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Article The Full-Length Transcriptome and Identification of Na+/H+ Antiporter Genes in Halophyte Nitraria tangutorum Bobrov

Liming Zhu 1, Lu Lu 1, Liming Yang 2, Zhaodong Hao 1, Jinhui Chen 1 and Tielong Cheng 2,*

1 Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education of China, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; [email protected] (L.Z.); [email protected] (L.L.); [email protected] (Z.H.); [email protected] (J.C.) 2 College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China; [email protected] * Correspondence: [email protected]; Tel.: +86-25-8542-8817

Abstract: Nitraria tangutorum Bobrov is a halophyte that is resistant to salt and alkali and is widely distributed in northwestern China. However, its has not been sequenced, thereby lim- iting studies on this particular species. For species without a reference genome, the full-length transcriptome is a convenient and rapid way to obtain reference information. To better study N. tangutorum, we used PacBio single-molecule real-time technology to perform full-length tran- scriptome analysis of this halophyte. In this study, a total of 21.83 Gb of data were obtained, and

 198,300 transcripts, 51,875 SSRs (simple sequence repeats), 55,574 CDS (coding sequence), and  74,913 lncRNAs (long non-coding RNA) were identified. In addition, using this full-length transcrip- + + Citation: Zhu, L.; Lu, L.; Yang, L.; tome, we identified the key Na /H antiporter (NHX) genes that maintain ion balance in plants and Hao, Z.; Chen, J.; Cheng, T. The found that these are induced to express under salt stress. The results indicate that the full-length Full-Length Transcriptome transcriptome of N. tangutorum can be used as a database and be utilized in elucidating the salt Sequencing and Identification of tolerance mechanism of N. tangutorum. Na+/H+ Antiporter Genes in Halophyte Nitraria tangutorum Keywords: full-length transcriptome; SMRT; Nitraria tangutorum; NtNHXs Bobrov. Genes 2021, 12, 836. https:// doi.org/10.3390/genes12060836

Academic Editor: Anca Macovei 1. Introduction Nitraria is a type of typical sand shrub in the Zygophyllaceae [1,2]. Nitraria tangutorum Received: 21 April 2021 is one of the species endemically distributed in the northwestern region of China [3,4]. Accepted: 27 May 2021 Published: 28 May 2021 N. tangutorum is one of the pioneer species that is resistant to salt, alkali, and sand burial, thereby playing an important role in maintaining the ecological balance of northwestern

Publisher’s Note: MDPI stays neutral China [3,5]. In addition, this species has both economic value and benefits. Its leaves can with regard to jurisdictional claims in be consumed, and its fruits can be used as food and medicine [6,7]. published maps and institutional affil- However, the current research on the of N. tangutorum is relatively scarce. iations. In recent years, omics has rapidly developed into a powerful tool for studying various biological processes. , transcriptomics, and other omics can provide a large amount of genetic information [8], which may be utilized to conduct comprehensive studies on N. tangutorum. The transcriptome is the sum of all mRNAs in a , tissue, or in a specific Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. environment [9]. Transcriptome sequencing is helpful in the discovery of potential genes This article is an open access article and can obtain a large number of gene sequences for molecular studies. The full-length distributed under the terms and transcriptome includes full-length mRNA sequences and complete transcripts with com- conditions of the Creative Commons plete structural information [10]. Due to the limitation of reading length, second-generation Attribution (CC BY) license (https:// sequencing technology can only obtain transcriptomes by splicing. Single-molecular real- creativecommons.org/licenses/by/ time technology (SMRT) based on third-generation sequencing has obvious advantages 4.0/). in the reading length of sequencing [11,12]. It does not need the splicing of transcripts to

Genes 2021, 12, 836. https://doi.org/10.3390/genes12060836 https://www.mdpi.com/journal/genes Genes 2021, 12, 836 2 of 15

obtain the full-length transcripts directly, which can more truly reflect the sequence infor- mation of a species and also help in the accurate analysis of the expression of homologous genes and family genes. Whole-genome sequencing can provide extremely rich genetic information [13]. How- ever, only 548 plant were sequenced before 8 March 2021 (https://www.plabipd. de/index.ep (accessed on 25 May 2021)). Species that do not have completed whole- genome sequencing often have limited genetic information that hinders further research. Full-length transcriptome sequencing can provide extensive genetic information that can be used as a database to provide support for molecular and gene function research [14]. In this study, we used PacBio SMRT sequencing technology to obtain the full-length transcriptome of N. tangutorum, which was used in analysis, including full- length transcriptome correction and analysis, CDS prediction factor analysis, SSR analysis, and lncRNA analysis, followed by functional annotation of the obtained transcripts based on the NR, Swiss-Prot, GO, COG, KOG, and KEGG databases. At the same time, we analyzed the full-length transcriptome, identified Na+/H+ antiporter genes, and verified their expression patterns under salt stress. These studies lay the founda- tion for elucidating the salt tolerance mechanism of N. tangutorum using molecular and omics technologies.

2. Materials and Methods 2.1. Plant Materials N. tangutorum seeds were collected from Dengkou County, Inner Mongolia. The seeds were mixed in sand at 4 ◦C for about two months and then were planted in a mixed soil matrix with peat soil: perlite at a ratio of 4:1 in a greenhouse with a 16 h-light/8 h-dark light cycle and 60% air humidity at 23 ◦C. When the seedlings were 2 months old, the roots, stems, and leaves of three individual seedlings showing similar growth rates were collected and stored at −80 ◦C for RNA extraction.

2.2. RNA Extraction, Library Construction and SMRT Sequencing Total RNA was extracted using an RNA extract kit (RNAiso Plus Co 9108, Takara, Japan), the qualified RNA of three seedings were mixed together, then used oligo dT as primer for cDNA synthesis using PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Shiga, Japan) and amplified by PCR, then the cDNA fragments were screened by a BluePippin nucleic acid recovery system (Sage , APG BIO, USA). Then, the full-length cDNA is nicked and repaired, end-repaired, and SMRT adapters were connected to complete the library construction. Then, Agilent 2100 was employed to detect the insert size of the library, qRT-PCR was performed to accurately quantify the library and then sequenced using SMRT technology.

2.3. SSR Analysis For SSR analysis, we screened sequences of > 1000 bp in length. The MISA software (http://pgrc.ipk-gatersleben.de/misa/ (accessed on 25 May 2021)) was used to identify six types of SSRs, namely, mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides.

2.4. CDS, TF, and lncRNA Analysis TransDecoder software was used to identify CDSs, iTAK software was used to predict TFs (transcription factors), and CNCI (Coding-Non-Coding Index) tools (https: //github.com/www-bioinfo-org/CNCI (accessed on 25 May 2021)), CPC (Coding Poten- tial Calculator) software (http://cpc2.gao-lab.org/ (accessed on 25 May 2021)), and Pfam ( family) were employed to predict the coding potential of sequencing data. Genes 2021, 12, 836 3 of 15

2.5. Gene Functional Annotation BLAST was used to compare unigene sequence with those in the NR (Non-Redundant protein sequence database, ftp://ftp.ncbi.nih.gov/blast/db (accessed on 25 May 2021)), Swiss-Prot (Swiss-Prot protein sequence database, http://www.uniprot.org (accessed on 25 May 2021)), GO (Gene Ontology Consortium, http://www.geneontology.org (accessed on 25 May 2021)), COG (Cluster of Orthologous Groups of , http://www.ncbi.nlm.nih. gov/COG (accessed on 25 May 2021)), KOG (euKaryotic Orthologous Groups, http://www. ncbi.nlm.nih.gov/KOG (accessed on 25 May 2021)), and KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/ (accessed on 25 May 2021)) databases to obtain unigene annotation information.

2.6. Identification and Multi-Segment Alignments of Na+/H+ Antiporter Genes Using the spliced sequence as the library, the BLAST program and Hmmer (v.3.0.1b) were used to search for genes containing the Na+/H+ antiporter domain (PF00999) with an E-value of <1 × 10−5, and then the SMAT online tool (http://smart.embl-heidelberg. de (accessed on 25 May 2021)) was employed to identify genes containing the domain. Multiple alignments were performed using DNAMAN v9.0 (Lynnon Corporation, San Ramon, CA, USA) with the deduced amino acid sequences.

2.7. Phylogenetic and Motif Analyses Phylogenetic reconstruction was performed using MEGA-X v10.1 (Temple, Philadel- phia, PA, USA) using the neighbor-joining method, and bootstrap values were calculated according to 1000 repetitions. For motif analysis, the online analysis software MEME (https://meme-suite.org/meme/tools/ (accessed on 25 May 2021)) was used to analyze protein conserved motifs, and the Tbtools [15] were used for drawing.

2.8. Gene Cloning Total RNA was extracted from N. tangutorum seedlings using RNA extraction kit (Eastep super total RNA extraction kit Co LS1040, Promega, China), and the first strand of the cDNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China). The specific primers used to amplify the target gene fragments were designed with Oligo 7.60 (Cascade, Co 80809, USA) and are shown in Supplementary Table S7.

2.9. QRT-PCR Analysis RNA was extracted from N. tangutorum seedlings exposed to 500 mM NaCl for 1, 6, 12, 24, and 48 h and then reverse transcribed for quantitative real-time-PCR (qRT-PCR). qRT-PCR was performed using a SYBR mix (AceQ qPCR SYBR Green Master Mix Co Q121-02, Vazyme, China). The PCR conditions were 94 ◦C for 15 s, 60 ◦C for 30 s, and 72 ◦C for 30 s. The primers used are shown in Supplementary Table S8, and each reaction was performed in triplicate. Relative expression values were calculated using the relative quantization method (2−∆∆CT).

2.10. Subcellular Localization ProtComp 9.0 (http://linux1.softberry.com/berry.phtml (accessed on 25 May 2021)) was employed for subcellular location prediction, and TMHMM (http://www.cbs.dtu.dk/ services/TMHMM/ (accessed on 25 May 2021)) was utilized for protein transmembrane analysis. For gene subcellular localization, pJIT-166 was used as the target vector, and XbaI and BamHI were employed to generate restriction sites to construct a fusion expression vector. The primers used are shown in Supplementary Table S9. The expression vector was transfected into onion epidermal cells by microprojectile bombardment and incubated for 24 h in the dark [16]. The cells were subsequently observed using a fluorescence microscope (X-cite 120Q, Carl Zeiss). Genes 2021, 12, 836 4 of 15

3. Results Genes 2021, 12, 836 3.1. Sequencing Data Statistics 4 of 15 Total RNA was extracted from the roots, stems, and leaves of N. tangutorum to con‐ struct a sequencing library, and SMRT was employed to sequence the qualified library, 3.removing Results errors and redundant consensus sequences in the original sequence and the ob‐ 3.1. Sequencing Data Statistics tained data statistics are shown in Table 1. A total of 21.83 Gb raw data were obtained, Total RNA was extracted from the roots, stems, and leaves of N. tangutorum to con- structrepresenting a sequencing a total library, of 17,951,056 and SMRT wassubreads. employed Most to sequenceof the lengths the qualified were library,within the size range removingof 1 to 2000 errors bp and(Figure redundant 1), the consensus average sequenceslength was in the1,216 original bp, and sequence the assembled and the N50 reached obtained1427 bp. data statistics are shown in Table1. A total of 21.83 Gb raw data were obtained, representing a total of 17,951,056 subreads. Most of the lengths were within the size range ofTable 1 to 20001. Full bp‐length (Figure 1transcriptome), the average length sequencing was 1216 data. bp, and the assembled N50 reached 1427 bp. Average Table 1. Full-lengthSubreads transcriptome sequencingSubreads data. Sample Length of N30 N50 N90 Base (G) Number Subreads Subreads Average Length Sample Subreads N30 N50 N90 Base (G) Number of Subreads N. tanguto‐ N. tangutorum 21.8321.83 17,951, 17,951, 056 056 1216 1216 17881788 1427 1427744 744 rum

FigureFigure 1. 1.Distribution Distribution of subread of subread length. length. The abscissa The is abscissa the length is of the the length subreads, of and the the subreads, ordinate and the ordi‐ is the number of subreads of a particular length. nate is the number of subreads of a particular length. 3.2. Data Correction 3.2. CCSData sequencing Correction analysis can effectively reduce the error rate of sequencing to obtain more accurateCCS sequencing data [17]. To analysis further correct can effectively the accurate reduce full-length the transcriptome error rate of data, sequencing we to obtain analyzed the statistical results of the consensus sequence distribution in Table2. At the samemore time, accurate we also data calculated [17]. To the further consensus correct length the and accurate constructed full a‐length consensus transcriptome length data, we distributionanalyzed the map statistical and found that results the main of the length consensus of the consensus sequence sequence distribution was 1–2000 in bp Table 2. At the (Figuresame 2time,). we also calculated the consensus length and constructed a consensus length distribution map and found that the main length of the consensus sequence was 1–2000 Table 2. CCS quantity statistics data. bp (Figure 2). Sample CCS Nfl-Reads Flnc-Reads Mean-Flnc Consensus Reads TableN. tangutorum 2. CCS quantity21.83 statistics179,510, 56 data. 1216 1788 1427 CCS (circular consensus sequence), flnc-reads (full-length non-chimeric reads), Mean-flnc (average length of full-length non-chimeric reads), Consensus-reads (reads of consensus sequence obtained after clustering). Consensus Sample CCS Nfl‐Reads Flnc‐Reads Mean‐Flnc Reads N. tangutorum 21.83 179,510, 56 1216 1788 1427 CCS (circular consensus sequence), flnc‐reads (full‐length non‐chimeric reads), Mean‐flnc (average length of full‐length non‐chimeric reads), Consensus‐reads (reads of consensus sequence obtained after clustering).

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Genes Genes2021,2021 12, ,83612, 836 5 of 15 5 of 15

Figure 2. Consensus length distribution. The abscissa is the length of consensus, and the ordinate is the number of the consensus length.

Figure3.3. CDS, 2. 2.Consensus Consensus TFs, lncRNA length length distribution. Analysis distribution The abscissa. The isabscissa the length is of the consensus, length of and consensus, the ordinate and is the ordinate the number of the consensus length. is the numberUsing TransDecoder of the consensus software length. sequencing data to perform coding sequence predic‐ 3.3.tion, CDS, a total TFs, lncRNAof 55,574 Analysis sequences were obtained. The length distribution of the sequences is 3.3.shown CDS,Using in TransDecoderTFs, Figure lncRNA 3, and software Analysis the distribution sequencing data frequency to perform is shown coding sequencein Table predic- S1. Using iTAK soft‐ tion, aUsing total of TransDecoder 55,574 sequences software were obtained. sequencing The length data distribution to perform of the coding sequences sequence predic‐ isware, shown a intotal Figure of 3,7453, and transcription the distribution factors frequency were is shown predicted in Table that S1. belonged Using iTAK to 75 gene families tion,software,(Figure a total a4), total includingof 55,574 of 3745 sequences transcriptionWRKY (157), were factors MYB obtained. were (236), predicted NACThe length, thatAP2/ERF belonged distribution (175), to 75 bZIP gene of the (155), sequences and other is shownfamiliestranscription (Figurein Figure4 ),factors. including 3, and CNCI, theWRKY distribution CPC(157), MYBsoftware, (236),frequency NACand, AP2/ERFPfam is shown database(175), inbZIP Table were(155), S1. used and Using to iTAKpredict soft the‐ ware,othercoding transcription a totalpotential of 3,745 factors. of thetranscription CNCI, sequencing CPC software, factors data. and wereCNCI Pfam predicted analysis database identifiedthat were belonged used to 74,913 predict to 75lncRNAs gene families (Table (FiguretheS2). coding The 4), potentialCPC including software of the WRKY sequencing was (157), used data. MYB to CNCIclassify (236), analysis NAC and, identifiedAP2/ERFidentify 74,913 lncRNA.(175), lncRNAs bZIP A (155),total ofand 139,217 other (Tabletranscription S2). The factors. CPC software CNCI, was CPC used software, to classify and and Pfam identify database lncRNA. were A total used of to predict the 139,217lncRNAs lncRNAs were were identified identified (Table (Table S3). UsingUsing the the Pfam Pfam database database to perform to perform mul- multiple se‐ codingtiplequence sequence potential alignments alignments of the and andsequencing HMMs HMMs searches data. CNCI on on PacBio PacBioanalysis sequencing sequencing identified data, a74,913data, total of alncRNAs total of (Table90,061 S2).90,061lncRNAs The lncRNAs CPC werewere software identified identified was (Table (Table used S4).S4). to Finally, Finally,classify a Venn aand Venn diagram identify diagram was lncRNA. constructed was constructed A tototal of to139,217 depict lncRNAsdepictthe lncRNAs the lncRNAs were identified identified identified by by (Table the three three S3). methods methods Using to identifythe to identifyPfam shared database shared sequences sequencesto (Figure perform5). (Figure multiple 5). se‐ quence alignments and HMMs searches on PacBio sequencing data, a total of 90,061 lncRNAs were identified (Table S4). Finally, a Venn diagram was constructed to depict the lncRNAs identified by the three methods to identify shared sequences (Figure 5).

FigureFigure 3. 3.Coding Coding sequence sequence length length distribution. distribution. The abscissa The is theabscissa predicted is the length predicted of CDSs, thelength left of CDSs, the ordinateleft ordinate is the number is the of number transcripts of of transcripts the CDSs, and of the the right CDSs, ordinate and is the the percentageright ordinate of transcripts. is the percentage of transcripts. Figure 3. Coding sequence length distribution. The abscissa is the predicted length of CDSs, the left ordinate is the number of transcripts of the CDSs, and the right ordinate is the percentage of transcripts.

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FigureFigureFigure 4. Transcription 4. 4.Transcription factor factor analysis analysis. analysis. The The. The ordinate ordinate shows shows shows different different different transcription transcription transcription factor factor families,factor families, families, andandand the the theabscissa abscissa abscissa depicts depicts depicts the the thenumber number number ofof the the of transcription thetranscription transcription factor factor families.factor families. families.

FigureFigureFigure 5. Coding 5. 5.Coding Coding ability ability ability prediction prediction prediction of VennVenn of Venn diagram. diagram. diagram. The The blue The blue circle, blue circle, greencircle, green circle, green circle, and circle, yellowand and yellow circle yellow cir‐ cir‐ cle representrepresentcle represent CNCI, CNCI, CNCI, CPC, CPC, Pfam,CPC, Pfam, respectively.Pfam, respectively. respectively. 3.4. SSR Analysis 3.4.3.4. SSR SSR Analysis Analysis Using MISA software to perform SSR analysis on of >1.000 bp in length, a totalUsing ofUsing 51,875 MISA MISA SSRs software weresoftware identified, to perform to perform most SSR of SSR analysis which analysis were on mononucleotides, unigeneson unigenes of >of 1.000 > dinucleotides, 1.000 bp bpin length, in length, a a totalandtotal of trinucleotides, 51,875 of 51,875 SSRs SSRs were accounting were identified, identified, for 71.68%, most most 15.04%, of which of which and were 12.13% were mononucleotides, of mononucleotides, the total number dinucleotides, of dinucleotides, SSRs, andrespectivelyand trinucleotides, trinucleotides, (Table accounting3), accounting whereas for tetranucleotides, for71.68%, 71.68%, 15.04%, 15.04%, pentanucleotides, and and 12.13% 12.13% of andthe of thetotal hexanucleotides total number number of SSRs, of SSRs, respectivelycollectivelyrespectively (Table accounted (Table 3), 3),whereas for whereas 11.47% tetranucleotides, of tetranucleotides, the total number pentanucleotides, pentanucleotides, of SSRs. At the and same and hexanucleotides time, hexanucleotides SSR collectivelydensitycollectively analysis accounted accounted also generated for for11.47% 11.47% similar of the of results thetotal total (Figurenumber number6). of SSRs. of SSRs. At theAt thesame same time, time, SSR SSR den den‐ ‐ sitysity analysis analysis also also generated generated similar similar results results (Figure (Figure 6). 6).

TableTable 3. Results 3. Results of SSR of SSR analysis. analysis.

FeatureFeature NumberNumber TotalTotal number number of sequences of sequences examined examined 71,08971,089 TotalTotal size size of examined of examined sequences sequences (bp) (bp) 111,431,918111,431,918 IdentifiedIdentified SSRs SSRs 51,87551,875 SSRSSR containing containing sequences sequences 29,24929,249 SequencesSequences containing containing more more than than 1 SSR 1 SSR 11,08211,082

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Table 3. Results of SSR analysis. Genes 2021, 12, 836 7 of 15 Feature Number Total number of sequences examined 71,089 Total size of examined sequences (bp) 111,431,918 SSRs presentIdentified in compound SSRs formation 51,875 12,283 SSR containing sequences 29,249 Sequences containingMononucleotides more than 1 SSR 11,082 37,182 SSRs present inDinucleotides compound formation 12,283 7804 Mononucleotides 37,182 DinucleotidesTrinucleotides 7804 6294 TrinucleotidesTetranucleotides 6294 330 Tetranucleotides 330 PentanucleotidesPentanucleotides 89 89 HexanucleotidesHexanucleotides 176 176

FigureFigure 6. 6.SSR SSR density density distribution. distribution. The abscissa The abscissa is the SSR is type, the andSSR the type, ordinate and isthe the ordinate number of is the number of SSRs of the corresponding type in each Mb of sequence. SSRs of the corresponding type in each Mb of sequence. 3.5. Functional Annotation 3.5. TheFunctional 65,361 unigenes Annotation were functionally annotated using the COG, GO, KEGG, NR, Swiss-Prot,The and65,361 KOG unigenes databases (Tablewere4 ),functionally with annotation annotated coverage rates using of 34.46%, the COG, 69.19%, GO, KEGG, NR, 31.43%, 99.53%, 78.99%, and 61.89%, respectively. Swiss‐Prot, and KOG databases (Table 4), with annotation coverage rates of 34.46%, Table69.19%, 4. Unigene 31.43%, annotation 99.53%, statistics. 78.99%, and 61.89%, respectively. Unigene annotation using the NR database revealed that these exhibited a high de‐ Annotated Databases Number of Unigenes 300–1000 bp ≥1000 bp gree of homology with Citrus sinensis and C. clementina (Figure 7). We compared the COG 26,526 3596 22,903 annotatedGO genes to the GO database 45,222 and divided 7626 them into three 37,501 independent classifica‐ tions: cellularKEGG component, molecular 20,548 function, and 3528 biological process, 16,970 respectively(Figure KOG 40,450 6479 33,887 8). Pfam 47,073 6746 40,303 COGSwiss-Prot analysis classified these 51,635 unigenes into 876025 categories. Amino 42,752 acid transport and metabolismNr was the most frequent 65,055 functional category, 12,596 followed 52,211 by cell motility (Figure All 65,361 12,728 52,376 The9). columnKEGG header annotation 300–1000 bp annotated indicates the number205,48 of unigenes unigenes annotated to 150 to pathways the database whose(Table length S5), which can be isdivided≥300 bases into and ≤ 221000 categories bases. The column (Figure header ≥10).1000 bp indicates the number of unigenes annotated to the database unigene number with lengths ≥ 1000 bases.

Figure 7. Homologous species distribution by Nr database. The best hits with an E‐value = 1.0E‐5 for each query were grouped according to species.

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SSRs present in compound formation 12,283 Mononucleotides 37,182 Dinucleotides 7804 Trinucleotides 6294 Tetranucleotides 330 Pentanucleotides 89 Hexanucleotides 176

Figure 6. SSR density distribution. The abscissa is the SSR type, and the ordinate is the number of SSRs of the corresponding type in each Mb of sequence.

3.5. Functional Annotation The 65,361 unigenes were functionally annotated using the COG, GO, KEGG, NR, Swiss‐Prot, and KOG databases (Table 4), with annotation coverage rates of 34.46%, 69.19%, 31.43%, 99.53%, 78.99%, and 61.89%, respectively. Unigene annotation using the NR database revealed that these exhibited a high de‐ gree of homology with Citrus sinensis and C. clementina (Figure 7). We compared the annotated genes to the GO database and divided them into three independent classifica‐ tions: cellular component, molecular function, and biological process, respectively(Figure Genes 2021, 12, 836 8 of 15 8). Genes 2021, 12, 836 8 of 15 COG analysis classified these unigenes into 25 categories. Amino acid transport and metabolism was the most frequent functional category, followed by cell motility (Figure Unigene annotation using the NR database revealed that these exhibited a high degree 9).of homology KEGG annotation with Citrus sinensis annotated and C. 205,48 clementina unigenes(Figure to7). 150 We compared pathways the (Table annotated S5), which can be dividedgenesTable to the4. into Unigene GO 22 database categories annotation and divided (Figure statistics them 10). into . three independent classifications: cellular component, molecular function, and biological process, respectively (Figure8). Annotated Databases Number of Unigenes 300–1000 bp ≥ 1000 bp COG 26,526 3596 22,903 GO 45,222 7626 37,501 KEGG 20,548 3528 16,970 KOG 40,450 6479 33,887 Pfam 47,073 6746 40,303 Swiss‐Prot 51,635 8760 42,752 Nr 65,055 12,596 52,211 All 65,361 12,728 52,376 The column header 300–1000 bp indicates the number of unigenes annotated to the database Figurewhose 7. 7.Homologous lengthHomologous is ≥ species300 species bases distribution distribution and by ≤ Nr1000 database. bybases. Nr The database. The best column hits withThe an bestheaderE-value hits ≥ =with 1.01000× an10 bp− E5‐ valueindicates = 1.0E the‐5 number forforof eacheach unigenes query query were annotatedwere grouped grouped according to the according database to species. to species.unigene number with lengths ≥ 1000 bases.

FigureFigure 8. GO 8. functionGO function classification. classification.

COG analysis classified these unigenes into 25 categories. Amino acid transport and metabolism was the most frequent functional category, followed by cell motility (Figure9). KEGG annotation annotated 20,548 unigenes to 150 pathways (Table S5), which can be divided into 22 categories (Figure 10).

Figure 9. COG annotation classification statistics of expressed genes.

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Table 4. Unigene annotation statistics.

Annotated Databases Number of Unigenes 300–1000 bp ≥ 1000 bp COG 26,526 3596 22,903 GO 45,222 7626 37,501 KEGG 20,548 3528 16,970 KOG 40,450 6479 33,887 Pfam 47,073 6746 40,303 Swiss‐Prot 51,635 8760 42,752 Nr 65,055 12,596 52,211 All 65,361 12,728 52,376 The column header 300–1000 bp indicates the number of unigenes annotated to the database whose length is ≥ 300 bases and ≤ 1000 bases. The column header ≥ 1000 bp indicates the number of unigenes annotated to the database unigene number with lengths ≥ 1000 bases.

Genes 2021, 12, 836 9 of 15 Figure 8. GO function classification.

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FigureFigure 9. 9.COG COG annotation annotation classification classification statistics statistics of expressed of genes. expressed genes.

FigureFigure 10. 10.KEGG KEGG metabolicmetabolic categoriescategories in the transcriptome of of Nitraria Nitraria tangutorum. tangutorum. The The ordinate ordinate is isthe the annotated annotated biological biological function, function, and and the the abscissa abscissa is the is the percentage percentage of these of these genes genes in the in total the total numbernumber of of annotated annotated genes. genes.

3.6.3.6. Identification Identification of of Na Na++/H/H++ Antiporter Genes in the Full Full-Length‐Length Transcriptome TheThe full-length full‐length transcriptome transcriptome has has a a resolution resolution accuracy accuracy of of a a single single nucleotide, nucleotide, thus thus allowingallowing the the discovery discovery of of gene gene transcripts. transcripts. N. N. tangutorum tangutorum is is a a typical typical halophyte, halophyte, but but its its molecularmolecular mechanism mechanism for for salt salt tolerance tolerance remains remains unclear. unclear. The The NHX NHX gene gene family family plays plays an an importantimportant role role in in stress stress resistance resistance in in plants. plants. However, However, its its distribution distribution in in N. N. tangutorum tangutorum is is unknown.unknown. UsingUsing the the full-length full‐length transcriptome transcriptome as as a a database, database, we we searched searched for for sequences sequences + + containingcontaining the the domain domain according according to to the the Na Na/H+/H+ antiporterantiporter (PF00999)(PF00999) PfamPfam domain domain and and manuallymanually compared compared it it to to SMAT SMAT online online analysis analysis for for confirmation. confirmation. Using Using these these steps, steps, we we havehave identified identified a a total total of of nine nine NtNHXs, NtNHXs, and and then then we we named named these these based based on theiron their homology homol‐ ogy to AtNHXs. Multiple sequence alignment shows that these all contain a Na+/H+ anti‐ porter domain (Supplemental Figure S2), then phylogenetic reconstruction of the AtNHXs was performed, which showed that these genes could be divided into three groups (Figure 11).

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to AtNHXs. Multiple sequence alignment shows that these all contain a Na+/H+ antiporter Genes 2021,, 12,, 836 10 of 15 domain (Supplemental Figure S2), then phylogenetic reconstruction of the AtNHXs was performed, which showed that these genes could be divided into three groups (Figure 11).

FigureFigure 11. 11. PhylogeneticPhylogenetic analysis analysis of NHX of NHX genes genes inin Nitraria in Nitraria tangutorumtangutorum tangutorum and Arabidopsisand thalianathaliana thaliana (Linn.)(Linn.)(Linn.) Heynh Heynh...

3.7.3.7. Gene Gene Conserved Conserved Motifs Motifs Analysis Analysis of of NHXs NHXs ToTo identifyidentify thethe conserved motifs motifs of of thesethese NtNHXs NtNHXs proteins, proteins, using using MEME MEME software, software, wewe determined determined thatthat that NtNHXs NtNHXs had had 14 14 mainly mainly conserved conserved motifs motifs (Figure(Figure (Figure 12). Almost12). Almost all ideniden all‐‐ tifiedtifiedidentified NtNHXs NtNHXs harbored harbored similar similar motif motifsequences, sequences, indicatingindicating indicating thatthat thesethese that motifs these were motifs highly were highly conserved. conserved.

Figure 12. Conserved motif analysis of NtNHXs. Figure 12. Conserved motif analysis of NtNHXs.. 3.8. Real-Time Quantitative PCR Analysis 3.8. Real‐‐Time Quantitative PCR Analysis NHXs play an important role in maintaining the balance of Na+/H+ and salt resistance NHXs play an important role in maintaining the balance of Na+/H+ and salt resistance in plantsNHXs [18 play,19]. an To important verify whether role in maintaining these NtNHX the genes balance play of Na a similar+/H+ and role salt in resistance the salt- in plants [18,19]. To verify whether these NtNHX genes play a similar role in the salt‐ intolerance plants [18,19]. process To of verify N. tangutorum, whether these the seedlings NtNHX genes of N. tangutorumplay a similar were role subjected in the salt to‐ tolerance process of N. tangutorum, the seedlings of N. tangutorum were subjected to tolerancestress using process 500 mM of N. NaCl tangutorum, solution and the plant seedlings materials of N. were tangutorum collected were at 1, 6,subjected 12, 24, and to stress using 500 mM NaCl solution and plant materials were collected at 1, 6, 12, 24, and stress48 h to using assess 500 NtNHX mM NaCl expression. solution Figure and plant 13 shows materials that the were NtNHX collected genes at 1, were 6, 12, expressed 24, and 48 h to assess NtNHX expression. Figure 13 shows that the NtNHX genes were expressed 48under h to saltassess stress, NtNHX indicating expression. that NtNHXs Figure 13 may shows play that an important the NtNHX role genes in the were salt expressed resistance underof N. tangutorum. salt stress, indicatingindicating thatthat NtNHXs may play an importantimportant role inin thethe salt resistance of N. tangutorum.tangutorum.

Genes 2021, 12, 836 11 of 15 GenesGenes2021 2021, 12, 12, 836, 836 1111 of of 15 15

Figure 13. Relative expression level of NtNHXs in Nitraria tangutorum under 500 mM NaCl salt stress.

Figure 13. Relative expression level of NtNHXs in Nitraria tangutorum under 500 mM NaCl salt stress. Figure3.9. Clone 13. Relativeand Subcellular expression Localization level of of NtNHX7 NtNHXs in Nitraria tangutorum under 500 mM NaCl salt stress3.9. Clone.In Arabidopsis and Subcellular, AtNHX7 Localization is also ofknown NtNHX7 as AtSOS1, which encodes a Na+/H+ antiporter thatIn is Arabidopsislocated in the, AtNHX7 plasma is membrane also known of as the AtSOS1, cell that which promotes encodes Na+ a efflux Na+/H and+ antiporter maintains 3.9.thatthe Clone isbalance located and of in Subcellularions the plasmainside and membrane Localization outside of the the of cell cellNtNHX7 [20]. that promotesTo explore Na whether+ efflux andNtNHX7 maintains has a similar effect and also to verify the accuracy of the predicted gene, we designed specific the balance of ions inside and outside the cell [20]. To explore whether NtNHX7 has a+ + similarprimersIn effectArabidopsis and andcloned also ,NtNHX7. AtNHX7 to verify We the is compared accuracy also known of the the sequence as predicted AtSOS1, difference gene, which we between designed encodes the specific acloned Na /H antiporter thatprimersgene is and located and the cloned reference in the NtNHX7. plasmasequence We membranefrom compared this transcriptome, the of sequence the cell and differencethat we promotes found between that Na their the+ efflux clonedsimilar and‐ maintains thegeneity balanceis and 99.8% the (Figure reference of ions S1), sequenceinside indicating and from that outside this the transcriptome, NtNHX7 the cell predicted and[20]. we byTo found the explore full that‐length their whether similarity transcrip NtNHX7‐ has a similaristome 99.8% has (Figureeffect higher and S1), accuracy. indicating also to verify that the the NtNHX7 accuracy predicted of the by thepredicted full-length gene, transcriptome we designed specific Real‐time quantitative PCR analysis revealed that NtNHX7 is expressed by salt stress primershas higher and accuracy. cloned NtNHX7. We compared the sequence difference between the cloned (FigureReal-time 13). Subsequently, quantitative PCR we used analysis the revealedsequencing that results NtNHX7 to analyze is expressed its expression by salt stress pat‐ gene(Figuretern. and First, 13). the Subsequently,ProtComp reference 9.0 we andsequence used TMHMM the sequencingfrom were this used transcriptome, results to predict to analyze the subcellular itsand expression we foundlocation pattern. that pat‐ their similar‐ ityFirst,tern is 99.8%of ProtComp NtNHX7, (Figure 9.0 which and S1), TMHMM showed indicating that were NtNHX7 usedthat the to has predict NtNHX7 a higher the subcellular probability predicted location of by localizing the pattern full to‐length ofthe transcrip‐ tomeNtNHX7,plasma has membrane which higher showed accuracy. of the that cell NtNHX7 (Table S6, has Figure a higher 14A). probability To further of determine localizing the to the subcellular plasma membranelocalizationReal‐ oftime of the NtNHX7, cellquantitative (Table we S6, constructed Figure PCR 14 analysisA). the To furtherNtNHX7 revealed determine‐GFP that vector the NtNHX7 subcellular and performed is localization expressed transi‐ by salt stress of NtNHX7, we constructed the NtNHX7-GFP vector and performed transient transformation (Figureent transformation 13). Subsequently, experiments we toused confirm the sequencingour findings. resultsAs shown to analyzein Figure its 14B, expression pat‐ experimentsNtNHX7 is tomainly confirm expressed our findings. on the As cell shown membrane, in Figure which 14B, also NtNHX7 verifies is mainly our previous expressed bio‐ tern.oninformatics the First, cell membrane, ProtComp prediction. which 9.0 also and verifies TMHMM our previous were bioinformaticsused to predict prediction. the subcellular location pat‐ tern of NtNHX7, which showed that NtNHX7 has a higher probability of localizing to the plasma membrane of the cell (Table S6, Figure 14A). To further determine the subcellular localization of NtNHX7, we constructed the NtNHX7‐GFP vector and performed transi‐ ent transformation experiments to confirm our findings. As shown in Figure 14B, NtNHX7 is mainly expressed on the cell membrane, which also verifies our previous bio‐ informatics prediction.

FigureFigure 14. 14.Subcellular Subcellular localization localization of of NtNHX7. NtNHX7. (A ()A NtNHX7) NtNHX7 transmembrane transmembrane helix helix domain domain prediction predic‐ bytion TMHMM. by TMHMM. (B) NtNHX7 (B) NtNHX7 subcellular subcellular localization localization by microprojectile by microprojectile bombardmen, bombardmen, 35S: GFP 35S: plants GFP asplants control. as control. Scale bar Scale =100 barµm. =100 μm.

Figure 14. Subcellular localization of NtNHX7. (A) NtNHX7 transmembrane helix domain predic‐ tion by TMHMM. (B) NtNHX7 subcellular localization by microprojectile bombardmen, 35S: GFP plants as control. Scale bar =100 μm.

Genes 2021, 12, 836 12 of 15

4. Discussion The SMRT technology is one of the relatively fast-developing bioinformatics technolo- gies in recent years [21]. It can be used to improve the gene annotation status of sequenced species and as a reference for unsequenced species to facilitate scientific research. For example, the annotation of the sorghum genome has been improved using the SMRT technology, which will help to further study sorghum [22]. In sugar beet, the SMRT technology has been used to predict new genes and has laid the foundation for further research on this species [23]. Currently, transcriptome sequencing is mainly performed through NGS sequenc- ing [24]. However, due to the limitation of sequencing read length in traditional next- generation sequencing, transcript information is mostly obtained by splicing sequences, and the full-length transcript sequence cannot be directly obtained. This hampers the analysis of transcriptome structure and may be accompanied by a high frequency of false-positive sequences. Compared with second-generation sequencing, third-generation sequencing has a significantly higher read length, reaching an average of > 10 kb, and the maximum can reach 60 kb [25]. The SMRT technology can obtain a complete transcript without splicing, which is of great significance for transcriptome structure analysis, prediction of new genes [26], and supplementary genome annotation [27]. At present, various species have completed whole-genome sequencing, but there are still numerous that have not completed the construction of a whole-genome map due to limited attention or sequencing cost, which also hinders further research on these species. For species that have not completed whole-genome sequencing, the full-length transcriptome provides a wealth of genetic information. Transcriptome data can be used to clone genes, develop SSR primers [28], and analyze homologous genes. N. tangutorum is a typical drought-tolerant and salt-tolerant plant that is of important ecological significance. However, whole-genome sequencing of this species has not been completed to date, and limited genetic information hinders related molecular research. Although some genes of N. tangutorum have been cloned and their functions have been verified, this is only the tip of the iceberg in terms of studying this species from a molecular perspective. Using its full-length transcriptome sequencing data, it is possible to analyze its CDS, transcription factors, and SSRs and at the same time perform functional annotations on its genes and develop SSRs primers for plant population classification [29]. Full-length transcriptomes can also be used as a database to identify target genes [30]. Nitraria tangutorum is a typical halophyte, and we would like to study the expression of salt-resistance-related genes in it. In the study of other plants such as Arabidopsis [31] and [18], NHXs have been shown to be involved in the process of salt stress toler- ance. Therefore, in this study, we used the full-length transcriptome data as a library to obtain sequence information on the NtNHXs using BLAST. Then we investigated whether these genes responded to salt stress, and we found they can be induced by salt stress, although they had different expression characteristics in different tissues after salt treat- ment. In general, their expression reached its maximum within 12 h and then decreased (Figure 13), which was similar to the study of Gossypium barbadense [32] and Pyrus betulaefo- lia [33], but there were also some differences, we suspect that the reason for this difference might be related to differences in salt stress concentration and species. In summary, the results indicated that the NtNHX gene might be involved in the process of resisting salt stress in Nitraria tangutorum, but further gene functional verification studies are needed. Genes 2021, 12, 836 13 of 15

In addition, we selected NtNHX7 to be cloned and sequenced and found that the cloned gene sequence was basically consistent with the sequence provided by the full- length transcriptome (Figure S1) through sequencing. Further, we constructed the NtNHX7- GFP gene fusion expression vector and observed its expression pattern through micro- projectile bombardment and found that the signal was mainly expressed on the plasma membrane and distributed inhomogeneously (Figure 14), which was similar to the expres- sion pattern of the AtNHX7 (also known as AtSOS1) in Arabidopsis [20]. According to the above results, we thought that the full-length transcriptome could be used as a gene database, although it may not be as complete when compared with the full-genome sequence. However, for unsequenced species, it can enrich the scarce omics information and also lay the foundation for further molecular research.

5. Conclusions In this study, we used SMRT technology to determine the full-length transcriptome of N. tangutorum. A total of 21.83 Gb of data were obtained, of which 198,300 transcripts, 51,875 SSRs, 55,574 CDS, and 74,913 IncRNAs were identified. In addition, using this full-length transcriptome, we identified the key NHX genes that maintain ionic balance in plants, and we induced their expression under salt stress. Based on the full-length transcriptome data, we also cloned the NtNHX7 gene and found a high similarity to the predicted result through sequencing, which indicates that the based on the full-length transcript is more accurate. In summary, this study obtained high-quality, full-length transcript information on N. tangutorum by full-length transcription sequencing and provided abundant transcript information, which promotes omics and molecular research on N. tangutorum.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/genes12060836/s1, Figure S1: The amino acid sequence comparison between predicted sequence and sequencing result of NtNHX7. The top row is the prediction result, and the second row is the sequencing result. Figure S2: The amino acid multiple fragment alignments of NtNHXs. Table S1: CDS length distribution and its frequency. Table S2: CNCI analysis for lncRNAs. Table S3: CPC analysis for lncRNAs. Table S4: Pfam analysis for IncRNAs. Table S5: KEGG annotation databases. Table S6: Prediction of NtNHX7 subcellular localization by ProtComp9.0 (http://linux1 .softberry.com/berry.phtml (accessed on 25 May 2021)). Table S7: Primers for gene cloning. Table S8: Primers for Quantitative RT-PCR. Table S9: Primers for subcellular localization. Table S10: amino acid sequence of NtNHXs. Author Contributions: L.Z. wrote sections of the manuscript, L.Z., L.L. performed the experiments, L.Y., Z.H. and J.C. carried out the statistical analysis; T.C. contributed to the design of this research. All authors contributed to manuscript revision and approved the submitted version. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (31770715 and 32071784), State Forestry and Grassland Administration (2020133106), Priority Academic Pro- gram Development of Jiangsu Higher Education Institutions. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analysed during this study are included in this published article and its Supplementary Information Files. Raw data generated for this study have been uploaded to NCBI with accession number is SAMN19103071. Acknowledgments: We acknowledge everyone who contributed to this article. Conflicts of Interest: The authors declare no conflict of interest. Genes 2021, 12, 836 14 of 15

Abbreviations

NHX Na+/H+ antiporter gene SRMT Single-molecule real-time CDS Coding sequence SSR Simple sequence repeat LncRNA Long non-coding RNA CPC Coding potential calculator CNCI Coding non-coding index CPC Coding potential calculator mRNA Messenger RNA qRT-PCR Quantitative real-time PCR TF Transcription factors cDNA Complementary DNA Pfam Protein family NR Non-redundant protein sequence database Swiss-Prot Swiss-Prot protein sequence database GO Gene Ontology Consortium COG Cluster of Orthologous Groups of proteins KOG EuKaryotic Orthologous Groups KEGG Kyoto Encyclopedia of Genes and Genomes

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