International Journal o f Molecular Sciences Article Genome-Wide Identification of ARF Transcription Factor Gene Family and Their Expression Analysis in Sweet Potato Isaac Seth Pratt and Baohong Zhang * Department of Biology, East Carolina University, Greenville, NC 27858, USA; prattis@ecu.edu * Correspondence: zhangb@ecu.edu Abstract: Auxin response factors (ARFs) are a family of transcription factors that play an important role of auxin regulation through their binding with auxin response elements. ARF genes are repre- sented by a large multigene family in plants; however, to our knowledge, the ARF gene family has not been well studied and characterized in sweet potatoes. In this study, a total of 25 ARF genes were identified in Ipomea trifida. The identified ItrARF genes’ conserved motifs, chromosomal locations, phylogenetic relationships, and their protein characteristics were systemically investigated using different bioinformatics tools. The expression patterns of ItfARF genes were analyzed within the storage roots and normal roots at an early stage of development. ItfARF16b and ItfARF16c were both highly expressed in the storage root, with minimal to no expression in the normal root. ItfARF6a and ItfARF10a exhibited higher expression in the normal root but not in the storage root. Subsequently, ItfARF1a, ItfARF2b, ItfARF3a, ItfARF6b, ItfARF8a, ItfARF8b, and ItfARF10b were expressed in both root types with moderate to high expression for each. All ten of these ARF genes and their prominent expression signify their importance within the development of each respective root type. This study   provides comprehensive information regarding the ARF family in sweet potatoes, which will be useful for future research to discover further functional verification of these ItfARF genes. Citation: Pratt, I.S.; Zhang, B. Genome-Wide Identification of ARF Keywords: sweet potato; auxin response factor; root development Transcription Factor Gene Family and Their Expression Analysis in Sweet Potato. Int. J. Mol. Sci. 2021, 22, 9391. https://doi.org/10.3390/ijms22179391 1. Introduction Academic Editor: Endang Indole-3-acetic acid (IAA), one type of auxin, is a plant growth hormone that elic- Septiningsih its developmental growth throughout the plant’s life cycle, including seed germination, vascular tissue formation, and reproductive and vegetative growth [1,2]. These auxin Received: 1 July 2021 plant hormones work in conjunction with auxin response factors (ARF), which are a Accepted: 24 August 2021 set of transcription factors. When ARFs are bound to auxin response elements (AuxRE, Published: 30 August 2021 TGTCNN), they work to activate or repress auxin gene expression and regulation [3–5]. General genome-wide AuxRE is TGTCNN and in particular TGTCGG for ARF5 but not Publisher’s Note: MDPI stays neutral TGTCTC [5]. ARF proteins can be broken down into three domain subunits: an amino- with regard to jurisdictional claims in terminal DNA-binding domain (DBD), a conserved carboxy-terminal dimerization domain published maps and institutional affil- (CTD), and a non-conserved middle domain (MD) [2]. The DBD for ARF’s are found in the iations. N-terminal region and bind specifically to the AuxRE TGTCNN in promoters for the regu- lation of auxin gene expression [6]. Carboxy-terminal dimerization domains facilitate both homo- and hetero-dimerization through protein interactions, relating themselves to the domain III and IV in Aux/IAA proteins [6]. The MD has a double function, acting as either Copyright: © 2021 by the authors. a repression domain (RD) or an activation domain (AD) [7,8]. ARF proteins that have a MD Licensee MDPI, Basel, Switzerland. rich in glutamine (Q) are constituted as an activator; this can be seen in AtARF5, 6, 7, 8, and This article is an open access article 19 [9,10], while ARF proteins that have an MD rich in Proline (P), Serine (S), and Threonine distributed under the terms and (T) are considered repressors; these repressors were found in AtARF1, 2, 3, 4 and 9 [8]. ARF conditions of the Creative Commons proteins have been identified in a variety of plants; 22 ARF genes and one pseudogene were Attribution (CC BY) license (https:// found in Arabidopsis [7], and 25 ARF genes were found in rice [11]. Subsequently, there have creativecommons.org/licenses/by/ been more ARFs identified in a multitude of other plant species, such as tomato (Solanum 4.0/). Int. J. Mol. Sci. 2021, 22, 9391. https://doi.org/10.3390/ijms22179391 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9391 2 of 13 lycopersicum) [12], kiwifruit (Actinidia chinensis) [13], longan (Dimocarpus longan L.) [14], tomato (Solanum lycopersicum) [15], sorghum (Sorghum bicolor) [16,17], soybean (Glycine max) [18], maize (Zea maize) [19], Chinese cabbage (Brassica rapa) [20], cotton (Gossypium raimondii) [21], sweet orange (Citrus sinensis) [22], peach (Prunus persica L.) [23], and alfalfa (Medicago truncatula) [24]. However, with all these identifications of auxin response factors, there has not been the identification of auxin response factors in sweet potato. The sweet potato (Ipomoea batatas) of the family Convolvulaceae is a highly desired food crop throughout the world and has been ranked the 7th largest crop. It serves as a staple diet in developing countries for its overall high nutritional and caloric values, ability to grow in most climates and conditions, and having a high production yield. The sweet potato (Ipomoea batatas) is a hexaploid with 90 chromosomes, making genomic research on the crop highly complicated [25]. However, there is an alternative to performing genomic research on sweet potatoes, and that is to use a diploid relative. The diploid Ipomoea trifida is a relative to the hexaploid I. batatas and is a model species of genomic research due to its small genome size and number of chromosomes [26–28]. In this study, all the potential ARF transcription factors from the I. trifida genome were identified using various bioinformatics tools. ItfARF proteins, once identified, underwent a phylogenetic analysis where they were compared among themselves and then amongst the model plant species: Arabidopsis, S. lycopersicum, O. sativa, and G. raimondii. Finally, the expression profiles of each ItfARF gene were analyzed using tissue samples of both the normal and storage roots of sweet potato plant I. trifida. 2. Materials and Methods 2.1. Identification of ARF Genes in I. trifida The genome sequence of Ipomoea trifida was downloaded from the Sweet potato genomic resource database (http://sweetpotato.plantbiology.msu.edu/, accessed on 1 June 2021) [29]. Both a BLAST and reciprocal BLAST search were performed for the conformation of I. trifida ARF gene families, and orthologues relations with the species: Arabidopsis, S. lycopersicum, G. raimondii, and O. sativa. For the identification of the genomic members of the ARF gene family, both HMMER 3.3.1 (http://hmmer.org/download.html, accessed on 1 June 2021) and Pfam 33.1 (http://pfam.xfam.org/, accessed on 1 June 2021) were used to search whole genome sequences with Pfam’s ARF (PF06507); all se- quences with an e-value < e-10 were selected after the homology search. All obtained sequences were further analyzed for B3, ARF, and AUX/IAA domains using NCBI’s CDD (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 1 June 2021). After identification of the ItfARF gene family, information on the ItfARF’s genome location, protein, and CDS length were obtained along with the gene, protein, and CDS sequences from the Sweet potato database. In addition, Expasy (https://web.expasy.org/compute_pi/, accessed on 1 June 2021) was used to compute the isoelectric point (PI) and the molecular weight (Mw) of the ItfARF proteins. To analyze the identified ItfARF genes for conserved motifs, the protein sequences were examined using the software MEME (Multiple Expectation maximizations for Motif Elicitation) (https://meme-suite.org/meme/tools/meme, accessed on 1 June 2021). The sequence search options were as follows: motif distribution among sequences was zero to one occurrence, the motif width range was from 6 to 50 amino acids, and the maximum motifs per sequences was 20. 2.2. Phylogenetic Analysis of ARF Genes To better understand the evolutionary relationship and homology between the se- quences, a phylogenetic analysis was performed using the protein sequence data [30]. For this analysis, the protein sequences from Arabidopsis, G. raimondii, I. trifida, O. sativa, and S. lycopersicum were analyzed with Molecular Evolutionary Genetics Analysis (MEGA-X 10.2) using the maximum-likelihood method and its built-in sequence alignment tool, Clustal-W [30]. Int. J. Mol. Sci. 2021, 22, 9391 3 of 13 2.3. Sweet Potato Culture, Tissue Collection, and RNA Extraction The sweet potatoes (Ipomoea batatas) were cultivated in the greenhouse with normal agronomic practices. Both storage root and normal root samples were collected from 5-weeks grown sweet potato plants. Root tissues were quickly sampled from the plants, immediately frozen in liquid nitrogen, and stored at ?80 ?C until RNA extraction. At least six biological replicates were collected for each type of root. Total RNAs were extracted from each root sample by using a mirVanaTM miRNA isolation Kit (Ambion, Austin, TX, USA) as performed in our previous studies [31–34]. Briefly, the tissues were ground into a fine powder in a mortar and pestle and then transferred into a 2 mL centrifuge tube with the Lysis/Binding buffer. For the normal roots, 400 µL of the Lysis/Binding buffer was used, and for the storage roots, 700 µL buffer was used; this is because the storage root contained higher levels of starch that absorbed large quantities of liquid. Then, the samples were sonicated for 15–20 s on ice. Post sonication, each sample was inverted for an accurate mixture of the Lysis buffer and the plant material. After a 10-min ice bath, 400 µL of Acid-Phenol/Chloroform was added for the separation of RNA from its cellular components. Carefully following the manufacturer’s protocols, consisting of aqueous phase extraction and several wash cycles, 95 °C nuclease-free water was added to the filter cartridge medium for the conclusion of RNA isolation. To test the concentration and purity of the RNA isolated, Nanodrop ND-1000 was used. Samples NR1, NR2, NR3, SR1, SR2, and SR3 were all collected, each having a ng/µL concentration in the range of 200–350 ng/µL. 2.4. qRT-PCR The RNA of each plant tissues sample was prepared for reverse transcription with specific primers designed from NCBI primer design tool. Then, reverse transcription was performed by following the instructions of the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, CA, USA). This kit included a MultiscribeTM Reverse transcriptase (50 U/µL), dNTPs with dTTP (100 nm), Reverse transcription buffer (10x), RNase inhibitor (20 µ/µL), nuclease-free water, and total RNAs; the volume for each reaction was 15 µL. Post reaction completion, 150 µL of nuclease-free water was added to the products and stored at ?20 ?C until ready for qRT-PCR. qRT-PCR was performed on a 96-well plate within the 7300 Fast Real-Time PCR System (Applied Biosystems, Waltham, CA, USA). The samples ran for each replicate went as follows: all ItfARF genes and 2 reference genes (EF1? and UBC). SYBR Green was used to analyze gene expression within the qRT-PCR system. A total of 6 reactions were run: three biological replicates for each root type, and each biological replicate had three technical replicates. The reaction temperature program settings were as follows: 10 min at 95 ?C, with 40 cycles of 15 s at 95 ?C, and 60 s at 60 ?C. 2.5. Data and Statistical Analysis Statistical analysis was performed after obtaining sample triplicate Ct values. Elonga- tion factor 1? (EF1?) and ubiquitin-c (UBC) served as the reference genes, and the average of each reference gene’s ?Ct values was combined and subtracted from all other ItfARF genes ?Ct values to obtain a second normalization. Differentially expressed genes were discovered using statistical calculation, p < 0.05. Then, fold change was calculated for each gene using the formula: 2?(??Ct). A hierarchical clustering analysis was performed using Multi Experiment Viewer (MeV) to create a heat map of gene expression for all the ItfARF genes. 3. Results 3.1. Identification and Sequence Analysis of ARF Genes in I. trifida To identify the ARF transcription factor genes in I. trifida, the ARF protein domain (PF00025) was used to blast search against the I. trifida genome. The first search led to a total of 282 possible protein sequences in I. trifida. After eliminating the redundant sequences and comparing with the ARF gene in other plant species (Figure 1), a total of 25 ItfARF were identified in the sweet potato genome (Supplementary Materials Table S1). Then, Int. J. Mol. Sci. 2021, 22, 9391 4 of 13 these 25 sequences were compared and categorized into 13 different ARF gene subfamilies, including ItfARF1, ItfARF2, ItfARF3, ItfARF4, ItfARF5, ItfARF6, ItfARF8, ItfARF9, ItfARF10 ItfARF11, ItfARF16, ItfARF18, and ItfARF19 named so after their similarities to Arabidopsis. The average number of exons in the gene sequences was 10, having a similar distribution to the Arabidopsis ARF genes. The length of the CDS varied from 1731 bp (ItfARF11) all the way up to 3375 bp (ItfARF19a). The 25 possible genes all produced adequate proteins ranging from 576 to 1124 amino acids in length. The predicted MW of the ItfARF were as low as 64.539 kDa and as high as 124.164 kDa. The PI values of the ItfARF genes were from 4.99 to 9.44; most of these values fell in the range of 5–7, suggesting they encode weak acid proteins, while those few that ranged from 7.56 to 9.44 encode for weak basic proteins. The 25 identified ARF genes were located among the 15 chromosomal pairs with the exclusion of Chr. 8, 13, and 14 as no ARF genes were mapped there. Most of the genes stacked onto a select few chromosomes (Figure 2), having five genes on chromosome 10 (19.2%), four genes on chromosome 6, three genes each on chromosomes 2, and 4, two genes each on chromosomes 1, 7, 9, and 11, and then one gene on chromosomes 3, 5, 12, and 15. The ARF genes, ItfARF8a and ItfARF8b and ItfARF1a and ItfARF1b, are the only two sets of duplicate genes that met the 80% sequence similarity for each of their respective nucleotide sequences. 3.2. Conserved Domains and Motif Analysis of ItfARF Proteins For the prediction of protein function, the analysis of both domains and subdomains was apparent [2]. Out of the twenty-five ItfARF proteins, there were four among them that lacked at least one of the three typical domains, and each of these four genes were lacking the Aux/IAA CTD domain; those four genes were ItfARF3a, ItfARF3b, ItfARF11, and ITFARF16c (Supplementary Materials Table S1). Regarding the domains, the middle domain (MD) gave insight into the sequences’ ability to either be a transcriptional activator or a repressor. The protein sequences that were rich in (Q) have been regarded as activators ItfARF5, 6a, 6b, 8a, 8b, 19a, and 19b, while the other protein sequences whose MD were rich in P, S, and T are regarded as transcriptional repressors. The conserved motif analysis allowed for further confirmation of these notions. As shown in Figure 3, motifs 1 and 2 correspond to a DNA-binding domain; motifs 8, 9, 10, and 11 correspond to an ARF domain, while motifs 17, 18, 19, and 20 correspond to an Aux/IAA domain. Lastly, all seven ItfARF transcriptional activators lack motif 16 while still maintaining motifs 18, 19, and 20, suggesting that this combination of motifs signifies a transcriptional activator. Cross-comparing Supplementary Materials Table S1 and Figure 3, we can see that these motifs correlate well with each other. Each protein sequence had a variable number of motifs, but the motifs for each domain were conserved with minimal variation preceding and succeeding them. 3.3. Phylogenetic Analysis of ItfARFs For better understanding the functional and evolutionary relationship of the ARF gene family, an unrooted phylogenetic tree was created by multiple sequence alignment in MEGA-X for all selected plant species, including sweet potato. This analysis used the 25 ItfARF protein sequences and all the ARF proteins from Arabidopsis, rice, cotton, and tomato. The 109 ARF transcription factors from all species fall into five major classifications ranging from I to IV; the classes were derived from their phylogenetic relationship. Out of all the classes, Class III was the largest, having 33 ARF members holding 30.27% of the total ARF genes analyzed. Class III was comprised of three subgroups: IIIa, IIIb, and IIIc; these contained 5, 12, and 16 ARF genes, respectively. Classes IIIa, IIIb, and IIIc contained ItfARFs with a Q-rich MD, which included ItfARF5 for IIIa, ItfARF6a, 6b, 8a, and 8b for IIIc, and 19a and 19b for IIIb. ItfARFs were in each class except for class V, suggesting that the ItfARF gene family arose before the lineage spilt. Int. J. Mol. Sci. 2021, 22, 9391 5 of 13 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 13 Figure 1. Phylogenetic tree comprised of ARF proteins found in Arabidopsis, cotton, I. trifida, tomato, and rice. This tree wFaigs ucroen1st.rPuhcytelodg uensientgic MtreEeGcAom-Xp rsiosfetdwoafreA uRsFinpgr ottheein ms faoxuimndumin-Alikraebliidhooposdis ,mcoettthoond, Iw. tirtihfi dtah,et oJmTTa tmo,oadnedl.r iTche.e Tphairsatmreeetwerass wceornes t1r0u0c0t ebdooutssitnragpMs aEnGdA p-Xairswofitswe agraepu dseinlegtitohnes.m Tahxei m10u9m A-RlikFe plirhooteoidnsm wetehreo dclwasistihfiethde inJTtoT fmivoed celal.ssTehse: Ip, aIrIa, mIIIe,t IeVrs, wanedre V1,0 a0n0db ocloatssstr IaIpI swaansd fupratihrewr idseivgiadpedd einlettoi oInIIsa.,T IhIIeb1, 0a9ndA RIIFIcp. roteins were classified into five classes: I, II, III, IV, and V, and class III was further divided into IIIa, IIIb, and IIIc. 3.4. TEhxep r2e5ss iidonenPtrifoifieldin AgRofFI tgfeAnReFs Gweenrees lioncaRtoeodt aTmissounegs the 15 chromosomal pairs with the exclusion of Chr. 8, 13, and 14 as no ARF genes were mapped there. Most of the genes stackeqdR oTn-PtoC aR swelaescte fmewp lcohyreodmtoosboemtteesr (uFnigduerres t2a)n, dhatvhiengex fpivrees gseionnesp oronfi clhesroomf oaslloImtfeA 1R0F (1g9e.n2%es),w fiotuhrin gsewneese topno ctahtroomnoorsmomaler o6o, ttsharened gsetonreasg eearcoho tosn. Tchhrroomugohsothmeegs e2n, eaenxdp 4re, stswioon gaennaelsy esiasc,hth oend cifhferorimngosfuomncetiso 1n,s 7o, f9a, lal nItdfA 1R1F, agnedn etshwene roenaep gpeanreen ot.nT chherroemwoesroemsoems 3e, I5tf,A 1R2,F agnedn 1e5s.w Tihthe AsimRFil agregneens,e IetfxApRreFs8saio annpda tIttefArnRsFb8ebt waneedn IttfhAeRtwF1oar aonotdt IytpfAesR, Fw1hbi,l earoet hthere goennlyes twshoo sweetsd ocfl edaurpdliifcfaetree ngceense, sw tihthath migehte trheex 8p0r%es ssieoqnuiennocne esirmooiltatryitpye foovr eeratchhe ooft htheeri(rF riegsupreec4-). tiFvoer neuxcalmeoptlied,eI tsfeAqRuFe8naceasn. d ItfARF8b were both highly expressed in both normal roots and storage roots. ItfARF2b, ItfARF3a, ItfARF6b, and ItfARF10b are all moderately expressed across both tissue types, suggesting that they may be primarly expressed under specific conditions. Only two of the 25 tested ItfARF genes were more highly expressed in the normal root than that in the storage root; those genes were: ItfARF6a and ItfARF10a. There Int. J. Mol. Sci. 2021, 22, 9391 6 of 13 were some genes that had little to no expression at all, such as ItfARF2c, ItfARF3b, ItfARF9b, ItfARF16a, ItfARF18, and ItfARF19a; these suggest that the rest of the ItfARF genes were expressed more highly in the storage roots as opposed to the normal roots, those genes being ItfARF1a, ItfARF1b, ItfARF2a, ItfARF4a, ItfARF4b, ItfARF5, ItfARF9a, ItfARF16b, Int. J. Mol. Sci. 2021, 22, x FOR PEER IRtfEAVRIE F W1 6c, and ItfARF19b. Out of those nine genes, ItfARF4a, ItfARF5, ItfARF9a, ItfAR6F 1of6 b1,3 ItfARF16c, and ItfARF19b had the highest level of expression in the storage roots. FFigiguurree2 2. .C Chhrroommoososommaal ld disitsrtirbiubutitoinono foAf ARFRFg egneenseisn iIn. tIr. itfirdifai,dsap, sapnanninngin1g2 1o2f tohfe th1e5 c1h5r cohmroomsoomsoesm, gese,n geednuep dliucpatliiocantiaonna laynsaisl- oyfsIitsf AofR IFtfwAaRsFp wreasse pnrteedsewntiethd awgitrha ya lginraey. line. 3.2. Conserved Domains and Motif Analysis of ItfARF Proteins For the prediction of protein function, the analysis of both domains and subdomains was apparent [2]. Out of the twenty-five ItfARF proteins, there were four among them that lacked at least one of the three typical domains, and each of these four genes were lacking the Aux/IAA CTD domain; those four genes were ItfARF3a, ItfARF3b, ItfARF11, and ITFARF16c (Supplementary Materials Table S1). Regarding the domains, the middle domain (MD) gave insight into the sequences’ ability to either be a transcriptional activa- tor or a repressor. The protein sequences that were rich in (Q) have been regarded as acti- vators ItfARF5, 6a, 6b, 8a, 8b, 19a, and 19b, while the other protein sequences whose MD were rich in P, S, and T are regarded as transcriptional repressors. The conserved motif analysis allowed for further confirmation of these notions. As shown in Figure 3, motifs 1 and 2 correspond to a DNA-binding domain; motifs 8, 9, 10, and 11 correspond to an ARF domain, while motifs 17, 18, 19, and 20 correspond to an Aux/IAA domain. Lastly, all seven ItfARF transcriptional activators lack motif 16 while still maintaining motifs 18, 19, and 20, suggesting that this combination of motifs signifies a transcriptional activator. Cross-comparing Supplementary Materials Table S1 and Figure 3, we can see that these motifs correlate well with each other. Each protein sequence had a variable number of motifs, but the motifs for each domain were conserved with minimal variation preceding and succeeding them. Int. J. Mol. Sci. 2021, 22, 9391 7 of 13 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 13 Figure 3. ItfARF proteins with twenty different identified conserved motifs through the MEME search tool. The motifs are Finguumreb3e.reItdfA aRndF prerporteeisnesnwtedit hbytw thene tdyifdfeifrfeenret nctoildoersn tinifi Medoctiofn Ssyemrvbeodl.m Aortriofswt h1r pouoignhtst htoe tMheE MDBEDs edaorcmhationo, lA. Trrhoewm 2o ptiofsinatrse to nuthmeb AerReFd daonmd areinp,r aesnedn Aterdrobwy t3h epodiinfftesr teon tthceo lCoTrsDi ndMomoatiifnS. ymbol. Arrow 1 points to the DBD domain, Arrow 2 points to the ARF domain, and Arrow 3 points to the CTD domain. 3.3. Phylogenetic Analysis of ItfARFs For better understanding the functional and evolutionary relationship of the ARF gene family, an unrooted phylogenetic tree was created by multiple sequence alignment in MEGA-X for all selected plant species, including sweet potato. This analysis used the 25 ItfARF protein sequences and all the ARF proteins from Arabidopsis, rice, cotton, and tomato. The 109 ARF transcription factors from all species fall into five major classifica- tions ranging from I to IV; the classes were derived from their phylogenetic relationship. Out of all the classes, Class III was the largest, having 33 ARF members holding 30.27% of the total ARF genes analyzed. Class III was comprised of three subgroups: IIIa, IIIb, and Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 13 IIIc; these contained 5, 12, and 16 ARF genes, respectively. Classes IIIa, IIIb, and IIIc con- tained ItfARFs with a Q-rich MD, which included ItfARF5 for IIIa, ItfARF6a, 6b, 8a, and 8b for IIIc, and 19a and 19b for IIIb. ItfARFs were in each class except for class V, suggest- ing that the ItfARF gene family arose before the lineage spilt. 3.4. Expression Profiling of ItfARF Genes in Root Tissues qRT-PCR was employed to better understand the expression profiles of all ItfARF genes within sweet potato normal roots and storage roots. Through the gene expression analysis, the differing functions of all ItfARF genes were apparent. There were some ItfARF genes with similar gene expression patterns between the two root types, while other genes showed clear differences, with higher expression in one root type over the other (Figure 4). For example, ItfARF8a and ItfARF8b were both highly expressed in both normal roots and storage roots. ItfARF2b, ItfARF3a, ItfARF6b, and ItfARF10b are all moderately expressed across both tissue types, suggesting that they may be primarly expressed under specific conditions. Only two of the 25 tested ItfARF genes were more highly expressed in the normal root than that in the storage root; those genes were: ItfARF6a and ItfARF10a. There were some genes that had little to no expression at all, such as ItfARF2c, ItfARF3b, ItfARF9b, ItfARF16a, ItfARF18, and ItfARF19a; these suggest that the rest of the ItfARF genes were expressed more highly in the storage roots as opposed to the normal roots, those genes being ItfARF1a, ItfARF1b, ItfARF2a, ItfARF4a, Int. J. Mol. Sci. 2021, 22, 9391 ItfARF4b, ItfARF5, ItfARF9a, ItfARF16b, ItfARF16c, and ItfARF19b. Out of thos8e onf i1n3e genes, ItfARF4a, ItfARF5, ItfARF9a, ItfARF16b, ItfARF16c, and ItfARF19b had the highest level of expression in the storage roots. . FFigiguurere4 4. .H Heeaattm maappo offA ARRFFg geenneee exxppreressisoionnw witihthinina alllls isxixb bioiolologgiciaclals asmampplelseso of fb booththt htheen noormrmalarl orotot aannddt hthees tsotorargageer oroot.t.T Thheet itsissusuees asammpplelsesa raeren namamededf rformomle lfetftto tor irgihght ta tatt htheet otpopo of ft htheefi fgiguurer,ea, nadndth tehe nnamameseso fotfh teh2e5 2g5e ngeesneasre adreir edcitrleycttolyt htoe rtihgeh triogfhtth eoffi tghuer ef.igTuhree.e xTphree sesxiopnrersasnioknin rgasnakriensghso awrne ushsionwgn aursainngge ao fracnogloer :ohf icgohloerr: gheingeheerx pgreenses ieoxnpirsersespiornes iesn rteepdrbeysegnrteeedn b; lyo wgreereenx; plorewsesiro enx,prereds;saionnd, mreedd; iaannd exmperdesiasnio enx, pbrlaecsski.o*n’s, bdleancokt. e*s’sw dheincohtegse nwehsiacrhe gsetanteiss taicrael lsytadtiisftfiecraelnlyt, d* ipff