Experimental materials and design
The mycorrhizal fungus Rhizophagus intraradices (R.intraradices (No. BGC BJ09)) used in this study was purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agricultural and Forestry Sciences, China. The inoculum was composed of spore (approximately 500 spores per gram inoculum), hypha, host root segment and culture medium.
Seeds of P. quinquefolius were sterilized with 10% H2O2 for 30 min, washed with sterilized water for three times, and then germinated at 25°C in an incubator after soaking for 24 h. There were two treatments in the present study, including control and AMF treatment (R. intraradices). For treatments, a mixture of soil and sand (2:1, V:V) was used as growth medium, then autoclaved at 121°C, 0.10 MPa for 2 h to kill indigenous AMF propagules and other microorganisms. The selected soil belongs to the Meadow soils (Semiaqueous soil Order) according to the China soil classification system. Approximately 3.50 kg of the medium and 30 g of inoculum were mixed, put in a plastic pot (180×145×155 mm) for the growth of P. quinquefolius. There were 3 replicates of each treatment. The non-AMF pot was added with the same amount of inactivated inoculum. We also added 30 ml of filtered inoculant (0.25mm filter membrane), which was free from mycorrhizal propagules to the non-AM treatment to provide the same microorganism biota. The plants were grown in a day/night temperature of 25/20°C, a relative day/night humidity of 70/65%, a day/night of 16/8 h and a photosynthetic photon flux density (PPFD) at the height of the plant of 100 μmol m−2 s−1. Light was provided by a fluorescent lamp. Pots were repositioned weekly to reduce environmental error. All pots were irrigated with 100 ml Hoagland solution with 1/10 P (0.1 mM) strength. The fresh roots of P. quinquefolius seedlings were harvested at 6 months old. Each plant of P. quinquefolius was divided into branch root and taproot The branch root of each P. quinquefolius was used to detect the colonization of AMF and the corresponding taproot was further divided equally into two parts, an aliquot of the taproot was used to determine the content of ginsenoside, and another aliquot was frozen in liquid nitrogen and stored at -80℃ for further analysis.
The analysis of AMF colonization in roots of P. quinquefolius
The AMF colonization in roots of P. quinquefolius was determined according to the method reported by Shu et al. (2016). The branch roots were removed from the FAA (Formalin 5 ml + Acetic Acid 5 ml + Alcohol 90 ml) solution, washed several times with distilled water, and then placed in a test tube. Subsequently, root samples were added with 10% (w/v) KOH at 99°C for 1.5 h, stained with 0.05% (w/v) trypan blue, and estimated the mycorrhizal colonization using a modified line intersect method (Rufykiri et al. 2000; McGonigle et al. 1990). 30 root segments per root sample were observed for the presence of AMF structures with repeated three times. The degree of root infection by AMF was assessed using a light microscope (Olympus-BH-2, Japan). The AMF infection was calculated according to the following formula: frequency of AM fungal colonization = (Infected root segments / All root segments)×100%. The AMF colonization intensity according to the following formula: AMF colonization indensity = (95×n5+70×n4+30×n3+5×n2+n1) / All root segments×100%, n5 represented the number of root segments infected at level 5; n4 represented the number of root segments infected by level 4; n3 represented the number of root segments infected at level 3, and so on.
Analysis of the content of ginsenoside
The content of ginsenoside was conducted using the methods reported by Liu et al. (2014) with slightly changes. A total of 0.50 g air-dried P. quinquefolius roots were ground into a powder, with 50 ml methanol for extraction. The extracts were obtained with ultrasound for 40 min, and then left to stand for 10 min. The supernatant was filtered with a 0.45 mm microporous membrane and injected with 10 ul for HPLC analysis. The content of ginsenoside was determined using a Kromasil C18 (4.60×250 mm, 5 mm) column. The mobile phase solution was (A) acetonitrile and (B) 0.10% phosphoric acid water with a speed of 1.00 ml min-1 and the following gradient program was used: 0-25 min, 19-20% A; 25-60 min, 20-40% A; 60-90 min, 40-55% A; 90-100 min, 55-60% A. The wavelength was detected at 203 nm, and the temperature of column was set at 40℃. The content of ginsenoside was calculated according to the regression equation of standard curve.
Total RNA Extraction, cDNA library construction and RNA Sequencing
Total RNA was extracted from control and AMF treated root samples using the RNA prep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) based on the manufacturer’s manual. DNA was then removed using the RNAclean Kit (Tiangen, Beijing, China). The quality and quantity of total RNA was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The library construction and RNA-Seq assay were performed by the Novogene Biotechnology Corporation (Beijing, China). The cDNA library was constructed using A TruSeq™ RNA Sample Preparation Kit (Illumina, Inc.). First, Poly-(A)-containing mRNA was purified from the total RNA using oligo (dT) magnetic beads and Oligotex mRNA kits (Qiagen, Germany), following the manufacturer’s instructions. Fragmentation was carried out using divalent cations. Fragmentary RNAs were used as template for first strand cDNA synthesis by an cDNA preparation kit (Illumina, San Diego, CA, USA).
Second-stranded cDNA was synthesized using RNase H and DNA polymerase I. Then cDNAs were subjected to end-repair, phosphorylation, and ligation to sequencing adapters. Afterward, the products enriched by PCR amplification were purified through 2% agarose gelelectrophoresis and quantified by TBS380 (Picogreen). Finally, cDNA libraries were subsequently sequenced using an Illumina HiSeq™ 2000 platform.
Transcriptome assembly, annotation and function enrichment
Before assembly, raw data were filtered to remove reads containing adapter, reads containing ploy-N, and low quality reads to generate highquality clean data. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. After removing the low quality reads, a denovo strategy was used to assemble the clean reads into distinct contigs using Trinity assembly software (Grabherr et al. 2011). All of the contigs were clustered, and the contigs with the longest sequences were defined as unigenes. For functional annotation, the unigenes, through a BLASTall algorithmbased program with a threshold of E-value < 10−10, were searched against different databases, including NCBI non redundant protein sequences (Nr), NCBI non-redundant nucleotide sequences (Nt), Protein family (Pfam), Protein primogenomic cluster (KOG/COG), Artificial annotated and reviewed protein sequence database (Swiss-Prot), KEGG Ortholog database (KO), Gene Ontology (GO).
Analysis of differential genes
The expression level of gene was determined using the the reads per kilobase per million mapped reads (RPKM) method (Trapnell et al. 2010) by RSEM software (Dewey and Li 2011). The transcript RPKM values were estimated using RNA-Seq by Expectation Maximization (RSEM) with Bowtie read mapping. Differentially expressed genes (DEGs) analysis in pair-wise comparisons was conducted using the DESeq software (Anders and Huber 2010), and the input data of the differentially expressed genes (DEGs) was based on the read counts. The false discovery rate (FDR) method was applied to correct the threshold of the P values in multiple tests for identifying the differences between two groups. A FDR < 0.001 and an absolute value of log2 (ratio) > 2 were applied as thresholds to identify significant differences between two groups (Mortazavi et al. 2008). Furthermore, the DEGs were then analyzed through GO and KEGG pathway enrichment analyses.
Simple sequence repeats (SSRs) detection and primer design
In order to check SSRs in P. quinquefolius, the microsatellite identification tool (MISA) was used to identify the SSR. The parameters were used to distinguish di-, tri-, tetra-, penta- and hexa nucleotide motifs with the lowest limit of repeats of 6, 5, 4, 4 and 4 respectively, and Primer 3 software was used to devise primers for each SSR. The design parameters of major primer were set as follows: 100 to 300 nt of PCR products, 18 to 24 nt of primer lengths, 60°C optimal annealing temperature, and 40% to 65% of GC content from (Zeng et al. 2019).
Quantitative real-time RT-PCR
A total of 5.0 ug RNA of each treatment root sample was used for first-stand cDNA synthesis using the RNA prep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) based on the manufacture’s instructions. The synthesised cDNA was used as a template for quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR reaction system with 20 ml volume of reaction mixture made up of 2 ml of dilute template cDNA, 2 ml of primer pairs, 10 ml of GoTaq® qPCR Master Mix (Promega, USA), and 6 ml of deionized water. The setting program of qPCR reaction was carried out as follows: denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, annealing at 57°C for 1 min, 72°C for 50 s and then extension at 72°C for 1 min (Wang et al. 2016). After the reactions, the specificity was assessed by melt curve and size estimation of the amplified product. Each gene was quantified using three biological replicates. The relative expression of the selected genes was normalized by using the 2−ΔΔCt method (Livak and Schmittgen 2001). β-Actin was used as internal standard and was amplified with the forward primer 5′-AGGAACCACCGATCCAGACA-3′and reverse primer 5′-GGTGCCCTGAGGTCCTGTT-3′. The gene primers used for RT-PCR are shown in Table S1.
The data were subjected to analysis of one-way ANOVA using Statistical Product and Service Solutions 17.0 software (SPSS Institute Inc. Chicago, IL, USA) and differences were compared by Duncan’s test with a significance level of P < 0.05.