全 文 ：GeneticAnalysisonChromosomeofHybridF1betweenElymuscanadensisandElymussibiricus
LIJing-huan1, 2 , YUNJing-feng1＊ , Durisihala2 , XINGJing-min2
1.ColegeofEcologyandEnvironmentalScience, InnerMongoliaAgriculturalUniversity, Hohhot010019;2.InstituteofLifeScienceand
Technology, InnerMongoliaNormalUniversity, Hohhot010022
Abstract　MorphologicandcytologicalcharacteristicsofhybridF1 betweenE.canadensisandE.sibiricuswerestudied.TheresultsshowedthathybridF1 wereobviouslyexceededparentsinplantgrowthvigor, andspiketypeofF1wasbetweenparents.ThekaryotypeformulaofhybridF1whichwastruehybridwas2n=4x=16M+10m+2st.Atmeioticanaphase, chromosomelaggingbehaviorswerefrequentlyobserved.Thefre-
quencyoflaggingchromosomesofF1 was87.37%.Atmeioticmetaphase, thereweremanyunivalentsandmultivalents, andtherodbivalentformedgreatly.
Keywords　E.canadensis;E.sibiricus;Distanthybrid;Chromosome
Received:August17, 2009　　Accepted:September4, 2009
SupportedbyResearchonDistantHybridizationBreedingofForage
(2008BADB3B02)intheEleventhFive-yearPlanPeriod, andthe
MajorScientificandTechnicalProjectofInnerMongoliaAutonomous
Region, ProductsDevelopmentofHighQualityAdequetGrass.
＊Correspondingauthor.E-mail:csgrass05@163.com
　　Thedistanthybridizationmeansthedistanthybridization
amongdiferentspecies, Generaorevenfamiliesthroughit
couldstudythespeciesevolution, geneticrelationship, be-
sides, thenewspeciescanbecreatedandthedesirable
genescanbetransferred.Therefore, thestudyofdistanthy-
bridizationplaysaveryimportantroleinplantgeneticresearch
andbreedingresearch, soitispaidmanyatentionsfrom
breedingscientistsinalongtime[ 1-4] .Whilehybrididentifica-
tionisakeylinkinwidecrossbreeding, besides, hybrididen-
tificationisamainmeanofhybrididentification, whichcon-
tainsmorphologicalmarker, cytologicalmarkerandbiochemi-
calmarker.Themorphologicalidentificationisthemainmean
forhybrididentification, whichcandirectlyrepresentthemor-
phologicaldifferentiationbetweenhybridandparents.When
thechromosomesinwholegroup, wholechromosomeorfrag-
mentsareintroducedintoplants, theplantmorphologywil
changesuchasplantheight, leafcolor, leafshape, leaf
length, tilering, panicletype, spikelength, shape, spikelets
density, waxiness, panicletype, hairglumeandawnetal,
becauseofgeneinteraction.Thecommoncytologicmarkers
includekaryotypeanalysis, ideogramanalysisandTelosome
testing-crossanalysis.Throughthestudiesofchromosome
morphology, chromosomenumberandstructure, itisfound
thatchromosomenumberandstructureofsomeindividuals
arevariedamongthewholepopulation.Alaneuploid, chromo-
someswithstructurevariationandheteromorphicchromosome
havespecialcytologicalcharacteristics[5-8] , therefore, thevari-
ationsofchromosomemorphology, chromosomenumberand
structurecanbetakenasameasuretoidentifyhybrid.
ThehybridF1 ofE.canadensisandE.sibiricusisidentified
tojudgethesuccessofhybridization.Thecombinationofmor-
phologyandcytologyisusedinthisexperimenttostudytheF1
ofE.canadensisandE.sibiricusforidentifyingthetruthandfake
ofhybrid.However, F1 ishighlysterile, sothesterilemecha-
nismofF1 ispreliminarilyexploredtoprovidebreedingmaterial
forusingheterosisandbreedingofnewvariety.
MaterialsandMethods
Generalsituationofexperimentalfield
Theexperimentalfieldwaslocatedinexperimentalforage
stationofInnerMongoliaAgriculturalUniversity.Itsposition
was111°42′E, 40°49′Nandaltitudewas1 063 m, soitwas
typicalcontinentalclimate, besides, itsannualprecipitation
wasaround400mmandfrostlessseasonwas140d.Theex-
perimentalfieldwasdarkchestnutsoilandthesoilwassandy
loamwithmediumfertility, besides, thesoilpHwasaround
7.5 withirrigationcondition.
Experimentalmaterials
Thesourceofmaterialsandtheirbiologicalcharacteris-
ticswerelistedinTable1.
Table1　Originandbiologicalcharacteristicsoftestedmaterials
Material Origin Biologicalcharacteristics
Elymus
canadensis
(♀)
NorthAmerica
Highyield, hugeleaves, coldresist-
ance, insectresistance, disease
resistance
Elymus
sibiricus
(♂)
InnerMongolia
Highseedsetingrate, coldresist-
ance, droughtresistance, goodfor-
agequalityF1 genera-
tion Goodgrowthpower
Experimentaldesign
TheElymuscanadensis, ElymussibiricusandF1 genera-tionwereseparatedfrom originalbundles, thentheywere
plantedseparatelyandeveryholehadfourplantswith60×60
cmplantingspacing.
Karyotypeanalysismethod
TheseedsofElymuscanadensisandElymussibiricus
wereputintoculturedish, whichwereplacedintoconstant
temperaturefosterboxat28.1 ℃ bystandardgermination
method.Whenthetenderrootsgrewto1.5 to2.0 cm, the
roottipswerecutoff.TheroottipsofF1 werecolectedintile-ringstageandputinto0.002M8-hydroxyquinoline.30 meta-
phaseswereselectedfromSlidesoftheroot-tipcelsoftested
materialstocount, andthen85%chromosomenumberofdi-
AgriculturalScience＆Technology, 2009, 10(5):67-71
Copyright 2009, InformationInstituteofHAAS.Alrightsreserved. Agronomy
DOI :10.16175/j.cnki.1009-4229.2009.05.022
visionphasewasusedtoconfirmchromosomenumberofma-
terials.Themethodwaslistedas:
(1)Pretreatment.Thematerialsweretreatedin0.002M
for4 to6 horincolchicinefor5 h.
(2)Fixation.Carnoyfixationfluidwasusedtofix24 h.
(3)Acidolysis.Afterwashedbydistiledwater, theroot
tipswereputinto1 mol/LHClat60 ℃ for8min.
(4)Enzymatichydrolysis.Theacidolysisroottipswere
washedbydistiledwater, andthenthewaterwasabsorbed
byfilterpaper.The2%mixedenzymeofPectinaseandcelu-
laseweredroppedonglassslide, thentheroottipswereput
initfor30minenzymatichydrolysisatroomtemperature.
(5)Staining.Theenzymesolutionwasabsorbed, and
thencarbolfuchsinwasaddedintoforstaining30 min.The
optimumstainingsolutionandstainingtimewerechosen.
(6)Squashingandmicroscopicexamination.Theroot
tipsandstainingsolutionwereplacedoncleanglassslide,
andthenoneabsorbentpaperwasputonitafterclosingglass
slide.Afterabsorbedwater, theforcepswasusedtoknock
coverglasstomakethecelsdisperse.
(7)Photography.Thepreparedslideswereobserved
throughmicroscopetochecktheslidescontaininggoodchro-
mosomespread, thentheyweretakenphotosbyJVCTK-
C1381 microphotographysystem, besides, MoticImagesAd-
vanced3.2 softwarewasusedtomeasurechromosome
lengthforcalculatingkaryotypeparametersofchromosome.
1:E.canadensis;2:F1generation;3:E.sibiricus.
Fig.1　EarmorphologyofE.canadensis, E.sibiricusandF1
generation
Observationofmeioticbehaviorofpollenmothercell
Inclearday, Elymuscanadensis, Elymussibiricusand
F1 generationnearheadingdatewereputintoglassbottlecontainingCarnoyforfixing6 hthenpreservedin75% etha-
nol.Theantherswereputinto2% celulasefordissociating5
to10 minthenstrainedbycarbolfuchsin.Themeiosisofpol-
lenmothercelwereobservedthroughmicroscope, everyma-
terialwasrecordedover100celsandtheclearglassslidewere
shotthroughJVCTK-C1381 microphotographysystem.
ResultsandAnalysis
ThemorphologicalobservationofparentsandhybridF1Itwasconcludedfrommorphologicalobservationthatthe
panicletypeofF1 wascrescentshapeandbetweentheseofparents.TheElymuscanadensiswaslightgreenanditsstem
wasrough, besides, itspaniclewasbigandbendedandever-
ynodehadtwotothreespikelet, whiletheglumeandlemma
hadshorthairs.TheElymussibiricuswaslightgreenandits
spikeswerelooselydrooped, besides, everynodehadtwo
spikeletsandeveryspikelethad3 to5 flowers.TheF1 was
celadonandrough, besides, lemmaandglumehadhairs, so
itwasmorelikeElymuscanadensis.
RoottipchromosomekaryotypeanalysisofparentsandF1ItwasconcludedfromFig.2 andTable2thatthechromo-
somenumberofElymuscanadensiswas2n=4x=28 andthe
relativelengthfrom 10 to4.65, whiletheratiobetweenthe
longestandshortestchromosomewas2.2∶1 andthearmratio
wasfrom1.14 to1.35.AccordingtoLevankaryotypeanaly-
sis, thechromosomesofElymuscanadensiswereconsisted
bymetacentricchromosomesandthekaryotypeformulawas
2n=4x=28m, however, accordingtoStebbinsclassification,
thechromosomesofElymuscanadensiswere1Atype.
ItwasconcludedfromFig.3 andTable3 thatsomatic
chromosomenumberofElymussibiricuswas2n=4x=28.
AccordingtoLevanclassification, thekaryotypeformulawas
listedas2n=4x=22m+6sm, soitbelongedto1Btype
basedonStebbinskaryotypeclassification.
Thekaryogramandtuberculousparameterswerelistedin
Fig.4 andTable4.ThechromosomenumberofF1 was2n=
4x=28 andtherelativelengthofchromosomewasfrom8.83
to4.42, whilearmratiowasfrom1.00to3.33.Thefirst, 5th,
7
th, 9th, 11th, 12th, 13th and14th pairofchromosomeswas
metacentricchromosomesatmiddle, whilethe3rdpairof
chromosomewasmetacentricchromosomeneartheendand
theleftweremetacentricchromosome.Thenumberofmeta-
centricchromosomesatmiddle(M)increasedandthenum-
berofmetacentricchromosomesnearthemiddle(sm)was
notexisted.L/Swas2∶1, soitskaryotypewas2Bandkaryo-
typeformulaewas2n=4x=16M+10m+2st.Therefore, F1wasnotElymuscanadensisneitherElymussibiricusaccording
tokaryotypeparameters.
Fig.2　ChromosomemorphologyandkaryotypeofE.canadensis
68 AgriculturalScience＆TechnologyVol.10, No.5, 2009
Table2　KaryotypeparametersofE.canadensis
Numberof
chromosome
Relativelength
ofchromosome∥% Ratioofarm
Ratioofchromo-
somelength(L/S)
Relativelength
rangeofchromosome∥%
Karyotypeform-
ulaeandtype
1 10.40 1.28
2 9.19 1.35
3 8.72 1.14
4 8.31 1.28
5 7.81 1.25
6 7.52 1.30 2n=4x=28m
7 7.13 1.27 2.23∶1 10.40-4.65 1A
8 6.65 1.25
9 6.42 1.29
10 6.25 1.32
11 5.94 1.16
12 5.67 1.23
13 5.34 1.28
14 4.65 1.25
Fig.3　ChromosomemorphologyandkaryotypeofE.sibiricus
Table3　KaryotypeparametersofE.sibiricus
Numberof
chromosome
Relativelength
ofchromosome∥% Ratioofarm
Ratioofchromo-
somelength(L/S)
Relativelength
rangeofchromosome∥%
Karyotypeform-
ulaeandtype
1 10.47 1.56
2 9.23 1.25
3 8.93 1.18
4 7.45 1.38
5 7.42 1.57
6 7.36 1.38 2n=4x=22m+6sm
7 7.36 1.83 2.32∶1 10.47-4.51 1B
8 7.25 1.28
9 7.05 1.00
10 6.99 1.41
11 6.02 1.40
12 5.06 1.79
13 4.90 1.01
14 4.51 1.72
Fig.4　ChromosomemorphologyandkaryotypeofhybridF1
Meioticbehaviorofpolenmothercel
ItwasconcludedfromTable5 thatthefrequencieslag-
gingchromosomeofElymuscanadensisandElymussibiricus
were0.96%and2.76% respectively.Thechromosomeseg-
regationofF1 inmeioticanaphaseappearedabnormalbehav-
iorofandchromosomeshadlaggingphenomenon, whilethe
laggingchromosomeshadhighcelfrequency, whichwas
87.37%.Duringmeioticmetaphase, somechromosomesof
69LIJing-huanetal.GeneticAnalysisonChromosomeofHybridF1 betweenElymuscanadensisandElymussibiricus
F1 couldalsomatchtoformbivalent, howeverchromosome
pairingwasdisorderly, besidesthebarbivalentsweremany
andringbivalentswerefew, besides, themultivalentwasalso
appeared.
Table4　KaryotypeparametersofhybridF1
Numberof
chromosome
Relativelength
ofchromosome∥% Ratioofarm
Ratioofchromo-
somelength(L/S)
Relativelength
rangeofchromosome∥%
Karyotypeform-
ulaeandtype
1 8.83 1.00
2 8.83 1.50
3 8.83 3.33
4 8.39 1.11
5 7.95 1.00
6 7.95 1.25
7 7.60 1.00 2∶1 8.83-4.42 2n=4x=16M+10m+2st
8 7.09 1.67 2B
9 6.71 1.00
10 6.63 1.14
11 6.18 1.00
12 5.30 1.00
13 5.30 1.00
14 4.42 1.00
Thenumberintheeachfiguremeansthenumberoflaggingchromosomes.
Fig.5　LaggingchromosomesofinhybridF1
Table5　Frequencyoflaggingchromosomesinhybrids
Materials Numberofcels Celnumberoflagg-ingchromosomes
Numberoflagging
chromosomes
Frequencyoflagging-
chromosomecels∥%
E.canadensis 126 5 0-1 3.96
E.sibiricus 110 3 0-1 2.76
F1 generation 103 90 0-6 87.37
ConclusionsandDiscussions
(1)TheElymuscanadensisandElymussibiricusareal-
lotetraploidinElymusL., sotheyarefaringeneticrelation-
shipandtheyaredistantspeciesgeographicaly.Throughdis-
tanthybridization, thenewspeciesofElymusL.canbecrea-
tedandthisprovidesbreedingmaterialsforutilizingheterosis
andnewspeciesbreeding.
(2)ThechromosomesofElymuscanadensisareconsis-
tedofmetacentricchromosomesandchromosomekaryotype
formulais2n=4x=28m.AccordingtoStebbinsclassification,
Elymuscanadensisbelongsto1Atype.Thechromosomekar-
yotypeformulaofElymussibiricusis2n=4x=22m+6smand
itbelongsto1BtypeaccordingtoStebbinsclassification.The
chromosomeofF1 is2n=4x=28 anditskaryotypeformulais
2n=4x=16M+10m+2st.
(3)ItispreliminarilyconcludedthatF1 istruehybridac-cordingtomorphologyanalysisandchromosomekaryotype
analysis, however, F1 isnotfruited.Thereasonsfornon-fruitingofplantdistanthybridizationF1 iscausedbythedifer-
70 AgriculturalScience＆TechnologyVol.10, No.5, 2009
enceofparentalsetofchromosome[ 9].Inmeioticmeta-
phase, chromosomescannotbeconnectednormalytoform
univalentandmultivalent, whileinmeioticanaphase, thelag-
gingchromosomesareshownupandthenormalgametesare
produced, sotheinfertilityisexisted[ 10-11] , whichhasbeen
provedbythisexperiment.
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Responsibleeditor:CHENXiu-chen　　　　Responsibletranslator:LIZhu-le　　　　Responsibleproofreader:WUXiao-yan
加拿大披碱草与老芒麦种间杂种 F1 的染色体遗传分析
李景环 1, 2 ,云锦凤 1＊ ,都日斯哈拉2 ,邢婧民 2　
(1.内蒙古农业大学生态环境学院 ,内蒙古呼和浩特 010019;2.内蒙古师范大学生命科学与技术学院 ,内蒙古呼和浩特 010022)
摘要　对加拿大披碱草与老芒麦种间杂种F1的形态学和细胞学特征进行研究。形态学观察结果表明 ,杂种F1的穗形介于双亲之间 ,呈半弯
曲形 ,其母本加拿大披碱草植株浅绿色 ,茎粗糙 ,穗粗大而弯曲 ,每节 2～ 3小穗 ,颖和外稃粗糙具短毛。父本老芒麦全株粉绿色 ,穗状花序疏
松下垂 ,每节 2小穗 ,每小穗 3 ～ 5小花。杂种F1植株灰绿色 ,粗糙 ,外稃和颖具柔毛 ,偏向母本加拿大披碱草。加拿大披碱草染色体都是由
中部着丝粒染色体组成 ,染色体相对长度范围在 10～ 4.65,最长染色体与最短染色体之比为 2.2∶1, 臂比介于 1.14～ 1.35。染色体核型公式
为 2n=4x=28m,属于 1A类型。老芒麦染色体核型公式为 2n=4x=22m+6sm,属于 1B型;F1的染色体数为 2n=4x=28,染色体相对长度的
变异范围在 8.83～ 4.42,臂比值变化在 1.00 ～ 3.33。其中 ,第 1、5、7、9、11、12、13、14对染色体为正中着丝粒(M)染色体 ,第 3对为近端着丝粒
染色体 ,剩下的染色体为中部着丝粒染色体 ,其中 ,正中着丝粒(M)染色体的数目增加 , 近中着丝粒染色体(sm)已不存在。L/S恰好为 2∶1,
核型类型为 2B。核型公式为 2n= 4x=16M+10m+2st。通过形态学和染色体的核型分析可以初步确定 , F1 是真杂种;但杂种F1不结实。加拿大披碱草和老芒麦含有落后染色体细胞的频率较低 ,分别为 0.96%和 2.76%;F1 减数分裂后期染色体的分配出现了异常行为 ,染色体有
落后现象 ,落后的染色体细胞频率较高 ,达 87.37%, F1在减数分裂中期一部分染色体也能配对形成二价体 ,但染色体配对杂乱无章 ,棒状二价体占多数 ,环状二价体较少 ,且出现多价体。
关键词　加拿大披碱草;老芒麦;远缘杂种;染色体
基金项目　十一五科技支撑项目 ,牧草远缘杂交育种技术研究(2008BADB3B02);内蒙古自治区重大科技项目 , “优质全价草产品开发”。
作者简介　李景环(1972-),女,内蒙古赤峰人,博士 ,讲师,从事植物分子生物学的教学与科研工作和牧草育种研究。 ＊通讯作者 ,教授,博士生导师。
收稿日期　 2009-08-17　　修回日期　 2009-09-04
71LIJing-huanetal.GeneticAnalysisonChromosomeofHybridF1 betweenElymuscanadensisandElymussibiricus