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Industrial Crops and Products 37 (2012) 270– 274

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h o me pag e: www.elsev ier .com/ locate / indcrop

hloroplast DNA molecular characterization and leaf volatiles analysis of mintMentha; Lamiaceae) populations in China

hen XiaoHuaa, Zhang FangYuanb, Yao Leia,∗

Aromatic Plant R&D Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, ChinaFudan-Sjtu-Nottingham Plant Biotechnology R&D Center, Sjtu Center, 200240, China

r t i c l e i n f o

rticle history:eceived 12 July 2011eceived in revised form 7 October 2011ccepted 14 November 2011vailable online 13 January 2012

eywords:

a b s t r a c t

Non-coding chloroplast DNA (cpDNA) was used to evaluate genetic diversity and relationships withinthe section Mentha collected from China. Leaf volatiles of Mentha spicata accessions and its parent wereanalyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). To thisend, genetic diversity of 30 Mentha accessions from different geographic origins of China, representingsix species and two hybrids, was assessed. Molecular studies grouped the samples mainly according togenetic relationship established by conventional methods. Cluster analysis based on the leaf volatiles

enetic diversityhemotypeenthaon-coding chloroplast DNA

chemical composition of M. spicata accessions defined two main chemotypes: 1,8-cineole-piperitenoneoxide type and limonene–carvone type. The results suggested that there was a high genetic variabilityamong individuals of Mentha in China. A distinct correlation between cpDNA marker and volatile oils inM. spicata accessions was revealed. Moreover, the chemical composition followed maternal inheritancein M. spicata accessions. In conclusion, the genetic diversity of Mentha populations as shown in this studyshould play a critical role in future selection and breeding programs.

. Introduction

Mentha is the most important genus in the Lamiaceae familyecause it contains a number of taxa, the essential oils of which haveigh economic value. The amount of the oils produced annually

s over 23,000 metric tonnes with a value exceeding $400 millionLawrence, 2006). This makes them the most economically impor-ant essential oils produced.

The genus Mentha includes five sections and 25–30 speciesHarly and Brighton, 1977). Although many varieties are existedn this genus, only four species (Mentha spicata, Mentha canadensis,

entha ×gracilis, Mentha ×piperita) are cultivated for the commer-ial oil produce in the world, and all of them belong to the sectionentha (Harly and Brighton, 1977). China is one of the largest mint

roducers in the world (Lawrence, 2006). At present, the presencef variety degeneration has created a big problem of decrease in oiluality and yield in this country. Recently, the rise in demand forint oil has led to expand mint production areas in China, which

n turn has put a lot of pressure on limited farmland resources. So

he selection of a mint cultivar with high growing rate and highontent of essential oil may be a good solution to overcome thoseroblems.

∗ Corresponding author. Tel.: +86 021 64785710; fax: +86 021 64785710.E-mail address: YaoLei@sjtu.edu.cn (L. Yao).

926-6690/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rioi:10.1016/j.indcrop.2011.11.011

Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

Knowledge of genetic diversity of a plant provides a basis forselection of superior parental combination (Schlotterer, 2004).Over the past decade, different types of molecular techniques havebeen used to assess the genetic diversity in genus Mentha (Voset al., 1995; Khanuja et al., 2000; Fenwick and Ward, 2001; Shasanyet al., 2010). The chloroplast genome, usually non-recombiningand uniparentally inherited, is useful to infer the maternal lineageand provided important information about the origin of polyploids(Gobert et al., 2006). The trnL intron and the two intergenic spacersincluding, trnL-trnF and psbA-trnH in non-coding regions of chloro-plast genome are used for phylogenetic studies of closely relatedspecies at the interspecific level (Gobert et al., 2006; Baraket Ghadaet al., 2010). In recent decades, chloroplast DNA (cpDNA) mark-ers have been widely used in investigations of genetic structure,phylogenetic study and phylogeography, for example in Oxytropis(Artyukova et al., 2011), Citrus (Lu et al., 2011) and Mentha (Gobertet al., 2006; Jiranan Bunsawat et al., 2004).

M. spicata (native spearmint) is the best known hybrid, whichare intensively cultivated for their essential oils. The compositionof the essential oil of M. spicata has been extensively investi-gated. For example carvone and menthone-rich oils (Sticher et al.,1968), carvone and neodihydrocarveol-rich oils (Nagasawa et al.,1976a,b), dihydrocarveol and carvone-rich oils (Sivropulou et al.,

1995), carvone and linalool-rich oils (Hadjiakhoondi et al., 2000),high carvone-rich oils and lower limonene-rich oils (Edris et al.,2003). However, the relationship between chemical compositionand genetic diversity of M. spicata has not been investigated.

ghts reserved.

X.H. Chen et al. / Industrial Crops and Products 37 (2012) 270– 274 271

Table 1Mentha accessions used in the present study.

No. Accession no. Stats Locality No. Accession no. Stats Locality

1 M. spicata-01 Wild Hainan 16 M. aquatica-07 Cultivar Shanghai2 M. spicata-02 Cultivar Shanghai 17 M. aquatica-08 Cultivar Shanghai3 M. spicata-03 Cultivar Shanghai 18 M. aquatica-09 Wild Xinjiang4 M. spicata-04 Cultivar Shanghai 19 M. aquatica-10 Wild Henan5 M. spicata-05 Cultivar Shanghai 20 M. aquatica-11 Wild Henan6 M. spicata-06 Wild Yunnan 21 M. ×piperita-01 Cultivar Henan7 M. spicata-07 Cultivar Shanghai 22 M. ×piperita-02 Cultivar Henan8 M. aquatica 01 Wild Henan 23 M. ×piperita-03 Cultivar Shanghai9 M.arvensis-02 Cultivar Shanghai 24 M. longifolia-01 Wild Jiangsu

10 M. aquatica-01 Cultivar Shanghai 25 M. longifolia-02 Wild Xinjiang11 M. aquatica-02 Cultivar Shanghai 26 M.suaveolens-01 Wild Shanghai12 M. aquatica -03 Wild Hunan 27 M.suaveolens-02 Wild Shanghai

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13 M. aquatica 04 Wild Henan

14 M.arvensis-05 Wild Henan15 M. aquatica -06 Cultivar Shanghai

In this study, we examined the genetic relationship among the0 mint accessions collected from China using cpDNA marker,

nvestigated the volatiles chemical diversity in M. spicata acces-ions, and defined the relationship between genetic and chemicalolymorphism in M. spicata accessions. Our study may be useful toint evolution study and future breeding programs.

. Materials and methods

.1. Plant materials and cpDNA amplification and sequencing

A total of 30 min populations were collected from differentain cultivation areas in China (Table 1). Young, emerging leaves

rom 30 plants randomly chosen from each population. DNA wassolated from fresh young leaves using a modified CTAB proto-ol (Doyle and Doyle, 1987). To investigate cpDNA variation weested three noncoding intergenic spacer regions of the chloroplastenome: psbA-trnH, the trnL intron-trnLF intergenic spacer (trnL-). To amplify these regions, we used previously published primerairs (Taberlet et al., 1991) and Polymerase Chain Reaction (PCR)mplification (Gobert et al., 2006). Sequencing of the PCR-amplifiedroducts was carried out in both directions under the sequencingonditions described by Gobert et al. (2006).

.2. Genetic diversity and chemotype analysis

Neighbour-joining tree based on pairwise sequence distancesere performed for cpDNA data with ClustalX ver.1.81 (Thompson

t al., 1997) and Mega ver. 4.0 software (Tamura et al., 2004) to esti-ate polymorphisms. To assess relative branch support from the

ata, phylogenetic trees were evaluated with the bootstrap analysisased on 1000 replicates. The percentage composition of the iso-

ated volatile oils was used to determine the relationship betweenhe different samples by cluster analysis using NTSYS (Rohlf, 2000).he way of data analysis was the same with Tonk et al. (2010).

.3. GC and GC–MS analysis of essential oil

GC analyses of essential oils were performed by an Agilent890. Oil was diluted in ethanol (1:10) and injected in a HP-

nnowax (30 m × 0.25 mm × 0.25 �m) to separate. The carrier gasas nitrogen at flow rate of 0.8 mL/min. Injector and detec-

or (FID) temperatures were 250 and 270 ◦C, respectively. Theolumn temperature was programmed from 60 to 250 ◦C at

◦C/min with the initial and final temperatures held for 5 min.iluted samples of 0.2 �L were injected in the split ratio of 1:50ode. Retention indices (RI) were determined by a series of n-

lkanes (C5–C26). GC/MS analyses were obtained on an Agilent

28 M. canadensis-01 Wild Shanghai29 M. canadensis-02 Cultivar Anhui30 M. ×gracilis-01 Cultivar Henan

Technologies S6890/5973N mass spectrometer using HP-Innowax(60 m × 0.25 mm × 0.25 �m). For GC/MS detection, an electron ion-ization system, with ionization energy of 70 eV, was used. The oventemperature programming was the same as in the GC analysis.

3. Results and discussion

3.1. Molecular evaluation

Neighbour-joining analysis of cpDNA generated two most clades(cluster I and cluster II) (Fig. 1). Cluster I included M. spicata, Menthasuaveolens, Mentha longifolia, M. ×gracilis, Mentha arvensis and Men-tha ×spiperita accessions and was supported by a 100% bootstrapvalue. Cluster II contained Mentha aquatica, and Mentha ×piperitaaccessions. There were three distinct subgroups in cluster I: (1) M.spicata–M. longifolia, (2) M. suaveolens–M. spicata, (3) M. arvensis–M.×gracilis (71% bootstrap value). Within the cluster II, two subgroupsare clearly defined: (1) M. canadensis–M. aquatica accessions, (2) M.×piperita (100% bootstrap value) (Fig. 1).

3.2. Chemical composition of the essential oil

The volatile fraction was isolated from M. spicata, M. longifoliaand M. suaveolens accessions. A total of 78 components were iden-tified in the essential oil of the three accessions, representing over90.2% of the total volatiles. The identified volatile components werelisted in Table 2 with the order of their elution on the innowax col-umn. The main constituents of the oil were limonene (1.4–9.2%),1,8-cineole (tr-28.7%), carvone (tr-74.6%) and piperitenone oxide(tr-58.9%).

Cluster analysis based on the volatiles chemical compositiongrouped these accessions in two main clusters (cluster A andcluster B) (Fig. 2). Cluster A contained M. spicata (M. spicata-01,M. spicata-02 and M. spicata-06) and M. longifolia-01 accessions.Cluster B included M. spicata (M. spicata-03, M. spicata-04, M.spicata-05 and M. spicata-07), M. longifolia-01 and M. suaveolensaccessions. Limonene (1.7–9.2%) and carvone content (45.8–74.6%)in the volatile oil from accessions grouped in cluster A were higherthan those of cluster B (1.4–5.0%) and (tr-0.7%), but cluster B sam-ples showed higher relative amounts of 1,8-cineole (tr-20%) andpiperitenone oxide (14.8–38.5%) than those of cluster A (1.6–8.2%)and (tr-0.2%).

In the present investigation, cpDNA analysis revealed that thegenetic basis of Chinese mint resources is highly polymorphic. This

assessment is fundamental because genetic diversity could be infuture exploited through molecular approaches or plant breedingtechniques to improve mint cultivars for disease resistance or toincrease essential oil yield (Gobert et al., 2006). According to Harly

272 X.H. Chen et al. / Industrial Crops and Products 37 (2012) 270– 274

Table 2Essential oil composition (%) of M. spicata, M.suaveolens and M. longifolia.

Compound RI M. spicata M. suaveolens M. longifolia

01 02 03 04 05 06 07 01 02 01 02

�-Pinene 1022 0.4 0.4 0.5 0.5 tr 0.1 1.4 0.4 0.2 0.6 0.2�-Pinene 1110 0.6 0.6 1.3 1.3 3.5 0.2 3.5 0.7 0.3 1.2 0.3Sabinene 1121 0.3 0.3 0.2 0.2 1.9 0.1 1.7 0.3 0.1 0.8 0.1Myrcene 1165 0.2 0.5 0.2 0.2 7.8 0.1 4.0 0.2 0.1 0.4 –�-Terpinene 1180 – tr 0.2 0.2 – – – tr – – trLimonene 1205 7.2 3.5 1.4 1.4 3.9 1.7 3.0 3.2 5.0 9.2 3.01,8-Cineole 1209 2.6 2.5 20.0 21.9 23.6 1.6 28.7 0.2 0.1 8.2 tr�-Terpinene 1240 – – – – 1.2 tr – 0.1 – – –(Z)-�-Ocimene 1248 tr – 0.2 0.2 0.3 – 0.3 – – – –Octanal 1299 – tr – – 0.2 – 0.1 – – – 0.23-Octyl acetate 1342 – 0.3 – – 0.3 tr 0.3 0.5 0.3 0.1 –1-Octenyl acetate 1385 – – – – 0.2 – 0.2 5.8 0.5 – –Nonanal 1399 0.1 0.4 0.3 0.3 0.6 0.2 0.6 0.1 0.4 0.3 –�-Thujone 1425 – – 0.1 0.1 0.1 – 0.1 – – tr –trans-Linalool oxide (furanoid) 1449 0.2 – 0.3 0.3 – tr – – 0.1 0.1 –1-Octen-3-ol 1457 0.1 – – – 0.1 – 0.1 0.6 2.2 – –Heptanol 1462 0.1 0.1 – – 0.1 0.1 0.1 – – – –�-Cubebene 1467 0.2 0.2 0.7 0.7 0.0 0.3 0.0 0.1 1.2 0.1 0.3trans-Sabinene hydrate 1469 0.1 2.3 0.3 0.3 1.2 0.2 1.6 0.1 0.3 6.4 0.5Menthone 1477 – – – tr 0.1 – 0.1 0.2 0.1 – 0.1�-Ylangene 1493 0.1 0.1 0.2 0.2 0.2 0.1 0.2 0.5 0.3 – 0.2Menthofuran 1496 – tr 0.1 – 0.1 – 0.1 tr – 0.1 –Chrysanthenone 1521 0.6 1.1 0.3 0.3 0.3 0.8 0.3 0.5 1.0 0.8 trLinalool 1553 0.1 0.2 0.5 0.6 0.2 0.3 0.3 0.1 0.2 0.4 0.3cis-Sabinene hydrate 1556 0.1 – – – 0.1 tr 0.1 – 0.6 – –Octanol 1562 – 0.1 – 0.1 – – – 0.1 0.2 – –Linalyl acetate 1569 – – 0.2 0.2 – – – – – 0.1 –Pinocarvone 1584 tr – 0.2 0.2 – – – 0.1 0.2 – 1.6Isomenthyl acetate 1591 – 0.2 – – – tr – 0.6 – – –trans-Isopulegone 1596 – – – – – 0.1 – – 0.6 0.2 –Bornyl acetate 1598 – – 0.3 0.4 1.2 0.2 0.8 0.5 0.3 – –Neomenthol 1604 0.1 0.9 – – tr – – – tr 1.7 1.8Terpinen-4-ol 1608 0.1 0.5 0.4 0.4 0.5 0.1 0.5 0.2 8.0 1.7 0.1�-Caryophyllene 1617 0.6 5.0 0.4 0.4 0.1 0.5 0.1 0.2 0.8 12.5 0.1Aromadendrene 1630 – tr – tr – 0.1 – 0.2 – tr –cis-p-Menth-2-en-1-ol 1635 0.3 0.2 0.9 0.8 0.3 0.6 0.3 – 0.3 0.3 –Myrtenal 1646 0.1 0.3 4.0 2.0 0.1 0.4 0.1 0.1 0.1 0.1 0.1�-Elemene 1653 0.9 0.6 1.8 1.5 0.2 1.0 0.2 0.6 0.8 0.1 3.6Menthol 1656 tr – – tr – tr – – 2.2 0.3 –Pulegone 1663 – – 0.5 0.6 0.1 – 0.1 – 4.0 – 0.1p-Mentha-1,5-dien-8-ol 1672 – 0.6 – tr tr – – 0.5 tr – 0.5cis-p-Mentha-2,8-dien-1-ol 1678 0.9 8.3 3.2 2.1 1.5 – 1.5 0.2 0.4 0.7 0.5Isomenthol 1680 – tr – tr – 1.0 – – 0.6 – –�-Terpineol 1685 – – – 0.2 0.1 0.3 0.1 tr – 0.1 –Neral 1694 – – 0.2 0.1 0.2 0.1 0.2 – 0.2 – –�-Terpineol 1704 0.1 0.4 – 0.6 0.2 – 0.4 0.2 – tr 0.3�-terpinylacetate 1709 0.4 – 0.1 0.1 0.1 1.0 0.1 0.1 1.0 0.9 0.4Borneol 1717 – 0.6 0.2 0.2 0.2 – 0.3 0.3 0.2 0.2 0.2Germacrane D 1729 – tr 0.1 0.1 0.1 tr 0.2 0.1 – tr –Geranial 1739 – 2.8 – 0.2 0.2 – 0.1 0.2 – 1.3 2.9Piperitone 1746 – – 18.5 16.7 1.2 – 1.3 5.9 3.8 – 11.3Carvone 1758 74.6 55.4 0.1 0.2 0.2 70.7 0.2 tr 0.2 45.8 0.7Decanol 1766 0.1 0.1 0.2 0.1 0.2 0.1 0.2 0.3 0.2 – 0.2�-Cadinene 1772 0.1 0.1 – – tr – – – tr 0.2 2.6�-Cadinene 1779 0.3 1.7 – 0.1 – 0.3 – tr 0.1 0.2 –Myrtenol 1800 0.1 – 0.5 0.4 0.2 0.1 0.2 tr – – trNerol 1810 0.2 0.1 0.3 0.3 – 0.6 – tr 0.1 0.1 –trans-Carveol 1842 0.2 0.9 0.2 0.3 0.3 0.3 0.3 0.1 1.8 0.2 0.9Calamenene 1847 0.9 0.1 – 0.2 – 1.7 tr – – 0.2 trCarvone oxide 1853 0.5 – 0.1 0.2 0.2 0.1 0.2 0.1 0.1 – 0.1p-Cymen-8-ol 1860 0.1 – – 0.4 0.1 0.1 0.1 0.5 1.6 0.1 2.8Dodecanol 1874 0.3 0.4 0.5 0.3 tr 0.2 – 0.1 0.5 0.2 –Nonadecane 1899 0.1 – – – – 0.4 – tr 0.5 0.1 tr4-Hydroxypiperitone 1938 0.8 0.1 4.0 0.6 – 1.2 0.1 0.6 0.2 0.1 1.3Piperitone 1949 – – – – – 0.1 – – 3.0 – –Cubebol 1956 0.1 0.5 0.2 0.2 – 0.2 – 5.4 3.0 0.1 –Dodecanol 1975 0.8 1.5 – tr – 1.4 tr tr – 0.3 –Piperitenone oxide 1983 tr tr 27.3 14.8 38.5 0.2 38.5 58.9 45.9 tr 36.72-Phenylethyl 2-methylbutyrate 1989 0.1 0.1 – – – 0.1 – 0.1 tr 0.2 –8,9-Dehydro thymol 1999 0.4 0.4 tr 4.2 – 0.4 tr – tr 0.8 –Isocaryophyllene oxide 2002 0.1 – tr – tr 0.1 tr 1.8 – – 15.7CaryophylleneOxide 2011 0.1 – – 0.5 – 0.2 – 0.4 tr – 0.3Ledol 2056 0.1 – tr – – 0.1 tr 0.1 – – 0.8Humulene 2073 0.4 – – 1.0 – 0.1 – – – – 0.8epi-�-Bisabolol 2087 tr – tr 0.6 – 0.1 tr 0.5 – 0.1 2.3Globulol 2098 – 0.8 – 0.3 – 0.1 – tr – 0.6 –Viridiflorol 2112 0.1 tr tr 0.2 – 0.1 tr – tr – trIdentification (%) 97.0 95.2 91.2 80.5 92.0 90.2 92.9 92.6 93.9 98.2 93.9

RI, retention index relative to C5–C26 n-alkanes on the HP-innowax column; tr, traces (<0.1); * – no detection.

X.H. Chen et al. / Industrial Crops and Products 37 (2012) 270– 274 273

M.canadensis-01 M.spicata-03 M.spicata-04 M.longifolia-02 M.spicata-05 M.spicata-07 M.longifolia-01

subcluster I - 1 (M.spicata - M.longicolia)

M.spicata-02 M.suaveolens-02 M.spicata-06 M.suaveolens-01 M.spicata-01

subcluster I - 2 (M.spicata - M.suaveolens)

M.arvensis-01 M.gracilis-01 M.arvensis-02

subcluster I - 3 (M.gracilis - M.arvensis)

M.piperita-03

cluster I

M.aquatica-08 M.aquatica-05 M.aquatica-11 M.aquatica-03 M.aquatica-06 M.aquatica-04 M.aquatica-01 M.canadensis-02 M.aquatica-02 M.aquatica-10 M.aquatica-07 M.aquatica-09

subcluster II - 1 (M.aquatica)

M.piperita-01ta-02

subcluster II - 2 (M.piperita)

cluster II

63

35

29

41

21

6

13

3

3

55

100

100

100

99

59

81

56

70

69

51

37

24

38

49

15

31

ccessi

aosgset

Fu

M.piperi96

Fig. 1. Hierarchical cluster analysis dendrogram of Mentha a

nd Brighton (1977), M. spicata is the hybrid between M. suave-lens and M. longifolia. Molecular studies showed that seven M.picata accessions were separated into two distinct subgroups androuped within their female parental respectively in cluster I. The

ame result was also gained in chemotype assessment, with anxception. Cluster analysis based on genetic diversity and phy-ochemical traits revealed a clear relationship between them in

ig. 2. Dendrogram obtained by cluster analysis of the percentage composition of volatinweighted pair-group method with arithmetic average (UPGMA).

ons based on cpDNA data using Neighbour-joining method.

M. spicata accessions. It is obvious that the major constituents ofoil followed the maternal inheritance in M. spicata accessions.

The most well-known hybrid, M. ×piperita (peppermint) resultsfrom a cross between M. spicata and M. aquatica. CpDNA showed

that M. ×piperita is closer to M. aquatica than M. spicata. M. ×gracilisis the hybrid between M. spicata and M. arvensis. Based on ourcpDNA data, we first found that two Chinese wild M. arvensis

les from M. spicata, M. suaveolens and M. longifolia based on correlation and using

2 ps and

aMttats

4

pactdcp

R

A

G

D

E

T

F

G

analysis tools. Nucl. Acids Res., 4876–4882.

74 X.H. Chen et al. / Industrial Cro

ccessions (M.arvensis-01 and M.arvensis-02) were close relate to. ×gracilis with a 70% bootstrap value. M. canadensis is thought

o be ancient stabilized allopolyploids, and has been hypothesizedo be a hybrid between M. arvensis and M. longifolia (Tuckernd Chambers, 2002). But our results could not strongly supporthe hypothesis. The M. canadensis accessions in our study wereeparated two clusters.

. Conclusions

Our results indicated that there was great genetic polymor-hism within Chinese mint resources. The present study of cpDNAnalysis of mint accessions from China supports current taxonomiclassification. Further detailed oil compositions give much informa-ion about the potential of different mint resources. Moreover, theiversity in M. spicata accessions correlated well with the chemi-al biodiversity, which will greatly be utilized in future breedingrograms to produce desired genotypes.

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