Transcriptomic and GC-MS metabolomic profiling analysis of

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Bioactive Constituents, Metabolites, and Functions

Transcriptomic and GC-MS metabolomic profiling analysis of epidermis provides insights into cuticular wax regulation in developing ‘Yuluxiang’ pear fruit Xiao Wu, Xinjie Shi, Mudan Bai, Yangyang Chen, Xiaolong Li, Kaijie Qi, Peng Cao, Mingzhi Li, Hao Yin, and Shaoling Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01899 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Journal of Agricultural and Food Chemistry

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Transcriptomic and GC-MS metabolomic profiling analysis of

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epidermis provides insights into cuticular wax regulation in

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developing ‘Yuluxiang’ pear fruit

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Short title: Wax synthesis in developing pear fruit

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Xiao Wu1, Xinjie Shi1, Mudan Bai2, Yangyang Chen1, Xiaolong Li1, Kaijie Qi1, Peng Cao1,

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Mingzhi Li3, Hao Yin1*, Shaoling Zhang1*

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1

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Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.

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2

Pomology Institute, Shanxi Academy of Agricultural Sciences, Shanxi, China.

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3

Genepioneer Biotechnologies Co. Ltd, Nanjing 210014, Jiangsu, China.

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Xiao Wu [email protected]

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Xinjie Shi [email protected]

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Mudan Bai [email protected]

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Yangyang Chen [email protected]

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Xiaolong Li [email protected]

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Kaijie Qi [email protected]

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Peng Cao [email protected]

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Mingzhi Li [email protected]

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Hao Yin [email protected]

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Shaoling Zhang [email protected]

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*Corresponding author: Hao Yin, Center of Pear Engineering Technology Research, Nanjing

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Agricultural University, Nanjing 210095, China; Email: [email protected]; Telephone:

Center of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and

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ACS Paragon Plus Environment


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+86-25-84396580

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Shaoling Zhang, Center of Pear Engineering Technology Research, State Key Laboratory of Crop

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Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China;

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Email: [email protected]; Telephone: +86-25-84396580

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Abstract

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The layer of cuticular wax covering fruits plays important roles in protecting against disease,

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preventing nonstomatal water loss and extending shelf life. However, the molecular basis of

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cuticular wax biosynthesis in pear (Pyrus) fruits remains elusive. Our study thoroughly

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investigates cuticular wax biosynthesis during pear fruit development from morphologic,

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transcriptomic and gas chromatography-mass spectrometry (GC-MS) metabolomic perspectives.

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Our results showed that cuticular wax concentrations increased during the early stage (20-80 days

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after full bloom, DAFB) from 0.64 mg/cm2 (50 DAFB) to 1.75 mg/cm2 (80 DAFB), and then

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slightly decreased to 1.22 mg/cm2 during the fruit ripening period (80-140 DAFB). Scanning

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electron microscopy (SEM) imaging indicated that wax plate crystals increased, and wax

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structures varied during the pear fruit development. The combined transcriptomic and

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metabolomic profiling analysis revealed 27 genes, including 12 genes encoding transcription

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factors (TFs) and a new structural gene (Pbr028523) encoding β-amyrin synthase, participating in

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the biosynthesis, transport and regulation of cuticular wax according to their expression patterns in

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pear fruit. The quantitative real-time PCR (qRT-PCR) experiments of 18 differentially expressed

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genes (DEGs) were performed, and confirmed the accuracy of the RNA-Seq derived transcript

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expression. A model of very-long-chain fatty acids (VLCFAs) and cuticular wax synthesis and

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transport in pear fruit is proposed, providing a mechanistic framework for understanding 2


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cuticular wax biosynthesis in pear fruit. These results and datasets provide a foundation for the

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molecular events related to cuticular wax in ‘Yuluxiang’ pear fruit, and may also help guide the

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functional analyses of candidate genes important for improving the cuticular wax of pear fruit in

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the future.

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Keywords: Cuticular wax; Pear fruit; Metabolome; Transcriptome; Morphology

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1 Introduction

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Pear (Pyrus) is a Rosaceae fruit species with high economic value, and has widespread cultivation

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throughout 80 countries and regions.1-2 The fruits of different pear cultivars vary in appearance,

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aroma, sugars, acids, stone cells and soluble solid contents. The cuticle is a hydrophobic layer on

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the primary surfaces of fruit, which is composed of a cutin polyester matrix and cuticular wax.3-4

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Cuticular wax is beneficial to fruit by reducing mechanical damage, diseases and insect pests, and

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also contributes to limiting water loss and extending postharvest storage.5-6 As such,

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understanding the molecular mechanism of cuticular wax synthesis will lay a solid foundation for

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enhancing the pear fruit quality. So far, the related functional genes involved in cuticular wax

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synthesis, transport and regulation were mainly focuses in the model plants Arabidopsis and

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tomato.7-8 In addition, several additional equivalent studies were obtained from other species, such

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as citrus, mango and apple.5,9-10 For example, very-long-chain fatty acids (VLCFAs) play

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important roles in wax synthesis, and VLCFAs biosynthesis can be regulated by fatty acid

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synthases (FASs), fatty acid elongases (FAEs), β-ketoacyl-CoA synthases (KCSs) and long-chain

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acyl-CoA synthetases (LACSs). Then VLCFAs are modified into different derivatives (aldehydes,

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alkanes, primer alcohols, ketones, esters, etc.) in the endoplasmic reticulum (ER) by the products

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of many genes, including CER1, CER4, CER17, WAX2 and MAH1. Finally, different wax 3



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molecules can be transported to the plant surface by lipid transfer proteins (LTPs) and ATP

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binding cassette (ABC) transporters. In addition, many transcription factors (TFs) are also

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involved in this process, including MYBs, WRKYs and WRIs, have been identified.8,10-15 To date,

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only several studies were focused on the fruit cuticular wax chemical composition or its crystal

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morphology during pear fruit development and storage or different treatments.16-18 Thus, the

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molecular mechanism of cuticular wax synthesis in pear fruit are still poorly understood.

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Fruit development is a complex process, that is subjected to various factors, such as temperature,

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hormones, light and genotypes.5,17,19 To maintain stability and for adaptation to various

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environments, the cuticular wax content, composition and structure are dynamic changed during

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the fruit development. For example, in the developing fruit of ‘Pingguoli’ pear, the wax

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concentration changed little (min to max: 10.23-11.91 μg/cm2) from 30 to 100 days after full

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bloom (DAFB), whereas the content dramatically increased from 10.23 μg/cm2 to 98.06 μg/cm2

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from 100 to 130 DAFB.17 In developing citrus fruit, the total wax concentration increased

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2.35-fold between 60 and 240 DAFB, and the total wax concentration of tomato fruit increased

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from 3.98 μg/cm2 to 16.36 μg/cm2 between the immature green and orange stages but then

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decreased to 13.75 μg/cm2 at the red overripe stage.5,7

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‘Yuluxiang’ is a popular newly promoted pear cultivar with high good quality in China, which is a

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hybrid progeny between ‘Kuerle’ (P. sinkiangensis) and ‘Xuehua’ (P. bretschneideri). In the

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present study, five developmental periods of ‘Yuluxiang’ pear fruits were used to characterize

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changes in cuticular wax and their relationships. We comprehensively analyzed the morphologic,

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metabolomic and transcriptomic characteristics of the cuticular wax to provide information about

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the dominant genes that regulate cuticular wax biosynthesis in pear. The key genes and regulatory 4


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factors participated in the pear fruit cuticular wax synthesis, transport and regulation were

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explored for the further functional characterization. Comprehensively, an important foundation

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should be provided for future molecular studies and the general regulatory mechanisms uncover of

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pear fruit cuticular wax based on these data.

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2 Materials and Methods

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2.1 Plant materials and experimental treatments

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‘Yuluxiang’ fruits were obtained from the Pomology Research Institutes of Shanxi in Jinzhong,

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Shanxi Province, China. Fruit samples from different developmental stages were collected at 20,

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50, 80, 120 and 140 DAFB. The fruits were collected from same three trees at different time

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periods, and each tree was considered one biological repeat. At least 10 fruits from each tree at

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each time periods were collected. All fruits were wrapped up through foam net bag and

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transported promptly to the laboratory. Samples of the peel tissue of the fruits collected at each

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time point were taken. After promptly freezing treated by liquid nitrogen and stored at −80 °C

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refrigerator.

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2.2 Chemicals and standards

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All solvents and reagents were of analytical grade in this research. N,O-bis (trimethylsilyl)

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trifluoroacetamide (BSTFA, containing 1% trimethylchlorosilane), pyridine and C7-C40 saturated

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alkane standards were bought from Sigma-Aldrich (Shanghai, China), and tetracosane was

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purchased from Macklin Co., Ltd. (Shanghai, China).

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2.3 Extraction and determination of cuticular wax concentrations

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The cuticular wax extraction was based on the method described by Li et al.17 with some

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modifications. Extraction was performed on three biological replicates, each consisting of five 5



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fruits. The whole pear fruits (five fruits per period) without trauma were immersed in 600 mL of

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chloroform and dipped twice for 1 min at ambient temperature in fume hood. The solvent was

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evaporated under a reduced pressure at 40°C.

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2.4 Gas chromatography-mass spectrometry (GC-MS) and electron microscopy

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Chemical analysis and electron microscopy were performed as described previously.20 The

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extracted wax (1 mg) was dissolved in 1.5 mL of chloroform; 10 μL (10 μg/μL) of n-tetracosane

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was added as an internal standard, and the solution was transferred to a GC sample bottle through

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organic filtration (Φ 0.45 μm). Samples were blown down under a gentle stream of nitrogen and

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dissolved in 40 μL of BSTFA and 40 μL of pyridine. After derivatization for 1 hour at 70°C, the

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residual BSTFA and pyridine were blown dry under nitrogen, and samples redissolved with 1.2

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mL of chloroform for GC-MS analysis was performed through a gas chromatograph (Bruker

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450-GC) equipped with a mass-selective detector (Bruker 320-MS) and capillary column (30

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m × 0.25 μm internal diameter, 0.25 μm film thickness, Bruker BR-5MS). The initial temperature

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was 50 °C (isothermal temperature for 2 minutes), 20 °C/min to 200 °C by for 2 min, and

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3 °C/min to 320 °C for 30 min. The inlet, MS transfer line and ion source temperature were set at

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280, 280 and 250 °C, respectively. The wax compounds were detected via the NIST 2013 library

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or by comparing their mass spectra and retention times with the standards. Saturated alkanes were

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detected by the external standard method, and the other compounds were identified by comparison

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with known amounts of the internal standard tetracosane.

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2.5 RNA preparation and transcriptome sequencing

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Total RNA was isolated from the peel tissues with the Column Plant RNA Out Kit (Fuji, China).

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The concentration and integrities of the RNA samples were detected using agarose gel 6


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electrophoresis (1.0%), NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and

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an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). On the basis of

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the Illumina standard procedure, six library preparations were sequenced on the Illumina HiSeq

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2500 platform with PE125, which was performed at Genepioneer Biotech Corporation (Nanjing,

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China). Each sample contained two biological replicates.

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2.6 RNA-seq data and differentially expressed gene (DEG) analysis

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Adapter sequences, low-quality and poly-N tails reads have been removed to obtain clean data.

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The clean reads from all three samples (two biological replicates each) were then mapped to the

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‘Dangshan suli’ pear reference genome (http://peargenome.njau.edu.cn).1 Fragments per kilobase

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per million reads (FPKM) values were calculated to evaluate the gene expression levels. The

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DESeq2 package was used to identify the DEGs.21 The thresholds of false discovery rate (FDR)

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≤0.05 and |log2ratio| ≥1 were set for significant gene expression differences between two samples.

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Each set of differentially expressed unigenes were performed Gene Ontology (GO) and Kyoto

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Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses. The singular

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enrichment analysis (SEA) method was used to do the GO enrichment analyses by agriGO, in

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which P < 0.01 and FDR < 0.05.22 We then perform two statistical tests (the hypergeometric

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Fisher exact test, P < 0.01 and the Benjamini test, FDR < 0.05) to identify statistically significant

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enrichment of KEGG pathways.

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2.7 Correlation network construction and visualization

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A correlation network between each metabolite and the transcriptome was constructed using all

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eight metabolite accumulation profiles (alkanes, primary alcohols, esters, fatty acids, terpenoids,

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aldehydes, ketones and other unclassified compounds) separately; the construction was performed 7



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with R using the ‘rsgcc’ package in conjunction with the Pearson correlation coefficient (PCC)

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method.23 |PCC| > 0.95 and P

Transcriptomic and GC-MS metabolomic profiling analysis of

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