Transcriptome sequencing and whole genome expression profiling of hexaploid sweetpotato under salt stress

Abstract

Background: Purple-fleshed sweetpotato (PFSP) is one of the most important crops in the word which helps to bridge the food gap and contribute to solve the malnutrition problem especially in developing countries. Salt stress is seriously limiting its production and distribution. Due to lacking of reference genome, transcriptome sequencing is offering a rapid approach for crop improvement with promising agronomic traits and stress adaptability.

Results: Five cDNA libraries were prepared from the third true leaf of hexaploid sweetpotato at seedlings stage (Xuzi-8 cultivar) treated with 200 mM NaCl for 0, 1, 6, 12, 48 h. Using second and third generation technology, Illumina sequencing generated 170,344,392 clean high-quality long reads that were assembled into 15,998 unigenes with an average length 2178 base pair and 96.55% of these unigenes were functionally annotated in the NR protein database. A number of 537 unigenes failed to hit any homologs which may be considered as novel genes. The current results indicated that sweetpotato plants behavior during the first hour of salt stress was different than the other three time points. Furthermore, expression profiling analysis identified 4, 479, 281, 508 significantly expressed unigenes in salt stress treated samples at the different time points including 1, 6, 12, 48 h, respectively as compared to control. In addition, there were 4, 1202, 764 and 2195 transcription factors differentially regulated DEGs by salt stress at different time points including 1, 6, 12, 48 h of salt stress. Validation experiment was done using 6 randomly selected unigenes and the results was in agree with the DEG results. Protein kinases include many genes which were found to play a vital role in phosphorylation process and act as a signal transductor/ receptor proteins in membranes. These findings suggest that salt stress tolerance in hexaploid sweetpotato plants may be mainly affected by TFs, PKs, Protein Detox and hormones related genes which contribute to enhance salt tolerance.

Conclusion: These transcriptome sequencing data of hexaploid sweetpotato under salt stress conditions can provide a valuable resource for sweetpotato breeding research and focus on novel insights into hexaploid sweetpotato responses to salt stress. In addition, it offers new candidate genes or markers that can be used as a guide to the future studies attempting to breed salt tolerance sweetpotato cultivars.

Keywords: Expression profile; Hexaploid sweetpotato; RNA-sequencing; Salt stress; Transcriptome.

Fig. 1

a Assembly result sequence length distribution map of transcripts and unigenes in Xuzi-8 sweetpotato cultivar. The horizontal axis represents the length intervals of the transcripts and unigenes, and the vertical axis represents the number of transcripts and unigenes. b Species distribution of the top BlastX matches of the transcriptome unigenes of Xuzi-8 sweetpotato cultivar in the non-redundant protein database (Nr) data base

Fig. 2

Gene ontology (GO) classifications in sweetpotato (Xuzi-8 cultivar), the percentage indicate the proportion of unigenes with the GO annotations

Fig. 3

Clusters of orthologous groups (COG) classification in Xuzi-8 sweetpotato cultivar. Genes from the same Orthologous have the same function, so that direct functional annotations to other members of the same KOG cluster

Fig. 4

The most enriched KEGG clusters in Xuzi-8 sweetpotato cultivar. The most enriched 22 clusters out of 123 clusters were presented in this figure

Fig. 5

Phenotypic variations in Xuzi-8 sweetpotato seedlings as related to fold change (FC) and false discovery rate (FDR) under salt stress (200 Mm NaCL). a, b, c and d; phenotypic variations at 0, 1, 6, 12 48 hours of salt stress, respectively. e, f, g and h; fold change (FC) and false discovery rate (FDR) at four libraries 1, 6, 12 48 hours, respectively as compared to control

Fig. 6

Comparison of four transcriptomes for classification of DEGs and statistics of sequence annotation of DEGs. a; Statistical chart of DEGs transcriptome in response to salt stress. Transcriptional level of five libraries including 1, 6, 12 and 48 hours of salt stress treatment as compared to control. b and c; Venn diagram analysis of up-regulated unigenes and all induced unigenes, respectively.

Fig. 7

Simple sequence repeat (SSRs) and single nucleotide polymorphism (SNPs) detected in the obtained sequences in Xuzi-8 sweetpotato cultivar under salt stress conditions (200mM NaCL) with 5 libraries (0, 1, 6, 12, 48 hours). a; Frequency and distribution of SSRs in coding sequence and untranslated region (UTRs). b; Frequency and substitution types of the identified SNPs in the five libraries (three replicates per each library)

Fig. 8

qRT-PCR validation of the salt induced fold changes detected in Xuzi-8 sweetpotato cultivar using RNA-seq. Standard error bars are showed for the expression values determined by qRT-PCR. The expression levels determined by qRT-PCR and DEG values of the genes (protein disulfide isomerase, Actin-12 (ACT12), nucleoredoxin 2 isoform X1, Thioredoxin-like domain, CTD small phosphatase-like protein 2 and Redoxinwere presented) at (a), (b), (c), (d), (e) and (f), respectively. Expression values determined by qRT-PCR are presented as columns and DEGs are presented as curves