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Isolating and location of Tandem Repetitive Afa-family Sequences from Mongolian Wheatgrass (Agropyron mongolicum Keng)

蒙古冰草Afa家族串联重复序列克隆及染色体定位



全 文 :!23" !3#
 Vol.23  No.3
! " # $
ACTA AGRESTIA SINICA
   2015$  5%
  May.  2015
犱狅犻:10.11733/j.issn.10070435.2015.03.002
犐狊狅犾犪狋犻狀犵犪狀犱犾狅犮犪狋犻狅狀狅犳犜犪狀犱犲犿犚犲狆犲狋犻狋犻狏犲犃犳犪犳犪犿犻犾狔犛犲狇狌犲狀犮犲狊犳狉狅犿
犕狅狀犵狅犾犻犪狀犠犺犲犪狋犵狉犪狊狊(犃犵狉狅狆狔狉狅狀犿狅狀犵狅犾犻犮狌犿 犓犲狀犵)
ZHAOYan1,2,DOUQuanwen3,YUNJinfeng1,2,WANGJunjie1,2
(1.EcologyandenvironmentcolegeofInnerMongoliaagriculturalUniversity(IMAU),Huhhot,InnerMongolia010019,China;
2.KeyLaboratoryofGrasslandResources(IMAU),MinistryofEducation,Hohhot,InnerMongolia010019,China;
3.NorthwestInstituteofPlateauBiology,ChineseAcademyofSciences,Xining,QinghaiProvince810008,China.)
犃犫狊狋狉犪犮狋:TandemrepetitiveAfafamilysequencesareknowntooccurinthewheatandrelated
speciesofTriticeae.AmemberoftheAfafamilysequencesisolatedfromMongolianWheatgrass
(犃犵狉狅狆狔狉狅狀犿狅狀犵狅犾犻犮狌犿 Keng)(2n=2x=14,PP)is233bp,namedas狆犃犿犃犳犪1,whichis
mostsimilarwithotherAfafamilysequencesin犜狉犻狋犻犮犲犪犲species.Thephylogenicanalysisresults
showthat狆犃犿犃犳犪1isclusteredwith狆犔狉犃犳犪3and狆犔狉犃犳犪5,andthefindingsindicatethatP
genomehashomologywithNandXgenomes.ToknowwhichthegenomeofMongolianwheat
grasscarriestheAfafamilysequence;FISHiscarriedoutusing狆犃犿犃犳犪1astheprobe.The
signalsappearinthetelomericregionsandsubtelomericregionsofalchromosomes.Thisfinding
indicatesthatPgenomescontainAfafamilyrepeats.
犓犲狔狑狅狉犱狊:MongolianWheatgrass;TandemrepetitiveAfafamilysequences;MolecularCloning;
FISH
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AuthorforCorrespondence,Email:csgrass@vip.163.com
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89:

hiê]
;Afa¹^å˜~¡uæ;ëì;ô=ûS õ
;<,-=
:S543.9    >?@AB:A     >CD=:10070435(2015)03044805
  ThefirstcloneoftandemrepetitiveAfafamily
sequences,pAs1,isclonedfrom犃犲犵犻犾狅狆狊狊狇狌犪狉狉狅
狊犪L.(2N=14,genomeDD),anddescribedasaD
genomespeciesrepetitivesequence[1],andthese
quenceshomologoustopAs1existin manyge
nomesofthetribeTriticeae[24].Therepeatunits
areabout340bplong.ThetandemarraysofAfa
familyrepeatsaredispersedinseveralsubtelomeric
andinterstitialchromosomalregions,and,there
fore,havebeenusedaschromosomemarkers[58].
TribeTriticeae (Gramineae)include many
wildspecies,althegenomesrecognizedinthis
tribe contain seven chromosomes. Mongolian
wheatgrass(犃犵狉狅狆狔狉狅狀犿狅狀犵狅犾犻犮狌犿 Keng)(2n=
!3# ­ Ð:hiê]Afa¹^å˜~¡uæëìQð–2ˆS
2x=14,PP)isoneofperennialwildrelativespe
ciesofwheat,thespeciesisanarrowspikeddip
loid(犃.犳狉犪犵犻犾犲ssp.犿狅狀犵狅犾犻犮狌犿)distributingin
desertgrasslandandtypicalgrasslandofChina.
Longtermevolutionandadaptationtoharshcondi
tionsmakeMongolianwheatgrassrichintolerance
genesforarangeofbioticandabioticstressessuch
aspestandfungalattacks,drought,cold,barren
andhighsalinity [911].Inviewofaltheseattrib
utes,Mongolianwheatgrasshasbeenproposedto
beavaluablegeneticresourceinforgegrassand
cropimprovementforresistancesortolerances.
ThereareabundantgermplasmresourcesofMon
golianwheatgrassinNorthwestChina.However,
uptonow,theevolutionallineagesofthespecies
andrelationship with wheathavenotbeenre
vealed.
Inthisstudy,Afafamilysequenceswereiso
latedfromMongolianwheatgrass,andthecharac
terswereexaminedinordertoprovidevaluableev
idenceforthe geneticevolution of Mongolian
wheatgrass.
1 犕犪狋犲狉犻犪犾狊犪狀犱犕犲狋犺狅犱狊
1.1 犘犾犪狀狋犿犪狋犲狉犻犪犾狊
WildMongolianwheatgrassspecies(2n=2x=
14,PP)werecolectedfromitsnaturalhabitatin
Xilingolprairier,InnerMongolian,China.
1.2 犆犾狅狀犻狀犵犪狀犱狊犲狇狌犲狀犮犻狀犵
GenomicDNAwasextractedfrom Mongolian
wheatgrassusingCTABmethod.GenomicDNAas
templatewasamplifiedbyPCRwithpAs1specific
primers(ASA5′GATGATGTGGCTTGAATGG
and ASB 5′GCATTTCAAATGAACTCTGA)
[2].Thefragments wereclonedintopUC19in
Escherichiacolistrain DH5αandsequencedby
ShanghaiSangonBiologicalEngineeringTechnolo
gy&Services(Shanghai,China).
1.3 犛犲狇狌犲狀犮犲犪狀犱狆犺狔犾狅犵犲狀犻犮犪狀犪犾狔狊犻狊
SequenceswereanalyzedwithDNAmanand
DNAusersoftware.Sequencehomologywiththe
nucleotidedatabaseofGenBankwasanalyzedusing
BLASTtools.Thephylogenyofthesequenceswas
analyzedbycomputersoftware,DNAMAN.ful.
version.v5.2.2.
1.4 犉犐犛犎犪狀犪犾狔狊犲狊
Slidesforfluorescenceinsituhybridization
(FISH)werepreparedbytheacetocarminesquash
methodusingroottipmeristemcels.ThepAm
Afa1waslabeledwithbiotin14dATPanddetec
tedwithavidinFITCaccordingtoMukai[12].Ima
gesweretakenwithacooledCCDcameraandana
lyzedwithIPLABSPECTRUMcomputersoftware
(SignalAnalytics).
2 犚犲狊狌犾狋狊
2.1 犕狅犾犲犮狌犾犪狉犮犾狅狀犻狀犵犪狀犱犛犲狇狌犲狀犮犲犪狀犪犾狔狊犻狊狅犳
狆犃犿犃犳犪1
AnAfafamilygenefrom Mongolianwheat
grasswas233bpandATrich(61.2%),namedas
狆犃犿犃犳犪1(GenBankaccessionno.KC990463),
whichwassimilartotheAfafamilysequencesof
otherTriticeaespecies[2].
NCBIBlastnresults(Fig.1)showedthatthe
maxidenticaldegreewas95% betweenthese
quenceof狆犃犿犃犳犪1and狆犘犼犃犳犪2(GenBankac
cessionno.AB022724.1),betweenthesequence
of狆犃犿犃犳犪1andcontigctg447from犜狉犻狋犻犮狌犿犪犲狊
狋犻狏狌犿chromosomearm3DSspecificBAClibrary
(GenBankaccessionno.HE774676.1),andwas
94%betweenthesequenceof狆犃犿犃犳犪1andAe
gilopstauschichromosome1Dsprolamingenelo
cus,completesequence (GenBankaccessionno.
JX295577.2).Blastanalysisshowedthatthese
quenceof狆犃犿犃犳犪1hadhigherhomologywiththe
AfafamilysequencesofotherTriticeaespecies.
2.2 犘犺狔犾狅犵犲狀犻犮犪狀犪犾狔狊犻狊
ToknowtherelationoftheAfafamilysequences
fromMongolianwheatgrasswiththoseofotherTrit
iceaespecies,27Afafamilysequencesfromtheseven
diploidandtwotetraploidspecieswerechosenatran
domfrom GENBANK(Table1).Thesesequences
944
! " # $ !23"
wereanalyzedbytheNJmethod,andclusteredby
DNAman(Fig.2).TheAfafamilysequencesfrom
Mongolianwheatgrasswasclusteredwith狆犔狉犃犳犪3
and狆犔狉犃犳犪5(Fig.1),犃犳犪犿狅狀2wasclustered
with狆狋狌犪犳犪1and狆狋狌犪犳犪3,theyhadthesamege
nome(AA).Since犃犳犪4犇犆犛犔5,犃犳犪4犇犆犛犔7,
犃犳犪4犇犆犛犔8,犃犳犪4犇犆犛犔9,犃犳犪5犇犆犛犔1,犃犳犪
6犇犆犛犔2,犃犳犪6犇犆犛犔2 and犃犳犪7犇犆犛犔1 were
characterizedtobeAABBgenomespecific(Table
1),theclusterwasdesignatedasAABB.Signifi
cantclusterswereindicatedbybarswiththename
ofthegenomefromwhichtheywerederived.
犉犻犵.1 犜犺犲犅犾犪狊狋狀狉犲狊狌犾狋狊狅犳狆犃犿犃犳犪1狊犲狇狌犲狀犮犲狊
犜犪犫犾犲1 犇犲狋犪犻犾犻狀犳狅狉犿犪狋犻狅狀狅犳犃犳犪犳犪犿犻犾狔狊犲狇狌犲狀犮犲狊
Afasequencename Origin Genomes GenBankaccessionNo.
犃犳犪4犇犆犛犔5 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003212
犃犳犪4犇犆犛犔7 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003214
犃犳犪4犇犆犛犔8 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB00325
犃犳犪4犇犆犛犔9 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003216
犃犳犪5犇犆犛犔1 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003217
犃犳犪6犇犆犛犔2 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003221
犃犳犪6犇犆犛犔2 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003221
犃犳犪7犇犆犛犔1 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003223
犃犳犪犱狌狉1 犜狉犻狋犻犮狌犿狋狌狉犵犻犱狌犿subsp.犱狌狉狌犿DNA AABB AB003235
犃犳犪犮犲狉3 犛犲犮犪犾犲犮犲狉犲犪犾犲DNA RR AB003228
犃犳犪犮犲狉4 犛犲犮犪犾犲犮犲狉犲犪犾犲DNA RR AB003229
犃犳犪狏狌狉2 犎狅狉犱犲狌犿狏狌犾犵犪狉犲DNA HH AB003252
犃犳犪狏狌狉4 犎狅狉犱犲狌犿狏狌犾犵犪狉犲DNA HH AB003254
犃犳犪狊狆犲3 犃犲犵犻犾狅狆狊狊狆犲犾狋狅犻犱犲狊DNA DD AB003242
狆犃狊犃犳犪2 犃犲犵犻犾狅狆狊狋狉犻狌狀犮犻犪犾犻狊var.狋狉犻狌狀犮犻犪犾犻狊DNA DD AB3256 
狆犜狌犃犳犪1 犜狉犻狋犻犮狌犿狌狉犪狉狋狌DNA AA AB003259
狆犜狌犃犳犪2 犜狉犻狋犻犮狌犿狌狉犪狉狋狌DNA AA AB003260
狆犜狌犃犳犪3 犜狉犻狋犻犮狌犿狌狉犪狉狋狌DNA AA AB003261
狆犜狌犃犳犪4 犜狉犻狋犻犮狌犿狌狉犪狉狋狌DNA AA AB003262
狆犘犼犃犳犪2 犘狊犪狋犺狔狉狅狊狋犪犮犺狔狊犼狌狀犮犲犪DNA NN AB022724
狆犘犼犃犳犪3 犘狊犪狋犺狔狉狅狊狋犪犮犺狔狊犼狌狀犮犲犪DNA NN AB022725
狆犔狉犃犳犪2 犔犲狔犿狌狊狉犪犮犲犿狅狊狌狊DNA NNXX AB022727
狆犔狉犃犳犪3 犔犲狔犿狌狊狉犪犮犲犿狅狊狌狊DNA NNXX AB022728
狆犔狉犃犳犪5 犔犲狔犿狌狊狉犪犮犲犿狅狊狌狊DNA NNXX AB022730
犃犳犪犿狅狀3 犜狉犻狋犻犮狌犿犿狅狀狅犮狅犮犮狌犿 DNA AA D82989 
犃犳犪犿狅狀1 犜狉犻狋犻犮狌犿犿狅狀狅犮狅犮犮狌犿 DNA AA D82987 
犃犳犪犿狅狀2 犜狉犻狋犻犮狌犿犿狅狀狅犮狅犮犮狌犿 DNA AA D82988 
狆犃犿犃犳犪1 犃犵狉狅狆狔狉狅狀犿狅狀犵狅犾犻犮狌犿 DNA PP KC990463
2.3 犉犐犛犎犪狀犪犾狔狊犲狊
Toknowifbothoronlyoneofthegenomesof
Mongolian wheatgrass carried the Afafamily se
quences,FISHwascarriedoutusing狆犃犿犃犳犪1asthe
probe.Thesignalsappearedanddispersedinthetelo
mericregionsandsubtelomericregionsofalchromo
somes(Fig.3),andwereverystrong.Thisfinding
indicatedthatPgenomescontainedAfafamilyrepeats.
054
!3# ­ Ð:hiê]Afa¹^å˜~¡uæëìQð–2ˆS
犉犻犵.2 犘犺狔犾狅犵犲狀犻犮狋狉犲犲狅犳狋犺犲犃犳犪犳犪犿犻犾狔狊犲狇狌犲狀犮犲狊犳狉狅犿犕狅狀犵狅犾犻犪狀狑犺犲犪狋犵狉犪狊狊
犉犻犵.3 犉犐犛犎狅犳犕狅狀犵狅犾犻犪狀狑犺犲犪狋犵狉犪狊狊犿犲狋犪狆犺犪狊犲
犮犺狉狅犿狅狊狅犿犲狊(2狀=2狓=14)狆狉狅犫犲犱狑犻狋犺狆犃犿犃犳犪1
犖狅狋犲:犜犺犲犪狉狉狅狑狊狊犺狅狑犅犮犺狉狅犿狅狊狅犿犲狊.犛犮犪犾犲犫犪狉=10μ犿.
3 犇犻狊犮狌狊狊犻狅狀
3.1 犌犲狀犲狋犻犮犪犾狆狉狅狆犲狉狋狔犪狀犱狆犺狔犾狅犵犲狀犻犮犪狀犪犾狔狊犻狊狅犳
犃犳犪犳犪犿犻犾狔狊犲狇狌犲狀犮犲
ApreviousinvestigationofNagaki[2]demon
stratedtheimportantpropertiesoftheAfafamily
sequences.Therewashighlyvariablecopynumber
pergenomeamongspecies;thesequencesinthe
genomesincluding morecopies were moreuni
form;therewasnochromosomespecificityinthe
sequenceswithingenomes;Nolargescaletranspo
sitionorconversiontookplacebetweengenomes
duringthepast7000years;theneighboringse
quenceswerethemostsimilarwitheachother.
  狆犃犿犃犳犪1sequenceclonedfrom犃.犿狅狀犵狅犾犻
犮狌犿KengwasthemostsimilartootherAfafamily
sequencesinTriticeaespecies.Ourfindingswere
consistentwiththeabove.TheDNAsequencesof
theAfafamilycloneschosenatrandomfromNCBI
databasewereanalyzed.Toknowtherelationof
the Afafamilysequencesfrom 犃.犿狅狀犵狅犾犻犮狌犿
KengwiththoseofotherTriticeaespecies,28re
peatedunitsamplifiedbyPCRwithASAandAS
BprimerswereanalyzedbytheNJmethod(Fig.
2).ThefindingindicatedthatPgenomehadho
mologywithNandXgenome,and犃.犿狅狀犵狅犾犻犮狌犿
Kengexistedearlierthan犔.狉犪犮犲犿狅狊狌狊and犘.
犼狌狀犮犲犪.SoitwassupportedthatPgenomewasa
154
! " # $ !23"
donorofNandXgenomes,ortherewasatranspo
sitionorconversionamongP,NandXgenomes.
3.2 犆犺狉狅犿狅狊狅犿犪犾犾狅犮犪犾犻狕犪狋犻狅狀狅犳犃犳犪犳犪犿犻犾狔狊犲
狇狌犲狀犮犲
Inrepetitivesequenceslocatedinspecificloca
tions,suchasribosomalRNAgenesandsubtelomeric
repetitivesequences,thegeneconversionprobably
playedanimportantroleforthehomogenizationofre
petitivesequencesbecausesuchsequenceswereclosely
positionedbytelomereassociationonnuclearmem
braneininterphasenuclei[7,1316].However,Afafami
lysequenceswerewidelydistributednotonlyinsubte
lomericbutalsoininterstitialregions.狆犎狏犃14,a
Afafamilyrepetitivesequencesisolatedfrom barley
distinguishedeachbarleychromosomebyinsituhy
bridization[8].
Toknowifbothoronlyoneofthegenomesof
犃.犿狅狀犵狅犾犻犮狌犿 KengcarriedtheAfafamilyse
quences,FISHwascarriedoutusing狆犃犿犃犳犪1as
theprobe,andthesequencehybridizedwithalte
lomericregionsandsubtelomericregionsofthe
chromosomes.Thesignalsappearedmostlyattwo
endsofthechromosomes.Thisfindingindicated
thatP genomecontained Afafamilyrepeatse
quences,andtherewasnochromosomespecificity
inthesequenceswithingenomes.
4 犆狅狀犮犾狌狊犻狅狀
狆犃犿犃犳犪1sequenceclonedfrom犃.犿狅狀犵狅犾犻
犮狌犿KengismostsimilarwithotherAfafamilyse
quencesinTriticeaespecies.Thissequencecanbe
usedforthephylogenicanalysisofTriticeaespe
cies.PgenomemusthavehomologywithNandX
genome;狆犃犿犃犳犪1sequencesarelocatedinthe
telomericregionsandsubtelomericregionofthe
everychromosomesof犃.犿狅狀犵狅犾犻犮狌犿 Keng.P
genomecontainsAfafamilyrepeatsequences,and
thereisnochromosomespecificityinthesequences
withingenomes.
犃犮犽狀狅狑犾犲犱犵犲犿犲狀狋
ThisworkwassupportedbyNationalNature
ScienceFoundationofChina(No.31260578,No.
31160479)
犚犲犳犲狉犲狀犮犲狊
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