Genetic and Chemical Diversity in Perovskia Abrotanoides KAR

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Genetic and Chemical Diversity in Perovskia abrotanoides KAR. (Lamiaceae) Populations Based on ISSRs Markers and Essential Oils Proﬁle Seyyed Hossein Pourhosseini,a Javad Hadian,a Ali Sonboli,b Samad Nejad Ebrahimi,c and Mohammad Hossein Mirjalili*a

aMedicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983969411 Tehran, Iran, e-mail: [email protected] bDepartment of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983969411 Tehran, Iran cDepartment of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, 1983969411 Tehran, Iran

Genetic and the essential oil composition variability among twelve Perovskia abrotanoides populations (PAbPs) growing wild in Iran were assessed by ISSR markers, GC-FID and GC/MS, respectively. Nine selected ISSR primers produced 119 discernible bands, of them 96 (80.7%) being polymorphic. Genetic similarity values among populations ranged between 0.07 and 0.79 which indicated a high level of genetic variation. Polymorphic information content, resolving power and marker index generated by ISSR primers were, 0.31, 6.14, and 3.32, respectively. UPGMA grouped PAbPs into four main clusters. Altogether, 38 chemical compounds were identiﬁed in the oils, and a relatively high variation in their contents was found. Camphor (11.9 – 27.5%), 1,8-cineole (11.3 – 21.3%), a-bisabolol (0.0 – 13.1%), a-pinene (5.9 – 10.8%), and d-3-carene (0.1 – 10.5%) were the major compounds. Oxygenated monoterpenes (32.1 – 35.8%) and monoterpene hydrocarbons (25.7 – 30.4%) were the main groups of compounds in the oils studied. Cluster analysis and principal-component analysis were used to characterize the samples according to oil components. Four main chemotypes were found to be Chemotype I (camphor/1,8-cineol), Chemotype II (1,8-cineole/camphor), Chemotype III (camphor/1,8-cineol/a-bisabolol), and Chemotype IV (camphor/d-3-carene/ a-bisabolol). The information, provided here on P. abrotanoides populations, will be useful to introduce this plant into agricultural systems.

Keywords: Perovskia abrotanoides, essential oils, genetic diversity, ISSR, chemotype.

conditions persist in the plant’s habitat, next genera- Introduction tions are chosen to adapt to the new environment Medicinal and aromatic plants (MAPs) changed their and this adaptation gradually becomes inherited and chemical profile in different ecological conditions in can be transmitted to these generations. In the other order to adapt to the environment.[1][2] Therefore, the hand, wild populations of MAPs are heterogeneous in populations of a medicinal species that are growing in morphological and chemical characteristics.[7][8] In this various natural habitats show the variability in the case, if a medicinal species is to be introduced into quantity and quality of active ingredients, which lead agricultural systems because of its economic impor- to differences in their pharmacological and biological tance and in particular, the risk of the occurrence of activity.[3 – 5] The genetic flexibility of plant popula- such populations, the genetic structure as well as tions makes this variation possible and then gradually chemical diversity of its natural populations in order leads to arise some individuals which are different in to provide the raw materials with security, stability chemical and botanical characteristics.[6] When the and proper function should be firstly investigated. plant is exposed to environmental changes, variability Because of the climate diversity, Iran has a vast and occurs in its physiological and chemical behavior to unique biodiversity especially for MAPs. Flora of Iran is adapt to the new environmental conditions. These represented by 7500 plant species, of which 1700 are changes are usually unstable, but if the environmental MAPs.[9] Of course, any efforts to evaluate the

DOI: 10.1002/cbdv.201700508 Chem. Biodiversity 2018, 15, e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508 phytochemical and genetic characteristics of each spe- population genetic,[39][40] and phylogenetic studies.[41] cies can lead to the introduction of susceptible spe- According to the literature information, ISSR markers cies for the production of new drugs. have been efficiently used for the genetic analysis of The Lamiaceae, with 46 genera and ca. 420 species several medicinal plant species.[42 – 44] and subspecies, have a great diversity and distribution Phytochemical and molecular markers have been in the flora of Iran.[10 – 12] Perovskia, a small genus simultaneously used to characterize the level of varia- from this family, is distributed in various regions of tion in several medicinal plant species, such as Witha- Asia, as Iran, Afghanistan, and Pakistan. The genus is nia spp.,[45] Ocimum basilicum,[46] Zataria multiflora,[47] represented in Iran by three species viz. Clitoria ternatea,[48] Salvia sclarea,[35] Ocimum selloi,[32] [49] [37][50] P. abrotanoides KAR., P. atriplicifolia BENTH., and Ocotea spp., and Satureja rechingeri. In the [13] P. artemisoides BOISS. Perovskia species are known to present study, ISSR markers were used for the first contain different class of compounds such as essential time to reveal the extent and distribution of the oils (EOs), phenolics, flavonones, irregular triterpenes, genetic diversity of twelve P. abrotanoides populations steroids and their glycoside and a large amount of (PAbPs) from Iran, as a first step towards gaining a abietane-type norditerpenoidquinones which called better knowledge of the genome diversity of the tanshinones.[14 – 18] P. abrotanoides, with the common plant. We also evaluated the EOs variability of the Persian name of ‘Brazambel’, is an aromatic erect herb wild-growing populations, which is important for phar- which is mainly growing in mountains from Northeast- maceutical and other related industries. ern Iran across Northern Pakistan to Northwestern India.[19] The plant is used by local communities for Results and Discussion treatment of leishmaniasis, typhoid, fever, headache, Habitats Characteristics of Perovskia abrotanoides gonorrhea, vomiting, motion, toothache, atherosclero- sis, cardiovascular diseases, liver fibrosis, and The geographic distribution of the studied cough.[20 – 23] It has sedative, analgesic, antiseptic and P. abrotanoides populations (PAbP1 – PAbP12) and cooling effect.[22][24] The plant herbal tea is used in P. atriplicfolia (PAtP) is presented in Table 1 and Fig- curing infection problems and painful urination.[25] ure 1. They belonged to the different geographical The essential oils of P. abrotanoides play an important zones. PAbP1 to PAbP12 and P. atriplicifolia population role in protection of stored grains and showed to be (PAtP) were located at the northeast and center of Iran effective in washing wounds, anti-ring worm, dermal with an inferior semi-arid climate characterized by a parasites, anti-fungus and anti-hypoxia.[26 – 28] The mean rainfall of 150 – 475 mm/year. The altitudes chemical composition of P. abrotanoides oil has been ranged from 1055 m (PAbP9) to 2220 m (PAbP2) previously investigated,[27 – 30] and 1,8-cineole, d-3- (Table 1). As can be shown in Figure 1, the studied carene, camphor, myrcene, and b-caryophyllene have Perovskia populations have grown within latitude of been reported as the major components of the oils. 33°350Nto37°250N and longitude of 50°590Eto Relationships among genetic and chemical variation 58°170E. have been studied at different levels in plants.[31 – 35] Species diversity in an environment depends on However, it should be noted that use of chemical pro- the ability to produce and sustain ecosystem.[51] file is most useful in taxonomic classification only Reducing biodiversity may be due to the environmen- when other complicating factors, such as environmental effects and reducing the fertility of the plant com- tal conditions, plant developmental stages, and extrac- munity. There are many reports in the literature tion methods are taken into account.[2][34] regarding the variation in the chemical profile of EOs Molecular markers are very useful in an early from various plant species collected from different breeding program for allowing germplasm screening geographical regions.[47][52] Such differences could be at any developmental stage of the plants. Among dif- linked to the varied environmental factors and possi- ferent markers, DNA-fingerprinting techniques are ble adaption response of different populations, result- independent from environmental effects, unlimited in ing in different chemical products being formed, number, and show high level of polymorphism. without morphological differences being observed in Molecular markers provide a powerful tool for proper the plants.[53] Altitude, temperature, annual precipita- characterization of germplasm and chemotypes and tion as well as soil texture has been reported as the their management. Among developed genetic mark- major environmental factors which affected the chem- ers, inter-simple sequence repeat (ISSR) markers have ical composition of the EOs.[54] Therefore, in the case been widely used for plant diversity analyses based of exploitation and introduction of a medicinal species on DNA finger-printing,[36] genetic diversity,[37][38] into the mass cultivation system, the study of its www.cb.wiley.com (2 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508

Table 1. Geographic location and climatic conditions of the studied Perovskia abrotanoides populations (PAbPs) from Iran Population Code Voucher Geographic location Climatic conditions name number Latitude Longitude Altitude Mean Max. mean Min. mean Rainfall (N) (E) [m] annual annual temp. annual [mm/year] temp. [°C] [°C] temp. [°C]

Hanjen PAbP1 2139 33°360 51°430 1537 17.2 20.6 9.2 191.5 32.5 Abyaneh PAbP2 2140 33°350 51°350 2220 13.4 16.6 14.4 210.7 42.5 Ghamsar PAbP3 2141 33°450 51°280 1740 15.3 21.6 11.2 152.3 26.2 Ghohroud PAbP4 2142 34°020 51°240 1810 14.8 21.1 14.9 159.8 34.2 Shahroud PAbP5 2143 36°190 54°480 1349 14.5 22.5 10.0 150.6 62.1 Abr PAbP6 2144 36°300 55°080 1366 14.48 22.48 10.3 164.5 39.2 Khosh-yeylagh1 PAbP7 2145 36°440 55°160 1415 14.45 22.45 10.3 412 91.4 Khosh-yeylagh2 PAbP8 2146 36°500 55°220 1302 12.2 16.9 4.5 457 62.1 Tilabad PAbP9 2147 36°550 55°270 1055 13.7 18.4 6.0 475 51.1 Chamanbid PAbP10 2148 37°250 56°370 1170 11.7 20.7 10.7 270 43.4 Mayami PAbP11 2149 36°240 55°440 1120 16.0 23.5 8.4 163 38.9 Bajestan PAbP12 2150 34°510 58°170 1265 17.5 22.5 6.5 176 26.2 Karaj PAtP 2068 35°480 50°590 1350 15.1 21.4 17.4 251 41.6

Figure 1. Geographical distribution of studied Perovskia abrotanoides populations. habitat characteristics is necessary. These data can be literature,[55][56] these results indicated that the geno- considered for any ecological modeling for the cultiva- mic DNA extracted from the Perovskia leaves pos- tion and sustainable production of a favorable chemo- sesses high quality and high yield. type in agricultural systems. Polymorphisms of Amplified Products Molecular Analysis and DNA Detection Genetic diversity assessment in various MAPs popula- The extracted genomic DNA of the thirteen Perovskia tions is important in their breeding and the conserva- populations was detected by agarose gel elec- tion of genetic resources; it is particularly useful in the trophoresis. The results showed that the spotting characterization of individual populations. The ability holes were clean, the DNA bands were clear and there to reliably distinguish MAPs populations could be were no diffusion phenomena. The extracted genomic invaluable for their diversity studies, and molecular DNA detected by nucleic acid protein detector markers offer an effective approach to manifest revealed that the ratio of D260 nm/D280 nm was genetic diversity based on DNA polymorphisms. ISSR between 1.625 – 1.830, the ratio of D260 nm/ markers have been successfully used for genetic diver- D230 nm was between 1.912 – 2.102, and the yield sity analysis of many other plant species like Salvia [57] [58] was between 85.2 – 112.29 lg/ll. Based on the miltiorrhiza BUNGE, Magnolia officinalis L., Fritillaria www.cb.wiley.com (3 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508 imperialis L.,[59] Satureja mutica,[60] Artemisia dracuncu- was the lowest (0.0714) between ‘Bajestan’ population lus L.,[61] and Satureja khuzistanica.[62] In the present of P. abrotanoides (PAbP12) and ‘Karaj’ population of study, nine highly polymorphic primers (Table 2) were P. atriplicifolia (PAtP), indicating the most remote kin- selected from the thirty ISSR primers to amplify the ship, and the highest (0.7931) between ‘Khosh-yey- thirteen Perovskia populations, and a total of 119 DNA lagh1’ population (PAbP7) and ‘Khosh-yeylagh2’ bands were amplified, 96 of which were polymorphic population(PAbP8), indicating the closest relationship. (the percentage of polymorphic bands (% P) = 80.7%). The thirteen Perovskia populations could be com- These results may be due to differences in the materi- pletely distinguished with the nine ISSR primers. Tak- als that were utilized in each study. The wide geo- ing the average GS (0.2817) as the threshold, the graphical distribution of Perovskia populations used in thirteen tested Perovskia populations could be clus- the present study determined that these have a high tered into four groups (Figure 3,a). Group I included level of variation at the DNA level. four populations: ‘Hanjen’ (PAbP1), ‘Abyaneh’ (PAbP2), The results showed that there was high genetic ‘Ghamsar’ (PAbP3) and ‘Ghohroud’ (PAbP4). Group II difference among Perovskia populations. The numbers comprised six populations: ‘Shahroud’ (PAbP5), ‘Abr’ of DNA bands amplified by these nine ISSR primers (PAbP6), ‘Khosh-yeylagh1’ (PAbP7), ‘Khosh-yeylagh2’ were from 8 to 18 with size of 250 – 3000 bp (Fig- (PAbP8), ‘Til-abad’ (PAbP9) and ‘Chaman-bid’ (PAbP10). ure 2). The average bands per primer was 13.2, while Group III included two populations: ‘Miami’ (PAbP11) ISSR9, ISSR13, ISSR23, and UBC868 were the least 13, and ‘Bajestan’ (PAP12). P. atriplicifolia population (PAtP) ISSR7, ISSR11, ISSR15, ISSR21, and UBC810 were the was clearly separated from other PAbPs. Principal coor- most 13 (Table 2). Values of polymorphic information dinates analysis (PCoA) describing the variability content (PIC) ranged between 0.25 (for ISSR13 primer) among these populations in a two-dimensional mode. and 0.36 (for ISSR15 primer) with an average of 0.31. The plot of the first two coordinates is shown (Fig- Resolving power (Rp) was varied from 4.0 (for ISSR13 ure 4,a). The first two components by PCoA based on primer) to 10.77 (for UBC810 primer) with an average ISSR data, accounted for 51.2% of variation observed of 6.14. Furthermore, marker index (MI) ranged from in the populations (data not shown). Two-dimensional 1.78 (for ISSR13 primer) to 4.43 (for UBC810 primer) plot generated from PCoA also supported the cluster- with an average of 3.32. Result of Rp and MI indicated ing pattern of UPGMA dendrogram and revealed that UBC810 primer contained higher genomic infor- intra-population relationships. The GS ranged from mation than other primers. 0.07 to 0.79, suggesting great genetic variation. The GS ranged from 0.45 to 0.94, indicating considerable distance and diversity in the studied germplasm.[63] Unweighted Pair-Group Method with Arithmetic Mean The GS of ISSRs among some samples of Satureja (UPGMA) Cluster rechingeri varied from 0.57 to 0.99.[37] Also, the GS of The results showed that the genetic similarity (GS)of random amplified polymorphic DNA (RAPD) between the thirteen Perovskia populations varied from 0.0714 local accessions of Satureja hortensis ranged from 0.34 to 0.7931, with an average of 0.2817 (Table 3). The GS to 0.95.[50] Furthermore, GS for several populations of

Table 2. Results of ISSR analysis on studied populations of Perovskia Primer Sequence AT[a] n[b] np[c] %np PIC[d] Rp[e] MI[f]

ISSR7 ACGACGACGACGACGG 52 18 16 88.9 0.26 7.23 4.12 ISSR9 TCGTCGTCGTCGTCGG 52 8 7 87.5 0.33 5.08 2.32 ISSR11 ACACACACACACACACG 52 14 12 85.7 0.31 5.08 3.67 ISSR13 AGAGAGAGAGAGAGAGY[g]T 49 11 7 63.6 0.25 4.00 1.78 ISSR15 ACACACACACACACACYG 52 14 11 78.6 0.36 5.54 3.91 ISSR21 AGAGAGAGAGAGAGAGRC 52 14 11 78.6 0.33 7.08 3.67 ISSR23 CTCCTCCTCCTCRC 49 12 8 66.7 0.31 4.15 2.51 UBC 810 GAGAGAGAGAGAGAGAT 52 16 14 87.5 0.32 10.77 4.43 UBC 868 GAAGAAGAAGAAGAAGAA 52 12 10 83.3 0.35 6.31 3.50 Total 119 96 Mean 13.2 10.7 80.7 0.31 6.14 3.32

[a] Annealing Temperature (°C). [b] Number of Bands. [c] Number of Polymorphic band. [d] Polymorphic Information Content. [e] Resolving Power. [f] Marker Index. [g] Y = C/T. www.cb.wiley.com (4 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508

Figure 2. ISSR proﬁle of thirteen populations of Perovskia with primer resolved on 1.5% agarose gel. M: DNA ladder and 1 to 13 = PAbP1 to PAtP.

Table 3. Genetic similarity coefﬁcients of the twelve Perovskia abrotanoides populations (PAbP)byJaccard coefﬁcient based on ISSR markers

Populations PAbP1 PAbP2 PAbP3 PAbP4 PAbP5 PAbP6 PAbP7 PAbP8 PAbP9 PAbP10 PAbP11 PAbP12 PAtP

PAbP1 1 PAbP2 0.6667 1 PAbP3 0.4500 0.4146 1 PAbP4 0.4359 0.4737 0.7419 1 PAbP5 0.2174 0.1667 0.2273 0.2093 1 PAbP6 0.2000 0.1739 0.1818 0.1628 0.5625 1 PAbP7 0.2174 0.2174 0.2273 0.2093 0.5294 0.3889 1 PAbP8 0.2727 0.2174 0.2558 0.2093 0.4857 0.4286 0.7931 1 PAbP9 0.2083 0.1837 0.1915 0.1489 0.3846 0.4857 0.6364 0.6875 1 PAbP10 0.2083 0.2083 0.1915 0.1489 0.2857 0.2381 0.4595 0.4595 0.4359 1 PAbP11 0.2391 0.2128 0.2500 0.1778 0.2045 0.2439 0.2045 0.2045 0.1957 0.3750 1 PAbP12 0.1875 0.1875 0.2500 0.2045 0.1778 0.1860 0.1522 0.1778 0.1702 0.4103 0.6364 1 PAtP 0.1250 0.1053 0.1091 0.0727 0.2041 0.1875 0.1569 0.1569 0.1509 0.1296 0.1538 0.0714 1

Figure 3. a) Genetic and b) chemical cluster’sofPerovskia populations. For a detailed description of populations PAbP1-PAbP12 and PAtP, cf. Table 1.

Figure 4. a) Two-dimensional plot of the principal coordinate analysis (PCoA) of thirteen populations based on nine ISSR markers along the ﬁrst two principal axes. b) PCA chemical diversity.

– Satureja bachtiarica was 0.08 0.63 using ISSR and Essential Oils Content 0.09 – 0.69 with RAPD.[64] The polymorphism information content (PIC) and The essential oils content of the studied samples was the polymorphism rate (P) were used to measure the ranging from 0.9% to 2.5 (% w/w based on dry genetic diversity in Perovskia populations. High, med- weight). The maximum EOs contents obtained from ium or low polymorphism is in accordance with ‘Ghamsar’ population (PAbP3)(Table 4). In previous PIC > 0.5, 0.5 > PIC > 0.25 and PIC < 0.25, respec- studies, the essential oil content of the plant has been tively.[63] PIC value using ISSR markers ranged from reported to be in ranged of 0.6 – 2.4%.[67][68] The pro- 0.13 to 0.35, Rp value for primers ranged from 2.06 to duction of the EOs in plants is a mechanism of adap- 8.78 with an average of 4.49 and MI ranged from 1.84 tation to environmental factors.[69] The studies on to 5.30 with an average of 3.20 in Satureja mutica.[60] different plant species such as Origanum vulgare,[52] PIC = 0.32, Rp = 7.80, and MI = 3.48 generated by ISSR and Satureja hortensis,[70] revealed that the EOs con- primers were higher than that of sequence-related tent tended to increase under water stress conditions. amplified polymorphism (SRAP) analysis (PIC = 0.30, = = Rp 5.61, and MI 1.88). The effectiveness of ISSR Chemical Diversity of the Essential Oils compared to the SRAP markers for assessing the degree of genetic variation in Kelussia odoratissima has The percentages and the retention indices of the already been confirmed.[65] Also, PIC value using ISSR identified compounds of EOs were listed in the order markers ranged from 0.22 to 0.36 and MI ranged from of their elution on the Rtx-5 column (Table 4). Analysis 1.10 to 2.88 per primer in Satureja rechingeri.[66] of the chemical composition of the EOs of twelve wild www.cb.wiley.com (6 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland w.bwlycm( f14) of (7 www.cb.wiley.com Table 4. Essential oil composition of the twelve Perovskia abrotanoides populations (PAbPs) from Iran Compound name CRI[a] LRI[b] Content [%]

Chemotype I[c] Chemotype II Chemotype III Chemotype IV

PAbP1 PAbP3 PAbP4 PAbP5 PAbP11 PAbP2 PAbP6 PAtP PAbP8 PAbP9 PAbP7 PAbP10 PAbP12

a-Thujene 925 924 0.4 0.4 0.6 0.5 0.4 0.4 0.5 0.3 0.5 0.5 0.4 0.4 0.1 a-Pinene 933 932 9.6 8.5 8.6 8.2 10.8 9.0 9.6 6.3 8.4 7.3 7.3 8.1 5.9 Camphene 948 946 2.7 3.1 3.5 3.2 3.4 1.9 3.3 2.8 3.1 3.4 3.2 2.9 2.0 Sabinene 972 969 0.2 0.2 0.4 0.2 0.3 0.2 0.4 3.8 0.3 0.2 0.3 0.3 0.0 b-Pinene 977 974 3.2 3.1 2.4 2.8 3.3 3.0 3.6 0.3 3.4 3.1 3.1 3.0 1.0 Myrcene 991 988 4.8 5.7 5.8 1.4 1.6 5.5 9.3 0.0 8.8 7.0 2.4 1.7 0.5 d-3-Carene 1009 1008 2.4 1.5 0.1 7.7 2.9 2.0 0.9 7.8 2.8 1.9 7.4 10.5 6.6 a-Terpinene 1012 1014 0.2 0.1 0.2 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.2 0.2 0.2

q-Cymene 1019 1020 1.2 1.1 1.1 1.5 2.5 0.8 1.4 0.0 1.6 1.3 1.6 1.8 1.7 Biodiversity Chem. 1,8-Cineole 1026 1026 17.3 21.0 18.6 18.2 14.0 18.0 16.8 21.3 17.8 17.1 15.1 14.9 11.3 (E)-b-Ocimene 1039 1044 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.3 0.0 0.0 e1700508 c-Terpinene 1046 1054 0.4 0.4 0.6 0.3 0.8 0.3 0.2 3.2 0.3 0.3 0.4 1.2 0.2 cis-Sabinene hydrate 1053 1065 0.2 0.4 0.2 0.4 0.4 0.3 0.3 0.9 0.4 0.2 0.4 0.3 0.2 Terpinolene 1070 1086 0.4 0.2 0.2 0.1 0.3 0.2 0.2 0.3 0.2 0.2 0.1 0.4 0.2

trans-Sabinene hydrate 1080 1098 0.1 0.1 0.5 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.0 0.3 3.2 2018 Camphor 1136 1141 23.4 24.4 27.1 24.7 27.5 15.6 14.9 11.9 18.2 24.0 20.6 16.8 19.9

Borneol 1162 1165 1.5 1.3 0.9 1.1 1.7 1.0 1.4 0.3 1.1 1.2 0.9 1.3 1.7 , 15

Terpinen-4-ol 1176 1174 0.4 1.4 0.7 0.2 2.0 0.6 0.9 1.4 1.0 1.0 0.3 0.5 1.8 e1700508 , a-Terpineol 1193 1186 0.3 0.3 1.4 0.5 0.7 0.4 0.3 0.4 0.2 0.6 0.5 0.5 1.0 Linalool acetate 1249 1254 0.1 0.0 0.2 0.1 0.0 0.1 0.0 0.5 0.1 0.0 0.1 0.2 0.0 Bornyl acetate 1295 1284 4.3 4.9 2.5 7.7 2.3 3.4 4.1 3.8 3.6 2.6 3.4 3.1 4.9 Carvacrol 1310 1298 0.2 0.1 0.1 0.2 0.1 0.3 0.2 2.6 0.1 0.1 0.1 0.1 0.1 © d-Terpinyl acetate 1324 1316 0.3 0.2 0.0 0.5 0.4 0.7 0.1 0.0 0.2 0.3 0.1 0.2 1.2 08WlyVC G uih Switzerland Zurich, AG, Wiley-VHCA 2018 a-Terpinyl acetate 1355 1346 2.8 2.8 2.5 3.9 0.5 4.0 2.9 4.3 2.6 1.3 2.4 2.6 3.4 a-Gurjunene 1413 1409 1.1 1.2 1.7 1.2 0.8 1.3 1.4 0.6 1.1 0.9 1.0 0.7 1.4 (E)-Caryophyllene 1424 1417 4.9 2.9 4.6 3.4 5.0 7.9 3.6 6.4 1.9 1.5 3.7 3.5 5.3 a-Humulene 1557 1452 4.2 5.1 3.6 2.9 4.2 6.0 3.0 6.0 3.0 2.5 3.2 3.5 4.4 c-Muurolene 1478 1478 0.1 0.1 0.1 0.3 0.1 0.0 0.1 0.0 0.3 0.3 0.2 0.2 0.3 b-Bisabolene 1507 1505 0.0 0.1 0.1 0.0 0.1 0.2 0.0 0.0 0.5 0.4 0.2 0.6 0.2 c-Cadinene 1515 1513 1.9 2.0 2.0 2.2 1.2 2.4 2.1 1.4 1.7 1.5 1.6 0.9 3.4 d-Cadinene 1524 1522 0.3 0.3 1.0 0.1 0.1 2.6 1.0 2.2 0.3 0.2 0.2 0.3 0.3 Caryophyllene oxide 1587 1582 0.4 0.5 0.2 0.3 0.3 0.5 0.2 1.3 0.4 0.5 0.5 0.7 1.0 b-Cedrene epoxide 1611 1621 3.8 0.3 0.2 3.2 5.4 1.4 6.8 0.0 1.5 1.3 2.4 2.5 3.5 c-Eudesmol 1635 1630 0.6 0.0 0.0 0.5 0.5 0.2 0.8 0.0 0.2 0.1 0.0 0.3 1.0 epi-a-Cadinol 1647 1638 1.7 1.0 0.6 0.4 0.4 2.6 3.0 0.0 0.7 0.8 0.9 1.8 2.6 a-Cadinol 1658 1652 0.8 0.3 1.0 0.3 0.6 0.4 0.6 4.8 0.5 0.3 0.2 1.1 0.5 a-Bisabolol 1692 1685 0.9 0.7 0.5 0.4 2.9 1.3 0.3 0.0 9.7 13.1 7.2 10.2 5.9 (2Z,6Z)-Farnesol 1700 1698 1.4 3.3 5.4 0.4 0.7 4.5 2.4 5.0 2.4 1.7 3.6 0.8 0.0 Chem. Biodiversity 2018, 15, e1700508

growing PAbPs and one cultivated population of P. atriplicifolia, resulted in the identiﬁcation of 28 – 37

Chemo- constituents. Altogether, representing 96.9% to 100% [c] of the total EOs, were identiﬁed and separated on the basis of their chemical structures into four classes (Table 4). The twelve main components were camphor, 1,8-cineole, a-pinene, a-bisabolol, d-3-carene, . bornyl acetate, b-caryophyllene, myrcene, a-humulene, camphene, (2Z,6Z)-farnesol, and b-pinene (Table 5). The obtained results were similar to the previously cf. Table 1

, published data for the plant from Iran, where d

PAtP 1,8-cineole, -3-carene, camphor, myrcene, and b-caryophyllene were identiﬁed as the major EOs and compounds.[27 – 30] Comparing the results obtained in : Literature retention index values.

LRI this study with the other reported for samples [71] PAbP12 [b]

- collected in Pakistan, a noticeable variation in composition and percentages of components can be obs-

PAbP1 erved. The major components of P. abrotanoides column. essential oil from Pakistan (stem and leaves) were (E)- 9-dodecenal (66.5%), hexadecanoic acid methyl ester DB-5 (27.79%), lupeol (21.5%), octadecenoic acid methyl ester (18.45%), eicosane (6.22%), tetradecane (5.19%), octadecanoic acid methyl ester (8.37%), and 2,2,5,

Chemotype II Chemotype III Chemotype IV 5-tetramethylhexane (3.96%). This chemical variability could be attributed to geographic and climatic varia- -alkanes on the n tion,[72] as well as different phenological stages and

24 [68][71] C plant parts. Monoterpenes and sesquiterpenes –

6 contents were different among populations. Maximum monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpene in EOs, were in PAbP7, PAtP, PAbP12 and PAbP6, respectively. Major oxygenated monoterpene compounds in EOs were camphor, and 1,8- cineole, whereas major monoterpene hydrocarbons compounds were myrcene and d-3-carene. While,

[c] a-humulene and b-caryophyllene were the major sesquiterpene hydrocarbons, b-cedrene epoxide and a-bisabolol were identiﬁed as the main oxygenated sesquiterpenes (Table 4). Literature survey on studies Content [%] Chemotype I PAbP1 PAbP3 PAbP4 PAbP5 PAbP11 PAbP2 PAbP6 PAtP PAbP8 PAbP9 PAbP7 PAbP10 PAbP12 related to the EOs obtained from the plants belonging to Lamiaceae family, showed high variability in their , camphor/1,8-cineol/bornyl acetate; for a detailed description of populations [b] II chemical composition depending on genetic, location, LRI and stages of plant development.[47][73 – 78] [a] -pinene; a CRI Characterization of Chemotypes

)] 2.2As 2.5 shown 1.9 in Figure 2.0 3,b 1.4, cluster 1.8 analysis 1.3 (CA) 1.1allowed 1.7 1.1 1.7 1.3 0.9 w /

w separating the thirteen Perovskia populations into four groups, each representing a chemotype. Chemotype I ed 98.6 99.2 99.1 99.1 98.3 98.9 96.9 100 98.9 98.7 99.4 98.5 96.9 ﬁ (camphor, 1,8-cineole, and a-pinene) was characterized

, camphor/1,8-cineol/ – I by a high content of camphor (23.4 27.5%), medium : Calculated Retention Indices determined in the present work relative to C content of 1,8-cineole (14.0 – 21.0%), and high con- CRI a – Table 4. (cont.) Compound name [a] Number of compoundsEssential oil [% ( 36.0 35.0 35.0 36.0 36.0 35.0 35.0 28.0 37.0 36.0 36.0 37.0 34.0 Monoterpene hydrocarbonsOxygenated monoterpenesSesquiterpene hydrocarbonsOxygenated sesquiterpeneTotal identi 27.4 35.6 13.7 28.4 34.0 21.9 17.0 28.3 34.0 19.8 17.0 27.5 35.8 19.8 13.8 27.3 32.8 22.0 16.4 28.3 21.8 33.9 14.1 27.7 33.2 22.6 13.8 28.6 22.2 42.9 29.4 10.7 32.1 16.0 17.8 27.4 32.9 21.4 16.5 30.4 33.1 21.9 16.6 26.6 34.6 19.3 16.0 25.7 21.3 34.2 17.1 20.0 types: tent of -pinene (8.2 10.8%). Five populations www.cb.wiley.com (8 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508

Table 5. Pearson’s correlation coefﬁcients among the main essential oil constituents (F01 – F12), essential oil content and altitude of the Perovskia sp. populations from Iran

Factors Code F01 F02 F03 F04 F05 F06 F07 F08 F09 F10 F11 F12 F13 F14 a-Pinene F01 ns[a] **ns* nsnsnsnsnsnsnsnsns Camphene F02 0.23 ns ns ns ns ns ns * * ns ns ns ns b-Pinene F03 0.70 0.41 * ns ns ns ns ns ns ns ns ns ns Myrcene F04 0.40 0.28 0.63 ** ns ns ns ns ns ns ns ns ns d-3-Carene F05 0.55 0.18 0.39 0.76 ns ns ns ns ns ns ns ns ns 1,8-Cineole F06 0.11 0.38 0.08 0.25 0.18 ns ns ns ns ns * ns ns Camphor F07 0.38 0.40 0.36 0.02 0.37 0.07 ns ns ns ns ns ns ns Bornyl F08 0.26 0.18 0.13 0.23 0.33 0.17 0.00 ns ns ns ns ns ns acetate (E)- F09 0.07 0.67 0.47 0.42 0.05 0.16 0.25 0.07 ** * ns ns ns Caryophyllene a-Humulene F10 0.04 0.63 0.51 0.34 0.01 0.19 0.29 0.04 0.80 ns ns * ns a-Bisabolol F11 0.30 0.10 0.19 0.10 0.24 0.38 0.05 0.37 0.57 0.52 ns ns ns (2Z,6Z)- F12 0.11 0.05 0.19 0.24 0.31 0.57 0.22 0.35 0.34 0.43 0.35 * ns Farnesol Altitude F13 0.19 0.41 0.05 0.24 0.41 0.25 0.03 0.03 0.55 0.56 0.55 0.65 * Essential F14 0.38 0.14 0.45 0.26 0.31 0.48 0.46 0.32 0.08 0.10 0.44 0.23 0.59 oil [%]

[a] Significance of the correlations is indicated as follows: **significance at the 1% nominal level; *significance at the 5% nominal level. ns, not significant.

(PAbP1, PAbP3, PAbP4, PAbP5, and PAbP11) belonged play an important role in future studies identifying the to this chemotype. Chemotype II (1,8-cineole and cam- genetic structure and its relationship with the essen- phor) included three populations (PAbP2, PAbP6, and tial oil profile. PAtP) and was characterized by a high content of It has been reported that camphor exhibits a number 1,8-cineole (16.8 – 21.3%) and very low content of of biological properties such as insecticidal, antimicro- camphor (11.9 – 15.6%). Chemotype III (camphor, 1,8- bial, antiviral, anticoccidial, anti-nociceptive, anticancer cineole, and a-bisabolol) consisted of two populations and antitussive activities, in addition to its use as a skin (PAbP8 and PAbP9) and was characterized by a med- penetration enhancer.[84] Additionally, the other EOs ium content of camphor (18 – 24%) and 1,8-cineole components such as 1,8 cineole, b-caryophyllene, and (17.1 – 17.8%), and high content of a-bisabolol a-humulene have been demonstrated to possess a (9.7 – 13.1%). Chemotype IV (camphor, 1,8-cineole, and potent anti-inflammatory activity by means of in vitro d-3-carene) was characterized by a low content of experiments and in vivo studies.[85][86] Therefore, the camphor (16.8 – 20.6%) and 1,8-cineole (11.3 – 15.1%) plant populations belong to the characterized chemo- and high content of d-3-carene (6.6 – 10.5%). Three type especially Chemotype I due to their higher content populations (PAbP7, PAbP10, and PAbP12) belonged to of the oil (Table 4) can be considered for further exploita- this chemotype. The influence of environmental fac- tion, domestication and breeding programs. tors on the gene expression and the incidence of a To determine the degree of phytochemical varia- variety of chemical responses is well-known.[79] In the tions, a principal component analysis (PCA) was per- present study, different clustering obtained from formed (Figure 4,b). The first two components of the essential oil data and molecular markers (Figure 3) can PCA accounted 61.7% of the total variation. The first be due to the soil conditions,[80] geographic,[76] and component (PC1) contributed to 48.2% of total vari- climatic[81] changes. Temperature, humidity and ance and included camphene and a-bisabolol. The sec- edaphic are the most important environmental factors ond component (PC2), explaining 13.5% of variance, affecting the EOs profiles.[80 – 83] Changes in tempera- contained a-pinene, b-pinene, myrcene, and camphor. ture, altitude and rainfall of P. abrotanoides habitats (Table 1) can have separate effects on genetic and Correlations Among Traits phytochemical traits. The dementia of all environmental and genotype factors change the essential oil pro- The relationships among the major compounds, alti- file. The implantation of all wild PAbPs in one or more tude and EOs content (Table 4) were examined using regions with the same environmental conditions can Pearson’s correlation coefficients. Significant positive www.cb.wiley.com (9 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508 correlations have been observed between: a-pinene populations into four distinct chemotypes. Cluster and b-pinene, b-pinene and myrcene, 1,8-cineol and analysis and principle component analysis results were (2Z,6Z)-farnesol, b-caryophyllene and a-humulene. Also similar to each other and grouped all populations in Significant positive correlations have been observed four classes. Due to the greater content of the essen- between altitude with a-humulene and (2Z,6Z)-farne- tial oil and biological properties value of the major sol and EOs content with altitude. Significant negative components, Chemotype I populations (PAbP1, PAbP3, correlations have been observed between: d-3-carene PAbP4, PAbP5, and PAbP11) can be considered for and a-pinene, d-3-carene and myrcene, b-caryophyl- in situ and ex situ domestication and cultivation. The lene and a-bisabolol (Table 5). information provided on the geographical characteristics of the habitats of this plant can be also used for ecological modeling for the cultivation of a favorable Conclusions chemical species in agricultural systems. The research on genetic diversity of species is funda- mental for our understanding of the origin and evolu- Experimental Section tion of species, and provides the basis on which we Plant Material can found our efforts at germplasm resources conservation, development and utilization and breeding.[87] A total of twelve natural populations of P. abrotanoides In this investigation, the applicability of ISSR markers KAR.(PAbP1–PAbP12) were collected across five pro- to characterize the thirteen populations of Perovskia vinces from the northeast to center of Iran, including was compared. The comparative results showed that the provinces of North Khorasan, Razavi Khorasan, Perovskia populations had high differences based on Golestan, Semnan, and Isfahan (Table 1). One culti- molecular markers. Nine ISSR primers were used to vated population of P. atriplicifolia (PAtP), as out group amplify all Perovskia populations. The results showed sample, was also collected from botanical garden of that the % P was 80.7%. Moreover, the mean value of Agricultural Faculty (35°480N, 50°590E at an altitude of the PIC obtained in this study was 0.31, indicating that 1238 m), University of Tehran, Karaj, Iran. The aerial the primers could develop medium polymorphism parts of the plants were collected during the flowering which is useful for genetic variation of genotypes stage in June and July 2013 from populations growing studied in this research. The Rp value for primers ran- in the mentioned collection sites (Figure 1), comprising ged from 4.0 to 10.77 with an average of 6.14, which various ecological environments. The samples were was able to differentiate all of the studied popula- identified by Dr. Ali Sonboli, and voucher specimens of tions. Furthermore, MI ranged from 1.78 to 4.43 with each population have been deposited with the Herbar- an average of 3.32. Our results demonstrate high ium of Medicinal Plants and Drug Research Institute levels of polymorphism among PAbPs from Iran. This (MPH), Shahid Beheshti University, Tehran, Iran. is the first attempt to use molecular markers to inves- tigate genetic relationships of P. abrotanoides popula- Molecular Analysis tions and the information generated herein may be useful for the plant breeders to improve of the plant DNA Extraction. Fresh leaves were taken from ten agro-morphological and phytochemical traits. The clas- individual plants of each Perovskia populations and sification of all populations derived from PCoA was were pooled in equal volume, and then 0.2 g was similar to the result of the UPGMA analysis. Groups 1- weighed out for DNA extraction. Genomic DNA was 2-3-4 of the plot by PCoA exactly corresponded to extracted from fresh leaves of Perovskia using a Clusters I-II-III-IV in the dendrogram by the UPGMA modified CTAB (cetyltrimethyl ammonium bromide) analysis. Totally, thirty-seven compounds were identi- protocol.[88] Agarose gel (0.8%) electrophoresis and fied in EOs of Perovskia populations. The main compo- the biospec-mini DNA/RNA/protein analyzer were nents were camphor (11.9 – 27.5%), 1,8-cineole applied to detect the quality and yield of DNA, which (11.3 – 21.3%), a-bisabolol (0.0 – 13.1%), a-pinene later was diluted to 100 ng/ll working solution and (5.9 – 10.8%), and d-3-carene (0.1 – 10.5%). Significant placed in a refrigerator at 4 °C for future use. positive and negative correlations have been observed between EOs compounds. EOs content increases with Primer Selection and ISSR-PCR Amplification. After increasing altitude. Also significant positive correla- prescreening of 30 ISSR primers, a total of nine primers tions have been observed between altitude with were selected. This selection was based on high a-humulene and (2Z,6Z)-farnesol, and EOs content polymorphisms and good reproducibility of the with altitude. Cluster analysis allowed grouping the fragments generated. The PCR procedure was carried www.cb.wiley.com (10 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508 out in the 20 ll mixture containing 10 ng/llof MS analyses as well as identification and quantifica- template DNA, 1 ll of primer (Padideh Ressalat, Armin tion of the oils components were carried out as Shegarf Co., Ltd., Tehran, Iran), 0.53 ll each dNTP, described previously.[93] 1.80 ll of MgCl2, and 0.30 U of Taq DNA polymerase in a2llof109 buffer. Amplification was performed on a Statistical Analysis BioRAD Thermal Cycler (USA). The following thermal cycling protocol was used: an initial denaturation at To classify and group the twelve populations of 94 °C for 5 min, followed by 35 cycles of 45 s P. abrotanoides based on their EOs components and denaturation at 94 °C, 1 min annealing (at the to identify the chemotypes, the oil composition data temperature shown in Table 2), and extension of 1 min matrix of the thirteen populations was analyzed using at 72 °C, and then a final 8 min extension at 72 °C, cluster analysis (CA) with SPSS version 16 (SPSS Inc., after which the samples were held at 4 °C. ISSR-PCR Chicago, USA).[94,95] Relationships among populations amplified products were analyzed by electrophoresis in were investigated by principal-component analysis 1.5% agarose gel (Ultrapure, Invitrogen) and 19 TAE (PCA). PCA was performed using SPSS statistics soft- buffer (Tris acetate) at 80 V/cm for 150 min, and then ware. Mean values were used to create a correlation observed and photographed by UV and visible analysis matrix from which standardized PC scores were devices. This was repeated once for each primer. The extracted. 100 – 3000 kb DNA ladder (Fermentas) as size marker to characterize the size of ISSR bands. Acknowledgements The authors thank the Shahid Beheshti University ISSR Data Scoring and Analysis Research Council for financial support of this project. When computing the polymorphic loci of DNA bands We also thank Mr. Ali Karimi and Mrs. Maryam Moham- on the ISSR-PCR amplified products, those which had madian for their kind collaboration in genetic diversity a band on the same migration position were scored experiment and GC/MS analyses, respectively. This as present (1) and those without were scored as work is part of S. H. Pourhosseini M.Sc. thesis. absent (0). In this way, a 1/0 binary matrix was gener- fi ated. Genetic similarity (GS) coef cient based on the Author Contribution Statement Jaccard coefficient was calculated by making a pair- wise comparison between all populations using the S. H. P. made contribution to conception of the study, Simqual Module of NTSYS-pc software version collection of plant materials, essential oil isolation and 2.02e.[89] The similarity coefficients obtained were analysis, interpretation of data and writing of the used to construct a dendrogram using UPGMA manuscript. J. H. contributed to the experimental work employing the SAHN algorithm in the same software especially to the genetic diversity. A. S. helped in the package. Polymorphic information content (PIC) and botanical identification and the localization of the MI were calculated according to a previously plant material as well as interpretation of data and described formula.[90] Ability of the primers to differ- made chemotype analysis. S. N. E. was involved in the entiate among the populations was assessed by calcu- GC-FID and GC/MS analysis and helped in the correc- lating their Rp.[91] Principal coordinate analysis (PCoA) tion of the manuscript. M. H. M. supervised the experi- was performed based on the variance covariance ments and wrote the manuscript. All authors read and matrix calculated from the ISSR marker data to detect approved the final manuscript. relationships between populations using NTSYS-pc software version 2.02e. Conflict of Interest The authors declare no conflict of interest. EOs Isolation and Analysis Dried aerial parts (20 g) of the pooled plant materials References of each population were ground and subjected to hydrodistillation for 3 h, using a Clevenger-type appa- [1] W. J. M. Lommen, H. J. Bouwmeester, E. Schenk, F. W. A. Verstappen, S. Elzinga, P. C. Struik, ‘Modelling Processes ratus according to the method recommended in Bri- [92] Determining and Limiting the Production of Secondary tish Pharmacopoeia. The isolated oils were dried Metabolites During Crop Growth: The Example of the Anti- over anhydrous sodium sulfate and stored in tightly malarial Artemisinin Produced in Artemisia annua’, Acta closed dark vials at 4 °C until analysis. GC-FID and GC/ Hortic. 2008, 765,87– 94. www.cb.wiley.com (11 of 14) e1700508 © 2018 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2018, 15, e1700508

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