A genome-wide transcriptome map of pistachio (Pistacia vera L.) provides novel insights into salinity-related genes and marker discovery

Abstract

Background: Pistachio (Pistacia vera L.) is one of the most important commercial nut crops worldwide. It is a salt-tolerant and long-lived tree, with the largest cultivation area in Iran. Climate change and subsequent increased soil salt content have adversely affected the pistachio yield in recent years. However, the lack of genomic/global transcriptomic sequences on P. vera impedes comprehensive researches at the molecular level. Hence, whole transcriptome sequencing is required to gain insight into functional genes and pathways in response to salt stress.

Results: RNA sequencing of a pooled sample representing 24 different tissues of two pistachio cultivars with contrasting salinity tolerance under control and salt treatment by Illumina Hiseq 2000 platform resulted in 368,953,262 clean 100 bp paired-ends reads (90 Gb). Following creating several assemblies and assessing their quality from multiple perspectives, we found that using the annotation-based metrics together with the length-based parameters allows an improved assessment of the transcriptome assembly quality, compared to the solely use of the length-based parameters. The generated assembly by Trinity was adopted for functional annotation and subsequent analyses. In total, 29,119 contigs annotated against all of five public databases, including NR, UniProt, TAIR10, KOG and InterProScan. Among 279 KEGG pathways supported by our assembly, we further examined the pathways involved in the plant hormone biosynthesis and signaling as well as those to be contributed to secondary metabolite biosynthesis due to their importance under salinity stress. In total, 11,337 SSRs were also identified, which the most abundant being dinucleotide repeats. Besides, 13,097 transcripts as candidate stress-responsive genes were identified. Expression of some of these genes experimentally validated through quantitative real-time PCR (qRT-PCR) that further confirmed the accuracy of the assembly. From this analysis, the contrasting expression pattern of NCED3 and SOS1 genes were observed between salt-sensitive and salt-tolerant cultivars.

Conclusion: This study, as the first report on the whole transcriptome survey of P. vera, provides important resources and paves the way for functional and comparative genomic studies on this major tree to discover the salinity tolerance-related markers and stress response mechanisms for breeding of new pistachio cultivars with more salinity tolerance.

Fig. 1

The workflow for pistachio transcriptome assembly and analysis. Two pistachio cultivars, salt-sensitive (Sarakhs) and salt-tolerant (Ghazvini), were selected. RNA was separately isolated from leaf, stem, and root of two cultivars after 0, 6, 24, and 48 h of salt treatment. Equal amount of extracted RNA was mixed to make a single pool for a deep sequencing on one lane of Illumina Hiseq 2000 as paired-end. Following quality control check and trimming, de novo assembly was carried out using Trinity, SOAPdenovo-Trans, and CLC genomics workbench softwares through two different strategies. After rigorous assembly quality assessment, final assembly was selected and exposed to functional annotation and SSR marker discover followed by gene ontology analysis and validation of some candidate genes by qRT-PCR

Fig. 2

Ortholog hit ratio of assembled transcripts for various assemblies. a Single assembly, CLC genomics workbench. b Single assembly, SOAPdenovo-Trans. c Single assembly, Trinity. d Merged assembly, CLC genomics workbench. e Merged assembly, SOAPdenovo-trans

Fig. 3

Graphical representations of functional annotations in P. vera transcriptome. a E-value distribution graph. b Similarity distribution graph. c Top-hit species distribution graph

Fig. 4

Venn diagram shows the BLAST results of P. vera against five databases, including NR, UniProt, KOG, TAIR10, and InterProScan. The number of transcripts with significant hits is presented in each intersection of the Venn diagram

Fig. 5

GO and enzyme classification of assembled pistachio transcripts. a GO level distribution of annotated sequences. b GO classification across three main categories. Biological process (BP), Molecular Function (MF), and Cellular Component (CC).c Catalytic activity distribution in annotated P. vera transcriptome

Fig. 6

KEGG classification of pistachio transcriptome. a KEGG distribution of annotated transcripts into biological categories. b The top 10 pathways with the highest transcript numbers

Fig. 7

Distribution of P. vera transcripts into different transcription factor families

Fig. 8

The proposed pathway for the biosynthesis of major flavonoids, including luteolin, quercetin, kaempferol and various types of anthocyanins (pelargonidin, delphinidin, and cyanidin) in pistachio. Phenylalanine ammonia-lyase (PAL), 4-coumarate CoA ligase (4CL), Trans-cinnamate 4-monooxygenase (C4H), Chalcone synthase (CHS), Chalcone isomerase (CHI), Flavone synthase (FNS), Flavonoid-3′-hydroxylase (F3’H), Flavone-3-hydroxylase (F3H), Flavonol synthase (FLS), Flavonoid 3′5′-hydroxylase (F3’5’H), Dihydroflavonol-4-reductase (DFR), Leucoanthocyanidin dioxygenase (LDOX), Anthocyanidin 3-O-glucosyltransferase (BZ1). The genes listed in this Fig. were all identified in P. vera transcriptome

Fig. 9

Fold change of selected salt-stress responsive genes over three time points of salt stress in root of P. vera L. cv Sarakhs and Ghazvini. The stressed samples were quantified to the non-stressed. EF1α was used as a reference gene for data normalization. Mean value and standard deviation (SD) were presented for three biological replicates. a ZEP, b NCED3, c PP2CA, d SOS1, e dehydrin, f CDPK11