gene family in Lycium ruthenicum

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Bioinformatics and Genomics

Introduction

Plants in arid desert regions are exposed to severe environmental conditions, and most desert plants are unique and precious. These plants possess constitutive and inducible defense barriers to counteract stresses and have formed various special structures, such as cuticles, waxy layers, trichomes, and spines, during the long process of evolution (Ménard et al., 2013). Lycium ruthenicum Murr. (black goji) belongs to the Solanaceae family, and is a unique nutritional and medicinal food mainly distributed in the salinized desert of northwestern China (Liu et al., 2012). Due to its nutritional, medicinal, and ecological value, L. ruthenicum has attracted widespread attention. Stem thorns are one of the important symbolic characters of L. ruthenicum. Lu, Wang & Fan (2014) found that compared with cultivated plants, wild L. ruthenicum had more and denser thorns. Furthermore, Zhang (2019) reported that L. ruthenicum did not grow stem thorns when cultured under the conditions of 100% and 80% water-holding capacity (WHC), while plants had a large number of stem thorns under 60% and 40% WHC treatments. These findings indicated that the formation of stem thorns was closely related to soil water content, and drought stress could promote the development of stem thorns in L. ruthenicum, suggesting that stem thorns may be a direct response mechanism to drought and one of the important strategies to resist drought stress. However, to date the molecular mechanism of thorn growth induced by drought has not been reported.

Stem thorns develop from axillary buds and can develop into branches under appropriate conditions. Therefore, factors affecting axillary bud development may play an important role in regulating the occurrence of stem thorns. Plant hormones play a key role in axillary meristem formation and the activation and growth of dormant axillary buds (Waldie & Leyser, 2018). Additionally, auxin influences nearly all aspects of plant growth and development by regulating cell division, expansion, differentiation, and patterning via regulating the expression of genes (Ren & Gray, 2015). Among auxin response genes, the small auxin-up RNA (SAUR) is the largest family of auxin early response genes, which are rapidly induced by auxin and encode plant-specific small proteins (Ren & Gray, 2015). SAURs are crucial regulators of diverse aspects of plant growth, development, and stress responses, such as root development, hypocotyl elongation, leaf growth and senescence, and response to drought, low temperature, disease and insect pests (Kant et al., 2009; Abbas, Alabadi & Blazquez, 2013; Vanhaeren et al., 2014; Ren & Gray, 2015). In Arabidopsis thaliana, the expression levels of AtSAUR36 and AtSAUR41 were closely related to cell expansion, and overexpression of these genes significantly elongated hypocotyl epidermal cells (Chae et al., 2012; Spartz et al., 2012; Spartz et al., 2017; Kong et al., 2013; Stamm & Kumar, 2013). SAUR32, SAUR19 and SAUR36 are mainly related to the formation of the apical hook, and overexpression seedlings have short hypocotyls in A. thaliana (Park et al., 2007; Spartz et al., 2012; Stamm & Kumar, 2013). AtSAUR41 and AtSAUR76 are related to root development, and the up-regulation of their transcription can promote taproot elongation and lateral root development (Kong et al., 2013; Markakis et al., 2013). The up-regulated expression of SAUR12, SAUR34, SAUR54, SAUR67, SAUR91, and SAUR97 in poplar improved the adaptability of seedlings to low temperature (Hu et al., 2018). In addition, SAUR family proteins are regulated by miR159, which promotes drought tolerance in wheat (Gupta et al., 2014). Moreover, SAUR30 is also related to drought adaptation in poplar (Chen, Hao & Cao, 2014). Research suggests that SAURs are crucial regulators of diverse aspects of plant growth and development. Furthermore, in A. thaliana, SAUR19 overexpression and SAUR19/23/24 amiRNA knockdown seedlings showed increased and decreased basipetal indole-3-acetic acid (IAA) transport in hypocotyls, respectively (Spartz et al., 2012). In contrast, the overexpression of OsSAUR39 in rice resulted in reduced IAA transport (Kant et al., 2009), indicating that SAUR proteins are capable of modulating IAA transport. Although SAUR family identification and expression analysis have been conducted in a variety of plants, the functions of the members of this gene family are largely unknown, and whether IAA and SAUR are involved in regulating the growth of stem thorns under drought conditions is not yet known. Hence, further study of SAURs to reveal their biological effects in L. ruthenicum is valuable.

In this study, the salt-xerophyte L. ruthenicum, which produces a large number of stem thorns under drought conditions, was used as a material. SAUR family members were identified and the tissue expression patterns of genes in L. ruthenicum were analyzed. The findings of this study provide valuable information on the stress-response profiles of SAUR genes in L. ruthenicum and lay a solid foundation for elucidating the functions of SAUR genes in regulating the generation of stem thorns.

Materials and Methods

Plant growth conditions and treatments

L. ruthenicum seeds were collected from Minqin County (101°59′E–104°12′E, 38°08′N–39°26′N), in Gansu Province of northwest China. Seedlings were cultured as described by Hu et al. (2022) with minor modifications: when seedlings grew to about 1 cm, they were transplanted into plant pots (20 cm3; three seedlings/container) filled with nutrient soil. Seedlings with consistent growth were selected after 20 days of cultivation and then sprayed with 0, 25, and 50 mg/L IAA every day for 60 days. To minimize the effects of possible environmental gradients in the greenhouse, pots were randomly reassigned to new positions every day.

Transcriptome analysis

Seedlings of L. ruthenicum were cultured for three weeks and then divided into two treatments: 80% WHC and 40% WHC. After treatment about for 60 days, seedlings were divided into treatment groups as follows: (i) D1: Non-thorn stem nodes near the apex under the treatment of 40% WHC. (ii) D2: Sprout-thorn stem nodes under the treatment of 40% WHC. (iii) D3: Long green thorn stem nodes under the treatment of 40% WHC. (iv) C: Stem nodes under the treatment of 80% WHC taken from the same part as that of group (iii).

Four groups of samples were mixed or used separately for third or second-generation transcriptome sequencing (Biomarker Technologies Company, China). This experiment was performed with three biological replicates. Raw data were submitted to the public NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra, accession number SRR22514555).

Sequence database search and identification of the LrSAURs in L. ruthenicum

The potential annotated nucleotide sequences of LrSAURs were downloaded from the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra, accession number SRR22514555). First, SAUR protein sequences from A. thaliana were collected (https://www.uniprot.org/) to construct a local BLASTP protein database (with an E-value cut off of 10−10 and an identity of 50%). Next, a hidden Markov Model (HMM) was constructed with the HMMER 3.0 program to find all predicted SAUR family members of L. ruthenicum. Then, the aligned sequences were considered as candidate SAUR family sequences, and the candidate LrSAURs were named beginning with “Lr” for L. ruthenicum. ProtParam (http://web.expasy.org/protparam/) was used to analyze the physicochemical parameters (length, molecular weight, and isoelectric point) of the candidate LrSAURs. Eighty-three A. thaliana protein sequences were downloaded from https://www.arabidopsis.org/, and fifty-eight rice (Oryza sativa) SAUR genes (OsSAURs) were searched according to the Data S1 in a previous report (Jain, Tyagi & Khurana, 2006). Multiple alignment using fast Fourier transform (MAFFT: http://mafft.cbrc.jp/alignment/software/) was used to analyze the multiple sequence alignment among these LrSAURs. A tree was constructed using the neighbor-joining (NJ) method in MEGA 7.0 with partial deletion and the p-distance model.

Structural characterization and heatmap analysis of LrSAURs

The MEME program was used to identify the conserved protein motifs of LrSAURs, with optimum motif widths of 6–50 residues and a maximum of 10 motifs. For the analysis of gene expression, the number of clean tags for each gene was calculated and normalized to fragments per kilobase of transcript per million mapped reads (FPKM): FPKM = {cDNA Fragments \over {Mapped Fragments (Millions) * Transcript Length (kb)}}. Heatmaps of genes in the different stem thorn development stages were generated based on their FPKM values using the TBtools (v0.67373) software (https://github.com/CJ-Chen/TBtools).

RNA extraction, cDNA synthesis, and quantitative real-time PCR (RT-PCR) analysis

Total RNA was extracted with a Trizol Kit R6827-01 (Omega Bio-Tek, USA) according to the manufacturer’s instructions from the roots, stems, stem thorns and leaves of three-month-old L. ruthenicum seedlings. A NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA) was used to evaluate the RNA quantity and quality (A260 nm/A280 nm: 1.8–2.0 for purity; 500–1000 ng/µL for concentration). The Evo M-MLV RT Kit AG11728 (Accurate Biotechnology, Changsa, China) was used to reverse-transcribe the total RNA into cDNA according to the instructions. Twenty-one LrSAURs were selected from the 33 genes according to the FPKM values (FPKM ≥ 5 at least one of the thorn developmental stages) for the RT-qPCR analysis of the tissue expression. The reference gene used was LrEF1 α (JX427553) (Tiika et al., 2020). The primer pairs are shown in Table S1. RT-qPCR analysis was performed using the SYBR Green Premix Pro Taq HS (Accurate Biotechnology, Changsha, China) according to the manufacturer’s protocol. Analysis was run on the QuantStudio 5 Real-Time PCR Instrument (ABI) (Life Technologies Holdings). The cycling parameters were 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The data was quantitated using the 2−ΔΔCt method (Livak & Schmittgen, 2001). This experiment was performed with three biological replicates.

Data analysis

Values of the gene expression levels were presented as the means ± SE (n = 3). Data were analyzed using one-way analysis of variance followed by Duncan’s multiple range tests (p ≤ 0.05) (SPSS statistical software, Version 25.0; SPSS Inc., Chicago, IL, USA).

Results

Effects of IAA on the stem thorn development of L. ruthenicum

The plant heights under the 25 and 50 mg/L IAA treatments were taller than those under the control (CK). A large number of stem thorns appeared in CK plants, a small number of thorns appeared later in 25 mg/L IAA plants, and fewer thorns were found in the 50 mg/L IAA plants than in the 25 mg/L IAA treatment (Fig. 1).

Identification of LrSAUR s

A total of 33 putative LrSAURs with a central domain (PF02519) were acquired and named LrSAUR1LrSAUR33 (Table 1). The obtained gene lengths ranged from 548 bp (LrSAUR13) to 7237 bp (LrSAUR19), and the predicted amino acids ranged from 82 aa (LrSAUR23) to 586 aa (LrSAUR1) (Table 1). In addition, the theoretical isoelectric point (pI) of the LrSAURs ranged from 5.27 to 10.18, and the molecular weight (Mw) ranged from 9202.04 to 67625.88 Da.

Phylogenetic analysis of SAUR genes is an effective way to unveil the functions of uncharacterized SAURs, and 174 SAUR protein sequences from L. ruthenicum, Arabidopsis and rice were aligned to perform a phylogenetic tree (Fig. 2). According to the phylogenetic tree, these proteins can be divided into three groups based on their sequence similarities with orthologs in other plants (Fig. 2). Group I, II and III contained 16, six and 11 LrSAUR members, respectively. The OsSAUR proteins were mainly placed in Group I and II, most of AtSAURs were in Group I and II.

Effects of indole-3-acetic acid (IAA) (0, 25, and 50 mg/L) on stem thorn development in Lycium ruthenicum.

Figure 1: Effects of indole-3-acetic acid (IAA) (0, 25, and 50 mg/L) on stem thorn development in Lycium ruthenicum.

The thorns are encircled with yellow lines.
Table 1:
Statistical information of LrSAURs in L. ruthenicum.
Gene name Amino acids MW (Da) pI Instability index Aliphatic index GRAVY
LrSAUR1 586 67625.88 7.15 47.64 84.06 −0.621
LrSAUR2 338 38866.58 7.00 46.50 80.24 −0.653
LrSAUR3 397 44228.90 5.52 46.89 75.29 −0.527
LrSAUR4 199 22906.93 5.27 41.02 75.53 −0.734
LrSAUR5 219 24647.28 5.71 51.05 45.43 −1.533
LrSAUR6 228 25699.56 5.82 45.33 45.79 −1.560
LrSAUR7 224 25184.98 5.81 45.96 46.61 −1.521
LrSAUR8 237 26828.93 6.19 48.95 47.30 −1.530
LrSAUR9 198 22342.65 5.38 54.71 48.79 −1.530
LrSAUR10 214 24108.67 5.49 48.55 44.67 −1.556
LrSAUR11 122 14354.17 6.47 41.11 71.89 −0.694
LrSAUR12 127 14870.92 7.79 58.51 91.26 −0.670
LrSAUR13 86 9666.26 8.93 35.45 88.37 −0.079
LrSAUR14 85 9607.08 6.71 43.13 88.24 −0.074
LrSAUR15 86 9781.42 5.73 42.32 96.28 0.016
LrSAUR16 138 16012.81 8.89 48.48 88.91 −0.319
LrSAUR17 102 11522.63 9.52 36.56 101.18 0.160
LrSAUR18 86 9637.22 7.92 39.81 87.33 −0.077
LrSAUR19 86 9781.42 5.73 42.32 96.28 0.016
LrSAUR20 148 16725.27 9.79 65.03 82.30 −0.295
LrSAUR21 133 15525.56 6.55 28.80 82.78 −0.616
LrSAUR22 138 16012.81 8.89 48.48 88.91 −0.319
LrSAUR23 82 9202.04 10.18 29.87 112.80 0.323
LrSAUR24 85 9657.30 8.89 41.11 88.24 −0.076
LrSAUR25 83 9489.96 8.89 38.91 77.47 −0.349
LrSAUR26 100 11767.53 7.92 46.95 93.50 −0.543
LrSAUR27 95 10704.51 7.95 37.44 92.21 0.083
LrSAUR28 160 18312.90 9.10 29.72 61.50 −0.559
LrSAUR29 100 11709.49 8.61 49.42 93.50 −0.505
LrSAUR30 111 12919.77 8.62 50.90 79.82 −0.638
LrSAUR31 164 19089.01 10.06 40.92 74.21 −0.514
LrSAUR32 83 9457.93 6.06 44.07 92.77 −0.095
LrSAUR33 83 9549.19 8.89 37.88 86.75 −0.081
DOI: 10.7717/peerj.15941/table-1
Phylogenetic relationship of LrSAURs, AtSAURs, OsSAURs.

Figure 2: Phylogenetic relationship of LrSAURs, AtSAURs, OsSAURs.

LrSAURs from Lycium ruthenicum, AtSAURs from Arabidopsis thaliana, and OsSAURs from rice. The different colored arcs indicate different groups, and red circles, blue triangles and yellow stars represent LrSAURs, AtSAURs and OsSAURs, respectively.

MEME software was used to analyze the LrSAUR sequences, and a total of 10 conserved motifs were identified: motif 1–motif 10 (Fig. 3). As shown in Fig. 3, the number and type of conserved motifs contained in 33 LrSAUR proteins were different: one LrSAUR protein contained one conserved motif, one LrSAUR protein contained two conserved motifs, 21 LrSAUR proteins contained three conserved motifs, nine LrSAUR proteins contained four conserved motifs, and one LrSAUR protein contained seven conserved motifs. Motif 1 and motif 3 were widely distributed.

Prediction of conserved motifs of LrSAURs in Lycium ruthenicum.

Figure 3: Prediction of conserved motifs of LrSAURs in Lycium ruthenicum.

Thirty-three LrSAURs are used to predict the motifs by MEME. Different motifs and their position in each LrSAUR sequence are represented by different colored boxes.

Expression analysis of LrSAURs under drought treatment

The heatmap illustration of expression profiles of LrSAURs in different thorn developmental stages is shown in Fig. 4. Thirty-three LrSAURs were found to exhibit different expression patterns in L. ruthenicum. The clustering results showed that these genes could be divided into six patterns. LrSAUR1, LrSAUR3, LrSAUR14, LrSAUR15, LrSAUR16, LrSAUR18, LrSAUR22, LrSAUR24, LrSAUR26, and LrSAUR28 were at low expression levels at all developmental stages. The expression level of LrSAUR21 was up-regulated once the first thorn had grown and down-regulated when thorns grew long. The transcript abundance of LrSAUR9 was higher in the control, but it was down-regulated gradually with the development of stem thorns. LrSAUR32, LrSAUR2, LrSAUR20, LrSAUR23, LrSAUR13, and LrSAUR31 gradually increased with the emergence of thorns and then showed a downward trend. The expression abundance of LrSAUR29, LrSAUR30, LrSAUR27, and LrSAUR28 decreased first and then increased with the growth of thorns. Moreover, LrSAUR4, LrSAUR5, LrSAUR6, LrSAUR7, LrSAUR8, LrSAUR12, LrSAUR17, LrSAUR19, LrSAUR25, and LrSAUR32 had high expression levels in different stages of stem thorn development of L. ruthenicum.

Heatmap of LrSAUR s in different developing thorns.

Figure 4: Heatmap of LrSAUR s in different developing thorns.

The expression profiles of LrSAUR s are based on FPKM-values from the RNA sequencing data. Low and high expression levels of genes are indicated by blue and red boxes.

Twenty-one genes were selected from the LrSAURs for tissue expression analysis using RT-qPCR (FPKM ≥ 5) (Fig. 5). All the LrSAURs had different expression patterns: LrSAUR31 was mainly expressed in roots, while LrSAUR13, LrSAUR23, and LrSAUR32 were primarily expressed in stems and leaves. LrSAUR4, LrSAUR5, LrSAUR6, LrSAUR7, LrSAUR10, LrSAUR21, LrSAUR25, LrSAUR29, and LrSAUR30 were predominantly expressed in leaves. LrSAUR2, LrSAUR8, LrSAUR9, LrSAUR11, LrSAUR12, and LrSAUR19 were mainly expressed in stems and stem thorns.

Tissue expression profiles of LrSAURs in Lycium ruthenicum based on Real-time qPCR.

Figure 5: Tissue expression profiles of LrSAURs in Lycium ruthenicum based on Real-time qPCR.

Different lowercase letters indicate significant differences (p < 0.05).

To further verify the genes mainly expressed in stems and thorns, this work analyzed the expression levels of LrSAUR2, LrSAUR8, LrSAUR9, LrSAUR11, LrSAUR12 and LrSAUR19 from the C and D3 transcriptome samples (Fig. 6). Compared with the C treatment, LrSAUR9 was significantly down-regulated under treatment D3, while LrSAUR12 and LrSAUR19 were significantly up-regulated, and LrSAUR2, LrSAUR8, and LrSAUR11 showed no significant changes.

Expression profiles of LrSAUR2, LrSAUR8, LrSAUR9, LrSAUR11, LrSAUR12, LrSAUR19 in the C and D3 treatments.

Figure 6: Expression profiles of LrSAUR2, LrSAUR8, LrSAUR9, LrSAUR11, LrSAUR12, LrSAUR19 in the C and D3 treatments.

The expression profiles of LrSAUR s are based on FPKM-values from the RNA sequencing data. Different lowercase letters indicate significant differences (p < 0.05).

Discussion

Plant hormones play important roles in axillary meristem formation and activation and axillary bud outgrowth. Studies have indicated that auxin, cytokinin, and strigolactone are involved in regulating shoot branching (Waldie, McCulloch & Leyser, 2014; Wang et al., 2018). The polar transport of IAA from the stem apex to the base inhibits the growth of axillary buds, resulting in apical dominance. If the terminal bud is removed, axillary bud growth is activated; when IAA is applied to the site of terminal bud removal, axillary bud growth will be re-inhibited (Müller & Leyser, 2011). In the present study, it was found that compared with the control, plants treated with IAA were significantly taller, and with the addition of an increased concentration of IAA, the number of stem thorns decreased and the outgrowth time was delayed. Therefore, auxin had an inhibitory effect on the growth of stem thorns of L. ruthenicum.

Axillary bud growth is affected by plant hormones, while the role of plant hormones is regulated by related genes. Most early regulated auxin responsive genes are classified into three families: Aux/IAA, Gretchen Hagen3 (GH3), and SAUR (Hagen & Guilfoyle, 2002). Among these genes, SAURs are considered to be the most abundant. Since the first SAUR gene was identified in elongating soybean hypocotyl sections (McClure & Guilfoyle, 1987), with the availability of genome sequences, genome-wide analysis has been broadly applied, including in the identification and expression profiling of SAUR genes in Arabidopsis (Hagen & Guilfoyle, 2002), rice (Jain, Tyagi & Khurana, 2006), sorghum (Glycine max; Wang et al., 2010), tomato (Solanum lycopersicum; Wu et al., 2012), potato (Solanum tuberosum; Wu et al., 2012), maize (Zea mays; Chen, Hao & Cao, 2014), citrus (Xie et al., 2015), and ramie (Boehmeria nivea; Huang et al., 2016). However, there was no reference genome database of L. ruthenicum available. In the present study, the SAUR family analysis in L. ruthenicum was mainly based on transcriptome data, and a total of 33 possible SAUR gene members were identified. Previous reports have indicated that SAURs contain a central domain (PF02519) (Marchler-Bauer et al., 2013), which is highly conserved. The results of the present study confirmed this finding and provided the first systematically identified SAUR family members in L. ruthenicum.

According to the MEME analysis results, motif 1 and motif 3 are highly conserved in the SAUR family in L. ruthenicum (Fig. 3), and may play an important role in the regulation of plant growth and development. The sequence from the N-terminal to the C-terminal was motif 1 and motif 3. However, no motif existed in all family members. Several studies showed that most members of the SAUR proteins have been found to have three highly conserved motifs, RFVIPJSFLSHPLFQDLLSQAEEEFGF, VPKGHFAVYVGE and VPKGHLAVYVG (Wu et al., 2012; Wang et al., 2020; Liu et al., 2022), which were consistent with our results. And RFVIPJSFLSHPLFQDLLSQAEEEFGF was the domain of auxin-inducible superfamily in LrSAURs (Gan, 2019). Kodaira et al. (2011) divided AtSAURs into three classes based on their evolutionary relationships in A. thaliana. In the present study, LrSAURs were also divided into three groups (Fig. 2). In addition, evolutionary tree analysis showed that LrSAURs in the same branch had a similar number of motifs and sequences, and there were significant differences among different subgroups (Fig. 2), which may have been due to the acquisition or loss of conserved motifs in the evolutionary process of the SAUR family. Previous studies have shown that stem thorns may be related to drought tolerance in L. ruthenicum (Lu, Wang & Fan, 2014; Zhang, 2019), and the absence of AtSAUR32 in A. thaliana may reduce drought tolerance (Kurihara et al., 2021). Guo et al. (2019) found that the overexpression of the SAUR gene could enhance tolerance to salt stress, drought stress, and low temperature stress in wheat. Therefore, the expression patterns of LrSAUR in response to drought stress were analyzed in combination with transcriptome data in L. ruthenicum, and a total of 14 LrSAURs were found to have significant changes (Fig. 4). Wang et al. (2012) showed that SAUR genes were regulated by IAA and brassinosteroid. Therefore, LrSAURs may play an important role in regulating the growth and development of stem thorns in L. ruthenicum under drought conditions. Furthermore, LrSAUR4, LrSAUR5, LrSAUR6, LrSAUR7, LrSAUR8, LrSAUR12, LrSAUR17, LrSAUR19, LrSAUR25 and LrSAUR32 had high expression levels in different stages of stem thorn development (Fig. 4). These results suggest that these genes may be involved in various physiological metabolic processes during the development of L. ruthenicum.

This study also investigated the gene expression patterns of 21 LrSAURs in various tissues. The number of LrSAURs expressed mainly in the leaves was the highest, while the number of genes expressed mainly in the roots was the lowest (Fig. 5), which was similar to previous results in tomato (Wu et al., 2012). In addition, in this study, six LrSAURs were highly expressed in stems and thorns (Fig. 5). In cotton, four of 12 SAURs were also mainly expressed in the stems (Li et al., 2017). Previous studies have shown that AtSAUR32 is mainly related to apical hook formation in Arabidopsis seedlings, and its overexpression causes the apical hook to disappear, while atsaur32 mutants restore the curved hook phenotype. Notably, AtSAUR32 is predominantly expressed on the inner side of the apical hook (Park et al., 2007), which provides direct evidence that SAURs function as important regulators of plant accessory structure formation. In the present study, it was found that LrSAURs were mainly expressed in stems and thorns, suggesting a putative role in regulating the growth of stem thorns in L. ruthenicum. Additionally, compared with the control (C), the expression levels of LrSAUR9, LrSAUR12, and LrSAUR19 changed significantly under the D3 treatment (Fig. 6). More study is necessary to further verify the functions of these three LrSAUR genes in the development of stem thorns.

Supplemental Information

Raw data of SAUR sequences and expression data

DOI: 10.7717/peerj.15941/supp-1

Primers of Real-time qPCR

DOI: 10.7717/peerj.15941/supp-2
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