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1of3ApplicationsinPlantSciences20153(8):1500040ApplicationsinPlantSciences20153(8):1500040;http://www.
bioone.
org/loi/apps2015Qinetal.
PublishedbytheBotanicalSocietyofAmerica.
ThisworkislicensedunderaCreativeCommonsAttributionLicense(CC-BY-NC-SA).
ApApplicatitionsonsininPlPlanttScienSciencescesIlexkaushueS.
Y.
Hu(Aquifoliaceae)isanevergreentreegrowingindenseforestsatelevationsbetween400and1000minsouthernChina(Chenetal.
,2008).
Thespeciesisoneoftheprimarysourcesofku-ding-cha(Haoetal.
,2013),ateausedintraditionalmedicinethathasbeenconsumedforthousandsofyearsinChina.
Modernmedicinalresearchhasdemonstratedthatku-ding-chahassignicantpharmacologicaleffects,in-cludingasanantidiabetesandantiobesitydrug,aswellasanantioxidant(Haoetal.
,2013).
Ilexkaushuelivesinafragmentedhabitatwithextremelysmallpopulationsizes,andwasincludedinaconservationprogramcarriedoutin2011bytheStateForestryAdministrationofChina(Chenetal.
,2014).
Furthermore,I.
kaushueisaneconomicallyimportantcropduetoitswide-spreadusefortea,andtherehasbeenrapiddevelopmentinitscultivationinsouthernChina(Guoetal.
,2005).
Evidencesupportsthatsmallnaturalpopulationsandmodernplantbreedingcanleadtoareductioninoverallgeneticdiver-sity(TanksleyandMcCouch,1997;Leimuetal.
,2006).
Lowlevelsofgeneticdiversityputwildpopulationsatriskandjeopardizethecontinuedabilitytoimprovecrops(Reifetal.
,2005).
Therefore,assessmentandpreservationofgeneticdiver-sityofI.
kaushueareimportantconcerns.
AlthoughZhangetal.
(2003)developedmolecularmarkers(RAPD)foruseinthestudyofI.
kaushue,furthergeneticdiversityresearchisnecessaryatthepopulationandspecieslevelstoassessandprotectitsgerm-plasmresources.
Assessmentandconservationofgeneticdiver-sityofaspeciesrequiresdevelopmentofefcientcodominantmicrosatellitemarkers.
Inthisstudy,16microsatellitelociforI.
kaushuewereisolatedandcharacterized,whichwillbeuse-fulforassessmentandconservationofgeneticdiversityofI.
kaushue.
METHODSANDRESULTSWesampled12I.
kaushuetreesinanaturalpopulation(BaishaCounty/HainanProvince,China[QS]:19°08′50.
38″N,109°16′14.
9″E)and10treesinacultivatedpopulation(DapuCounty/GuangdongProvince,China[DM]:24°16′40.
31″N,116°28′02.
83″E).
VoucherspecimensofeachpopulationweredepositedintheGuangxiInstituteofTraditionalMedicalandPharmaceuticalSciencesherbarium(GXMI;accessionnumbersIk-012-ZQWandIk-008-HYF,respectively;Appendix1).
GenomicDNA(gDNA)wasextractedfromsilicagel–driedleavesusingthecetyltrimethylammoniumbromide(CTAB)method(DoyleandDoyle,1987).
WemixedgDNAofallwild-collectedindividualstobeshotgunsequencedbySangonBiotech(Shanghai,China)using454GSFLXTitanium(RocheAppliedScience,Branford,Connecticut,USA).
The454sequencingtechniqueisdescribedindetailinMarguliesetal.
(2005).
Weobtained29,247readsrangingfrom32to691bpwithanaveragereadlengthof401bp,foratotalof11,736,223bases.
AllreadswerefurtherscreenedformicrosatellitemotifsimplementedintheprogramSSRHunter1.
3withthedefaultparameters(LiandWan,2005).
Atotalof1109sequencescontaining1104dinucleotide,259trinucleotide,andninetetranucleotiderepeatswereob-tained.
Ofthesesequences,thosecontainingatleastsixdinucleotideortrinu-cleotiderepeatsandsufcientlengthsateitherendoftherepeatmotifwerechosenforprimerdesignusingPrimerPremier5.
0(ClarkeandGorley,2001);atotalof631sequences,containing691dinucleotideand82trinucleotidere-peats,weresubjectedtoprimerdesign.
ThesettingsforPrimerPremierwereasfollows:(i)eachsearchrangeofsenseprimerandantisenseprimerwasateachendoftherepeatmotif;(ii)theprimerlengthwasbetween17and25bp;(iii)thePCRproductsizewasbetween100and400bplong;(iv)theannealingtemperatureofprimerswasbetween50°Cand64°C,andthedifferenceinannealingtemperaturebetweentheforwardandreverseprimerswas<4°C;(v)theGCcontentwasbetween40%and60%;(vi)therewasnotobvioushairpinstructurewithintheprimer;and(vii)otherparametersfollowedthedefaultsettingsof"High"stringencyinthesearchcriteria.
Atotalof78primerpairsweresuccessfullydesignedforatotalof99repeatsincluding65dinucleotide,1Manuscriptreceived3April2015;revisionaccepted6May2015.
ThisworkwassupportedbytheKeyProjectsintheNationalScienceandTechnologyPillarProgramduringtheTwelfthFive-YearPlanPeriod(2011BAI01B04)andbytheGuangxiScienticResearchandTechnologyDevelopmentProgram(14124002-3).
4Authorforcorrespondence:zqw_21@163.
comdoi:10.
3732/apps.
1500040PRIMERNOTEDEVELOPMENTOFMICROSATELLITEMARKERSINILEXKAUSHUE(AQUIFOLIACEAE),AMEDICINALPLANTSPECIES1LAN-FANGQIN2,XIN-CHENGQU2,GANGHU3,YUN-FENGHUANG2,ANDQI-WEIZHANG2,42GuangxiInstituteofTraditionalMedicalandPharmaceuticalSciences,Naning530022,People'sRepublicofChina;and3SchoolofEnvironmentandLifeSciences,GuangxiTeacher'sEducationUniversity,Naning530001,People'sRepublicofChinaPremiseofthestudy:MicrosatellitemarkersweredevelopedforIlexkaushue(Aquifoliaceae),amedicinalplantwithex-tremelysmallwildpopulationsthatexistsinfragmentedhabitats,toassessandprotectitsgeneticdiversity.
MethodsandResults:Using454GSFLXTitaniumsequencing,16microsatelliteprimersetswereisolatedandcharacterized.
Fifteenofthesemarkerswerepolymorphic.
Thenumberofallelesperlocusrangedfromonetonineacross22individualsfrombothcultivatedandwildpopulations.
Theobservedandexpectedheterozygosityinthesetwopopulationsrangedfrom0.
000to1.
000andfrom0.
000to0.
785,respectively.
Conclusions:ThesemarkerswillbeusefulinstudiesongeneticdiversityofI.
kaushue.
Keywords:Aquifoliaceae;geneticdiversity;Ilexkaushue;microsatellitemarker.
2of3ApplicationsinPlantSciences20153(8):1500040Qinetal.
—Ilexkaushuemicrosatellitesdoi:10.
3732/apps.
1500040http://www.
bioone.
org/loi/apps45s;andanalextensionstepat72°Cfor5min.
PCRproductswereresolvedon6%polyacrylamidedenaturinggelusinga10-bpor25-bpDNAladder(Invitrogen,Carlsbad,California,USA)asareferenceandwerevisualizedbysilverstaining.
Sixteenprimerpairsweresuccessfullyamplied;theseproductsexhibitedtheexpectedsizesandshowedclearlydenedbandingpatternswithamaximumoftwoallelesineachlocusperindividual.
Thenumberofallelesperlocus(A)andtheobservedandexpectedheterozygosity(HoandHe)ofthetwopopulationswereestimatedbyGenAlExversion6(PeakallandSmouse,2006).
Linkage18trinucleotide,and16compoundrepeats.
Theseprimersweretestedforpoly-morphismin22individualsfromthetwopopulations.
PCRreactionswereperformedina20-μLreactionvolumecontaining50–100ngofgDNA,0.
5μMofeachprimer,and10μLof2*TaqPCRMasterMix(0.
1U/μLTaqpolymerase,0.
5mMdNTPeach,20mMTris-HCl[pH8.
3],100mMKCl,and3mMMgCl2[TiangenBiotech,Beijing,China]).
PCRamplicationswereconductedunderthefollowingconditions:95°Cfor5min;followedby35cyclesat94°Cfor45s,attheannealingtemperatureforeachspecicprimer(optimizedforeachlocus,Table1)for45s,72°CforTABLE1.
Characteristicsof16microsatellitemarkersinIlexkaushue.
LocusPrimersequences(5′–3′)RepeatmotifTa(°C)Allelesizerange(bp)GenBankaccessionno.
KDC1F:CTTACTCCCTTTGGTGCTC(AG)1360181–191KP943496R:CTCTTTTAGTCATTTTGCCCKDC10F:GGCCCTCCTGTAATTTTTC(TA)758133–139KP943497R:GGTCGGTCCCATTCTTGTKDC11F:TCTCAGGGTGCCTAAATA(GA)756122–138KP868632R:AACTAAGGTGTTTAAGGTCCKDC12F:GTAGACGACAATAATGGCGG(TGG)660329–335KP868633R:CTCCACCGATTGCTACTATTGKDC16F:CGAGCGGAAAGCAGAAATC(GTG)660238KP943498R:AGCCGAGGCAGAGGTAAAGAKDC27F:GACAACCAAACACAGAAAAG(AG)857186–192KP868635R:CAAAAGGACCAGTAACCCKDC29F:GAGTGGTTTGTATGGTCTTGT(TG)7(GT)560203–207KP868636R:CAGTGGTTAGCCTTTGATTCKDC32F:AGGTGATAAAGGAGAGGTCG(AT)5(AG)760127–135KP868637R:CTCCCTCTCGTATACCACCTKDC41F:CACTAGTTGCATTGGTGCT(TTC)1058282–306KP868638R:TGTTTAATGAACCCACCTCKDC49F:CAACTAACCCTATGTGTC(AG)1453120–142KP868639R:TTGTTAGAAAATCCTCGKDC50F:GCATGGTCTTTTGAAAACGA(GA)1458272–286KP943499R:GGGACGGCATAGAACTGTAATKDC58F:AGAGGACAACGAAGATTAGG(CT)960346–350KP868641R:GAGAGGGTGGACTGAGAGATKDC61F:CATTCCACTGACACAACCG(GA)860238–244KP868642R:GAGCCTCCTCCTTCATTGTKDC62F:GTGTTGTTGATGGTGGGTT(GA)5(GA)759166–176KP868643R:ACGTTAGACCCACTCTCATCKDC63F:CGACATTTACAGTCTAGC(GT)856170–174KP868644R:CTCAACCTTTAACTCTCTCKDC66F:CCAACAAATCAATAGGGAC(GA)956142–156KP868645R:AACTTTTAAGAGCAGTGCCNote:Ta=annealingtemperaturewhenrunindividually.
TABLE2.
ResultsofinitialprimerscreeningintwopopulationsofIlexkaushue.
LocusQSpopulation(natural,N=12)DMpopulation(cultivated,N=10)ATAHoHePvalueAHoHePvalueKDC130.
6670.
6531.
00020.
3000.
3750.
4804KDC1030.
4170.
5690.
19320.
6000.
5000.
9764KDC1140.
6670.
5590.
60730.
3000.
5850.
0576KDC1230.
4170.
5690.
50420.
6000.
4801.
0003KDC161———1———1KDC2740.
7500.
7261.
0001———4KDC291———30.
8000.
6601.
0003KDC3240.
8330.
7120.
28620.
5000.
4951.
0005KDC4150.
8330.
7080.
53330.
8000.
6551.
0006KDC4960.
9170.
7640.
84650.
9000.
6750.
9539KDC5061.
0000.
7850.
79140.
7000.
6550.
8497KDC5830.
5830.
5691.
0001———3KDC6130.
5830.
5311.
00020.
5000.
4951.
0004KDC6250.
6670.
7260.
80920.
6000.
5001.
0005KDC6320.
3330.
5000.
28230.
6000.
6451.
0003KDC6640.
9170.
6910.
54730.
8000.
6650.
0246Note:A=numberofalleles;AT=totalnumberofalleles;He=expectedheterozygosity;Ho=observedheterozygosity;N=samplesizeforeachpopulation;Pvalue=testfordeviationfromHardy–Weinbergexpectations.
ApplicationsinPlantSciences20153(8):1500040Qinetal.
—Ilexkaushuemicrosatellitesdoi:10.
3732/apps.
15000403of3http://www.
bioone.
org/loi/appsdisequilibrium(LD)anddeviationfromHardy–Weinbergequilibrium(HWE)werecalculatedbyGENEPOPversion4.
2(RaymondandRousset,1995).
Acrossthecultivatedandwildpopulations,Avariedfromonetonine,andatotalof73alleleswerescoredin22individuals(Table2).
HoandHeinthenaturalandcultivatedpopulationsrangedfrom0.
000to1.
000andfrom0.
000to0.
785,respectively.
NopairsoflocishowedsignicantLD.
ThePvalueoftestsforHWErangedfrom0.
024to1.
000(Table2).
OnlylocusKDC66inpopulationDMsignicantlydeviatedfromHWE(P<0.
05),whichmayduetooverdominantselectionoradmixturefromdifferentresourcesgiventhehighlevelofheterozygosityforthislocus.
CONCLUSIONSAtotalof16nuclearmicrosatellitemarkersweredevelopedforI.
kaushue.
Fifteenofthesemarkersshowedvaryinglevelsofpolymorphismandonemarkerexhibitedmonomorphism.
TheselociwillbeusefulforassessmentandconservationofgeneticdiversityofI.
kaushue.
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APPENDIX1.
VoucherinformationforIlexkaushueusedinthisstudy.
PopulationVoucherspecimenaccessionno.
aCollectionlocalitybGeographiccoordinatesNQSIk-012-ZQWQingsongTownship,BaishaCounty,HainanProvince19°08′50.
38″N,109°16′14.
91″E12DMIk-008-HYFDamaTown,DapuCounty,GuangdongProvince24°16′40.
31″N,116°28′02.
83″E10Note:N=numberofindividuals.
aVouchersdepositedintheGuangxiInstituteofTraditionalMedicalandPharmaceuticalSciencesherbarium.
ZQW=Qi-WeiZhang,collector;HYF=Yun-FengHuang,collector.
bLocalityandChineseprovince.