Authentication of Zanthoxylum Species Based on Integrated Analysis

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Authentication of Zanthoxylum Species Based on Integrated Analysis of Complete Chloroplast Genome Sequences and Metabolite Profiles Hyeon Ju Lee, Hyun Jo Koo, Jonghoon Lee, Dong Young Lee, Vo Ngoc Linh Giang, Minjung Kim, Hyeonah Shim, Jee Young Park, Ki-Oug Yoo, Sang Hyun Sung, and Tae-Jin Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04167 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Journal of Agricultural and Food Chemistry

Authentication of Zanthoxylum Species Based on Integrated Analysis of Complete Chloroplast Genome Sequences and Metabolite Profiles

Hyeon Ju Lee1,4, Hyun Jo Koo1,4, Jonghoon Lee1,4, Sang-Choon Lee1, Dong Young Lee2, Vo Ngoc Linh Giang1, Minjung Kim1, Hyeonah Shim1, Jee Young Park1, Ki-Oug Yoo3, Sang Hyun Sung2*, Tae-Jin Yang1*

1

Department of Plant Science, Plant Genomics and Breeding Institute, and Research

Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea 2

College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National

University, Seoul, 08826, Republic of Korea 3

Department of Biological Sciences, Kangwon National University, Chuncheon,

Gangwon, 24341, Republic of Korea 4

These authors contributed equally to this work.

*Corresponding authors Email: [email protected] Tel: +82-2-880-4547 Fax: + 82-2-873-2056

1

ACS Paragon Plus Environment


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Abstract

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We performed chloroplast genome sequencing and comparative analysis of two Rutaceae

3

species, Zanthoxylum schinifolium (Korean pepper tree) and Z. piperitum (Japanese pepper

4

tree), which are medicinal and culinary crops in Asia. We identified more than 837 single

5

nucleotide polymorphisms and 103 insertions/deletions (InDels) based on a comparison of

6

the two chloroplast genomes and developed seven DNA markers derived from five tandem

7

repeats and two InDel variations that discriminated between Korean Zanthoxylum species.

8

Metabolite profile analysis pointed to three metabolic groups, one with Korean Z. piperitum

9

samples, one with Korean Z. schinifolium samples and the last containing all the tested

10

Chinese Zanthoxylum species samples, which are considered to be Z. bungeanum based on

11

our results. Two markers were capable of distinguishing among these three groups. The

12

chloroplast genome sequences identified in this study represent a valuable genomics

13

resource for exploring diversity in Rutaceae, and the molecular markers will be useful for

14

authenticating dried Zanthoxylum berries in the marketplace.

15 16

Key words: Zanthoxylum, Z. schinifolium, Z. piperitum, chloroplast genome, marker

17 18

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Introduction

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The chloroplast is an essential cytoplasmic organelle in plant cells, serving as the

21

location for photosynthesis to produce energy via CO2 assimilation.1 Chloroplasts retain an

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autonomous organellar genome that encodes, among other proteins, the large subunit of the

23

key

24

carboxylase/oxygenase, rbcL).2 The circular chloroplast genome ranges in size from

25

approximately 120–217 kb and is maternally inherited in most angiosperms.3 The

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chloroplast genome is usually divided into four parts including a large single copy (LSC)

27

region and a small single copy (SSC) region separated by a pair of inverted repeats (IRs).4-6

28

Compared to nuclear and mitochondrial genomes, chloroplast genomes are highly

29

conserved7, and there is little variation within a single species. Although chloroplast

30

genomes are highly conserved, small nucleotide variations offer enough information to

31

distinguish among different species and sometimes between different variants or cultivars

32

within a species.

enzyme

in

photosynthesis,

RuBisCO

(Ribulose-1,5-bisphosphate

33

Sequence variations in chloroplast genomes can be found in both protein-coding

34

genes (e.g., matK, rpoB, rpoC1 and rbcL) and intergenic regions (e.g., psbK-psbI, trnL-trnF

35

and atpF-atpH). These variations have been used to study plant genetic diversity and

36

evolution and to develop markers for authenticating plant species.8-13 Due to recent

37

advances in sequencing and assembly technologies, the complete chloroplast genome

38

sequences from more than 1800 species have been deposited in GenBank14. Phylogenetic

39

analyses based on chloroplast genome information have shed light on plant evolution. In

40

phylogenomic studies using chloroplast genes, the selection of the proper sequence datasets,

41

taxon sampling techniques and methods for phylogenetic analysis (Bayesian analysis,

42

maximum likelihood and so on) is important because different methods can produce

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different results; therefore, the correct methodology is still under debate15. To date,

44

comprehensive surveys of genetic diversity using chloroplast genomes have been

45

performed in many plant species using close relatives16-19 or within subspecies in plants

46

such as Oryza sativa20 and Panax ginseng14.

47

The Zanthoxylum genus, which belongs to the Rutaceae family, comprises

48

approximately 250 species of aromatic trees and shrubs.21 In Africa, the Americas and Asia,

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many Zanthoxylum species are traditionally used as food supplements or drugs due to the

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valuable aromatic oil compounds obtained from their pericarps and leaves.22-26 Some Asian

51

species such as Z. piperitum (Japanese pepper), Z. schinifolium (Korean pepper) and Z.

52

bungeanum (Szechuan pepper) are also used as condiments and spices due to their strong

53

taste, especially in Eastern Asian countries including Korea, Japan and China22-25, 27-29,

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while American and African Zanthoxylum species are not used for culinary purposes.25

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Essential oils from Z. piperitum and Z. schinifolium contain beneficial compounds with

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anti-microbial, anti-inflammatory and antioxidant activities.23,

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schinifolium and Z. piperitum plants appear similar, they can be distinguished based on the

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arrangement of their spikes on branches, which is alternate in Z. schinifolium and

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symmetrically opposite in Z. piperitum (Figure 1A, B). However, it is not easy to

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discriminate between fruits and seeds harvested from these plants due to their similar

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morphology, especially when their dried and ground pericarps are distributed in the

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marketplace. The chemical components of these species differ, including aromatic

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compounds (especially their isopulegol contents), but their metabolic profiles sometimes

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differ within a single species, such as in samples from different countries.22, 23, 30 It is even

65

more difficult to distinguish between Z. schinifolium and Z. piperitum based on their

66

metabolic profiles when the pericarp powders from the species are mixed. Differences in

25, 27, 28, 30

Although Z.

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DNA sequences could be used to differentiate/identify these two Zanthoxylum species;

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however, the limited availability of genetic and genomic resources for both species

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represents an obstacle for the establishment of a clear molecular authentication system.31

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Therefore, a reliable tool is needed for authenticating the pericarps from these Zanthoxylum

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species at the species level.

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Several efforts have focused on developing markers to distinguish Z. schinifolium

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from Z. piperitum based on sequence variations in their internal transcribed spacer (ITS)

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regions in nuclear ribosomal DNA (nrDNA),31,

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However, complete chloroplast genome sequences exhibit less variation than nrDNA within

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species and can therefore provide important information for comprehensive analysis of

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genetic diversity and establishment of a clear molecular authentication system.17

32

a well-known barcoding region.14,

33

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In this study, we obtained the complete chloroplast genome sequences of Z.

79

piperitum and Z. schinifolium by de novo assembly of whole-genome sequencing (WGS)

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data using next-generation sequencing (NGS) technology. We also carried out metabolic

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profiling of Korean and Chinese Zanthoxylum species, and developed practical molecular

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markers that distinguish Z. schinifolium, Z. piperitum and Chinese Zanthoxylum species.

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Materials and Methods

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Plant materials and DNA preparation

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Twenty-three individual samples of Zanthoxylum species collected from Korea and

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China were used in this study, including 11 Chinese Zanthoxylum species (CZ), eight

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Korean Z. piperitum (KZP) samples and four Korean Z. schinifolium (KZS) samples; their

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geographical origins are described in Table 1 and Figure 1C. Total genomic DNA was

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extracted from fresh leaves of KZP-08, KZS-03 and KZS-04 and from freeze-dried fruits

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from the other samples using a modified cetyltrimethylammonium bromide (CTAB)

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method.34 The quality and quantity of the extracted DNA samples were examined using a

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NanoDrop ND-1000 (Thermo Scientific, Wilmington, MA).

94 95

Whole-genome shotgun sequencing

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To generate the chloroplast genome sequences, genomic DNA was extracted from

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the leaves of KZS-03 (Z. schinifolium) and KZP-08 (Z. piperitum) and used for whole

98

genome shotgun sequencing on the Illumina MiSeq platform (Illumina, San Diego, CA)

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and Illumina NextSeq500 (Illumina, San Diego, CA), respectively (Table 1). A paired-end

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genomic library was constructed following the manufacturer’s instructions. Library

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construction and sequencing were carried out by Lab Genomics Co. (Seongnam, Korea).

102 103

Chloroplast genome assembly and gene annotation

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The generated sequencing data with Phred scores of 20 or less were filtered and de

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novo assembled using CLC genome assembler (v. beta 4.6, CLC Inc., Rarhus, Denmark)

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according to the dnaLCW method described in Kim et al.14,

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representing the chloroplast genome were combined into a draft sequence based on the

33

Principal contigs

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linkages of overlapping contig sequences. Annotation of protein-coding genes in the

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chloroplast genome was carried out using the DOGMA program35 and manually confirmed

110

using BLAST searches. Circular gene maps of the complete chloroplast genomes were

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drawn using OGDRAW (http://ogdraw.mpimp-golm.mpg.de/)36.

112 113

Comparative analysis of the chloroplast genomes of Zanthoxylum species

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The assembled chloroplast genome sequence of Z. schinifolium was compared to

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the complete chloroplast genome sequence of Z. piperitum obtained from sample KZP-08

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(GenBank No.: KT153018).37 The two sequences were aligned and compared using

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MAFFT

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(http://genome.lbl.gov/vista/mvista/submit.shtml)39. Annotation information for mVISTA

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was obtained using DOGMA35 and tRNAscan-SE40, followed by manual curation that also

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included a comparison with published chloroplast genome sequences. In addition, tandem

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repeats (TRs) were identified from the chloroplast genomes of the two Zanthoxylum species

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using the Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html)41 and

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compared to identify the different regions between Z. schinifolium and Z. piperitum. The

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rates of nonsynonymous substitutions per nonsynonymous sites (Ka) over synonymous

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substitutions

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(http://www.bork.embl.de/pal2nal/)42.

(http://mafft.cbrc.jp/alignment/server/)38

per

synonymous

site

(Ks)

were

and

calculated

using

mVISTA

PAL2NAL

127

To compare the ndhG sequences from Z. piperitum, Z. schinifolium and Z.

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bungeanum, these sequences and their translated sequences were aligned and compared

129

using MAFFT (http://mafft.cbrc.jp/alignment/server/)38.

130 131

Molecular marker analysis

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To validate the inter-species polymorphisms in the chloroplast genomes and to

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develop DNA markers for discriminating these Zanthoxylum species, specific primers were

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designed based on polymorphic regions derived from InDels and copy number variation of

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the TRs between Z. piperitum and Z. schinifolium using the Primer 3 program

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(http://bioinfo.ut.ee/primer3-0.4.0/).43, 44 Seven molecular markers were developed based on

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the sequence variation between the Z. piperitum and Z. schinifolium chloroplast genomes.

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PCR amplifications were performed in a total volume of 25 µl containing 20 ng of genomic

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DNA template, 1× PCR buffer, 10 pM of each primer, 0.2 mM dNTPs and 1 U Taq DNA

140

polymerase (Vivagen, Korea). The amplified PCR fragments were analyzed via size

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separation in 1.5% agarose gels or 9.0% polyacrylamide gels or by capillary electrophoresis

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using a Fragment Analyzer (Advanced Analytical Technologies Inc., Ankeny, IA),

143

depending on the sizes of the PCR products.

144 145

Principle Component Analysis based on near infrared reflectance spectroscopy

146

analysis

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The pericarp samples were cleaned, air-dried, placed into a stoppered glass vial and

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dried for 12 h in an oven at 60°C to remove the moisture in the samples prior to near-

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infrared reflectance (NIR) spectroscopy analysis. NIR spectra were obtained from the

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samples using an NIR system (MPA; Bruker Optics, Ettlingen, Germany) over a

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wavelength range of 10,000–4000 cm-1 using 32 scans at a resolution of 8 cm-1 per

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spectrum. Each spectrum represents an average of 32 scanned spectra. Approximately 1 g

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of sample was placed into a single glass sample vial. The spectra were acquired in the

154

reflectance mode using a glass sample vial as a reference standard. Each sample spectrum

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was measured three times and the final spectra were averaged. NIR spectra are affected by 8


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both the chemical and physical properties of samples; the latter properties contribute to the

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majority of unwanted variance among spectra. Therefore, spectral pre-processing must be

158

performed to reduce systematic noise, such as light scattering, path length differences,

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baseline variation and so on. In this study, several spectral preprocessing methods were

160

used comparatively to obtain the optimum results, including first derivative, second

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derivative, standard normal variate (SNV) and multiplicative scatter correction (MSC). To

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avoid noise enhancement, which occurs as a consequence of derivative analysis, a

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Savitzky-Golay smoothing filter was employed. NIR spectral data acquisition and spectral

164

preprocessing were performed with OPUS 7.0 software (Bruker Optics, Ettlingen,

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Germany). SIMCA 13 software (Umetrics, Malmö, Sweden) was used for PCA. The data

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sets were in Pareto scaling mode prior to PCA.

167 168

Phylogenetic analysis

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The whole chloroplast genome sequences from 16 plant species were aligned using

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ClustalW, and a maximum likelihood tree was generated with very strong branch swap

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filter options using MEGA5 (version 5.2.2)45. To measure clade support, 1000 bootstrap

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replicates were generated. A Bayesian tree was generated from the same sequence

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alignment using BEAST (version 1.8.4)46 with the following options: substitution model,

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HKY; base frequencies, Estimated; site heterogeneity model, None; tree prior, Coalescent -

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Constant Size; tree model, Random starting model. The length of the chain for Two Markov

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Chain Monte Carlo searches was 10,000,000 generations, with trees samples every 1000

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generations. TreeAnnotator was run with the following options: burn-in (as trees), 100;

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posterior probability limit, 0; target tree type, Maximum clade credibility tree; node heights,

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Mean heights. A final tree with posterior probability values at the clade nodes was

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generated with FigTree version 1.4.3 and edited with MEGA5 (version 5.2.2)45.

181 182

Results and Discussion

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Complete chloroplast genome sequence of Z. schinifolium

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We obtained approximately 3.26 Gb and 4.23 Gb of paired-end sequences from

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whole-genome sequencing of KZP-08 (Z. piperitum) and KZS-03 (Z. schinifolium),

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respectively (Table 2), using low-coverage WGS, an efficient method that has been used to

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produce complete chloroplast genome sequences in many plant species.14,

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Compared to the raw sequence data for KZP-08, the sequencing data for KZS-03 contains

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many more raw sequence reads corresponding to the chloroplast genome (164.87x

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chloroplast coverage from 3.26 Gbp in KZP-08 and 1069.04x chloroplast coverage from

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4.23 Gbp in KZS-03) (Table 2). Following de novo assembly of the KZS-03 data, five

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contigs were produced for the chloroplast genome, which were ordered based on the

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complete chloroplast genome sequence of Z. piperitum (GenBank No.: KT153018). The

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contigs were merged into a single circular draft sequence by combining overlapping

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sequences. After putative assembly errors were curated by mapping raw reads onto the draft

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sequence, we obtained 158,963 bp of the complete chloroplast genome sequence, with 38.4%

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GC content. The chloroplast genome of Z. schinifolium has a typical quadripartite structure

198

with a pair of inverted repeat regions (IRa and IRb, each 27,085 bp) separated by a large

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single copy (LSC) region (86,528 bp) and a small single copy (SSC) region (18,265 bp)

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(Table 2). In addition, analysis of GC contents (calculated based on the GC composition in

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100 bp sliding windows) and raw read mapping depth revealed that parts of the IR regions

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next to SSC have relatively high GC contents with lower sequencing depth in the

19, 20, 33, 37

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chloroplast genomes of both Zanthoxylum species (Figure S1). In total, we identified 111

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genes in the Z. piperitum and Z. schinifolium chloroplast genomes, including 78 protein-

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coding genes, 29 tRNA genes and 4 rRNA genes, including 18 genes containing introns

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(Table S1). When counting gene numbers, duplicated genes in IRa and IRb were considered

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to be one gene instead of two. The complete chloroplast genome sequence of Z.

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schinifolium was deposited in GenBank under accession number KT321318.

209 210

Comparative analysis of the chloroplast genomes of Z. piperitum versus Z.

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schinifolium

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The chloroplast genome sequences of the two Zanthoxylum species are 97.1%

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identical, and their GC contents are also very similar (38.5% and 38.4% in Z. piperitum and

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Z. schinifolium, respectively) (Table 2). Compared to the Z. piperitum chloroplast genome,

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the Z. schinifolium chloroplast genome is 809 bp longer, with shorter IR regions (27,644 bp

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in Z. piperitum and 27,085 bp in Z. schinifolium) but longer LSC and SSC regions (85,340

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bp and 17,526 bp, respectively, in Z. piperitum and 86,528 bp and 18,265 bp, respectively,

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in Z. schinifolium) (Table 2). Both chloroplast genomes contain 112 identical genes, which

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are present in the same order in the genome (Figure 2). The IR regions in both genomes

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contain completely duplicated genes, including eight protein-coding genes (rps19, rpl2,

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rpl23, ycf2, ycf15, ndhB, rps7 and rps12), seven tRNA genes (trnI-CAU, trnL-CAA, trnV-

222

GAC, trnI-GAU, trnA-UGC, trnR-ACG and trnN-GUU) and four rRNA genes (rrn16, rrn23,

223

rrn4.5 and rrn5) (Table S1).

224

We performed comparative analysis using mVISTA to determine the level of

225

sequence divergence, finding that intergenic regions are more divergent than genic regions

226

(Figure S2). The nucleotide and amino acid sequences of protein-coding genes are highly

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similar, with an average sequence similarity of 98.6 and 98.5%, respectively (Table S2).

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When we aligned both chloroplast sequences, there were two notably large InDels: in the

229

rps16–trnQ (UUG) (473 bp) intergenic region in the LSC and in ycf1 (582 bp) in IRa (see

230

red arrowheads in Figure 2 and red dashed boxes in Figure S2). The ycf1 genes are located

231

in the borders between SSR and two IR regions, and an extended IR region in Z. piperitum

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(approximately 560 bp) caused the latter large InDel (Figure 3).

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We compared TRs within the chloroplast genome between Z. piperitum and Z.

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schinifolium. Nineteen TR regions differ between the two Zanthoxylum species, and TRs

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ranging from 15–38 bp in length were repeated from 0.5 to 3 times (Table 3). All TRs were

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found in the 14 intergenic regions, including 13 located in LSC and one in an IR region

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(blue arrowheads in Figure 2).

238

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Divergence of coding gene sequences

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Between the two Zanthoxylum species, 17 and 34 genes share identical nucleotide

241

and amino acid sequences, respectively (Table S2). Several genes with higher Ka, Ks or

242

Ka/Ks values are indicated in Figure S3. The average Ks values between the two

243

Zanthoxylum species are 0.0185, 0.0250 and 0.0059 in the LSC, SSC and IR regions,

244

respectively, with an average ratio of 0.0165 (Table S2, Figure S3). This result is in

245

agreement with the previous finding that IR regions are more conserved than other regions

246

because they frequently compensate for each other19. However, small variations were

247

observed even in highly conserved coding regions. Genes in SSC regions had higher rates

248

of changes in non-synonymous sites and higher average Ka/Ks ratios, indicating that genes

249

in the SSC region are relatively more variable between the two Zanthoxylum species than

250

those in other regions. Only one gene had a Ka/Ks ratio >1 (ndhG in the SSC region had a 12


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Ka/Ks ratio of 1.4324) (Table S2). NdhG is a subunit of the NADPH dehydrogenase

252

complex, which provides electrons for cyclic electron flow47 and helps protect the plant

253

against photo-oxidative stress.47, 48 NdhG might be structurally similar to the NuoJ subunit

254

of Escherichia coli complex I (NADH: ubiquinone oxidoreductase) and the Nqo10 subunit

255

of Thermus thermophiles complex I, and it appears that NdhG offers part of a

256

plastoquinone-binding site on its surface and is not involved in electron transport.49

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Therefore, NdhG is likely to be more of a structural subunit than a functional subunit for

258

the NADPH dehydrogenase complex, and ZpNdhG and ZsNdhG, with four amino acid

259

differences, might both be functional. These result imply that accumulation of non-

260

synonymous mutations not affecting protein function sometimes can result in high value of

261

Ks/Ka even without positive selection.

262 263

Validation of inter-species polymorphism and development of authentication markers

264

We performed molecular classification of the two Zanthoxylum species by

265

designing TR markers based on InDel and copy number variations. We designed seven

266

primer sets derived from the two large InDels and five intergenic regions harboring TRs

267

and confirmed them by PCR analysis of Zanthoxylum species using two accessions each of

268

Chinese Zanthoxylum species (CZP-03, CZP-11), Korean Z. piperitum (KZP-01, KZP-08)

269

and Korean Z. schinifolium (KZS-03, KZS-04) (Table 3 and Table 4). Schematic diagrams

270

of the five TR markers are shown in Figure 4A–E. The lengths of genes used as markers are

271

varied due to tandem repeats and insertions. The sizes of PCR products from these markers

272

were varied according to their size expected from their sequences (Figure 4F–J). Although

273

3 markers (2 TR markers and 1 InDel marker) were the same between Z. piperitum and Z.

274

bungeanum, all seven markers revealed inter-specific polymorphism and clearly

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discriminated between Z. piperitum and Z. schinifolium (see Figure 4 for TR markers,

276

Figure S4 for InDel markers and Figure S5 for all samples).

277 278

Authentication of Zanthoxylum species using the newly developed markers and

279

metabolite profile analysis

280

The sizes of PCR products from KZP and KZS amplified using the TR markers and InDel

281

markers (Figure 4, Figure S4 and Figure S5) were as expected (Table 4). Since the CZ

282

samples were purchased from markets in China, their tentative production area was known

283

but their scientific names were uncertain, although we thought they could have been

284

obtained from Z. piperitum or Z. schinifolium plants grown in China. When we analyzed

285

metabolite data using near infrared reflectance spectroscopy analysis, principle component

286

analysis (PCA) indicated that the CZ samples harbored distinct metabolites and therefore

287

might have been different species from the KZP and KZS samples (Figure 5). Several

288

Zanthoxylum species grow in China, and the chloroplast genome sequence of one of these

289

species, Z. bungeanum, has been reported (GenBank No. KX497031). Notably, the sizes of

290

PCR products from CZ matched the sizes expected from the Z. bungeanum chloroplast

291

genome (Figure 4, Figure S4, Figure S5 and Table 4). Several varieties of Z. bungeanum

292

also grow in Szechuan province; the fruits of these varieties, as well as of Z. armatum, are

293

commonly referred to as Szechuan peppers.29 Of the 11 CZ samples, three were also

294

obtained from Szechuan province, and we believe that the CZ samples are Szechuan

295

peppers. To date, the chloroplast genomes of all Chinese Zanthoxylum species except Z.

296

bungeanum have yet to be sequenced, so it is unclear if the chloroplast genomes of several

297

Chinese Zanthoxylum species are highly similar. However, we confirmed that the sequence

298

lengths of all CZ samples collected from China match that expected from Z. bungeanum.

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Therefore, our two TR markers, IMZanTR-1 and IMZanTR-3, can be used to distinguish Z.

300

piperitum, Z. schinifolium and Z. bungeanum and to identify Korean pepper, Japanese

301

pepper and some Szechuan peppers in the marketplace.

302 303 304

Zanthoxylum species in East Asia

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Comparative analysis of metabolites revealed different profiles between Z.

306

piperitum and Z. schinifolium. While the Z. piperitum pericarp produces oleic acid as a

307

major fatty acid, the Z. schinifolium pericarp produces linolenic acid instead.22 Among

308

terpenoids, isopulegol is produced at the highest levels in Z. piperitum pericarp, followed

309

by myrcene, whereas myrcene is produced at the highest levels in Z. schinifolium pericarp,

310

followed by citronellal.22 Since isopulegol can be synthesized from citronellal by squalene

311

hopene cyclase,50 perhaps Z. piperitum contains high levels of oxidosqualene cyclase for

312

this conversion. However, Z. piperitum fruit from Japan contains high levels of limonene as

313

a major terpene product and produces very little isopulegol,51 and Z. schinifolium from

314

China produces linalool as a major product.52 The metabolite profiles of plants can vary

315

based on the environment, developmental stage, storage conditions after harvest, metabolite

316

extraction method and so on. To identify the differences in metabolite profiles among

317

Zanthoxylum species, it is best to perform experiments using the same method. Unlike

318

metabolite analysis, genetic information is highly reproducible and digitalized.

319

We constructed a maximum likelihood (ML) tree for several Rutaceae species

320

using whole chloroplast genome sequences with a clade of Anacardiaceae species as an

321

outgroup (Figure 6). In this phylogenetic tree, Z. piperitum and Z. bungeanum are closer to

322

each other than to Z. schinifolium. The ndhG sequence data support this result; there are

15



Page 16 of 35

323

four amino acids difference between Z. piperitum and Z. schinifolium but no difference

324

between Z. piperitum and Z. bungeanum. The Bayesian (B) tree has the same structure as

325

the ML tree (Figure S6). Interestingly, Korean Z. piperitum is genetically closer to Chinese

326

Z. bungeanum than to Korean Z. schinifolium. Perhaps Z. piperitum and Z. bungeanum

327

diverged after their ancestor split from Z. schinifolium; this hypothesis is well supported by

328

both bootstrap and posterior probability values from the ML and B trees, respectively.

329

Z. piperitum is preferred for use as a spice over Z. schinifolium in Korea and Japan,

330

and Z. bungeanum is also widely used as a spice in China. There are additional

331

Zanthoxylum species in East Asia, some of which also produce culinary seeds. More

332

sequencing data from these species, such as Z. armatum and Z. simulans, will shed light on

333

the evolution of the Zanthoxylum species used as spices in this area.

334

The sizes of the PCR products obtained using the newly developed markers were

335

more similar between the KZP and CZ samples versus the KZS samples (Figure 4, Table 3).

336

However, these markers must be much more broadly applicable to other Zanthoxylum

337

species when they are used to discriminate species other than Z. piperitum, Z. schinifolium

338

and Z. bungeanum. The availability of additional sequencing data from other Zanthoxylum

339

species will also facilitate the development of a system for discriminating all of these

340

species in the marketplace.

341 342 343 344

16


Page 17 of 35


345 346

Abbreviations Used

347

LSC, large single copy; SSC, small single copy; IR, inverted repeat; TR, tandem repeat;

348

InDel, insertion or deletion; KZP, Korean Zanthoxylum piperitum; KZS, Korean

349

Zanthoxylum schinifolium; CZ, Chinese Zanthoxylum species

350 351

Acknowledgments

352

We thank all members of the Laboratory of Functional Crop Genomics and Biotechnology,

353

Seoul National University and Phyzen Genomics Institute for their technical assistance.

354 355

Funding Sources

356

This research was supported by the Next-Generation BioGreen21 Program for Agriculture

357

and Technology Development (Project No. PJ01103001) of the Rural Development

358

Administration and the Bio and Medical Technology Development Program of the NRF

359

funded by the Korean government, MSIP (NRF-2015M3A9A5030733), Republic of Korea.

360 361

17



Page 18 of 35

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Figure captions Figure 1. Zanthoxylum species used in this study, and areas of origin of leaf samples or seed products collected from various markets. (A, B) Morphological characteristics of Z. piperitum and Z. schinifolium, respectively, used for genome and RNA sequencing. Opposite or alternately arranged spikes are indicated by orange circles. (C) Collection areas of seed products utilized for food or oriental medicine production. Collection areas for Korean Z. piperitum (KZP), Korean Z. schinifolium (KZS) and Chinese Zanthoxylum species (CZ) are indicated by circles on the map.

Figure 2. Circular gene maps of the chloroplast genomes of the two Zanthoxylum species. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise. The genes are colored according to their functions, as shown in the legend. Polymorphic sites between two Zanthoxylum species derived from the copy number variation of tandem repeat sequences are denoted with blue arrowheads at 14 locations, and the two InDel sites are marked by red arrows; those used for marker development are indicated by “*”.

Figure 3. Comparison of the borders of SSC and IR regions between the chloroplast genomes of the two Zanthoxylum species. Compared to the Z. piperitum sequences, IR regions in Z. schinifolium are shorter, and part of ycf1 in the SSC region (indicated with a triangle) was removed.

Figure 4. Schematic diagram of TRs and insertions in five TR markers (A–E), and confirmation of these markers for the discrimination of Zanthoxylum species (F–G). PCR products from two individuals each of CZ, KZP and KZS are of the predicted sizes using TR23



Page 24 of 35

type markers. The primer sequences and expected sizes are shown in Table 4, and PCR results from whole samples (11 CZ, 8 KZP and 4 KZS) are shown in Figure S5.

Figure 5. Comparison of the metabolite profiles of Zanthoxylum seed samples. Principle Component Analysis of pericarp metabolites based on near infrared reflectance spectroscopy analysis data formed three groups: Chinese Zanthoxylum species (CZ), Korean Z. piperitum (KZP) and Korean Z. schinifolium (KZS).

Figure 6. Phylogenetic tree including sequences from Z. piperitum, Z. schinifolium and Z. bungeanum. The maximum likelihood tree was generated using whole chloroplast genome sequences with 1000 bootstrap replicates. Bootstrap values are shown in percentages, and posterior probability values from the Bayesian tree (adapted from Figure S6) are in parentheses. A clade of Anacardiaceae species was used as an outgroup.

24


Page 25 of 35


Table 1. List of Zanthoxylum species used in the study Species Chinese Zanthoxylum species (CZ)

Korean Z. piperitum (KZP)

Korean Z. schinifolium (KZS)

Sample CZ-01

Geographical origin Szechuan province

CZ-02

Szechuan province

CZ-03

Szechuan province

CZ-04

Shantung province

CZ-05

Shantung province

CZ-06

Shanxi province

CZ-07

Shanxi province

CZ-08

Shanxi province

CZ-09

Shanxi province

CZ-10

Shanxi province

CZ-11

Shanxi province

KZP-01

Geochang, Gyeongsangnam-do

KZP-02

Mungyeong, Gyeongsangbuk-do

KZP-03

Gurye, Jeollanam-do

KZP-04

Gurye, Jeollanam-do

KZP-05

Gurye, Jeollanam-do

KZP-06

Gimje, Jeollabuk-do

KZP-07

Imsil, Jeollabuk-do

KZP-08*

Geoje, Gyeongsangnam-do

KZS-01

Hongcheon, Gangwon-do

KZS-02

Yangpyeong, Gyeonggi-do

KZS-03*

Chuncheon, Gangwon-do

KZS-04 Yongin, Gyeonggi-do * Plant materials used for complete chloroplast genome sequencing

25



Page 26 of 35

Table 2. Summary of whole genome sequencing and chloroplast genome assembly in Zanthoxylum species Species (Sample No.)

Raw data amount (Gbp)

Z. piperitum* 3.26 (KZP-08) Z. schinifolium 4.23 (KZS-03) Sequence identity (%) * cited from Lee et al. 53

GenBank No.

Cp coverage (x)

KT153018 KT321318

Length (bp)

GC content

LSC

SSC

IR

Total

164.87

85,340

17,526

27,644

158,154

38.5%

1069.04

86,528

18,265

27,085

158,963

38.4%

96.4

89.9

97.1

97.1

26


Page 27 of 35


Table 3. Intergenic regions containing tandem repeats with copy number variation between Zanthoxylum piperitum and Z. schinifolium No. 1 2 3 4 5 6

Position trnH(GUG) - psbA psbK - psbI trnS(GCU) trnG(UCC) trnR(UCU) - atpA atpH - atpI petN - psbM

Sequence (length) TAATTTTCTTAGTAGTATTC (20 bp) AGAGCCAACCACAATGT (17 bp) GTTACATTGTTACATTACACA (21 bp)

TTATATATTTATATT (15 bp) AAAGAAAATATTAAG (15 bp) AGTAATTTCATTATA (15 bp) TTTAATTCAGTAATTCAATT (20 bp) CCATTTAGAATTTTTCAGTAATTTAATT (28 bp) 7* psbM - trnD(GUC) AATACTAAAATACTAATA (18 bp) CTTTTTTTTATTTATCATT (17 bp) 8* psbZ - trnG(GCC) AAATAAATATTAATATAATAATT (23 bp) TTATTAATAGAAATATATATTATTTATA (28 bp) 9* trnS(GGA) - rps4 GGTGAAAGGGGAAATTTGTACGAGCCCGTTATTTTAGT (38 bp) 10 trnT(UGU) - trnL(UAA) TCTTAATCTATTCTA (15 bp) 11 ndhC - trnV(UAC) TAGTTTCGTTTGTTTGTTGT (20 bp) TTTTGATTCTATTCTATA (18 bp) 12* rpl33 - rps18 TTATTTCATATATTTAAATAGAAACAA (27 bp) 13 rpl16 - rps3 TTTAGAGATAATCTCAA (17 bp) 14 rrn4.5 - rrn5 ATTGTTCAACTCTTTGACAACATGAAAAAACC (32 bp) * Regions where PCR markers were developed for validation

Copy Number KZP KZS 2 1 1 2 1.5

2

1 2 0.5 2 1 1.5 1 1 1 1 1 1 1 1 1 1

2 1 2 1 2 2 2 2 3 2 2 2 2 3 2 2

27



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Table 4. Newly developed markers for validation of the polymorphic sites among Zanthoxylum species Product size (bp) Marker name

IMZanTR-1 IMZanTR-2 IMZanTR-3 IMZanTR-4 IMZanTR-5 IMZanInDel-1 IMZanInDel-2

Primer sequence (5´- 3´)

Region

Forward

AATTGAGTTGGGAAATCAAACTGTA

Reverse

CTCGCTAGAATCCAAGACAATAGAA

Forward

GATCTTTTATCCACACACCGAATAC

Reverse

GAAAAGACAGAATGGAAAAGAATGA

Forward

GGGATCAAACTTCTGGAACTTGA

Reverse

TTATCCCGAGTTAGGCCAGATAC

Forward

CAATTCCCAGTTTCTGTGATACG

Reverse

CTCGTCAGACTTAAACCTAACTAAAAT

Forward

GTGCTTGTGTGTCACCCTTG

Reverse

GAGTCGCTTGGTTTTATCCAT

Forward

AGTGGTAAGGCAACGGGTTT

Reverse

GATACAAAGACAAAAAGTCCCACA

Forward

CAAAATCGAGGAAACGGAAGAGA

Reverse

TTGATGGAATTACGAATGGGGTC

Specific to

Type

290

CZ/KZP/KZS

TR & InDel

133

163

KZS

TR

389

405

520

CZ/KZP/KZS

TR & InDel

trnS(GGA) - rps4

210

210

248

KZS

TR

rpl33 - rps18

132

132

258

KZS

TR & InDel

rps16 - trnQ(UUG)

594

120

593

KZP

InDel

ycf1

943

943

361

KZS

InDel

CZ*

KZP

KZS

petN - psbM

196

214

psbM - trnD(GUC)

131

psbZ - trnG(GCC)

* Predicted CZ product size based on Zanthoxylum bungeanum (GenBank No. KX497031)

28


Page 29 of 35


Figure 1. Zanthoxylum species used in this study, and areas of origin of leaf samples or seed products collected from various markets. (A, B) Morphological characteristics of Z. piperitum and Z. schinifolium, respectively, used for genome and RNA sequencing. Opposite or alternately arranged spikes are indicated by orange circles. (C) Collection areas of seed products utilized for food or oriental medicine production. Collection areas for Korean Z. piperitum (KZP), Korean Z. schinifolium (KZS) and Chinese Zanthoxylum species (CZ) are indicated by circles on the map.

29



Page 30 of 35

Figure 2. Circular gene maps of the chloroplast genomes of the two Zanthoxylum species. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise. The genes are colored according to their functions, as shown in the legend. Polymorphic sites between two Zanthoxylum species derived from the copy number variation of tandem repeat sequences are denoted with blue arrowheads at 14 locations, and the two InDel sites are marked by red arrows; those used for marker development are indicated by “*”.

30


Page 31 of 35


Figure 3. Comparison of the borders of SSC and IR regions between the chloroplast genomes of the two Zanthoxylum species. Compared to the Z. piperitum sequences, IR regions in Z. schinifolium are shorter, and part of ycf1 in the SSC region (indicated with a triangle) was removed.

31



Page 32 of 35

Figure 4. Schematic diagram of TRs and insertions in five TR markers (A–E), and confirmation of these markers for the discrimination of Zanthoxylum species (F–G). PCR products from two individuals each of CZ, KZP and KZS are of the predicted sizes using TR-type markers. The primer sequences and expected sizes are shown in Table 4, and PCR results from whole samples (11 CZ, 8 KZP and 4 KZS) are shown in Figure S5.

32


Page 33 of 35


Figure 5. Comparison of the metabolite profiles of Zanthoxylum seed samples. Principle Component Analysis of pericarp metabolites based on near infrared reflectance spectroscopy analysis data formed three groups: Chinese Zanthoxylum species (CZ), Korean Z. piperitum (KZP) and Korean Z. schinifolium (KZS).

33



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Figure 6. Phylogenetic tree including sequences from Z. piperitum, Z. schinifolium and Z. bungeanum. The maximum likelihood tree was generated using whole chloroplast genome sequences with 1000 bootstrap replicates. Bootstrap values are shown in percentages, and posterior probability values from the Bayesian tree (adapted from Figure S6) are in parentheses. A clade of Anacardiaceae species was used as an outgroup.

34


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For Table of Contents Only

35


Authentication of Zanthoxylum Species Based on Integrated Analysis

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