Differential gene expression analysis and SNP/InDel marker discovery in resistant wild Asparagus kiusianus and susceptible A. officinalis in response to Phomopsis asparagi infection

This data article reports de novo transcriptome analysis of resistant wild Asparagus kiusianus and susceptible A. officinalis plants 24 and 48 h post-inoculation (24 and 48 hpi) with Phomopsis asparagi. Differential gene expression (DGE) analysis demonstrated that several genes involved in secondary metabolites and plant-pathogen interactions are up-regulated in resistant wild A. kiusianus relative to susceptible A. officinalis. The assembled contig sequences generated in this study were used to search single nucleotide polymorphism (SNP) and insertion/deletion (InDel) distribution in A. kiusianus and A. officinalis plants. SNP and InDel data developed from this transcriptome analysis will be used to generate a high-density linkage map to facilitate further development of molecular marker-assisted selection in A. officinalis.


a b s t r a c t
This data article reports de novo transcriptome analysis of resistant wild Asparagus kiusianus and susceptible A. officinalis plants 24 and 48 h post-inoculation (24 and 48 hpi) with Phomopsis asparagi. Differential gene expression (DGE) analysis demonstrated that several genes involved in secondary metabolites and plantpathogen interactions are up-regulated in resistant wild A. kiusianus relative to susceptible A. officinalis. The assembled contig sequences generated in this study were used to search single nucleotide polymorphism (SNP) and insertion/deletion (InDel) distribution in A. kiusianus and A. officinalis plants. SNP  DEG analysis provides a valuable information about defense responsive genes in Asparagus species against Phomopsis disease.
SNP and InDel variants provided in this study will be useful for researchers involved in the future development of high-density linkage maps associated with Phomopsis disease resistance.
SNP and Indel data can be further used for phylogenetic analysis of different Asparagus populations.

Data
In this study, the plant defense response in resistant wild A. kiusianus and susceptible A. officinalis was investigated 24 and 48 hpi with P. asparagi in comparison with non-inoculated control plants. Our recent study [1] showed that A. kiusianus, a wild relative of cultivated A. officinalis, displayed significantly reduced disease symptoms compared with susceptible A. officinalis upon artificial inoculation with P. asparagi [1]. In this study, we conducted de novo transcriptome analysis of resistant wild A. kiusianus and susceptible A. officinalis 24 and 48 hpi with P. asparagi. In total, 390,811,866 and 432,232,432 read counts with 100 bp read length were generated from 18 cDNA libraries. After removing adaptors and low-quality reads, more than 98% of the raw reads were clean reads. The high-quality reads were de novo assembled using Trinity software, and more than 95.68% and 95.74% were successfully mapped for the A. officinalis and A. kiusianus samples, respectively. In total, 206,164 and 213,950 contigs (average length, 973 bp) were obtained from A. officinalis and A. kiusianus, respectively. The quality of transcriptome assemblies was assessed, and the length distribution of the contigs in both A. officinalis and A. kiusianus is shown in Fig. 1A and B. Principal component analysis (PCA) of the transcriptome data (two Asparagus species Â three biological replicates Â three treatments (control, 24 and 48 hpi)) demonstrated a significant segregation in the wild resistant A. kiusianus (accumulation of variance 54,3%) and A. officinalis (accumulation of variance 58.3%) 24 and 48 hpi with P. asparagi relative to untreated control plants ( Fig. 1C and D). The DEG analyses revealed that the total number of up-regulated transcripts in resistant wild A. kiusianus (7728) was relatively higher than that (7499) in susceptible A. officinalis (Fig. 1E and F). However, down-regulated transcripts (10,713) in susceptible A. officinalis was relatively higher than that (6789) in resistant wild A. kiusianus (Fig. 1G and H), suggesting that gene expression responses to P. asparagi infection differed between susceptible A. officinalis and resistant wild A. kiusianus. Further, we conducted SNP and InDel distribution in the resistant wild A. kiusianus and susceptible A. officinalis transcriptome using the recently released A. officinalis reference genome in NCBI (Tables S1-S4).

Experimental designs, materials and methods
Male wild A. kiusianus (AK0501 strain) and female A. officinalis 'Mary Washington 500W' were cultivated under greenhouse conditions at Kagawa Prefectural Agricultural Experiment Station, Kagawa, Japan. Total RNA was extracted from 5-year-old A. officinalis and wild A. kiusianus 24 and 48 hpi with P. asparagi and from non-inoculated Asparagus plants grown under the same conditions (two Asparagus species Â three biological replicates Â three treatments (control, 24 and 48 hpi)). RNA concentration and quality were assessed using gel electrophoresis and UV/VIS Beckman DU 730 spectrophotometer (Beckman Coulter Inc., San Diego, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA) instruments. Paired-end reads were generated by TaKaRa Bio (TaKaRa Bio, Kusatsu, Japan) using an Illumina HiSeq. 2500 instrument (Illumina Inc., USA). The raw reads were trimmed using Cutadapt v.1.339 and Sickle v.1.200. After trimming, 26.5 Gb of data were used for de novo transcriptome assembly using Trinity package v.2.0.6 [2,3]. After assembly, 213,950 and 206,164 contigs were obtained from A. officinalis and A. kiusianus, respectively. These contigs were further used for DEG analysis. Sequencing read counts were calculated using RSEM v1.2.15 [4]. Gene expression from different samples was normalized by the TMM method [5]. DEGs were determined using the edgeR program. Genes with false discovery rate (FDR) o 0.05 and fold change 4 2 were considered to be differentially expressed. PCA analysis was carried out by R statistics v3.4 (https://www.r-project.org/) using PCA-based unsupervised gene expression of A. officinalis and A. kiusianus.
SNPs and InDels between A. officinalis, wild A. kiusianus, and the recently released A. officinalis reference genome were precisely pinpointed using a variant calling process. RNA-Seq reads were aligned to the reference genome using TopHat v. The output BAM files were subjected to SNP/InDel calling using PICARD and GATK (http://www.broadinstitute.org/gatk/) using the default parameters. In each condition, SNPs with reading depth 4 5 and quality 4 20 were identified as putative homozygous SNPs. The read depth at each locus was calculated using BED tools.