Research Article Endangered Uyghur Medicinal Plant Ferula ...

Research ArticleEndangered Uyghur Medicinal Plant Ferula Identificationthrough the Second Internal Transcribed Spacer

Congzhao Fan1 Xiaojin Li1 Jun Zhu1 Jingyuan Song23 and Hui Yao2

1 Xinjiang Institute of Chinese Materia Medica and Ethnical Materia 9 Xinming Road Urumqi Xinjiang 830002 China2The National Engineering Laboratory for Breeding of Endangered Medicinal Materials Institute of Medicinal Plant DevelopmentChinese Academy of Medical Sciences and Peking Union Medical College Beijing 100193 China

3 Chongqing Institute of Medicinal Plant Cultivation Chongqing 408335 China

Correspondence should be addressed to Xiaojin Li xjlxj126com

Received 10 June 2014 Revised 6 October 2014 Accepted 6 October 2014

Academic Editor Robert Henry

Copyright copy 2015 Congzhao Fan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The medicinal plant Ferula has been widely used in Asian medicine especially in Uyghur medicine in Xinjiang China Given thatvarious substitutes and closely related species have similar morphological characteristics Ferula is difficult to distinguish basedon morphology alone thereby causing confusion and threatening the safe use of Ferula In this study internal transcribed spacer2 (ITS2) sequences were analyzed and assessed for the accurate identification of two salable Ferula species (Ferula sinkiangensisand Ferula fukangensis) and eight substitutes or closely related species Results showed that the sequence length of ITS2 rangedfrom 451 bp to 45 bp whereas guanine and cytosine contents (GC) were from 536 to 562 A total of 77 variation sites weredetected including 63 base mutations and 14 insertiondeletion mutations The ITS2 sequence correctly identified 100 of thesamples at the species level using the basic local alignment search tool 1 and nearest-distance method Furthermore neighbor-joining tree successfully identified the genuine plants F sinkiangensis and F fukangensis from their succedaneum and closely relatedspeciesThese results indicated that ITS2 sequence could be used as a valuable barcode to distinguish Uyghurmedicine Ferula fromcounterfeits and closely related species This study may broaden DNA barcoding application in the Uyghur medicinal plant field

1 Introduction

The endangered Uyghur medicinal herb Ferula has a longhistory of application in China It was first utilized in theTang Materia Medica during the Tang Dynasty (AD 659)because of its effectiveness in removing food residues as wellas its dispersed stuffiness and chlordime form [1] To dateFerula is traditionally employed for treating different diseasessuch as asthma epilepsy stomach ache flatulence intestinalparasites weak digestion and influenza in many countries[2 3] Pharmacological and biological studies have shownthat Ferula has antioxidant [4] antiviral [5] antifungal [6]cancer chemopreventive [7] antidiabetic [8] antispasmodic[6] hypotensive [9] and molluscicidal effects [10] Ferulais now included in the Uyghur Medicine Criteria [11] andcalendar version of the Chinese Pharmacopoeia [12] Theoriginal plants for this medicine are Ferula sinkiangensis andFerula fukangensis In addition to a close relationship with

traditional Chinesemedicine Uyghurmedicine has absorbedthe essences of Indian Arabian Iran and ancient Greekmedicine thereby forming a complete system of medicinewith distinctive ethnic characteristics However itsmedicinalparts and functions are not precisely the same For instancetraditional Chinese medicine utilizes Ferula resin whereasUyghur medicineutilizes Ferula resin roots and seeds [13] asclinical parts

In China the Ferula genus is mostly distributed in Xin-jiang but has been destroyed in this area as a result of exces-sive collection In addition to seed plant breeding changein reclamation irrigation road building and deteriorationof the original habitat contribute to the annual shrinking ofFerula resources Currently F fukangensis is on the verge ofextinction andF sinkiangensis is listed as a class 3 endangeredplant and class 2 protected wild medicinal species Givenits high cost and scarce resources various substitutes andadulterants have emerged F sinkiangensis and F fukangensis

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 479879 6 pageshttpdxdoiorg1011552015479879

2 Evidence-Based Complementary and Alternative Medicine

have beenmisused and substituted by other species such as FferulaeoidesKorov [14] Furthermore numerous species fromthe Ferula genus have been utilized as traditional Chinesemedicines and ethical materials For example F soongaricahas been utilized byKazakhs in treating headaches colds andstomach aches F caspica in treating nervous breakdown andF lehmannii in treating parasitic malnutrition and cold painin the heart and abdomen [15] Whenmixed with substitutesadulterants and closely related species the originalmedicinalplant could not be traced to the medicinal herb market Afamous line that is widely circulated in the herbal medicinefield is ldquothere is no false Scutellaria no true Ferulardquo Thusinvestigating the authenticity of the Uyghur medicinal herbFerula is urgently required

The Ferula genus is usually recognized as monophyleticthe delimitation of species requires examination of completespecimens with roots stem bases basal leaves inflorescenceflowers and ripe fruits based on the observations of livingplants [16] Given that members are similar in habit andmorphology flowers inflorescences and fruit anatomy areoften hardly discernible taxa [17 18] Thus the infragenericclassification method of the Uyghur medicinal herb Ferulaurgently requires a solution To address this problem micro-scopic identification and powder identification techniqueshave been utilized for F sinkiangensis and its closely relatedspecies [19ndash21] However these techniques could not solvethe problem of authenticating the Uyghur medicinal herbs Fsinkiangensis andF fukangensis fromother traditionalmedic-inal herbs and closely related Ferula species By comparisonDNA barcoding employs a short DNA sequence from astandard locus as a species identification tool [22] whichhas been widely applied in forage identification [23] andanimal genetic relationship identification [24] The internaltranscribed spacer (ITS) region of this barcode which com-prises ITS1 58S and ITS2 has been widely utilized as a coreDNA barcode to identify different herbal medicinal materials[25] Researchers have proposed ITS2 as a standard DNAbarcode formedicinal plant authentication [26 27] Howevermolecular identification of F sinkiangensis and F fukangensisfrom their closely related species utilizing the ITS2 sequencehas not been reported In the present study DNA barcodingtechnology was applied for the first time to distinguish Fsinkiangensis and F fukangensis from their succedaneumand other closely related species The application of DNAbarcoding in trade andmarket management could ensure thesafe use of the pharmacopoeia herb Ferula

2 Samples and Methods

21 Materials and Methods for Sampling of Plant MaterialsSeventy-three leaves and root samples which represented10 Ferula species were collected from different locationsNine Ferula species were acquired from Xinjiang and onespecies of Ferula litwinowiana was obtained from Kaza-khstan The 10 species were given specimen collection num-bers (Table S1) (see Supplementary Material available onlineat httpdxdoiorg1011552015479879) and authenticatedby Researcher Guanmian Shen (Xinjiang Institute of EcologyandGeography Chinese Academy of Sciences) and Associate

Fellow Guoping Wang (Xinjiang Institute of Chinese Tradi-tional Medical and Ethical Materia Medica) All correspond-ing voucher samples were deposited in the herbarium of theXinjiang Institute of Chinese Traditional Medical and EthicalMateria Medica In addition one published sequence wasdownloaded from GenBank for analysis (Table S1)

22 DNA Extraction Amplification and Sequencing Ge-nomic DNA was isolated from 30mg of leaves or rootsaccording to the protocol of the Plant Genomic DNA Kit(Tiangen Biotech Co China) Specific regions of the ITS2sequences were amplified from dried leaves and root extractsuniversal primer and PCR conditions for ITS2 were obtainedfrom previous studies [28] The purified PCR products weresequenced using a 3730XL sequencer (Applied BiosystemsFoster City CA USA)

23 Sequence Alignment and Analysis The CodonCodeAligner v 404 was utilized to edit the sequences andassemble the contigs and shear sequence using universalprimers (51015840-31015840 ATTCACACCAAGTATCGCAT and 31015840-51015840ATTGTAGTCTGGAGAAGCGTC) The ITS2 core regioncould be obtained using the HMMer annotation methodbased on the hidden Markov model (HMM) to remove the58S and 28S sections at both ends of the sequences [29]The interspecificintraspecific variation of the samples wascalculated according to Kress et al [22] A phylogenetictree was constructed utilizing MEGA 50 Neighbor-joiningalgorithm (NJ tree) was used to evaluate the capability of theITS2 sequence to authenticate the studied species Bootstraptests were conducted utilizing 1000 resampling to assess theconfidence of the phylogenetic relationships using employedMEGA 50 [30 31] BLAST1 and nearest-distance methodswere performed to identify species as previously described[32 33]

3 Results

31 Universality and Sequence Characteristics DNAwas suc-cessfully extracted from 73 samples Gel electrophoretic anal-ysis revealed that all sample extractions emitted bright bandsITS2 sequences were successfully amplified and sequencedhigh-quality bidirectional sequences were obtainedThe ITS2sequence including partial the 58S ITS2 region and partial28S ranged from 451 bp to 455 bp and GC contents were536 to 562 The partial 58S length was 84 bp the GCcontents ranged from 571 to 583 The length of the ITS2region was 226 bp to 230 bp the GC contents ranged from530 to 568 The partial 28S was 141 bp and the GCcontents ranged from 518 to 539

32 IntraspecificInterspecific Variations Sequence lengthvariable sites and average interspecific K2P distance of ITS2regions were analyzed and summarized (Table 1) Most ofthe 10 Ferula species in this study were represented byseveralsamples because they were collected from differentlocalities F sinkiangensis was represented by 13 samples(Table S1) F fukanensis by 6 samples F ferulaeoides by 14

Evidence-Based Complementary and Alternative Medicine 3

11111111111111111111111111111111122222222222222222222222222222333334444444

59900000001111112222233466677788899902233444555555666667777889999000192444445

43701246890135670378939435726835612864525689012457127890145890579234572127895

Ferula fukanensis KF793024 TGCCTGCAACATCTCTGATCCTGTGTTCCCAGACTTGAGTTTTATGC--GCTTCA-GCACGCAAGTGATCTAGTACT

Ferula lehmannii KF793028 C---

Ferula caspica KF793015 TTTCT--C-

Ferula ferulaeoides KF793002 CTT---C-

Ferula soongarica KF792996 CTT---C-T

Ferula dubjanskyi KJ804107 C-TTATTA--ACAAC

Ferula syreitschikowi KJ804110 CAATT--C-ATC

Ferula sinkiangensis KF792984 CTCAA--C-----

Ferula akitschkensis KJ804122 CCACAACCTCAACTGATGAAAACTGCT-CC-CCCGAATGTTCAG-GCTAGCCC---CCGTCTC

Ferula litwinowiana KF793035 TCC---

Figure 1 Variable sites in the haplotypes of the 10 Ferula species

samples F soongarica by 6 samples F caspica by 11 samplesand so on (Table 1) The results showed that each Ferulaspecies only possesses a haplotype and their intraspecificdistances were all zero

Among the 74 ITS2 sequences a total of 77 variation siteswere detected in the region including 63 base mutations and14 insertiondeletion mutations The following contents weredetected among the 63 base mutation sites CT (26) CG(5) AC (12) GA (10) TA (5) TG (4) CGA (1) andCTA (4) substitutions A total of 76 distinctive variable siteswere discovered among the 77 variation sites which couldprecisely authenticate different Ferula species (Figure 1) Forinstance the CndashT transition at site 133 and the AndashG transi-tion at site 206 could distinguish F sinkiangensis from theothernine Ferula species The CndashT transition at the 397 bpsite could distinguish F soongarica and the CndashT transition atsite 176 could individually classify F caspicaThe interspecificdivergence calculated utilizing the K2P model indicated thatthe ITS2 sequences of F sinkiangensis possessed an averageinterspecific distance of 0030 F fukanensis possessed anaverage intraspecific distance of 0027 (Table 1)

33 Species Identification Capability of ITS2 Barcode andNJ Tree Analysis BLAST1 nearest distance and tree-basedmethods were utilized to determine whether the certifiedmedicinal herb Ferula could be identified by ITS2 sequencesThe results indicated that ITS2 correctly identified 100 ofthe samples at the species level through the BLAST1 methodThe nearest genetic distance method also resulted in 100success rate of identification

Up to 74 ITS2 sequences of Ferula species were chosento build the trees NJ trees (Figure 2) were constructed basedon the ITS2 sequences The results showed that the Uyghurmedicinal herbs F fukangensis and F sinkiangensis presentedgood monophyly they could be correctly distinguished fromtheir succedaneum F ferulaeoides and other closely relatedspecies Furthermore the remaining Ferula species could

be distinguished from one another Thus the ITS2 barcodecould correctly identify two Uyghur medicinal herb plantsfrom their adulterants and closely related species

4 Discussion

41 ITS2 Region Is a Useful DNA Barcode to Differentiate 10Ferula Species The genus Ferula is an important ephemeralplant that possesses highly ecological and medicinal val-ues Our results emphasized the advantages of using theITS2 region as a DNA barcode including high interspecificdivergence but small intraspecific variation favorable uni-versality and short length which lead to easy amplificationand sequencing efficiency Given that pharmacologists oftentraded Ferula genus plants internationally for their medicinalvalues our research provides a convenient tool for validatingthe quality products of Ferula In a previous study [18] 83accessions (74 species) of Ferula were used to ascertain thephylogenetic position of the genus within the family Mostspecies had one sample hence the intraspecific divergencecould not be determined The ITS1 and ITS2 regions areseparately amplified and then assembled to successfullyredress several misplaced Ferula genus plants Phylogeneticanalyses of tree-based method successfully corrected thethree accessions of misplaced Ferula species In our study 74ITS2 sequences of 10 Ferula species were used to authenticateF sinkiangensis and F ferulaeoides from their succedaneumand closely related species most of which were newlyreported sequences and each species was represented by twoormore samplesTherefore intraspecific variationwas clearlydisplayed

Morphological characteristics are an important basis foridentifying the original Ferula plants However morphologi-cal identification often relies on abundant experience whichcould be easily affected by biocoenosis and geographical envi-ronment [17] As a result identification of traditional Chinesemedicine Ferula has been limited Genomic sequence is not


Feru

la so

onga

rica

KF7

9299

7Fe

rula

soon

garic

a K

F792

998

Feru

la so

onga

rica

KF79

2999

Feru

la so

onga

rica

KF79

3001

Feru

la so

onga

rica K

F792

996

Feru

la so

onga

rica K

F793

000

63

Feru

la fer

ulae

oides

KF79

3007

Feru

la fer

ulaeo

ides K

F793

006

Feru

la fer

ulaeo

ides K

F793

004

Ferula

ferulae

oides

KF7930

08

Ferula fer

ulaeoides K

F793005

Ferula fer

ulaeoides KF793010

Ferula fer

ulaeoides KF793009

Ferula ferulaeoides KJ804106

Ferula ferulaeoides KF793002






68

Ferula sinkiangensis KF792985Ferula sinkiangensis KF792986Ferula sinkiangensis KF792987

Ferula sinkiangensis KF792990









Ferula sinkiangensis KJ804121

95

Ferula caspica KF793015

Ferula caspica KJ804105






Ferula caspica KF793018Ferula caspica KF793036


96

Feru

la sy

reits

chik

owi K

J804

116

Feru

la syr

eitsch

ikowi

KJ8

0411

8

Feru

la syr

eitsch

ikowi K

J8041

10

Feru

la syr

eitsch

ikowi K

J8041

17

Ferula

syreit

schiko

wi KJ80

4111

Ferula syr

eitschikowi K

J804114

Ferula syreit

schikowi K

J804112

Ferula syreit

schikowi KJ804119

Ferula syreitschikowi KJ804113


96Ferula lehmannii KF793028Ferula lehmannii KF793029Ferula lehmannii KF793031Ferula lehmannii KF793032

Ferula lehmannii KF793030

64

Ferula fukanensis KJ804103

Ferula fukanensis KF793026





Ferula litwinowiana KF793035

Ferula litwinowiana DQ379423

96

51

9264

Ferula dubjanskyi KJ804108



99

Ferula akitschkensis KJ804122




Figure 2 NJ tree of F sinkiangensis and F fukangensis and their closely related species constructed with the ITS2 sequence The bootstrapscores (1000 replicates) are shown (ge50) for each branch

Table 1 ITS2 sequence characteristics and K2P distances of Ferula used in this research

Species Sample number GC content () Sequence length (bp) Distinctive variable site Interspecific K2P distance (mean)F sinkiangensis 13 550 451 7 0009ndash0163 (0030)F fukanensis 6 547 455 0 0002ndash0161 (0027)F ferulaeoides 14 546 454 0 0002ndash0150 (0023)F soongarica 6 544 454 1 0002ndash0154 (0025)F caspica 11 543 455 3 0009ndash0161 (0030)F lehmannii 5 549 455 0 0002ndash0157 (0028)F syreitschikowi 10 543 455 3 0007ndash0145 (0028)F akitschkensis 4 562 452 53 0145ndash0170 (0156)F dubjanskyi 3 536 455 5 0016ndash0151 (0035)F litwinowiana 2 549 455 4 0007ndash0170 (0033)


influenced by individual characteristics and developmentalstages therefore DNA barcode technology has been widelyused in recent years [34] DNA barcoding is an effectivesupplement to traditional morphological methods and inour study the phylogenetic tree (Figure 2) demonstrated thatthe ITS2 region has an effective role in authenticating theUyghur medicinal herb Ferula and differentiating 10 Ferulaspecies from each other

42 Identification of UyghurMedicine Utilizing ITS2 SequenceAccording to the records Uyghur medicine includes 1000types of herbal medicines and up to 450 types are fre-quently utilized Nevertheless limited by rare resources andconfusing plant origins only about 200 species of Uyghurmedicinal herbs have been formulated based on national andlocal criteria Aside from the Uyghur medicinal herb Ferulanumerous other Uyghur medicinal herbs such as Cichoriumintybus L and Cichorium glandulosum Boiss et Huet arefacing the same dilemma [14]These situations are significantobstacles to the standardization and industrialization ofUyghurmedicine Althoughmorphological examination andchemical analysis are routine practices in identifying Uyghurmedicinal herbs these methods are influenced by biologicaland physical factors [35] DNA barcoding and forensicallyinformative nucleotide sequencing are less affected by theseelementsThe ITS2 barcode has been proposed as a universalDNA barcode for medicinal herb and animal identification[26 27 36 37] Thus based on this study DNA barcodingwould have a broad prospect in authenticating more Uyghurmedicinal herbs which provides a new method for the Foodand Drug Administration to control Uyghur medicine andensure its safe and efficient use and has a considerable role indeveloping the Uyghur medicine industry

43 Significance of DNA Barcode in Food Safety ControlAlthough several Ferula genus plant roots are used traditionalmedicine or folk medicine in Xinjiang few Ferula speciesare edible Starch extracted from F ferulaeoides and Fdubjanskyi could be made into food [2] The tender leavesof F lehmannii could be used as wild herbs to make colddishes and dumplings [38] DNAbarcoding has been used forfood safety control [39ndash41]TheNJ tree demonstrated that theITS2 barcode could accurately perform food safety controlSequencing the ITS2 DNA barcode region represents a newtechnique that guarantees food and drug safety in Ferulaspecies This technology is easy to learn and utilized by foodmanagers [42]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor S L Chen from the Institute ofMedicinal Plant Development Chinese Academy of Medic-inal Sciences for his valuable comments This study was

supported by the National Natural Science Foundation ofChina (Grant nos 81060265 and 81373922) and the NaturalScience Foundation of the Xinjiang Uygur AutonomousRegion (Grant no 2011211B44)

References

[1] E P Korovin ldquoFerula Lrdquo in Flora Uzbekistana vol 4 pp 399ndash438 1959

[2] M Iranshahy and M Iranshahi ldquoTraditional uses phytochem-istry and pharmacology of asafoetida (Ferula assa-foetida oleo-gum-resin)mdasha reviewrdquo Journal of Ethnopharmacology vol 134no 1 pp 1ndash10 2011

[3] P Mahendra and S Bisht ldquoFerula asafoetida traditional usesand pharmacological activityrdquo Pharmacognosy Reviews vol 6no 12 pp 141ndash146 2012

[4] S Ben SalemA Jabrane FHarzallah-Skhiri andH Ben JannetldquoNew bioactive dihydrofuranocoumarins from the roots of theTunisian Ferula lutea (Poir) Mairerdquo Bioorganic and MedicinalChemistry Letters vol 23 no 14 pp 4248ndash4252 2013

[5] Z E Nazari and M Iranshahi ldquoBiologically active sesquiter-pene coumarins from Ferula speciesrdquo Phytotherapy Researchvol 25 no 3 pp 315ndash323 2011

[6] G Kavoosi A Tafsiry A A Ebdam and V Rowshan ldquoEval-uation of antioxidant and antimicrobial activities of essentialoils from Carum copticum Seed and Ferula assafoetida latexrdquoJournal of Food Science vol 78 no 2 pp T356ndashT361 2013

[7] M Iranshahi A Sahebkar S T Hosseini M Takasaki TKonoshima and H Tokuda ldquoCancer chemopreventive activityof diversin from Ferula diversivittata in vitro and in vivordquoPhytomedicine vol 17 no 3-4 pp 269ndash273 2010

[8] A S Abu-Zaiton ldquoAnti-diabetic activity of Ferula assafoetidaextract in normal and alloxan-induced diabetic ratsrdquo PakistanJournal of Biological Sciences vol 13 no 2 pp 97ndash100 2010

[9] M Fatehi F Farifteh and Z Fatehi-Hassanabad ldquoAntispas-modic and hypotensive effects ofFerula asafoetida gumextractrdquoJournal of Ethnopharmacology vol 91 no 2-3 pp 321ndash3242004

[10] S Kumar D K Singh andV K Singh ldquoTertiary combination offreeze-dried urine of Indian breeds of cow with plant productsagainst snail Lymnaea acuminatardquo Pakistan Journal of BiologicalSciences vol 15 no 20 pp 992ndash996 2012

[11] Xinjiang Food and Drug Administration The Xinjiang UygurAutonomous Region Traditional Chinese Medicine and UygurMedicine Pieces Concoct Standard Xinjiang Peoplersquos SaitaryPress 2010

[12] Chinese Pharmacopoeia CommissionChinese Pharmacopoeiavol 1 Chinese Medicine Science and Technology Press 2010

[13] Chinese Materia Medica Editorial Committee The ChineseMateria Medica (Uyghur Volume) Shanghai Science and Tech-nology Press 2005

[14] C Liu W J Yang Z Y Gu et al ldquoResearch progress of Uygurmedicine easy mixed species arrangement and the applicationofmolecular biologyrdquoNorthwest Pharmaceutical Journa vol 28no 4 pp 440ndash442 2013

[15] D Bandyopadhyay B Basak A Chatterjee et al ldquoSaradaferina new sesquiterpenoid coumarin from Ferula assafoetidardquoNatural Product Research vol 20 no 10 pp 961ndash965 2006

[16] E P KorovinGeneris Ferula (Tourn) LMonographia illustrataAcademiae Scientiarum UzRSS Taschkent Uzbekistan 1947


[17] Y Ajani A Ajani J M Cordes M FWatson and S R DownieldquoPhylogenetic analysis of nrDNA ITS sequences reveals rela-tionships within five groups of Iranian Apiaceae subfamilyApioideaerdquo Taxon vol 57 no 2 pp 383ndash401 2008

[18] R Kurzyna-Młynik A A Oskolski S R Downie R KopaczA Wojewodzka and K Spalik ldquoPhylogenetic position of thegenusFerula (Apiaceae) and its placement in tribe Scandiceae asinferred fromnrDNAITS sequence variationrdquoPlant Systematicsand Evolution vol 274 no 1-2 pp 47ndash66 2008

[19] Y Tan T-T Gao Y Ma X Ding and Y-H Cheng ldquoPhar-macognostic identification of Ferula syreitschikowiirdquo Journal ofChinese Medicinal Materials vol 34 no 11 pp 1694ndash1696 2011

[20] H Y Han J M Xu G Y Li et al ldquoMicroscopic identification ofFerula dissecta (Ledeb) Ledebrdquo Modern Chinese Medicine vol12 no 12 pp 17ndash19 2010

[21] X Ding Y Tan Y Zhang et al ldquoPharmacognostic identificationofFerula sinkiangensisrdquoChina Pharmacy vol 35 pp 3307ndash33092011

[22] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005

[23] I Ganopoulos P Madesis and A Tsaftaris ldquoUniversal ITS2barcoding DNA region coupled with High-Resolution Melting(HRM) analysis for seed authentication and adulteration testingin Leguminous forage and pasture speciesrdquo Plant MolecularBiology Reporter vol 30 no 6 pp 1322ndash1328 2012

[24] R Zomuanpuii L Ringngheti S Brindha G Gurusubra-manian and N Senthil Kumar ldquoITS2 characterization andAnopheles species identification of the subgenus Celliardquo ActaTropica vol 125 no 3 pp 309ndash319 2013

[25] M Li H Cao P P H But and P C Shaw ldquoIdentification ofherbal medicinal materials using DNA barcodesrdquo Journal ofSystematics and Evolution vol 49 no 3 pp 271ndash283 2011

[26] S Chen H Yao J Han et al ldquoValidation of the ITS2 region asa novel DNA barcode for identifying medicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010

[27] H Yao J Song C Liu et al ldquoUse of ITS2 region as the universalDNA barcode for plants and animalsrdquo PLoS ONE vol 5 no 10Article ID e13102 2010

[28] Z Sun and S Chen ldquoIdentification of cortex herbs using theDNA barcode nrITS2rdquo Journal of Natural Medicines vol 67 no2 pp 296ndash302 2013

[29] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009

[30] C Koetschan T Hackl T Muller M Wolf F Forster andJ Schultz ldquoITS2 Database IV interactive taxon sampling forinternal transcribed spacer 2 based phylogeniesrdquo MolecularPhylogenetics and Evolution vol 63 no 3 pp 585ndash588 2012

[31] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[32] L Xiang J Song T Xin et al ldquoDNA barcoding the commercialChinese caterpillar fungusrdquo FEMSMicrobiology Letters vol 347no 2 pp 156ndash162 2013

[33] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008

[34] C Liu D Liang T Gao et al ldquoPTIGS-IdIt a system for speciesidentification by DNA sequences of the psbA-trnH intergenicspacer regionrdquo BMC Bioinformatics vol 12 no 13 article S42011

[35] P C Shaw F N Ngan and P P H But Authenticationof Chinese Medicinal Materials by DNA Technology WorldScientific Publishing Singapore 2002

[36] P D N Hebert S Ratnasingham and J R DeWaard ldquoBar-coding animal life cytochrome c oxidase subunit 1 divergencesamong closely related speciesrdquo Proceedings of the Royal SocietyB Biological Sciences vol 270 supplement 1 pp S96ndashS99 2003

[37] M Li P P H But and P C Shaw ldquoMolecular identification oftraditional medicinal materialsrdquo in Molecular Pharmacognosypp 45ndash66 Springer Dordrecht The Netherlands 2013

[38] China Flora Editorial Board of Cas Flora China vol 14Sciences Press Beijing China 1992

[39] L Jaakola M Suokas and H Haggman ldquoNovel approachesbased on DNA barcoding and high-resolution melting ofamplicons for authenticity analyses of berry speciesrdquo FoodChemistry vol 123 no 2 pp 494ndash500 2010

[40] M A Pavon I Gonzalez R Martın and T G Lacarra ldquoITS-based detection and quantification of Alternaria spp in rawand processed vegetables by real-time quantitative PCRrdquo FoodMicrobiology vol 32 no 1 pp 165ndash171 2012

[41] T Xin H Yao H Gao et al ldquoSuper food Lycium barbarum(Solanaceae) traceability via an internal transcribed spacer 2barcoderdquo Food Research International vol 54 no 2 pp 1699ndash1704 2013

[42] M Y Stoeckle C C Gamble R Kirpekar G Young S Ahmedand D P Little ldquoCommercial teas highlight plant DNA barcodeidentification successes and obstaclesrdquo Scientific Reports vol 1article 42 2011

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


have beenmisused and substituted by other species such as FferulaeoidesKorov [14] Furthermore numerous species fromthe Ferula genus have been utilized as traditional Chinesemedicines and ethical materials For example F soongaricahas been utilized byKazakhs in treating headaches colds andstomach aches F caspica in treating nervous breakdown andF lehmannii in treating parasitic malnutrition and cold painin the heart and abdomen [15] Whenmixed with substitutesadulterants and closely related species the originalmedicinalplant could not be traced to the medicinal herb market Afamous line that is widely circulated in the herbal medicinefield is ldquothere is no false Scutellaria no true Ferulardquo Thusinvestigating the authenticity of the Uyghur medicinal herbFerula is urgently required

The Ferula genus is usually recognized as monophyleticthe delimitation of species requires examination of completespecimens with roots stem bases basal leaves inflorescenceflowers and ripe fruits based on the observations of livingplants [16] Given that members are similar in habit andmorphology flowers inflorescences and fruit anatomy areoften hardly discernible taxa [17 18] Thus the infragenericclassification method of the Uyghur medicinal herb Ferulaurgently requires a solution To address this problem micro-scopic identification and powder identification techniqueshave been utilized for F sinkiangensis and its closely relatedspecies [19ndash21] However these techniques could not solvethe problem of authenticating the Uyghur medicinal herbs Fsinkiangensis andF fukangensis fromother traditionalmedic-inal herbs and closely related Ferula species By comparisonDNA barcoding employs a short DNA sequence from astandard locus as a species identification tool [22] whichhas been widely applied in forage identification [23] andanimal genetic relationship identification [24] The internaltranscribed spacer (ITS) region of this barcode which com-prises ITS1 58S and ITS2 has been widely utilized as a coreDNA barcode to identify different herbal medicinal materials[25] Researchers have proposed ITS2 as a standard DNAbarcode formedicinal plant authentication [26 27] Howevermolecular identification of F sinkiangensis and F fukangensisfrom their closely related species utilizing the ITS2 sequencehas not been reported In the present study DNA barcodingtechnology was applied for the first time to distinguish Fsinkiangensis and F fukangensis from their succedaneumand other closely related species The application of DNAbarcoding in trade andmarket management could ensure thesafe use of the pharmacopoeia herb Ferula

2 Samples and Methods

21 Materials and Methods for Sampling of Plant MaterialsSeventy-three leaves and root samples which represented10 Ferula species were collected from different locationsNine Ferula species were acquired from Xinjiang and onespecies of Ferula litwinowiana was obtained from Kaza-khstan The 10 species were given specimen collection num-bers (Table S1) (see Supplementary Material available onlineat httpdxdoiorg1011552015479879) and authenticatedby Researcher Guanmian Shen (Xinjiang Institute of EcologyandGeography Chinese Academy of Sciences) and Associate

Fellow Guoping Wang (Xinjiang Institute of Chinese Tradi-tional Medical and Ethical Materia Medica) All correspond-ing voucher samples were deposited in the herbarium of theXinjiang Institute of Chinese Traditional Medical and EthicalMateria Medica In addition one published sequence wasdownloaded from GenBank for analysis (Table S1)

22 DNA Extraction Amplification and Sequencing Ge-nomic DNA was isolated from 30mg of leaves or rootsaccording to the protocol of the Plant Genomic DNA Kit(Tiangen Biotech Co China) Specific regions of the ITS2sequences were amplified from dried leaves and root extractsuniversal primer and PCR conditions for ITS2 were obtainedfrom previous studies [28] The purified PCR products weresequenced using a 3730XL sequencer (Applied BiosystemsFoster City CA USA)

23 Sequence Alignment and Analysis The CodonCodeAligner v 404 was utilized to edit the sequences andassemble the contigs and shear sequence using universalprimers (51015840-31015840 ATTCACACCAAGTATCGCAT and 31015840-51015840ATTGTAGTCTGGAGAAGCGTC) The ITS2 core regioncould be obtained using the HMMer annotation methodbased on the hidden Markov model (HMM) to remove the58S and 28S sections at both ends of the sequences [29]The interspecificintraspecific variation of the samples wascalculated according to Kress et al [22] A phylogenetictree was constructed utilizing MEGA 50 Neighbor-joiningalgorithm (NJ tree) was used to evaluate the capability of theITS2 sequence to authenticate the studied species Bootstraptests were conducted utilizing 1000 resampling to assess theconfidence of the phylogenetic relationships using employedMEGA 50 [30 31] BLAST1 and nearest-distance methodswere performed to identify species as previously described[32 33]

3 Results

31 Universality and Sequence Characteristics DNAwas suc-cessfully extracted from 73 samples Gel electrophoretic anal-ysis revealed that all sample extractions emitted bright bandsITS2 sequences were successfully amplified and sequencedhigh-quality bidirectional sequences were obtainedThe ITS2sequence including partial the 58S ITS2 region and partial28S ranged from 451 bp to 455 bp and GC contents were536 to 562 The partial 58S length was 84 bp the GCcontents ranged from 571 to 583 The length of the ITS2region was 226 bp to 230 bp the GC contents ranged from530 to 568 The partial 28S was 141 bp and the GCcontents ranged from 518 to 539

32 IntraspecificInterspecific Variations Sequence lengthvariable sites and average interspecific K2P distance of ITS2regions were analyzed and summarized (Table 1) Most ofthe 10 Ferula species in this study were represented byseveralsamples because they were collected from differentlocalities F sinkiangensis was represented by 13 samples(Table S1) F fukanensis by 6 samples F ferulaeoides by 14


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43701246890135670378939435726835612864525689012457127890145890579234572127895

















4 Discussion




Feru

la so

onga

rica

KF7

9299

7Fe

rula

soon

garic

a K

F792

998

Feru

la so

onga

rica

KF79

2999

Feru

la so

onga

rica

KF79

3001

Feru

la so

onga

rica K

F792

996

Feru

la so

onga

rica K

F793

000

63

Feru

la fer

ulae

oides

KF79

3007

Feru

la fer

ulaeo

ides K

F793

006

Feru

la fer

ulaeo

ides K

F793

004

Ferula

ferulae

oides

KF7930

08

Ferula fer

ulaeoides K

F793005

Ferula fer

ulaeoides KF793010

Ferula fer

ulaeoides KF793009








68












95










96

Feru

la sy

reits

chik

owi K

J804

116

Feru

la syr

eitsch

ikowi

KJ8

0411

8

Feru

la syr

eitsch

ikowi K

J8041

10

Feru

la syr

eitsch

ikowi K

J8041

17

Ferula

syreit

schiko

wi KJ80

4111

Ferula syr

eitschikowi K

J804114

Ferula syreit

schikowi K

J804112

Ferula syreit

schikowi KJ804119





64









96

51

9264




99














Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















11111111111111111111111111111111122222222222222222222222222222333334444444

59900000001111112222233466677788899902233444555555666667777889999000192444445

43701246890135670378939435726835612864525689012457127890145890579234572127895

















4 Discussion




Feru

la so

onga

rica

KF7

9299

7Fe

rula

soon

garic

a K

F792

998

Feru

la so

onga

rica

KF79

2999

Feru

la so

onga

rica

KF79

3001

Feru

la so

onga

rica K

F792

996

Feru

la so

onga

rica K

F793

000

63

Feru

la fer

ulae

oides

KF79

3007

Feru

la fer

ulaeo

ides K

F793

006

Feru

la fer

ulaeo

ides K

F793

004

Ferula

ferulae

oides

KF7930

08

Ferula fer

ulaeoides K

F793005

Ferula fer

ulaeoides KF793010

Ferula fer

ulaeoides KF793009








68












95










96

Feru

la sy

reits

chik

owi K

J804

116

Feru

la syr

eitsch

ikowi

KJ8

0411

8

Feru

la syr

eitsch

ikowi K

J8041

10

Feru

la syr

eitsch

ikowi K

J8041

17

Ferula

syreit

schiko

wi KJ80

4111

Ferula syr

eitschikowi K

J804114

Ferula syreit

schikowi K

J804112

Ferula syreit

schikowi KJ804119





64









96

51

9264




99














Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Feru

la so

onga

rica

KF7

9299

7Fe

rula

soon

garic

a K

F792

998

Feru

la so

onga

rica

KF79

2999

Feru

la so

onga

rica

KF79

3001

Feru

la so

onga

rica K

F792

996

Feru

la so

onga

rica K

F793

000

63

Feru

la fer

ulae

oides

KF79

3007

Feru

la fer

ulaeo

ides K

F793

006

Feru

la fer

ulaeo

ides K

F793

004

Ferula

ferulae

oides

KF7930

08

Ferula fer

ulaeoides K

F793005

Ferula fer

ulaeoides KF793010

Ferula fer

ulaeoides KF793009








68












95










96

Feru

la sy

reits

chik

owi K

J804

116

Feru

la syr

eitsch

ikowi

KJ8

0411

8

Feru

la syr

eitsch

ikowi K

J8041

10

Feru

la syr

eitsch

ikowi K

J8041

17

Ferula

syreit

schiko

wi KJ80

4111

Ferula syr

eitschikowi K

J804114

Ferula syreit

schikowi K

J804112

Ferula syreit

schikowi KJ804119





64









96

51

9264




99














Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Endangered Uyghur Medicinal Plant Ferula ...

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