全 文 :!23" !6#
Vol.23 No.6
! " # $
ACTA AGRESTIA SINICA
2015$ 11%
Nov. 2015
犱狅犻:10.11733/j.issn.10070435.2015.06.025
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(犆犻犮犲狉犪狉犻犲狋犻狀狌犿)ISSRXnÓIlóôs¾³ëîJòBCçV|}~x£、çVûçbóeZ
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§DECTABòÂul³ëîDNA¾OÇ,©¢L16(45)vÔùxMx£
ò
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h³ëîISSRPCRXn=l4[%w(Mg2+dÐ,PdÐ,TaqDNA&°dÐ,dNTPsdÐ)4[
㻼z]
,
ßzISSRPCRXnÓIlZøhP UBC826lßu\´tл¼%±。ÆÇÈÉ:
Mg2+ePdÐhPCRÐÆÇÙ!"ém,dNTPseTaqldÐk]hPCRÐÆÇy!"ém。³ë
îISSRPCRz]XnÓI(25μL)¾:3mmol·L
-1 Mg2+,0.2mmol·L-1dNTP,0.24μmol·L
-1
P
,2UTaq
DNA&°。UBC826ßu\´tо56℃。
STU
:
³ëî
;ISSR;XnÓI;vÔù;z]
VWXYZ
:Q341 [\]^_:A [`aZ:10070435(2015)06130307
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
JINXiaoli,TIANXinhui,DU Wenhua
(ColegeofGrasslandScience,GansuAgriculturalUniversity/KeyLaboratoryofGrasslandEcosystem,MinistryofEducation/
SinoU.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.AnISSRPCRamplificationsystemforchickpea
wasoptimizedbyvisualanalysisandanorthogonaldesignL16(45)involving4levelsof4mainfactors(con
centrationsofMg2+,Primer,TaqDNApolymerase,anddNTPs).Theannealingtemperatureofthe
primerUBC826wasoptimizedbasedontheoptimalISSRPCRamplificationsystem.Theresultsindicated
thattheconcentrationsofMg2+andprimeraffectedthePCRamplificationsignificantly,whiletheconcen
trationsofdNTPsandTaqDNApolymerasehadnoeffectsonit.ThesuitableISSRPCRamplification
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
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Table1 Nameandoriginofchickpeagenotype
³ëîJò
Chickpeagenotype
qC
Sources
³ëîJò
Chickpeagenotype
qC
Sources
ICCV05111 :ÐIndia ICCV06108 :ÐIndia
ICCV98818 :ÐIndia Jimbour
p(K
Australia
ICCV96852 :ÐIndia Howzat
p(K
Australia
ICCV98801 :ÐIndia Bumper
p(K
Australia
ICCV98816 :ÐIndia Jimbour#1
p(K
Australia
ICCV06107 :ÐIndia Flipper
p(K
Australia
ICCV96853 :ÐIndia Yorker
p(K
Australia
ICCV98813 :ÐIndia FLIP97-114C
p(K
Australia
Ô:JimboureJimbour#1ð£¤?R°Xl¤-³ëîþ
J
Note:JimbourandJimbour#1arethesamechickpeavarieties
colectedfromdifferentlocations
1.2 ghhi{Rgj
PCRÐ9(BioRad-MyCycler,L>);¦^
¿À7ÄIJ
(UVPGDS08000,L>);Uð9
(JY300,/VÜ®zU®]DE)。ISSRPÎ
¾µHoK()
(UniversityofColumbia)D¹l
Î
,
s3G¿G®]DE°7
,TaqDNA&°
、dNTPse DNA Marker¸(XG¿G¿U
(
3
)
ÚºÙDE
,
Ò°X¾>Ýx£³
。
1.3 ghef
1.3.1 UVW DNA XYeßfZ¢ Z%6
DNAÂu©¢»lCTABò,ÂulDNAy
H100μLTE=d¢,3000bpDNA Marker+
:;
,
¢0.8%mè¿ÀUðhZ%6DNAld
Ðeòº»¼[N
。
uZ%6DNA}þ3μLA
DNAMarker¤ÞUð,UðÆ]>ho DNAj
l4Ð
,
F DNA }þldÐ,qÈ}þ
DNAdÐ25ng·μL
-1,-20℃å`。
1.3.2 ISSRUàáâDJ±´ã ZøXnU
©¢³ëî!l25μLXnÓI
[16],
FXn
}dо
:2.5mmol·L-1 Mg2+,0.20mmol·L-1
dNTPs,0.24μmol·L
-1ISSRP,1UTaqDNA
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&°
,1×PCRBuffer。°T=OÇDNAdи
©¢25ng·μL
-1。PCRÐU¾:94℃·k~
2min;Y>94℃k~30s,50℃\´30s,72℃
。
1.3.3 \äÇ sù±u2[¥Haêd(
l³ëîJò
(ICCV05111eJimbour#1)¢HP
%±
,
¢ISSRZøXnÓIeU,uöH³
ëî!l13vPUBC807,UBC809,UBC811,
UBC817,UBC821,UBC825,UBC826,UBC
834,UBC856,UBC858,UBC880,UBC884e
UBC885=,z%±S7vÐvj²、|H
~|lP
,
x2¾ UBC807,UBC809,UBC
811,UBC817,UBC825,UBC826eUBC834,
=PUBC826(åZÎ5′→3′¾(AC)8C)Èa
ß|
,
o¾vÔùl¼oP
。
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H2%mè¿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、PdÐe
TaqDNA&°dÐ4%w»¼4ã%±。F
%wãóÈ2,°TóÈ3,516[¾@,3
¾@24QY,h»¼PCRXn,cælÝ
»¼Uð
。
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×ß|l¾@o¾16x,ßlo¾1x,4Ðx
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Table2 Factorsandlevelsforchickpea
ISSRPCRreactionsystem
ã
Level
Mg2+dÐ
Mg2+
concentration
/mmol·L-1
dNTPsdÐ
dNTPs
concentration
/mmol·L-1
PdÐ
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
chickpeaISSRPCRreactionsystem
¾@
Treatment
Mg2+dÐ
Mg2+
concentration
/mmol·L-1
dNTPsdÐ
dNTPs
concentration
/mmol·L-1
PdÐ
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,
ßXnÓIlZø
,
hz%±Sl7vPl
\´tл¼¯Ð°T
,
oFPlßu\´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 ISSRPCRáâìèDJ]@í ©¢z
]>lISSRPCRXnÓI,¢z%±l7vP
h16º³ëîJò»¼Ð,[TISSRPCRX
nÓIl@o~
。
1.4 X
©¢Photoshophûo»¼:Y,Excel2003
»¼×YÈ@
,
¢SPSS16.0=l Generallinear
modelhISSRz]ÓIF%wãvxÆÇ»¼
ax£
,
¢Duncanòh»¼!"~x£。
2
dX
2.1 g7&¬¶î
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-È3ùl16[¾@»¼ PCRXn
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cælÝ»¼Uð
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lPCRÝUðÆÇ0û1F6。û1=F¾
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W1 cde犐犛犛犚fJ6Çg7&¬gh
(æ>犝犅犆826)
Fig.1 TheelectrophoresisresultsfortheISSRPCRorthogonaldesign(PrimerUBC826)
Ô
:
WX116x2hnÈ3=F¾@6°,M:3000bpDNA
Note:Numbers116areconsistenttothetreatmentnumberintable3,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
vxÆÇlax£ÈÉ
,
F%whPCRX
ném(æg4¾
:Mg2+ dÐ、PdÐ、
dNTPsdÐeTaqDNA&°dÐ。Mg2+eP
dÐhXnÆÇÙé!"ém
(犘<0.01),
dNTPseTaqDNA&°dÐhXnÆÇy!
"ém
。
W2 cde犐犛犛犚fJ6Ç 犕犵2+
µdÙX
¾¿@SÇ
Fig.2 Relationshipbetweentheconcentrationof
Mg2+andthemeanvalueofgradingresult
2.2.1 Mg2+îª l Mg2+dо3mmol·L-1
Þ
,
vxÆǸIåæß(I
,
!"^HÒdÐ
¯Ð
;
dо1.5mmol·L-1Þ¸Iß;2e2.5
mmol·L-1edÐùy!"aê(û2)。ÐÆ
Çûç
(
û1)=,l Mg2+dо1.5mmol·L-1
(
¾@1~4)Þ,ñ8yÐvj\Ðvjd)、
4У
;
dо2~2.5mmol·L-1Þ(¾@5~
12),vj×|,4ÐË,oO(@o900~
2000bp ñ ù,¢ v j ¾ d |;d Ð ¾ 3
mmol·L-1(¾@13~16)Þ,vjß¾²Î,
ÐoO(900~3000bpñù。%F,25
μLXnÓI=±Ë 3 mmol·L
-1
¾³ëî
ISSRPCRXn= Mg2+lßdÐ。
W3 cde犐犛犛犚fJ6Çæ>
µdÙX
¾¿@SÇ
Fig.3 Relationshipbetweentheconcentrationof
primerandthemeanvalueofgradingresult
2.2.2 \îª %w|QodÆÇÈÉ,P
dо0.24μmol·L
-1
ÞvxÆÇß^
,
A0.12
e0.48μmol·L
-1
dÐùaê!"
,
4ð0.36
μmol·L
-1,
!"^H0.12μmol·L
-1(
û3)。
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Ð Æ Ç û ç
(
û 1)=,P d Ð ¾0.12
μmol·L
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¾0.48μmol·L
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¾@4,7,10,13)Þ,vj¾,
Oº£²
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ÙT¾@eM¬æ900bp69loO,
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Ð(
,
¢|H~þ)
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%F
,25μlXnÓI=±Ë
0.24μmol·L
-1
¾³ëîISSRPCRXn=P
lßdÐ
。
2.3 ¢l$/cdeISSRfJ6Ç@AB
PUBC826l\´tо55.3~56℃Þ,Ð
Çd|
;
\´tÐsH54.1℃Þ,Ê\tÐÎ
s
,
vj|H~jkþ)
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4ÐÎsýôõkOº
£²
(
û4)。ö°Tß}oPUBC826lß
u\´tо56℃。§°T%±Slu°³ëî
ISSRPCRXnÓIlP¶ß\´tÐ0È
5F6,£¤Plß\´tУ¤,UBC807l
\´tÐds
(47℃),UBC817e UBC826\´t
Ðd^
(56℃),Ì UBC809l\´tÐѾHÁ
ñù
。
FPlß\´tÐeTmIùyÉ!l
ÚÛ
。
W4 ¢l$/cde
犐犛犛犚犘犆犚é#
@AB(æ>犝犅犆826)
Fig.4 Effectsofannealingtemperatureon
ISSRamplification(PrimerUBC826)
Ô
:
û=18È6\´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
P
Primer
Î
(5′→3′)
Sequence(5′→3′)
ß\´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 ISSRPCRfJ6Ç@hm
z]>lISSRXnÓIlZø,¢z
%±l7vPx2h16º³ëîJò»¼Ð,
¸eÐS²Î
、
lvj
。
P UBC809h16
º³ëîJòlÐûç0û5F6。þPeÉ
!xSICCV98818,JimboureFLIP97114C¡
Jò
,
|H~o²å66.67%。sFÈÉ,z]>l
ISSRPCRXnÓI@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
theoptimizedISSRPCRreactionsystem(primerUBC809)
Ô
:1ICCV05111;2ICCV98818;3ICCV96852;
4ICCV98801;5ICCV98816;6ICCV06107;7ICCV96853;
8ICCV98813;9ICCV06108;10Jimbour;11Howzat;12Bumper;
13Jimbour#1;14Flipper;15Yorker;16FLIP97114C
3
¢vÔùxMx£òhPCRXnÓIz
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。
¢L16(45)vÔùból³ëîISSRPCRß
lXnÓI¾
:Mg2+3 mmol·L-1,dNTPs0.2
mmol·L-1,P0.24μmol·L
-1,TaqDNA&
°2U。
DNAOÇðISSRPCRXn=lò[Q%
w
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,
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s£¾QRz]hw
。
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¤F¡
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£¤
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。
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、
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,
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ém¾(
[25]。
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,
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,
h3vPlß\´tл¼%±øxÂ
,
ñ§DEh³ëîISSRPCRßXnÓIl
P\´tл¼+¯Ð°T
。
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,
sHÐl#ê~¦
,
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;
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,
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,
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çæÐ
,
¢£¤lPÐÆÇaêf(
,
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tÐsÞ
,
(xܺoO`ç%)jOº£²
,
XÌ
tÐd^Þ(xܺoOvjd²Î
[26]。
§°
T=
,
£¤Plß\´tÐ^saêf(
,
A
ÒDEÆÇò*
。
ïÿ¡h(ÎISSRXnÓI
lz]=úa
,
F±PÐS²ÎjÞl´tÐò9^H@¢TmI1~5℃[36],̧°T
=F±Plß\´tÐA@¢TmIñùyÉ
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,
A8(
[20]
e?»
[26]
lDEÆÇò*
,
se
A±ulPeDElJ1£¤Ù=
。
4
§DEövÔùh³ëîISSRXnÓI»
¼+z]
,
zóô+³ëîISSRPCRßlXn
ÓI
:Mg2+3mmol·L-1,dNTPs0.2mmol·L-1,P
0.24μmol·L
-1,TaqDNA&°2U,h»
¼+T
,
Éz]>lISSRPCRXnÓI@os
9
,
s¢H³ëîçV|}~x£
,
¤ÞÉ+vÔ
ù³ëîISSRÓIz]ls¼~。
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Az]
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[25]¡ÆÉ,µ7,(¦,¡.5ýISSRXnÓIlóô¶vÔ
ùz]
[J].4),2007,21(5):470473
[26]?»,$>²,_À.¦³`ISSRXnÓIlóôA
z]
[J].`ª),2011,20(5):142150
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[J].G®],2012,22(3):6165
[28]Lfæ,Fî,Eï,¡.vÔùz]ÇISSRXnÓI
[J].Çý),2009,26(1):108112
[29]ïãG,f¦ç,5ó,¡.·ISSRXnÓIlz]eÄ´
ûçlbó
[J].Çý),2005,22(6):626629
[30]ç,5Vã,ïL,¡.6ýISSRPCRXnÓIlz]
[J].GH)ZA,2003,22(3):9193
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(î<)
,2010,29(3):510513
[32]OÄÒ,ÞLe,5=+.?ç`ISSRXnÓIlóôAz]
[J].=5())(XY<)C),2004,43(3):8084
[33]ëôF,l],lK6,¡.#ISSRxÜ:Y®]lóô
AÓIz]
[J].=``,2011,42(2):353357
[34]lM,1?G.);`ISSRxÜ:Y®]lóôeÓIz]
[J].=``,2009,40(7):11311135
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[J]..Å4ª())(XY<)C),2010,36(4):414417
[36]ïÿ,Åã,RLæ,¡.(ÎISSRXnÓIlóôAz]
[J].=>`?),2010,32(4):8085
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