Dev Genes Evol DOI 10.1007/s00427-015-0506-3
ORIGINAL ARTICLE
Identification and expression dynamics of three WUSCHEL related homeobox 13 (WOX13) genes in peanut Pengfei Wang 1,2 & Changsheng Li 1,2 & Cui Li 1,2 & Chuanzhi Zhao 1,2 & Han Xia 1,2 & Shuzhen Zhao 1,2 & Lei Hou 1,2 & Chao Gao 1,2 & Shubo Wan 1,2 & Xingjun Wang 1,2
Received: 17 March 2015 / Accepted: 9 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract WUSCHEL-related homeobox (WOX) genes play key roles in plant stem cell maintenance and development. WOX genes showed specific expression patterns which are important for their functions. WOX13 subfamily genes as the ancestor genes of this family were less studied in the past. In this study, we cloned three Arachis hypogaea (peanut) WOX13 (AhWOX13) subfamily genes from peanut: WOX13A and WOX13B1, 2. WOX13B1 encoded a same protein as WOX13B2, and there were only two-base difference between these two genes. Differential expression patterns were observed for these three AhWOX13 subfamily genes in different tissues and developmental stages. Phylogenic trees analysis showed that these AhWOX13 subfamily genes were the most conserved WOX genes and belonged to the ancient clade of WOX family. This was also supported by the conserved motif analysis. Selective pressure analysis showed that the WOX family genes mainly underwent weak purifying selection (ω=0.58097), while many positive mutations accumulated during the evolution history. Under the purifying Communicated by Sureshkumar Balasubramanian Pengfei Wang and Changsheng Li contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00427-015-0506-3) contains supplementary material, which is available to authorized users. * Shubo Wan [email protected] * Xingjun Wang [email protected] 1
Bio-Tech Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, People’s Republic of China
2
Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan 250100, People’s Republic of China
selection, gene duplication event and loss of duplicated gene play important roles in the expansion and evolution of WOX family. Keywords Peanut . WOX13 subfamily gene . Stem cell . Evolution . Selective pressure
Introduction Peanut (Arachis hypogaea L.) is native in South America and grown worldwide. The world annual production of peanut is about 38 million tons (Bertioli et al. 2011). Cultivated peanut is allotetraploid (AABB, 4n=4x=40) which originated from a single hybridization event between AA and BB wild species and subsequently underwent spontaneous genome duplication (Stalker and Simpson 1995; Moretzsohn et al. 2013; Kochert et al. 1996; Ferguson et al. 2005; Freitas et al. 2007; Gregory and Gregory 1979; Hammons 1982; Knauft and Gorbet 1995). Peanut gynophore is a specialized and unique organ that carries and eventually pushes the fertilized ovules into the soil in order for seed and fruit development to occur (Xia et al. 2013; Moctezuma 2003). The peanut gynophore has a typical shoot anatomy and responds positively to gravity, similar to a typical primary root (Moctezuma and Feldman 1998; Moctezuma and Feldman 1999). The activity of an intercalary meristem located proximal to the seed region contributes to the elongation and downward growth of the gynophore (Jacobs 1947; Shushu and Cutter 2011). The peanut gynophore intercalary meristem resembles the shoot apical meristem (SAM) and root apical meristems (RAM). These meristems are responsible for organ formation (Laux 2003; Hedman et al. 2013), and the maintenance of meristem depends on selfrenewal of the stem cell population (Schoof et al. 2000).
Dev Genes Evol
WUSCHEL-related homeobox (WOX) genes including WUSCHEL (WUS) and WOX1-WOX14 genes play key roles in stem cell maintenance. Members of the WOX family are involved in the stem cell maintenance in meristems, embryo apical-basal polarity patterning, and development of lateral organs (Zhao et al. 2009a, b; Lin et al. 2013; Ikeda et al. 2009; Lie et al. 2012; Liu et al. 2014). Furthermore, several studies suggest that the WOX gene family may be involved in the evolution of developmental processes, such as the evolution of novel shoot-leaf mixed organs in Podostemaceae (Hedman et al. 2013). Based on phylogenetic analysis and their distribution in different plant, WOX family genes have been classified into three clades. The first clade is the modern clade (found in seed plants) including WUS, WOX1, WOX2, NS/WOX3, WOX4, WOX5, WOX6, and WOX7 orthologue. The second is the intermediate clade (found in vascular plants including lycophytes), including WOX8, 9, 11, and 12 orthologue. The third one is the ancient/basal clade (found in vascular and nonvascular plants including mosses and green algae), including WOX13 subfamily (wox10, 13, and 14) (Hedman et al. 2013; Nardmann et al. 2009; Zhang et al. 2010; Van der Graaff et al. 2009). WUS gene is involved in the stem cell maintenance in SAM and functions in a non-cell autonomous manner to control the stem cell fate (Mayer et al. 1998). WOX3 gene is involved in the stem cell maintenance in leaf marginal meristems (Shimizu et al. 2009; Nakata et al. 2012) and in development of lateral sepals and stipules (Lin et al. 2013; Nardmann et al. 2004; Matsumoto and Okada 2001). WOX4 gene is involved in the stem cell maintenance in the vascular (cambial meristem, CAM) (Hedman et al. 2013; Hirakawa et al. 2010; Ji et al. 2010). WOX5 gene plays key roles in stem cell maintenance in the RAM (Wu et al. 2005a). WOX8 and WOX9 redundantly regulate zygote development and embryo axis formation, and WOX9 has an additional role in shoot meristem regulation (Wu et al. 2005a). All WOX genes examined show specific expression patterns, both spatially and temporally, which are important for their specific functions (Hedman et al. 2013). AtWUS gene expressed specifically in a restricted region of the meristem called the organizing center located below the stem cells of the SAM. It was required for maintaining stem cells via a feedback loop with CLV3, a peptide ligand that interacts with leucine-rich repeat receptor kinases in neighboring cells to restrict the SAM size (Schoof et al. 2000; Liu et al. 2014). AtWOX4 was strongly expressed in the CAM and played key roles in regulation of vascular stem cell maintenance (Hirakawa et al. 2010; Suer et al. 2011). AtWOX5 expressed in root quiescent center (QC) which is surrounded by the stem cells (Liu et al. 2014; Sarkar et al. 2007). Evidence showed that AtWOX5 was essential for stem cell maintenance via a negative feedback signal provided by CLE40 (Liu et al. 2014; Stah et al. 2009). The specific expression of the WOX genes
in different plant organs and cell types suggested an important role for them during organogenesis (Deveaux et al. 2008). WOX13 subfamily genes as the ancestor genes of WOX family were less studied in the past. WOX13 OG might be a good candidate to study the functional diversification of WOX genes (Chandler et al. 2008). The WOX13 subfamily genes were proposed to be involved in replum development, lateral root formation, and vegetative to floral growth transition (Chandler et al. 2008; Romera-Branchat et al. 2012). Romera-Branchat et al. (2012) found that WOX13 expressed mainly in the meristematic tissue in Arabidopsis (RomeraBranchat et al. 2012). In this study, we identified three WOX13 genes (WOX13A, wox13B1, and wox13B2) in peanut. The expression pattern, their origin from the wild type peanut species and the evolution model of the WOX family were analyzed. The possible functions of these genes were discussed.
Material and methods Plant material Root, shoot, leaf, flower, seed, and three stages of gynophore apex (5 mm of gynophore apex) were collected from the cultivated peanut Luhua-14. SAM enrichment region, shoot without the SAM, RAM enrichment region, root without the RAM, and three different stages gynophores apex were collected from Luhua-14 too. Stage 1 gynophores, aerial grown with green or purple color (3–5 cm in length); stage 2 gynophores, after soil penetration before pod enlargement; stage 3 gynophores, dark grown gynophores with 2–3 mm small pod. Total RNA isolation and cloning of WOX13 genes in peanut Cetyltrimethylammonium bromide (CTAB) method was used to extract total RNA from 0.2 g frozen tissues. The samples were incubated at 65 °C for 15 min. Equal volume of chloroform and water saturated phenol (1:1) was added and mixed thoroughly. After centrifugation, the supernatant was collected and equal volume chloroform was added and mixed by vortex. After centrifugation, the aqueous phase which containing the RNA was transferred to a clean tube and was precipitated with equal volume of isopropyl alcohol, stored for 8 h at −20 °C and pelleted by centrifugation at 12,000g for 20 min at 4 °C. RNA pellet was washed with 75 % ethanol, air dried, and resuspended with RNAase-free H2O. For reverse transcription, the first-strand complementary DNA (cDNA) was synthesized with an oligo (dT) primer using a PrimeScript™ first-strand cDNA synthesis kit (D6110A; TaKaRa, Dalian, China).
Dev Genes Evol
cDNA from peanut was used to perform subsequent PCR amplification. Primers used to amplify WOX13A were 5′ATGGTGGTGAAGATGATAGAA-3′ and 5′-CCTCTCAA CATGGAATAAGTAAC-3′. The PCR reactions were run using the following conditions: 5 min at 94 °C, 35 cycles (30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C), and 7 min at 72 °C. Primers used to amplify WOX13B1 and WOX13B2 were 5′-TCATCATCTTCTTCCTTTTGCT-3′ and 5′-TATGCTATGCCACATCACT TGT-3′. The PCR reactions were run using the following conditions: 5 min at 94 °C, 35 cycles (30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C), and 7 min at 72 °C. The three gene sequences from this study have been submitted to the gene under accession no. KM502259 (AhWOX13A), KM502260 (AhWOX13B1), and KM502261 (AhWOX13B2). Gene expression and promoter analysis Equal amounts of cDNA from peanut tissues were used to perform subsequent qRT-PCR. Primers used to amplify WOX13A were 5′-GCAGATTG CGGTTTATGCCACT-3′ and 5′-CCACCTTTGCCTAGACGTTATT-3′. Primers used to amplify WOX13B were 5′-AGAAGCAGAAGCAGAT GTAGAAGTAG-3′ and 5′-AAGAACCATAGC AGCCTC CAGT-3′. Primers used to amplify the control, actin 1, were 5′-GTCATCGTCATCCTCTTCTC-3′ and 5′-CATTCCTGTT CCATTGT CAC-3′. Primers used to amplify WOX13B1 were 5′-TGGACGCCGACGCCTC TT-3′ and 5′-TTGATG AATTGTTTCTTGAATTTCT-3′. Primers used to amplify WOX13B2 were 5′-TGGACGCCGACGCCTCTG-3′ and 5′-TTGATGAATTGTT TCTTGAAT TTCA-3′. The PCR reactions were run using the following conditions: 30 s at 95 °C, 40 cycles (5 s at 95 °C and 30 s at 60 °C), 15 s at 95 °C, 1 min at 60 °C, 30 s at 95 °C, and 15 s min at 60 °C. qRT-PCR was carried out using FastStart Universal SYBR Green Master (ROX) with a 7500 real-time PCR machine (ABI). Data were quantified using the 2−ΔΔCt method based on Ct values of AhWOX13 subfamily genes and β-actin. Values were considered to be significant at P < 0.05. Additionally, we analyzed the promoters of WOX13B1 and WOX13B2 using the online software Plantcare (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/). Data collection We collected full length WOX proteins sequences and WOX gene ORF sequences from the following plant species: monocots (Orazy sativa, Zea may, and Sorghum bicolor), dicots (A. hypogaea, Populus trichocarpa, and Arabidopsis thaliana), Gymnospermae (Ginkgo biloba), Bryophyta (P. trichocarpa), and green alga (Ostreococcus tauri). The
accession number of WOX proteins sequences and WOX gene ORF sequences were shown in the Supplemental Table 1. Domain, conserved motifs and phylogenetic analysis Peanut WOX13A and WOX13B domain was identified based on the Pfam database (http://pfam.sanger.ac.uk/). In order to identify shared motifs among the WOX protein sequences, we used the MEME online software and domain conservation was predicted by Pfam online software. Conserved motifs were analyzed online using a MEME system Version (http:// meme.nbcr.net/meme/cgi-bin/meme.cgi) (Parameters set: minimum width; 6 aa, maximum width; 200 aa, and maximum number of motifs to find; 20). Neighbor-joining (NJ) and maximum likelihood (ML) trees were constructed using MEGA 4.0. To assess the support for the calculated relationships, 1000 bootstrap samples were generated. CLUSTALX2.1 software was used for WOX proteins alignment. Codon-based sequence analysis of WOX family genes The ratio of nonsynonymous/synonymous substitution rates ω (dn/ds) can provide a measurement for the change of selective pressure. Values of ω=1, 1 indicate neutral evolution, purifying selection, and positive selection on the target gene, respectively. The codon-substitution model implemented in the CODEML program in the PAML4.4 software package was used to analyze changes in selective pressure. The presence of sites under positive selection was detected with the M0, M1a (nearly neutral), M2a (positive selection), M7 (beta), and M8 (beta and ω) model. The presence of codons evolving under positive selection was further tested by comparing M1a and M2a models, and M7 and M8 models by likelihood ratio tests. To explore the divergence of different branches of WOX family genes in the evolutionary history, CODEML software was used to compute the nonsynonymous/synonymous substitution rate ratio of different branches. Analysis of gene duplication and synteny analysis Based on all-vs-all local BLASTP and BLASTN of all WOX proteins and gene sequences, we collected for gene duplication analysis. The duplicated gene pair was identified according to the following criteria: the alignable nucleotide sequence covered >70 % of the longest gene and the identity of amino acid sequences was >70 % (Zhou et al. 2004). Genome data from plantGDB (http://www.plantgdb.org/) were downloaded for Intraspecies and Interspecies synteny analysis. Homology gene pair was identify by local BLASTP (E-value
ORIGINAL ARTICLE
Identification and expression dynamics of three WUSCHEL related homeobox 13 (WOX13) genes in peanut Pengfei Wang 1,2 & Changsheng Li 1,2 & Cui Li 1,2 & Chuanzhi Zhao 1,2 & Han Xia 1,2 & Shuzhen Zhao 1,2 & Lei Hou 1,2 & Chao Gao 1,2 & Shubo Wan 1,2 & Xingjun Wang 1,2
Received: 17 March 2015 / Accepted: 9 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract WUSCHEL-related homeobox (WOX) genes play key roles in plant stem cell maintenance and development. WOX genes showed specific expression patterns which are important for their functions. WOX13 subfamily genes as the ancestor genes of this family were less studied in the past. In this study, we cloned three Arachis hypogaea (peanut) WOX13 (AhWOX13) subfamily genes from peanut: WOX13A and WOX13B1, 2. WOX13B1 encoded a same protein as WOX13B2, and there were only two-base difference between these two genes. Differential expression patterns were observed for these three AhWOX13 subfamily genes in different tissues and developmental stages. Phylogenic trees analysis showed that these AhWOX13 subfamily genes were the most conserved WOX genes and belonged to the ancient clade of WOX family. This was also supported by the conserved motif analysis. Selective pressure analysis showed that the WOX family genes mainly underwent weak purifying selection (ω=0.58097), while many positive mutations accumulated during the evolution history. Under the purifying Communicated by Sureshkumar Balasubramanian Pengfei Wang and Changsheng Li contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00427-015-0506-3) contains supplementary material, which is available to authorized users. * Shubo Wan [email protected] * Xingjun Wang [email protected] 1
Bio-Tech Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, People’s Republic of China
2
Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan 250100, People’s Republic of China
selection, gene duplication event and loss of duplicated gene play important roles in the expansion and evolution of WOX family. Keywords Peanut . WOX13 subfamily gene . Stem cell . Evolution . Selective pressure
Introduction Peanut (Arachis hypogaea L.) is native in South America and grown worldwide. The world annual production of peanut is about 38 million tons (Bertioli et al. 2011). Cultivated peanut is allotetraploid (AABB, 4n=4x=40) which originated from a single hybridization event between AA and BB wild species and subsequently underwent spontaneous genome duplication (Stalker and Simpson 1995; Moretzsohn et al. 2013; Kochert et al. 1996; Ferguson et al. 2005; Freitas et al. 2007; Gregory and Gregory 1979; Hammons 1982; Knauft and Gorbet 1995). Peanut gynophore is a specialized and unique organ that carries and eventually pushes the fertilized ovules into the soil in order for seed and fruit development to occur (Xia et al. 2013; Moctezuma 2003). The peanut gynophore has a typical shoot anatomy and responds positively to gravity, similar to a typical primary root (Moctezuma and Feldman 1998; Moctezuma and Feldman 1999). The activity of an intercalary meristem located proximal to the seed region contributes to the elongation and downward growth of the gynophore (Jacobs 1947; Shushu and Cutter 2011). The peanut gynophore intercalary meristem resembles the shoot apical meristem (SAM) and root apical meristems (RAM). These meristems are responsible for organ formation (Laux 2003; Hedman et al. 2013), and the maintenance of meristem depends on selfrenewal of the stem cell population (Schoof et al. 2000).
Dev Genes Evol
WUSCHEL-related homeobox (WOX) genes including WUSCHEL (WUS) and WOX1-WOX14 genes play key roles in stem cell maintenance. Members of the WOX family are involved in the stem cell maintenance in meristems, embryo apical-basal polarity patterning, and development of lateral organs (Zhao et al. 2009a, b; Lin et al. 2013; Ikeda et al. 2009; Lie et al. 2012; Liu et al. 2014). Furthermore, several studies suggest that the WOX gene family may be involved in the evolution of developmental processes, such as the evolution of novel shoot-leaf mixed organs in Podostemaceae (Hedman et al. 2013). Based on phylogenetic analysis and their distribution in different plant, WOX family genes have been classified into three clades. The first clade is the modern clade (found in seed plants) including WUS, WOX1, WOX2, NS/WOX3, WOX4, WOX5, WOX6, and WOX7 orthologue. The second is the intermediate clade (found in vascular plants including lycophytes), including WOX8, 9, 11, and 12 orthologue. The third one is the ancient/basal clade (found in vascular and nonvascular plants including mosses and green algae), including WOX13 subfamily (wox10, 13, and 14) (Hedman et al. 2013; Nardmann et al. 2009; Zhang et al. 2010; Van der Graaff et al. 2009). WUS gene is involved in the stem cell maintenance in SAM and functions in a non-cell autonomous manner to control the stem cell fate (Mayer et al. 1998). WOX3 gene is involved in the stem cell maintenance in leaf marginal meristems (Shimizu et al. 2009; Nakata et al. 2012) and in development of lateral sepals and stipules (Lin et al. 2013; Nardmann et al. 2004; Matsumoto and Okada 2001). WOX4 gene is involved in the stem cell maintenance in the vascular (cambial meristem, CAM) (Hedman et al. 2013; Hirakawa et al. 2010; Ji et al. 2010). WOX5 gene plays key roles in stem cell maintenance in the RAM (Wu et al. 2005a). WOX8 and WOX9 redundantly regulate zygote development and embryo axis formation, and WOX9 has an additional role in shoot meristem regulation (Wu et al. 2005a). All WOX genes examined show specific expression patterns, both spatially and temporally, which are important for their specific functions (Hedman et al. 2013). AtWUS gene expressed specifically in a restricted region of the meristem called the organizing center located below the stem cells of the SAM. It was required for maintaining stem cells via a feedback loop with CLV3, a peptide ligand that interacts with leucine-rich repeat receptor kinases in neighboring cells to restrict the SAM size (Schoof et al. 2000; Liu et al. 2014). AtWOX4 was strongly expressed in the CAM and played key roles in regulation of vascular stem cell maintenance (Hirakawa et al. 2010; Suer et al. 2011). AtWOX5 expressed in root quiescent center (QC) which is surrounded by the stem cells (Liu et al. 2014; Sarkar et al. 2007). Evidence showed that AtWOX5 was essential for stem cell maintenance via a negative feedback signal provided by CLE40 (Liu et al. 2014; Stah et al. 2009). The specific expression of the WOX genes
in different plant organs and cell types suggested an important role for them during organogenesis (Deveaux et al. 2008). WOX13 subfamily genes as the ancestor genes of WOX family were less studied in the past. WOX13 OG might be a good candidate to study the functional diversification of WOX genes (Chandler et al. 2008). The WOX13 subfamily genes were proposed to be involved in replum development, lateral root formation, and vegetative to floral growth transition (Chandler et al. 2008; Romera-Branchat et al. 2012). Romera-Branchat et al. (2012) found that WOX13 expressed mainly in the meristematic tissue in Arabidopsis (RomeraBranchat et al. 2012). In this study, we identified three WOX13 genes (WOX13A, wox13B1, and wox13B2) in peanut. The expression pattern, their origin from the wild type peanut species and the evolution model of the WOX family were analyzed. The possible functions of these genes were discussed.
Material and methods Plant material Root, shoot, leaf, flower, seed, and three stages of gynophore apex (5 mm of gynophore apex) were collected from the cultivated peanut Luhua-14. SAM enrichment region, shoot without the SAM, RAM enrichment region, root without the RAM, and three different stages gynophores apex were collected from Luhua-14 too. Stage 1 gynophores, aerial grown with green or purple color (3–5 cm in length); stage 2 gynophores, after soil penetration before pod enlargement; stage 3 gynophores, dark grown gynophores with 2–3 mm small pod. Total RNA isolation and cloning of WOX13 genes in peanut Cetyltrimethylammonium bromide (CTAB) method was used to extract total RNA from 0.2 g frozen tissues. The samples were incubated at 65 °C for 15 min. Equal volume of chloroform and water saturated phenol (1:1) was added and mixed thoroughly. After centrifugation, the supernatant was collected and equal volume chloroform was added and mixed by vortex. After centrifugation, the aqueous phase which containing the RNA was transferred to a clean tube and was precipitated with equal volume of isopropyl alcohol, stored for 8 h at −20 °C and pelleted by centrifugation at 12,000g for 20 min at 4 °C. RNA pellet was washed with 75 % ethanol, air dried, and resuspended with RNAase-free H2O. For reverse transcription, the first-strand complementary DNA (cDNA) was synthesized with an oligo (dT) primer using a PrimeScript™ first-strand cDNA synthesis kit (D6110A; TaKaRa, Dalian, China).
Dev Genes Evol
cDNA from peanut was used to perform subsequent PCR amplification. Primers used to amplify WOX13A were 5′ATGGTGGTGAAGATGATAGAA-3′ and 5′-CCTCTCAA CATGGAATAAGTAAC-3′. The PCR reactions were run using the following conditions: 5 min at 94 °C, 35 cycles (30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C), and 7 min at 72 °C. Primers used to amplify WOX13B1 and WOX13B2 were 5′-TCATCATCTTCTTCCTTTTGCT-3′ and 5′-TATGCTATGCCACATCACT TGT-3′. The PCR reactions were run using the following conditions: 5 min at 94 °C, 35 cycles (30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C), and 7 min at 72 °C. The three gene sequences from this study have been submitted to the gene under accession no. KM502259 (AhWOX13A), KM502260 (AhWOX13B1), and KM502261 (AhWOX13B2). Gene expression and promoter analysis Equal amounts of cDNA from peanut tissues were used to perform subsequent qRT-PCR. Primers used to amplify WOX13A were 5′-GCAGATTG CGGTTTATGCCACT-3′ and 5′-CCACCTTTGCCTAGACGTTATT-3′. Primers used to amplify WOX13B were 5′-AGAAGCAGAAGCAGAT GTAGAAGTAG-3′ and 5′-AAGAACCATAGC AGCCTC CAGT-3′. Primers used to amplify the control, actin 1, were 5′-GTCATCGTCATCCTCTTCTC-3′ and 5′-CATTCCTGTT CCATTGT CAC-3′. Primers used to amplify WOX13B1 were 5′-TGGACGCCGACGCCTC TT-3′ and 5′-TTGATG AATTGTTTCTTGAATTTCT-3′. Primers used to amplify WOX13B2 were 5′-TGGACGCCGACGCCTCTG-3′ and 5′-TTGATGAATTGTT TCTTGAAT TTCA-3′. The PCR reactions were run using the following conditions: 30 s at 95 °C, 40 cycles (5 s at 95 °C and 30 s at 60 °C), 15 s at 95 °C, 1 min at 60 °C, 30 s at 95 °C, and 15 s min at 60 °C. qRT-PCR was carried out using FastStart Universal SYBR Green Master (ROX) with a 7500 real-time PCR machine (ABI). Data were quantified using the 2−ΔΔCt method based on Ct values of AhWOX13 subfamily genes and β-actin. Values were considered to be significant at P < 0.05. Additionally, we analyzed the promoters of WOX13B1 and WOX13B2 using the online software Plantcare (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/). Data collection We collected full length WOX proteins sequences and WOX gene ORF sequences from the following plant species: monocots (Orazy sativa, Zea may, and Sorghum bicolor), dicots (A. hypogaea, Populus trichocarpa, and Arabidopsis thaliana), Gymnospermae (Ginkgo biloba), Bryophyta (P. trichocarpa), and green alga (Ostreococcus tauri). The
accession number of WOX proteins sequences and WOX gene ORF sequences were shown in the Supplemental Table 1. Domain, conserved motifs and phylogenetic analysis Peanut WOX13A and WOX13B domain was identified based on the Pfam database (http://pfam.sanger.ac.uk/). In order to identify shared motifs among the WOX protein sequences, we used the MEME online software and domain conservation was predicted by Pfam online software. Conserved motifs were analyzed online using a MEME system Version (http:// meme.nbcr.net/meme/cgi-bin/meme.cgi) (Parameters set: minimum width; 6 aa, maximum width; 200 aa, and maximum number of motifs to find; 20). Neighbor-joining (NJ) and maximum likelihood (ML) trees were constructed using MEGA 4.0. To assess the support for the calculated relationships, 1000 bootstrap samples were generated. CLUSTALX2.1 software was used for WOX proteins alignment. Codon-based sequence analysis of WOX family genes The ratio of nonsynonymous/synonymous substitution rates ω (dn/ds) can provide a measurement for the change of selective pressure. Values of ω=1, 1 indicate neutral evolution, purifying selection, and positive selection on the target gene, respectively. The codon-substitution model implemented in the CODEML program in the PAML4.4 software package was used to analyze changes in selective pressure. The presence of sites under positive selection was detected with the M0, M1a (nearly neutral), M2a (positive selection), M7 (beta), and M8 (beta and ω) model. The presence of codons evolving under positive selection was further tested by comparing M1a and M2a models, and M7 and M8 models by likelihood ratio tests. To explore the divergence of different branches of WOX family genes in the evolutionary history, CODEML software was used to compute the nonsynonymous/synonymous substitution rate ratio of different branches. Analysis of gene duplication and synteny analysis Based on all-vs-all local BLASTP and BLASTN of all WOX proteins and gene sequences, we collected for gene duplication analysis. The duplicated gene pair was identified according to the following criteria: the alignable nucleotide sequence covered >70 % of the longest gene and the identity of amino acid sequences was >70 % (Zhou et al. 2004). Genome data from plantGDB (http://www.plantgdb.org/) were downloaded for Intraspecies and Interspecies synteny analysis. Homology gene pair was identify by local BLASTP (E-value
