- Research article
- Open Access
Transcriptomic profiling of the salt-stress response in the halophyte Halogeton glomeratus
- Juncheng Wang†1, 2,
- Baochun Li†1, 3,
- Yaxiong Meng1, 2,
- Xiaole Ma1, 2,
- Yong Lai1, 2,
- Erjing Si1, 2,
- Ke Yang1, 2,
- Panrong Ren1, 2,
- Xunwu Shang1 and
- Huajun Wang1, 2Email author
© Wang et al.; licensee BioMed Central. 2015
- Received: 23 August 2014
- Accepted: 20 February 2015
- Published: 11 March 2015
Halogeton glomeratus (H. glomeratus) is an extreme halophyte that is widely distributed in arid regions, including foothills, the Gobi desert of northwest China, and the marginal loess of Central Asia. However, research on the salt-tolerant mechanisms and genes of this species are limited because of a lack of genomic sequences. In the present study, the transcriptome of H. glomeratus was analyzed using next-generation sequencing technology to identify genes involved in salt tolerance and better understand mechanisms of salt response in the halophyte H. glomeratus.
Illumina RNA-sequencing was performed in five sequencing libraries that were prepared from samples treated with 200 mM NaCl for 6, 12, 24, and 72 h and a control sample to investigate changes in the H. glomeratus transcriptome in response to salt stress. The de novo assembly of five transcriptomes identified 50,267 transcripts. Among these transcripts, 31,496 (62.66%) were annotated, including 44 Gene Ontology (GO) terms and 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Compared with transcriptomes from the control and NaCl-treated samples, there were 2,223, 5,643, 7,510 and 10,908 genes that were differentially expressed after exposure to NaCl for 6, 12, 24, and 72 h, respectively. One hundred and eighteen salt-induced genes were common to at least two stages of salt stress, and 291 up-regulated genes were common to various stages of salt stress. Numerous genes that are related to ion transport, reactive oxygen species scavenging, energy metabolism, hormone-response pathways, and responses to biotic and abiotic stress appear to play a significant role in adaptation to salinity conditions in this species. The detection of expression patterns of 18 salt-induced genes by quantitative real-time polymerase chain reaction were basically consistent with their changes in transcript abundance determined by RNA sequencing.
Our findings provide a genomic sequence resource for functional genetic assignments of an extreme halophyte, H. glomeratus. We believe that the transcriptome datasets will help elucidate the genetic basis of this species’ response to a salt environment and develop stress-tolerant crops based on favorable wild genetic resources.
- Halogeton glomeratus
- Illumina sequencing
Salinity stress is one of the most serious factors that severely affect crop growth, development, and yield [1,2]. A better understanding of plant salt tolerance mechanisms is crucial for the sustainable development of agriculture worldwide . Halophytes constitute approximately 1% of the world’s flora. They are adapted to highly saline soil conditions  and thus represent ideal materials to understand complex physiological and genetic mechanisms of salt tolerance . Several halophytes have been investigated in physiological and molecular biological studies of the mechanisms of salt tolerance, such as Thellungiella halophila [3,5,6], Crithmummaritimum , Suaeda fruticosa , Cakile maritime , Centaurea tuzgoluensis , and Spartina alterniflora Loisel . Especially Thellungiella halophila, it is a close relative of Arabidopsis with strong ability to adapt to saline environments, which make it suitable halophytic model for studying mechanisms of salt tolerance [5,6]. Genes may contribute to its salt-tolerance, such as related to cation transport, abscisic acid signaling, and wax production have been identified . Adaption of salt tolerance in halophyte may involve a global network adjustment of multiple regulatory mechanisms [6,9,11]. However, unclear are whether all halophytes are similarly salt-tolerant and whether halophytes have specific salt-tolerant mechanisms . Our understanding of the molecular basis of non-genomic halophytes is limited. The identification and isolation of novel genes using genomic approaches will advance our understanding of the salt-tolerance mechanisms of halophytes .
The halophyte Halogeton glomeratus (H. glomeratus) is a chenopodiaceae succulent annual herbaceous plant that is naturally distributed in arid and desert regions of northwest China, from Mongolia to Central Asia, and plays an important role in sustaining local ecosystems [13,14]. During the process of adaptation to drought, salinization, and desertification environments, H. glomeratus has evolved many strategies, including high tolerance to salt at the germination stage , stems that form multiple branches, increased leaf and stem succulents, the storage of large amounts of water, the compartmentalization of salinity in mesophyll cells, and specialized stomatal regulation to cope with environmental stress . However, little effort has been made to elucidate the salt tolerance mechanisms of H. glomeratus. Previous studies have focused only on its morphological and physiological characteristics [13,15]. We recently reported that this species can grow normally under conditions of a NaCl concentration up to 500 mM, and a detailed analysis of the molecular mechanisms that underlie salt tolerance in H. glomeratus is required.
RNA transcript profiling is an important strategy for studying the expression of a large number of genes in a given tissue at a given time point . RNA sequencing (RNA-Seq) technology is a major quantitative platform for transcriptome analysis that efficiently and economically enables investigations of transcriptomes in various gene expression studies, even for species that lack a reference genome [17,18]. This sequencing method has been widely applied to transcript profiling in numerous non-model species, and related candidate genes, single-nucleotide polymorphisms (SNPs), and simple-sequence repeats (SSRs) have been identified [19-21].
In the present study, paired-end sequencing technology was used to examine the leaf transcriptomes of H. glomeratus under salt stress conditions. We constructed five libraries that were sequenced using Illumina HiSeq 2000 and 2-month-old seedlings that were treated with 200 mM NaCl for 0, 6, 12, 24, and 72 h. We then analyzed the expression profiles of 50,267 identified unigenes during different stages of salt stress compared with the expression of unigenes in the control library (0 h). A total of 409 candidate genes were identified to be potentially related to the salt response and tolerance (118 salt-induced and 291 upregulated with salt stress at all stages). We validated salt-induced unigenes based on different stress stages using quantitative real-time polymerase chain reaction (qRT-PCR). This research will facilitate elucidation of the salt tolerance mechanisms of this species and provide an important wild gene resource to improve salt tolerance in crops through genetic manipulation.
Na+ accumulation response to NaCl stress
De novo assembly and quantitative assessment of IIIumina sequence
Statistical summary of sequencing and assembly results
Total Raw Reads
Total Clean Reads
Total Clean Nucleotides (nt)
Total Length (nt)
Mean Length (nt)
Total Length (nt)
Mean Length (nt)
Total Number: 50,267; Total Length (nt): 43,714,924; Mean Length (nt): 870; N50: 1441
Several complementary approaches were utilized to annotate functional information about these assembled unigenes, including protein sequence similarities, clusters of orthologous groups (COG), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway information. All of the unigenes were first compared with the NCBI Nr database, non-redundant nucleotide sequence (Nt) database, and the Swiss-Prot protein database by BLASTing with E values < 1e-5. Using the best hits found by BLAST, an inferred putative function was assigned to the sequences. The 29,588 (58.8%) unigenes were significantly matched to the known genes in Nr, a total of 22,810 (45.4%) unigenes were identified in Nt, and 19,117 (38.0%) sequences had best hits in the Swiss-Prot database.
To further predict the genes with different expression levels under salt-stressed conditions, the GO functional analysis was performed on the differentially expressed genes. The differentially expressed genes (DEGs) were defined as those with an FDR ≤ 0.001 and log2 ratio ≥ 1 (RPKMs > 2).
We obtained KEGG pathway annotation for 29,558 unigenes. A total of 17,410 sequences were assigned to 128 pathways. “Metabolic pathways (ko01100)” represented the largest group (4,422; 25.50%), followed by “Biosynthesis of secondary metabolites (ko01110)” (2,160; 12.41%), “Plant-pathogen interaction (ko04626)” (915; 5.26%), “Plant hormone signal transduction (ko04075)” (781; 4.48%), and “RNA transport (ko03013)” (732; 4.20%; Additional file 3: Table S3). Compared with the control, a total of 17,410 differentially expressed unigenes were assigned to 115, 126, 124, and 126 KEGG pathways, respectively (Additional file 4: Table S4, Additional file 5: Table S5, Additional file 6: Table S6 and Additional file 7: Table S7). After multiple-testing corrections, we chose pathways with Q values ≤ 0.05 as significantly enriched among the DEGs. A total of 27, 39, 31, and 44 significant enrichment pathways were detected in the salt-treated samples at 6, 12, 24, and 72 h, respectively. The enrichment pathways with more genes than the other pathways were “Metabolic pathways (ko01100)”, “Biosynthesis of secondary metabolites (ko01110)”, “Endocytosis (ko04144)”, “Ether lipid metabolism (ko00565)”, “Glycerophospholipid metabolism (ko00564)”, “Plant hormone signal transduction (ko04075)”, “Plant-pathogen interaction (ko04626)”, and “Starch and sucrose metabolism (ko00500)”. These annotations are a valuable resource for comparisons of processes, functions, and pathways in salt-tolerant H. glomeratus research. Additionally, 29,245 CDS mapped to the protein database. The number of predicted CDS was 2,209, and the total number of CDS was 31,454 (Additional file 8: Figure S2).
Detection of salt-induced genes related to salt tolerance
Verification of differential gene expression by qRT-PCR
SSR and SNP identification
For further application of H. glomeratus, SSRs and SNPs were discovered using the assembled transcriptomes. A total of 23,876 SSRs was detected in transcriptomes, and the major types of the identified SSRs were mono-nucleotide (3,378), di-nucleotide (1,962), tri-nucleotide (5,489), quad-nucleotide (397), penta-nucleotide (418), and hexa-nucleotide (511). The most frequent SSR motif was AT/TA (350), followed by TA/AT (328), AG (282), GAA (292), TC (272), and TGA (272). We identified a total of 172,064 SNPs between transcriptomes, among which 61,785 were transitions, and 110,279 were transversions (Additional file 12: Figure S3). The SSRs and SNPs identified in this study provided a valuable resource for future studies on genetic linkage mapping and the analysis of interesting traits in H. glomeratus.
Numerous physiological [22,23], functional genomics [3,24], and proteomics  studies have identified physiological and molecular mechanisms of plant salt tolerance. However, our current understanding of the complex mechanisms of salt tolerance in plants remains limited. Recently, next-generation sequencing technology has provided a powerful tool for transcriptome analysis, and the de novo assembly of transcript sequences offers a rapid approach to obtain expressed gene catalogs for non-model organisms . In the present study, we performed deep sequencing analysis of H. glomeratus in response to salt stress using the Illumina HiSeq 2000 platform and de novo transcriptome assembly. We identified 50,265 unigenes with an average length of 870 bp and N50 length of 1,441 bp using blast against the Nr, Nt, Swiss-Prot, GO, COG, and KEGG databases. We identified 31,486 (62.64%) unigene hits with known plant species, and the remaining 18,779 (37.36%) failed to hit any homologs, which might be considered novel genes. Gene function and expression studies are needed to further investigate the genes that are involved in the process of salt tolerance in H. glomeratus.
In the present study, the top seven species (Figure 3C) were the most highly related species with known genomes to H. glomeratus, and 21,534 (72.86%) annotated unigenes in H. glomeratus hit protein sequences in these species. However, none of these species belong to chenopodiaceae plants, which is one of the main generas of halophytes or xerophytes , and none of these species belong to herbage halophytes. There were 21,809 unigenes with GO annotation and 17,410 unigenes mapped to 128 pathways with KEGG annotation (Additional file 3: Table S3, Additional file 4: Table S4, Additional file 5: Table S5, Additional file 6: Table S6 and Additional file 7: Table S7; Additional files 13, 14, 15, 16 and 17). The deep sequencing and exhaustive annotation results will improve genome annotation and facilitate the exploitation of genetic resources that are responsible for salt tolerance in the halophyte H. glomeratus.
The high tolerance of H. glomeratus to salinity enabled us to identity a succession of gene expression changes and trace the underlying salt-responsive genes under different salt treatment conditions . The enriched dominant GO terms that were identified during salt stress included “cellular process”, “metabolic process”, “single-organism process”, and “response to stimulus”, which are mostly in agreement with other analyses of DEGs under salt stress conditions [20,28]. However, the enriched term “single-organism process” was firstly reported as one of the enriched dominant GO terms under salt stress conditions, suggesting that H. glomeratus may have special genes that regulate the salt-response.
To identify significant gene expression changes with NaCl treatment, differentially expressed unigenes were analyzed by comparing the 6, 12, 24, and 72 h libraries with the control library. We focused on the unigenes that were strongly induced by NaCl or upregulated after salt stress. In the present study, a large number of unigenes were identified, thus clarifying the mechanisms of salt tolerance and identifying candidate genes that are responsible for salt adaptation in H. glomeratus. Of the candidate genes, 118 salt-induced genes and 291 up-regulated genes under salt-treatment conditions were analyzed to elucidate salt adaptation strategies. However, there were only a few genes know its function of these genes, a total of 31 (26.27%) and 196 (67.35%) were successfully identified in the salt-induced and up-regulated genes, respectively (Additional file 9: Table S8, Additional file 10: Table S9 and Additional file 11: Table S10). It is necessary to verify whether these genes of unknown function play important roles in response to salt stress in the further study.
The mechanisms of salinity tolerance involve three strategies, including tolerance to osmotic stress, Na+ exclusion from leaf blades, and tissues tolerance . The tolerance of tissue mainly occurs through the compartmentalization of Na+ and Cl− in vacuoles in mesophyll cells in the leaf to avoid toxic concentrations within the cytoplasm, and this process is implemented by transporting ions both through plasma and across tonoplasts . H. glomeratus is a leafy, highly succulent halophyte. It does not have salt glands or salt bladders in the leaves and stores Na+ in leaves under salinity conditions (Figure 1). In the present study, V-ATPase (V-type H+-transporting ATPase; Unigene8306 and CL4328.Contig1) transcripts were present at high levels in the salt-stress libraries. V-ATPase is essential for establishing an electrochemical H+-gradient across tonoplasts to energize tonoplasts for efficient ion uptake into vacuoles via the tonoplast Na+/H+ antiporter (NHX) . Transcript profiling of the salt-tolerant Festuca rubra ssp. litoralis revealed that V-ATPase mediated ion transport under salinity stress conditions, and enhanced V-ATPase activity can contribute to improvements in salt tolerance in transgenic crops [32,33]. Our results showed that salt-induced transcript of the V-ATPase was enhanced by prolonging the salt treatment time. However, vacuolar Na+/H+ antiporters in leaves are mainly involved in compartmentalizing Na+ in vacuoles. In contrast to our expectation, this gene was not shown significantly up-regulated under typical salinity condition. Similarly, we did not find Na+/H+ antiporters exhibited increased abundance at the levels of protein in response to salt stress . Few reports have indicated that vacuolar Na+/H+ antiporters and V-ATPase play a coordinated role in sequestering Na+ in vacuoles in the process of the salt stress response in plants [33,34]. Apse reported that the overexpression of vacuolar Na+/H+ antiporters in Arabidopsis thaliana enhanced salt tolerance and the vacuolar antiporter activity of transgenic plants but did not detect an increase in AtNHX1 transcript levels in response to NaCl stress at 50-250 mM . This is consistent with our results. Perhaps the increase in vacuolar Na+/H+ antiporter activity in response to NaCl stress in H. glomeratus requires a higher concentration of NaCl. Our transcriptome sequencing revealed that the higher transcript levels of V-ATPase contributes to providing more energy and forming a stronger proton gradient to drive excessive cytoplasmic Na+ into vacuoles. Therefore, a reasonable assumption is that H. glomeratus has tighter control over ion compartmentalization by adjusting the driving force of ion transport.
The exposure of plants to abiotic stress, such as high salinity, drought, and low temperatures, causes a dramatic increase in reactive oxygen species (ROS) production , including superoxide radicals (O-2), hydrogen peroxide (H2O2), and hydroxyl radicals (-OH), which can perturb cellular redox homeostasis and result in oxidative damage to cellular structures and eventually lead to cell death . A large number of unigenes that are associated with in ROS scavenging-related genes were identified, including glutathione S-transferases (GSTs; Unigene6753), which have glutathione peroxidase (GPX) activity and can use glutathione (GSH) to prevent the degradation of organic hydroperoxides to cytotoxic aldehyde derivatives. The activity of GSTs is upregulated in salt-stressed plants , and the overexpression of GST activity improves abiotic stress tolerance in plants . Peroxidases (PODs; CL5820.Contig1), which are also important in H2O2 scavenging, exhibit increased levels in salt-stressed seedlings, consistent with the salt-treated halophyte Aeluropus littoralis . Serine hydroxymethyltransferase (SHMT; Unigene2603 and Unigene1010) are involved in the photorespiratory pathway and plays an important role in plants to minimize the production of ROS in chloroplasts and mitigate oxidative damage . Therefore, antioxidant enzymes that are involved in the ROS scavenging pathway protect cells from oxidative damage under salt stress conditions in H. glomeratus.
The adjustment of energy metabolism under salt stress conditions is an important strategy to cope with salinity stress . We identified three unigenes that were related to energy metabolism. One (Unigene5334) was an homolog to ATP-citrate synthase, the expression of which was induced under salt stress conditions. Two unigenes were homologs to ATP synthase (CL5295.Contig2), adenosine diphosphate (ADP), and ATP carrier protein (Unigene6293) and upregulated under salt stress conditions. One unigene (CL5099.Contig1) was found to be homologous to cytochrome c oxidase, which is an electrostatically coupled energy transducer that contributes to the formation of ATP in aerobic life . Efficient ATP generation is crucial for salt tolerance during ion exclusion and tissue tolerance in plants.
Plants integrate multiple hormone-response pathways for adaptation to environmental stress, including the abscisic acid (ABA), cytokinin, auxin, and strigolactone pathways . Abscisic acid signaling regulation enables plants to survive under environmental stress conditions . In the present study, we found that numerous upregulated unigenes were related to the hormone-response pathway, such as abscisic stress-ripening protein (ASR; Unigene3863) in the ABA signaling regulation pathway. ASR is transiently upregulated when plants are exposed to salt or water stress . Cytokinins (CKs) regulate several plant growth and developmental processes, such as cell division, leaf senescence, and nutritional signaling. We identified a CK hydroxylase (Unigene9103) that is involved in the regulation of CK metabolism and signaling of Arabidopsis . Calreticulin (CR; Unigene6139) was upregulated under salt stress conditions and is essential for integrin-mediated calcium signaling and cell adhesion . Additionally, seven unigenes (Unigene1081, Unigene3770, Unigene4073, Unigene4177, Unigene6237, Unigene6742, and Unigene8963) were strongly upregulated in response to salt stress and found to be homologs of the proline-rich receptor-like protein kinase (PERK) family in Arabidopsis, which plays an important role in hormone signal transduction for plant growth and development, stress responses, and disease resistance .
We also found numerous unigenes that are involved in the response to biotic and abiotic stress. The expression of cysteine proteinase (Unigene6468) is strongly induced in leaves in response to salinity and drought stress, and it is also involved in signaling pathways and the response to biotic and abiotic stress [48,49]. Aspartate aminotransferase (AST) catalyzes the bidirectional conversion of aspartate into glutamate, and its activity significantly increased under salt shock . Recent reports showed that this gene was involved in amino acid metabolism, which might interact with plant defense responses . The transcript (Unigene4102) was identified as betaine aldehyde dehydrogenase (BADH), a key enzyme that catalyzes the final reaction in the glycine betaine synthetic pathway of the plant response to drought and salt stress , thus helping maintain osmotic homeostasis in response to water deficit, osmotic stress, and salt stress.
Aquaporins play an important role in regulating transmembrane water transport during plant growth and development and under osmotic stress and salinity stress conditions . The overexpression of aquaporins was recently reported to increase the salt stress tolerance of transgenic tobacco .
A number of unigenes were identified as vegetative cell wall proteins (Unigene7663, Unigene3984, Unigene4128, Unigene6186, Unigene6746). These proteins have many functions, including structural functions, and participate in plant morphogenesis . Notably, the transcripts of vegetative cell wall proteins that changed under salinity conditions are involved in cell adaptations to salt, but our understanding of the nature of these changes is still limited.
Finally, we also focused on some genes that are involved in the photosynthetic pathway. In the present study, two genes that encode key enzymes of carbon assimilation in the C4 photosynthetic pathway were present in the transcriptomic dataset from the KEGG annotation (ko00170), including phosphoenolpyruvate carboxylase (PEPC; Unigene338) and NADP-malate dehydrogenase (NADP-MDH; Unigene4014). Two genes that encode major C4 decarboxylation enzymes were also found in the transcriptomic KEGG dataset (ko00620), including NADP-malic enzyme (NADP-ME; Unigene216, Unigene6517, and CL5690.contig1/2) and NAD-malic enzyme (NAD-ME; CL6152.contig1/2). This is consistent with our previous anatomical research on H. glomeratus. The anatomical structure analysis of leaves showed that the palisade tissue, bundle sheath tissue, and vascular bundle form the kranz anatomy as a typical feature the C4 photosynthetic pathway . No studies of which we are aware have reported the photosynthetic pathway of H. glomeratus. C4 plants have many advantages over C3 plants in photosynthetic performance, nitrogen and water use efficiency, and the response to environmental stress . The presence of C4 enzymes in leaves is the first transcriptomic evidence to support the existence of C4 pathways in the H. glomeratus response to environmental stress.
In the present study, we surveyed five transcriptomes of H. glomeratus leaves during various stages of salt stress, and 50,267 unigenes were generated with the Illumina HiSeq 2000 platform. We identified 118 salt-induced genes and 291 upregulated unigenes that are involved in ion transport, ROS scavenging, energy metabolism, hormone-response pathways, and response to biotic and abiotic stress in the response of this species to salt stress. Our findings substantially enrich the existing sequence resources of H. glomeratus and will greatly promote research on salt-tolerant mechanisms of halophytes. This work also provides numerous salt-tolerant candidate genes for further functional analyses to improve the salt tolerance of crops.
Seeds of H. glomeratus were collected in a salinized river shoal in Huining County, Gansu Province, in Northwest China and planted in plastic pots that were filled with a mixture of sand and vermiculite (1:1, v/v). The plants were placed under long-day (16 h light/8 h dark cycle) conditions at a temperatures of 25°C (light) and 18°C (dark) with irradiation intensity of approximately 300 μmol m−2 s−1. The plants were irrigated daily with half-strength Hoagland solution. Two months after germination, seedlings were treated with 200 mM NaCl for 6, 12, 24, and 72 h. Untreated seedlings were used to construct a reference library for de novo sequencing. Leaves were immediately harvested and frozen in liquid nitrogen for the extraction of total RNA.
Na+ accumulation analyses of seedlings after treated with NaCl
For salt crystals of leaf surfaces assays, the leaf abaxial surfaces of seedlings treated with 200 mM NaCl for 72 h were scanned with an scanning electron microscopy (SEM) (JSM-5600LV, JEOL Ltd., Japan) with 20.0 kV current. For Ion concentration determination of the leaves, leaf samples harvested from the seedlings were treated with 200 mM NaCl for 0 h and 72 h. Three biologically independent replicates were prepared. Then, K+ and Na+ contents were measured using an atomic absorption spectrophotometer (AA240; Varian Medical Systems, USA).
RNA extraction and quality determination
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. The RNA samples were digested using DNase I at 37°C for 30 min to remove potential genomic DNA contamination. The concentration and quality of each sample were determined using an Agilent 2100 bioanalyzer. High-quality RNA from three replicates at each time point at equal quantities were pooled for cDNA preparation and RNA-Seq.
IIIumina cDNA library preparation and sequencing
Poly (A) mRNA was purified from total RNA using magnetic beads with oligo (dT). The mRNA was then broken into short fragments using divalent cations at an elevated temperature. Using these cleaved RNA fragments as templates, random hexamer primer was used for first-strand cDNA synthesis, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments were subjected to purification, end repair, and ligation to sequencing adapters. The products were then amplified by PCR to construct the cDNA library.
Sequencing and de novo assembly
Sequencing libraries were sequenced using the Illumina HiSeq2000 system at the Beijing Genomics Institute (Shenzhen, China). Raw sequencing image data were transformed by base calling into raw reads. The raw reads were cleaned by discarding adaptor sequences and reads with unknown nucleotides that were larger than 5% and low-quality reads. The rate of reads with quality ≤ 10 was > 20%. The subsequent analysis was based on these clean, high-quality reads. The transcriptome de novo assembly of clean reads was performed using the short-read assembling program Trinity . Short reads with overlapping sequences were first assembled to form the longest sequences, referred to as contigs. These contigs were then pooled to build de Brujin graphs. Contigs from the same transcript and the distances between these contigs were detected based on the paired-end read. Finally, sequences that lacked Ns and could not be extended on either end were defined as unigenes. Unigenes from each sample’s assembly were taken for further processing, including sequence splicing and redundancy removal using sequence clustering software to acquire non-redundant unigenes that were as long as possible. After clustering, the unigenes were divided into clusters (CL prefix) and singletons (Unigene prefix).
The expression profile of each unigene was calculated using the RPKM method (reads per kb per million reads) and normalized using ERANGE 3.1 software. If more than one transcript was identified for a given gene, then the longest transcript was used to calculate its expression level and coverage. The false discovery rate (FDR) method was used to determine the threshold of the p value in multiple tests. Unigene annotations provided functional annotations for all of the unigenes, together with their expression levels. All of the generated unigene sequences were aligned by BLASTX (E value < 0.0001) to public proteins databases, such as the National Center for Biotechnology Information non-redundant protein (Nr) database, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and clusters of orthologous groups (COG) database, with an E cutoff value of 1e-5. The best-aligning results were used to determine the sequence direction of the unigenes. If the results from different databases conflicted with each other, then the following priority order was followed when deciding the sequence orientation of the unigenes: NCBI nr, Swiss-Prot, KEGG, and COG.
With Nr annotation, we used the Blast2GO program (https://www.blast2go.com/blast2go-pro; accessed July 12, 2014) to obtain the annotation of unigenes with GO terms. We then used WEGO software (http://wego.genomics.org.cn; accessed July 12, 2014) for GO functional classification for all of the unigenes and determine the distribution of gene functions of the species at the macro level. KEGG pathway annotation was performed using blastall software against the KEGG database (http://www.genome.jp/kegg/; accessed July 12, 2014).
Prediction of unigene coding regions
For coding region prediction (CDS) of the unigenes, all of the unigenes were first aligned by blastx (E value < 0.00001) to protein databases in the priority order NCBI nr, Swiss-Prot, KEGG, and COG. Unigenes that were aligned to a higher-priority database did not align to a lower-priority database. Proteins with the highest ranks in the BLAST results were taken to determine the CDS of the unigenes, and the CDS was translated into amino acid sequences with the standard codon table. Both the nucleotide sequences (5′ → 3′) and amino acid sequences of the unigene coding region were acquired. When a unigene happened to be unaligned to none of the above databases, then ESTScan software was introduced to decide its sequence orientation and protein coding region prediction (CDS) .
Identification of different expressed genes, SSRs, and SNPs
To identify genes that are regulated by salt stress, the significance of differences in gene expression between salt-treated and control samples was determined using a threshold FDR ≤ 0.001 and an absolute log2 ratio ≥ 1 (at least two-fold change). The genes that were expressed at different levels across samples were further annotated by GO enrichment analysis and KEGG pathway enrichment analysis to ascertain biological significance. The self-organizing tree algorithm of hierarchical clustering of gene express level profiles was performed on the log (RPKM) values of genes using Cluster software (version 3.0; http://rana.lbl.gov/EisenSoftware).
The SSR detection of unigenes was preformed with MicroSAtellite (MISA) software (http://pgrc.ipk-gatersleben.de/misa/misa.html; accessed July 12, 2014). Primers were designed using Primer 3 software (http://primer3.sourceforge.net; accessed July 12, 2014). We used SOAPsnp (http://soap.genomics.org.cn/soapsnp.html; accessed July 12, 2014) to identify SNPs in the transcripts of H. glomeratus. The SNPs could then be identified based on the consensus sequence by comparisons with the unigenes.
Gene validation and express analysis
Samples were prepared, and total RNA was isolated using the same method mentioned above. The salt-induced expression of unigenes with potential roles in the salt stress response were chosen for validation using qRT-PCR. A total 18 unigenes were selected for qRT-PCR with RPKM values > 5.5 in the 72 h sample. Three independent biological samples of each were used in the analysis. Reverse transcription reactions were performed using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) with 2 μg total RNA according to the manufacturer’s instructions. The actin gene of H. glomeratus (GI: 567319963) was used as an internal control for all of the experiments. The primers for RT-PCR were designed using Primer Express 2.0 software to amplify 100- to 150-bp regions of the chosen genes. The gene-specific primers are listed in Additional file 18: Table S1. qRT-PCR was performed using an ABI ViiA 7 Real Time PCR System (Applied Biosystems, Carlsbad, USA) and SYBR-Green Mastermix (Qiagen, California, USA). The relative expression levels of the selected unigenes normalized to the expression level of actin were calculated from cycle threshold values using the 2-ΔΔCt method.
This work was supported by National Basic Research Program of China (973 program, 2014CB160313), National Natural Science Foundation of China (No.31171558, 31460347), China Agriculture Research System (CARS-05), and Fostering Foundation for the Excellent Ph.D. Dissertation of Gansu Agricultural University (YBPY2014001).
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