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Orthogonal Optimization of ISSR Reaction System for Chickpea

鹰嘴豆ISSR反应体系的正交优化



全 文 :!23" !6#
 Vol.23  No.6
! " # $
ACTA AGRESTIA SINICA
   2015$  11%
  Nov.  2015
犱狅犻:10.11733/j.issn.10070435.2015.06.025
cde犐犛犛犚fJ6Ç@g7Þ£
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=L`?95ªs€çDE=n,¥¦ £1730070)
QR

³ëî
(犆犻犮犲狉犪狉犻犲狋犻狀狌犿)ISSRXnÓIlóôs˜¾³ëîJòBCçV|}~x£、çVûçbóeZ
%oÌÂÃ@¢gYe®]Ö¤

§DE˜CTABòÂul³ëîDNA¾OÇ,©¢L16(45)vÔùxMx£
ò

h³ëîISSRPCRXn=l4[Š‹%w(Mg2+dÐ,PdÐ,TaqDNA&°†dÐ,dNTPsdÐ)”4[
Œã—»¼z]

”ßzISSRPCRXnÓIlZø—hP UBC826lßu\´tл¼%±。ÆÇÈÉ:
Mg2+ePdÐhPCRНÆÇÙ!"ém,dNTPseTaq†ldÐk]hPCRНÆÇy!"ém。³ë
îISSRPCRz]XnÓI(25μL)¾:3mmol·L
-1 Mg2+,0.2mmol·L-1dNTP,0.24μmol·L
-1

,2UTaq
DNA&°†。UBC826ßu\´tо56℃。
STU

³ëî
;ISSR;XnÓI;vÔù;z]
VWXYZ
:Q341    [\]^_:A     [`aZ:10070435(2015)06130307
犗狉狋犺狅犵狅狀犪犾犗狆狋犻犿犻狕犪狋犻狅狀狅犳犐犛犛犚犚犲犪犮狋犻狅狀犛狔狊狋犲犿犳狅狉犆犺犻犮犽狆犲犪
JINXiaoli,TIANXinhui,DU Wenhua
(ColegeofGrasslandScience,GansuAgriculturalUniversity/KeyLaboratoryofGrasslandEcosystem,MinistryofEducation/
SinoU.SCentersforSustainableDevelopmentofGrasslandandAnimalHusbandry,Lanzhou,GansuProvince730070,China)
犃犫狊狋狉犪犮狋:TheestablishmentofthechickpeaISSRreactionsystem wouldprovidetheoreticalbasesand
technicalreferencesfortheanalysisofchickpeadiversity,theconstructionofthegenemapandthelocali
zationofimportanttraitsforchickpeagenotypesusingISSR markers.TheDNAtemplateforchickpea
(犆犻犮犲狉犪狉犻犲狋犻狀狌犿)wasextractedusingCTABmethod.AnISSRPCRamplificationsystemforchickpea
wasoptimizedbyvisualanalysisandanorthogonaldesignL16(45)involving4levelsof4mainfactors(con
centrationsofMg2+,Primer,TaqDNApolymerase,anddNTPs).Theannealingtemperatureofthe
primerUBC826wasoptimizedbasedontheoptimalISSRPCRamplificationsystem.Theresultsindicated
thattheconcentrationsofMg2+andprimeraffectedthePCRamplificationsignificantly,whiletheconcen
trationsofdNTPsandTaqDNApolymerasehadnoeffectsonit.ThesuitableISSRPCRamplification
system (25μL)forchickpeaincluded3mmol·L
-1Mg2+,0.2mmol·L-1dNTP,0.24μmol·L
-1primer
and2UTaqDNApolymerase.ThesuitableannealingtemperaturefortheprimerUBC826was56℃.
犓犲狔狑狅狉犱狊:Chickpea;ISSR;Reactionsystem;Orthogonaldesign;Optimization
CDEF
:20140707;GHEF:20141214
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(1986),¼,ÂÅ‘’“ Authorforcorrespondence,Email:duwh@gsau.edu.cn
  ³ëî(犆犻犮犲狉犪狉犻犲狋犻狀狌犿 L.)ð²^!Á(î
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² ^ J Ü Ý º Œ 1980 $ l 500
kg·hm-2Â^æ2007$l798kg·hm-2。tY
ݺÙFÂ^

¢A(î
(犌犾狔犮犻狀犲犿犪狓犮 Merr)
(1468.7kg·hm-2)Mo,³ëîJÜݺ‡ðM
hds
[1]。
+y³ëîJòlçVkê#~eŠÞ
=IÒÇ

َH»¼þJˆe<)±˜ZԊ
§
[2]。
Ê \ x Ü G  ) l ú «
,RAPD[3],
RFLP[46],STMSs[710]eSSR[11]xÜ:YQ¡¢n
¢H³ëîþJŽ2¶çV|}~vw¡ˆ‰

Ahmad¡[12]Ž¢SSReRAPDxÜ:Y,Æ°³
ëîJòl¥H#$eSDSPAGEG]x£,hç
V|}~»¼+x£
。ISSR:Y(InterSimpleSe
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quenceRepeat,mÆQY†Î–ùН)ðs˶
(©#Žß()Zietkiewicz¡[13]H1994$ÂS
l

ð”SSR(SimpleSequenceRepeat,mÆQY†
Î

Zø—qól

ZHPCRНlòJ_xÜ
:Y®]
。Ratnaparke¡[1415]¦4Ž¢ISSR:Y
oÌ+³ëîläT¨YZ%

Ê>
,Iruela¡[16]
eChowdhury¡[17]x2cISSR®]¢HDE³
ëîl»]=IeG2£¤þJ¶ŠÞ=I

A
RAPD®]»¼+x£od。ÆÇÈÉ,ISSR¸È
aS+d^lDNA|H~,ý@o~¦、®]º’


ðŽçV|}~eJòBCGolٗÂ
O

YÌISSR®]Нçjtd RAPD:Y@
o

¢¤}™XnvweНU†k]

˜¶£¤
Jlém
[18]。
%Fóôò[u°H³ëîlISSR
PCRXnÓI,Â^ISSRx£ÆÇls9~eQ
Y~

c¾³ëîJòlxÜGoeçV|}~D
E¶oZø

v¨¡
[19]
H1998$¦4ÂS+v
ÔùxMx£ò

c°TJ)—lvÔùn
¢æPCRXnÓIz]=,l°¤•F%w¶žÔ
¯’¢

öٍ°T

Œ|%w=î±2æFé
m%wlߙŒã6°

AÆ%wùMo


ùþœ+,|¾@6°

öò4°T¥e„
æߙXnÓI

£ðs((r»Þù

ÌýeÎ
s°T¹¢
[20]。
Ç$q
,PCRvÔùxMx£
òQ”Æ ì  Ü
[21]、
Œ § Ç
[22]、
¦ œ
[23]、
  ¢
·
[24]
¡Ž—„æ¡¢n¢

ý•ô+d¾s
9lISSRPCRXnÓI。§DEŽ¢vÔ°T
ù

h³ëîISSRXnÓI TaqDNA&°†

、dNTPsdÐ、PdÐe Mg2+dÐ4[%
w

”4[Œã»¼z]x£,hPCRXnU†
=l\´tл¼¯ÐNT

˜#S„³ëîIS
SRXnÓI,¾â>Ž¢ISSR®]»¼³ëî
JòBCvw

çV|}~DE

çVûçbóe
Z%o̶oZø

1 bcdef
1.1 ghbc
ð½)¾16º³ëîJò(È1),2011$
3%29\JŽH¥¦4ª()5`°Tµ,°T
Ê z ± u Jimbour¢ H ISSR Ó I z ],IC
CV05111¢HP\´tÐ%±。4%25\©X
Ô.³o

¢­¦å`jOSTU

úH-80℃1
‰=d¢

v1 µgcde¦Â9:Sž
Table1 Nameandoriginofchickpeagenotype
³ëîJò
Chickpeagenotype
qC
Sources
³ëîJò
Chickpeagenotype
qC
Sources
ICCV05111 :ÐIndia ICCV06108 :ÐIndia
ICCV98818 :ÐIndia Jimbour
p(ŽK
Australia
ICCV96852 :ÐIndia Howzat
p(ŽK
Australia
ICCV98801 :ÐIndia Bumper
p(ŽK
Australia
ICCV98816 :ÐIndia Jimbour#1
p(ŽK
Australia
ICCV06107 :ÐIndia Flipper
p(ŽK
Australia
ICCV96853 :ÐIndia Yorker
p(ŽK
Australia
ICCV98813 :ÐIndia FLIP97-114C
p(ŽK
Australia
  Ô:JimboureJimbour#1ðŒ£¤?R°Xl¤-³ëîþ
J
Note:JimbourandJimbour#1arethesamechickpeavarieties
colectedfromdifferentlocations
1.2 ghhi{ˆRgj
PCRН9(BioRad-MyCycler,L>);¦^
¿À7ÄIJ
(UVPGDS08000,L>);Uð9
(JY300,/VÜ®zU®]DE)。ISSRP†Î
¾µHoK()
(UniversityofColumbia)D¹l†
Î

s—3G¿G®]DE°7
,TaqDNA&°
†
、dNTPse DNA Marker¸(XG¿G¿U

—3

ںٍDE

žÒ°X¾>Ýx£³

1.3 ghef
1.3.1 UVW DNA XYeßfZ¢ Z%6
DNAÂu©¢»lCTABò,ÂulDNA‘y
H100μLTE=d¢,˜3000bpDNA Marker+
:;

¢0.8%m脿ÀUðhZ%6DNAld
Ðeòº»¼[N

uZ%6DNA}þ3μLA
DNAMarker¤ÞUð,UðÆ]>ho DNAj
l4Ð

˜F‚ DNA }þldÐ,qÈ}þ
DNAdЕ25ng·μL
-1,-20℃å`。
1.3.2 ISSRUàáâDJ±´ã ZøXnU†
©¢³ëî!Žl25μLXnÓI
[16],
FXn
}dо
:2.5mmol·L-1 Mg2+,0.20mmol·L-1
dNTPs,0.24μmol·L
-1ISSRP,1UTaqDNA
4031
!6# ´[M¡:³ëîISSRXnÓIlvÔz]
&°†
,1×PCRBuffer。°T=OÇDNAdи
©¢25ng·μL
-1。PCRНU†¾:94℃·k~
2min;Y>94℃k~30s,50℃\´30s,72℃2min,354Í&;ß>H72℃

1.3.3 \‡äÇ Œsù±u2[¥Haêd(
l³ëîJò
(ICCV05111eJimbour#1)¢HP
%±

¢ISSRZøXnÓIeU†,ŒuöH³
ëî!l13vPUBC807,UBC809,UBC811,
UBC817,UBC821,UBC825,UBC826,UBC
834,UBC856,UBC858,UBC880,UBC884e
UBC885=,z‰%±S7vНvj²、|H
~|lP

x2¾ UBC807,UBC809,UBC
811,UBC817,UBC825,UBC826eUBC834,ž
=PUBC826(åZ†Î5′→3′¾(AC)8C)Èa
ß|

•ož¾vÔùl¼oP

1.3.4 PCRC‡ðåZ¢ PCRНݏ(8μL)
H2%m脿À=Uð[N,¢DM3000’:;x
ܺh-

UéË¿­¾1×TAE,UðÞù¾40
min。UðÆ]>”¿À7Ä9¦^¸ÍM¬-M。
1.3.5 ISSRáâæçèg ©¢L16(45)vÔ°
Tù

h Mg2+ dÐ、dNTPsdÐ、PdÐe
TaqDNA&°†dÐ4%w»¼4Œã%±。F
%wŒãóÈ2,°Tˆ„óÈ3,516[¾@,3
¾@24QY,hž»¼PCRXn,c„ælݏ
»¼Uð

×YНçj#ê~Aîï~

_çj


4Ц)eÙy¹Ñaw

h16[¾@24
QYlPCRݏUðÆÇx2vx,çj€|、4
Ѐ¦

y¹Ñawvx€^

Xñ€s
[19]。
vj
×ß|l¾@o¾16x,ߜlo¾1x,4Ðx
¾¦

=

)eèÙèy4[{2,x2­x0,-1,
-2,-3。
v2 cde犐犛犛犚犘犆犚fJ6Ç`+“½v
Table2 Factorsandlevelsforchickpea
ISSRPCRreactionsystem
΋
Level
Mg2+dÐ
Mg2+
concentration
/mmol·L-1
dNTPsdÐ
dNTPs
concentration
/mmol·L-1
PdÐ
Primer
concentration
/μmol·L-1
TaqDNA
&°†dÐ
TaqDNA
polymerase
concentration/U
1 1.5 0.1 0.12 0.5
2 2.0 0.15 0.24 1.0
3 2.5 0.2 0.36 1.5
4 3.0 0.25 0.48 2.0
v3 cde犐犛犛犚犘犆犚fJ6Ç犔16(45)g7gh&¬
Table3 OrthogonaldesignL16(45)for
chickpeaISSRPCRreactionsystem
¾@
Treatment
Mg2+dÐ
Mg2+
concentration
/mmol·L-1
dNTPsdÐ
dNTPs
concentration
/mmol·L-1
PdÐ
Primer
concentration
/μmol·L-1
TaqDNA
&°†dÐ
TaqDNA
polymerase
concentration/U
1 1.5 0.10 0.12 0.5
2 1.5 0.15 0.24 1.0
3 1.5 0.20 0.36 1.5
4 1.5 0.25 0.48 2.0
5 2.0 0.10 0.24 1.5
6 2.0 0.15 0.12 2.0
7 2.0 0.20 0.48 0.5
8 2.0 0.25 0.36 1.0
9 2.5 0.10 0.36 2.0
10 2.5 0.15 0.48 1.5
11 2.5 0.20 0.12 1.0
12 2.5 0.25 0.24 0.5
13 3.0 0.10 0.48 1.0
14 3.0 0.15 0.36 0.5
15 3.0 0.20 0.24 2.0
16 3.0 0.25 0.12 1.5
1.3.6 \‡éêëªäÇ ×YvÔ°TÆÇ,”
ߙXnÓIlZø—

hz‰%±Sl7vPl
\´tл¼¯Ð°T

•oFPlßu\´t
Ð

×YP Tm I,ùo\´tо47~56℃,
PCR9XDG78[tЯÐ:47,47.6,48.7,50.3,
52.4,54.1,55.3,56℃。
1.3.7 ISSRPCRáâìèDJ]@í ©¢z
]>lISSRPCRXnÓI,¢z‰%±l7vP
h16º³ëîJò»¼Ð,[TISSRPCRX
nÓIl@o~

1.4 ‚ƒX„
©¢Photoshophûo»¼:Y,Excel2003
»¼×YÈ@

¢SPSS16.0=l Generallinear
modelhISSRz]ÓIF%wŒãvxÆÇ»¼
ˆax£

¢Duncanòhž»¼!"~x£。
2 …†dX„
2.1 g7&¬¶î…†ókÙX
‡-È3ùl16[¾@»¼ PCRXn
>

c„ælݏ»¼Uð
,16[¾@24QY
lPCRݏUðÆÇ0û1F6。û1=F¾
@lxMvxÆÇ0È4F6,¾@15vxß
5031
! " # $ !23"
^

ž4ð¾@14,•o¾@15¾ISSRXnß ™ÓI。
W1 cde犐犛犛犚fJ6Çg7&¬gh…†(æ>犝犅犆826)
Fig.1 TheelectrophoresisresultsfortheISSRPCRorthogonaldesign(PrimerUBC826)
Ô

WX116x2hnÈ3=F¾@6°,M:3000bpDNA
Note:Numbers116areconsistenttothetreatmentnumberintable3,M:3000bpDNA
v4 cde犐犛犛犚fJ6Çg7gh&¬ÙX…†
Table4 Gradingresultsoftheorthogonaldesignsystem
¾@ Treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
QY1Replication1 1 6 4 3 10 4 5 7 5 10 2 10 5 12 13 1
QY2Replication2 1 3 3 1 10 4 5 2 7 7 1 1 6 12 13 7
2.2 È`+/PCRfJ@AB
vxÆÇlˆax£ÈÉ

F%whPCRX
némŒ(æg4¾
:Mg2+ dÐ、PdÐ、
dNTPsdÐeTaqDNA&°†dÐ。Mg2+eP
dÐhXnÆÇÙé!"ém
(犘<0.01),
dNTPseTaqDNA&°†dÐhXnÆÇy!
"ém

W2 cde犐犛犛犚fJ6Ç 犕犵2+
µdÙX…†¾¿@SÇ
Fig.2 Relationshipbetweentheconcentrationof
Mg2+andthemeanvalueofgradingresult
2.2.1 Mg2+îª l Mg2+dо3mmol·L-1
Þ

vxÆǸIåæß(I

!"^HžÒdÐ
¯Ð

dо1.5mmol·L-1Þ¸Iߝ;2e2.5
mmol·L-1edÐùy!"aê(û2)。”НÆ
Çûç

û1)=,l Mg2+dо1.5mmol·L-1

¾@1~4)Þ,ñ8yНvj\Нvjd)、
4У’

dо2~2.5mmol·L-1Þ(¾@5~
12),vjם|,4НË,oO(@o”900~
2000bp ñ ù,¢ v j  ¾ d |;d Ð ¾ 3
mmol·L-1(¾@13~16)Þ,vjß¾Š‹²Î,
НoO(”900~3000bpñù。%F,”25
μLXnÓI=±Ë 3 mmol·L
-1
’¾³ëî
ISSRPCRXn= Mg2+lߙdÐ。
W3 cde犐犛犛犚fJ6Çæ>
µdÙX…†¾¿@SÇ
Fig.3 Relationshipbetweentheconcentrationof
primerandthemeanvalueofgradingresult
2.2.2 \‡îª %wŸ|QodÆÇÈÉ,P
dо0.24μmol·L
-1
ÞvxÆÇß^

A0.12
e0.48μmol·L
-1
dÐùaê!"

ž4ð0.36
μmol·L
-1,
!"^H0.12μmol·L
-1(
û3)。”
6031
!6# ´[M¡:³ëîISSRXnÓIlvÔz]
Ð  Æ Ç û ç

û 1)=,P  d Ð ¾0.12
μmol·L
-1(
¾@1,6,11,16)Þ,yНvj\vj


eM¬ælvj”1000~3000bpñù;dÐ
¾0.48μmol·L
-1(
¾@4,7,10,13)Þ,vj¾,
Oº£²

ÙT¾@eM¬æ900bp69loO,
1000bp ˜ — l o O v j d );d Ð ¾ 0.24
μmol·L
-1(
¾@2,5,12,15)Þ,vjŠ‹、4Ðd
(

dо0.36μmol·L
-1(
¾@3,8,9,16)Þ,4
Ð(

¢|H~þ)

%F
,25μlXnÓI=±Ë
0.24μmol·L
-1
’¾³ëîISSRPCRXn=P
lߙdÐ

2.3 ¢l$/cdeISSRfJ6Ç@AB
PUBC826l\´tо55.3~56℃Þ,Ð
—Çd|

\´tÐsH54.1℃Þ,Ê\tÐÎ
s

vj|H~jkþ)

4ÐÎsýôõk„Oº
£²

û4)。ö°Tß}•oPUBC826lß
u\´tо56℃。§°T%±Slu°³ëî
ISSRPCRXnÓIlP¶žß™\´tÐ0È
5F6,£¤Plߙ\´tУ¤,UBC807l
\´tÐds
(47℃),UBC817e UBC826\´t
Ðd^
(56℃),Ì UBC809l\´tÐѾHÁ“
ñù

FPlߙ\´tÐeTmIùyÉ!l
ÚÛ

W4 ¢l$/cde
犐犛犛犚犘犆犚é#…†@AB(æ>犝犅犆826)
Fig.4 Effectsofannealingtemperatureon
ISSRamplification(PrimerUBC826)
Ô

û=18È6\´tÐ,
1:56℃,2:55.3℃,3:54.1℃,4:52.4℃,
5:50.3℃,6:48.7℃,7:47.6℃,8:47℃
Note:Thenumber1to8means
theannealingtemperature,
1:56℃,2:55.3℃,3:54.1℃,4:52.4℃,
5:50.3℃,6:48.7℃,7:47.6℃,8:47℃
v5 cde犐犛犛犚fJõxæ>9:³´¢l$
Table5 Theoptimalannealingtemperatureof
selectedprimersforISSR

Primer
†Î
(5′→3′)
Sequence(5′→3′)
ߙ\´tÐ
Optimizedannealing
temperature/℃
TmI
Tmvalue
/℃
UBC807 (AG)8T 47.0 49.0
UBC809 (AG)8G 52.4 49.9
UBC811 (GA)8C 47.6 48.9
UBC817 (CA)8T 56.0 52.5
UBC825 (AC)8T 47.6 54.0
UBC826 (AC)8C 56.0 55.3
UBC834 (AG)8YT 47.6 50.3
2.4 ISSRPCRfJ6Ç@hm
”z]>lISSRXnÓIlZø—,¢z‰
%±l7vPx2h16º³ëîJò»¼Ð,
¸eНS²Î

Š‹lvj

P UBC809h16
º³ëîJòlНûç0û5F6。þPeÉ
!–xSICCV98818,JimboureFLIP97114C¡


|H~o²å66.67%。sFÈÉ,z]>l
ISSRPCRXnÓI@os9,s¢H³ëîJò
BClçV|}~x£

W5 Þ£,@犐犛犛犚犘犆犚fJ6Ç/
16Ûcde¦Â@犐犛犛犚é#…†(æ>犝犅犆809)
Fig.5 Amplicationresultsof16chickpeagenotypesusing
theoptimizedISSRPCRreactionsystem(primerUBC809)
Ô
:1ICCV05111;2ICCV98818;3ICCV96852;
4ICCV98801;5ICCV98816;6ICCV06107;7ICCV96853;
8ICCV98813;9ICCV06108;10Jimbour;11Howzat;12Bumper;
13Jimbour#1;14Flipper;15Yorker;16FLIP97114C
3™š
  Ž¢vÔùxMx£òhPCRXnÓIz
]lôd|

¢(|×DEðcß|eßa¾@
’¾:;

£cžÒ¾@lçjÆÇAIe[:;
od

g4­x
[20,2326],
IJˆòŠM~d¦

£e
f|l‚°T_a
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›[[1] FAOSTAT_FAO2009AgriculturalProductionDomain.Last
updateJune11,2008
[2] MehmetAS.InterandintraspeciesInterSimpleSequence
8031
!6# ´[M¡:³ëîISSRXnÓIlvÔz]
Repeat(ISSR)variationsinthegenus犆犻犮犲狉[J].Euphytica,
2004,135(2):229238
[3] TalebiR,FayazF,MardiM,犲狋犪犾.Geneticrelationshipsamong
chickpea(犆犻犮犲狉犪狉犻犲狋犻狀狌犿)elitelinebasedonRAPDandagro
nomicmarkers[J].InternationalJournalofAgricultureand
Biology,2008,10(3):301305
[4] WeisingK,KaemmerD,WeigandF,犲狋犪犾.Oligonucleotidefin
gerprintingrevealsvariousprobedependentlevelsofinforma
tivenessinchickpea(犆犻犮犲狉犪狉犻犲狋犻狀狌犿)[J].Genome,1992,35
(3):436442
[5] SharmaPC,WinterP,BüngerT,犲狋犪犾.Abundanceandpoly
morphismofdi,tri,andtetranucleotidetandemrepeatsin
chickpea(犆犻犮犲狉犪狉犻犲狋犻狀狌犿 L.)[J].TheoreticalandApplied
Genetics,1995,90(1):9096
[6] SerretMD,UdupaSM,WeigandF.Assessmentofgeneticdi
versity ofcultivated chickpea using microsatelitederived
RFLPmarkers:implicationsfororigin[J].PlantBreeding,
1997,116(6):573578
[7] SantVJ,PatankarAG,SarodeND,犲狋犪犾.PotentialofDNA
markersindetectingdivergenceandinanalyzinghysteresisin
Indianelitechickpeacultivars[J].TheoreticalandAppliedGe
netics,1999,98(8):12171225
[8] UdupaSM,RobertsonLD,WeigandF,犲狋犪犾.Alelicvariation
at(TAA)nmicrosatelitelociinaworldcolectionofchickpea
(犆犻犮犲狉犪狉犻犲狋犻狀狌犿 L.)germplasm[J].MolecularandGeneral
Genetics,1999,261(2):354363
[9] WinterP,PfaffT,UdupaS M,犲狋犪犾.Characterizationand
mappingofsequencetaggedmicrosatelitesitesinthechickpea
(犆犻犮犲狉犪狉犻犲狋犻狀狌犿L.)genome[J].MolecularandGeneralGe
netics,1999,262(1):90101
[10]WinterP,BenkolsepponAM,HüttelB,犲狋犪犾.Alinkagemap
ofthechickpea(犆犻犮犲狉犪狉犻犲狋犻狀狌犿L.)genomebasedonrecom
binantinbredlinesfromaC.犪狉犻犲狋犻狀狌犿andC.狉犲狋犻犮狌犾犪狋狌犿
cross:localizationofresistancegenesforfusariumwiltraces4
and5[J].TheoreticalandAppliedGenetics,2000,101(7):
11551163
[11]CastroP,MilanT,GilJ,犲狋犪犾.Identificationofchickpeaculti
varsbymicrosatelitemarkers[J].JournalofAgriculturalSci
ence,2011,149(4):451460
[12]AhmadZ,MumtazAS,NisarM,犲狋犪犾.Diversityanalysisof
chickpea(犆犻犮犲狉犪狉犻犲狋犻狀狌犿L.)germplasmanditsimplications
forconservationandcropbreeding[J].AgriculturalSciences,
2012,3(5):723731
[13]ZietkiewiczE.Genomefingerprintingbysimplesequencere
peat(SSR)anchoredpolymeraseChainreactionamplification
[J].Genomes,1994,20(2):176183
[14]RatnaparkheMB,SantraDK,TuluA,犲狋犪犾.Inheritanceof
intersimplesequencerepeatpolymorphismsandlinkagewitha
fusariumwiltresistancegeneinchickpea[J].Theoreticaland
AppliedGenetics,1998,96(34):348353
[15]RatnaparkheMB,TekeogluM,MuehlbauerFJ.Intersimple
sequencerepeat(ISSR)polymorphismsareusefulforfinding
markersassociatedwithdiseaseresistancegeneclusters[J].
TheoreticalandAppliedGenetics,1998,97(4):515519
[16]IruelaM,RubioJ,CuberoJI,犲狋犪犾.Phylogeneticanalysisin
thegenus犆犻犮犲狉andcultivatedchickpeausingRAPDandISSR
markers[J].TheoreticalandAppliedGenetics,2002,104(4):
643651
[17]ChowdhuryM A,VandenbergBert,WarkentinT.Cultivari
dentificationandgeneticrelationshipamongselectedbreeding
linesandcultivarsinchickpea(犆犻犮犲狉犪狉犻犲狋犻狀狌犿 L.)[J].Eu
phytica,2002,127(3):317325
[18]$óG.ISSRxÜ:Y¶ž”ŽçV)DE=ln¢[J].
çV
,2002,24(5):613616
[19]v¨,ï“G,äô³,¡.vÔùxMx£òz]PCR
vw
[J]..Å;<()),1998,23(4):403404
[20]8(,DF,5¼,¡.½¾!ŽISSRXnÓIlóôez
]
[J].Çý),2009,26(2):180185
[21]$Ò³,ž:,,TÉX.vÔùz]£ÆìÜISSRXn
ÓIDE
[J].6/Ž),2004,24(5):899902
[22]O“3,)C<,Šç,¡.Ž¢vÔùz]Œ§ÇISSR
PCRXnÓI[J].x܎±J,2005,3(3):445450
[23]}ôÄ,ᅡ,f‹M,¡.¦œISSRPCRXnÓIlóô
Az]
[J].,ª<),2006,42(6):2631
[24]¤F,Ñ[J].x܎±J,2009,7(6):12371224
[25]¡ÆÉ,µ7,€(¦,¡.5ýISSRXnÓIlóô¶vÔ
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[27]ï™,á*,H1.vÔùz]¦ÁÂ<ŽlISSRPCR
XnÓI
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[28]Lfæ,Fî,Eïœ,¡.vÔùz]ǖISSRXnÓI
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[29]ïãG,f¦ç,5óœ,¡.·ISSRXnÓIlz]eÄ´
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[30]Šç,5Vã,ïLŠ,¡.6ýISSRPCRXnÓIlz]
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[31]3(,á*,ž7Ã.(îISSRPCRXnÓIlz][J].
(î<)
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[32]OÄÒ,ÞLe,5=+.?ç`ISSRXnÓIlóôAz]
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[33]ëôF,l],lK6,¡.ƒ„#ISSRxÜ:Y®]lóô
AÓIz]
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[34]lM,1?G.);`ISSRxÜ:Y®]lóôeÓIz]
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[35]7ç,Áó<,+M,¡.¸ýISSRPCRXnÓIlvÔz]
[J]..Å4ª())(XY<)C),2010,36(4):414417
[36]ïÿ,Åã,RLæ,¡.(ÎISSRXnÓIlóôAz]
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