免费文献传递   相关文献

Comparison of Genetic Diversity of Irradiated Sophora viciifolia Using ISSR and RAPD Markers

辐射白刺花遗传多样性的ISSR和RAPD比较分析



全 文 :!23" !5#
 Vol.23  No.5
! " # $
ACTA AGRESTIA SINICA
   2015$  9%
  Sep.  2015
犱狅犻:10.11733/j.issn.10070435.2015.05.033
lmœ<÷ø†[30犐犛犛犚a犚犃犘犇./;n
Y¾9
1,
[||
1,
/ÒÓ
2,
! Ô1,Մ|1
(1.p2*+Q9°++0N)°+A,p2 pà550025;2.p22N)˜™],p2 pà550025)
45

B¥ISSR=RAPD¬±K8e60Coγ)*§¨*nê‡o(犛狅狆犺狅狉犪狏犻犮犻犻犳狅犾犻犪);iÁ‹&gz{。l
mPJ
:20ISSR=6RAPD½9B犘犘犔‹5zÆG51.37%=21.03%,ISSRBNei’s«p=L–Shan
non«p–*¦RAPD;ISSRB¬±«p(1.28)5RAPD(0.20)B6.4[,PJISSRŒšìB¬±i5。Man
telIjPJ,ISSR+RAPD¬±8ISSR¬±Blm—rƒ@(犘<0.05)。é@z{PJê‡o¥ISSR¬±|I
jŸìB‹g

Ÿâ»µ;ê‡o‘“BÁÂ@A

678

ê‡o

)**n
;ISSR;RAPD;Á‹&g
9:;<=
:S548    >?@AB:A     >CD=:10070435(2015)05111405
犆狅犿狆犪狉犻狊狅狀狅犳犌犲狀犲狋犻犮犇犻狏犲狉狊犻狋狔狅犳犐狉狉犪犱犻犪狋犲犱犛狅狆犺狅狉犪狏犻犮犻犳狅犾犻犪
犝狊犻狀犵犐犛犛犚犪狀犱犚犃犘犇犕犪狉犽犲狉狊
LIFengtao1,ZHAOLili1,WANGPuchang2,CHENChao1,WUXiaoli1
(1.ColegeofAnimalSciences,GuizhouUniversity,Guiyang,GuizhouProvince550025,China;
2.GuizhouPractacultureResearchInstitute,Guiyang,GuizhouProvince550006,China)
犃犫狊狋狉犪犮狋:TheRAPAandISSRmarkerswereusedinanalyzingthegeneticdiversityofmutagenic犛狅狆犺狅狉犪
狏犻犮犻犻犳狅犾犻犪Franchwitheightdifferent60Coγraysgradients.Theresultsshowedthatthepercentageof
polymorphicbandsof20ISSRprimersand6RAPDprimerswere51.27%and21.03%,respectively.The
Nei’sgeneheterozygosityandShannon’sinformationindexofISSRweregreaterthanRAPD.For8mate
rials,ISSRsgotaMIvalue(1.28)thatwas6.4timeshigherthanthatofRAPDs(0.20),indicatingthat
ISSRshadahigherMIefficiency.TheManteltestrevealedthatthecorrelationwassignificantbetweenIS
SR+RAPDsandISSRs(狉=0.9791,犘<0.05).ComprehensiveresearchshowedthatISSRscoulddetect
thehigherpolymorphismof犛狅狆犺狅狉犪狏犻犮犻犻犳狅犾犻犪,andmoreaccuratelyreflectthegeneticrelationshipamong
犛狅狆犺狅狉犪狏犻犮犻犻犳狅犾犻犪.
犓犲狔狑狅狉犱狊:犛狅狆犺狅狉犪狏犻犮犻犻犳狅犾犻犪;Radiantmutagenesis;ISSR;RAPD;Geneticdiversity
EFGH
:20140804;IJGH:20150122
KLMN

p22°–J$H9{T./²³

2Ä@B
(2012)142™];¬­®¯°+‡±(31260572);p2*+½;»9°˜²³

p*»‡@B
(2010)036™];p2*+–¿²³(JG2013067)´ µ
ÅƔ

Ö!ø
(1989),Ë,p2pà»,¼½,¾D¿ÀNñ:8X,Email:lft23520@163.com;ÇÈÅÆ Authorforcorre
spondence,Email:zhaolili_0508@163.com
  *m)*5*¥X*m、γ*m!NO:9)*¡¢¤Yˆ@:³Ÿ

¿ˆÓA]“AB½ê
DNAl%Bn<[1]。z7¬±5¼½‡EàDNA
”•$%B‹gˆ@kê…B

|ÓA;:9
e\•DNAKL‚DoBŠyÁ¬±[2]。‹$
…

˜™»È*¥)**nH͋,N

CìRp
(犉犲狊狋狌犮犪犪狉狌狀犱犻狀犪犮犲犪)[3]、k·7(犔犲狊狆犲犱犲狕犪犫犻犮狅犾
狅狉)[4]!,G0¬N)@kÃHCDħ。
ê‡o
(犛狅狆犺狅狉犪狏犻犮犻犻犳狅犾犻犪Franch)GPO—
©¤h

O¦oo½š89

ŒzÎ.

ížgï
=²Öâç›!R:
[5],
Òo

©=Y7+%̋Y
%‡s

I:F†¯ù®F



ê‡o•Ràñ
PØè

ÖLKËè“

óÆ­O!N/ŒšìB
U@*¥ôõ

 vG5:i‹=()i‹–š
ïBãöÖY
[6]。
³0

@¦ê‡oB˜™¾D§
+•Y7\=
[7]、
oB<+áz
[8]
!N/

@ÒY
7)**nB˜™Ÿ¾?w‡

˜™Ò)*iž

›
LZYùK ¸3ãöYùB*¥ŒCDVÛ

T˜™B¥
60Coγ*m)*":ê‡oY7,ÃÈ
šISSR=RAPD¬±•ê‡oÁ‹&g˜™+
!5# Ö!ø!:)*ê‡oÁ‹&gBISSR=RAPDȚz{
B|ž¥g

Gê‡oB)*TYΏXWÔ½8
˜™‡Ã

1 STYUV
1.1 ^šST
UV ê ‡ o Y 7 B ® p 2 2 K Œ º

E104°44′13″,N26°17′08″,341140m。
1.2 ^E
10×TaqPCRMasterMix(Mix1mL,ddH2O
1mL,MgCl20.5mL)、DNA Marker、ùx、Tris
–æ‚3:9Kªe

1.3 UV
1.3.1 ¹ºÖ׍Ž ¦B§BÜY7Z¦ë
K=7t

ÈÊ1∶1)Bܒ‰‚L¼Kz×13%,
•p22±()}°•KYªe;iWXÊë
B
60Coγ*m)*。æZ8e)}ÊëzÆG0

K}
),20,40,60,80,100,120,140kR,Êë5G
110R·min-1,”Êë^XY7pG300±,CM3
ü

2013$5%19+¦)*BY7ÿY¦ë&
ô

Qj

-ˆùË|1R1R1ÈÊo@BoÉ
+

Z¦p2*+N)°+A_º‚;i+nX

1.3.2 DNAØ€Ùú ”e^Xx10iê
©®o@

¥89‡EàUÊñ

î4¥c¼:<
°•Kªe

ÎxDNA,¥0.8%Bùxg{
³{IjDNAùë,¦DNA¡#×10ng·μL
-1
Õ¥

1.3.3 ڲۏӻ°”m ½9§Œ:}K}‡
EàDNAGéh,;i½9§Œ,¿100ISSR
=130RAPD½9+(ë‚3:.:9./•–
ªe@á

§Œ&<‹

Çkgï

‹g$%
Bí1ISSR½920,RAPD½96。
æñž\AG20μL:10×Mix10μL,½9
(0.8μM )1μL,ddH2O7.5μL,DNA(10ng)1.5
μL。æñ/º:94℃Cng4min;94℃ng30s,
50℃,‡30s,72℃?1min,35e½F;72℃?
10min̞Sž,4℃û”。
³{Ij

x5μLPCRú9•1×TAE"í
‘š;i1.5%ùxg{³{(Ì1%ŸsΑ),
100V™d³{40min,g{áRAÀT}±²。
1.4 hi;n
¦³{׆+B”1&–UGŠez7¬
±

&B±G
“1”,V&B±G“0”,¿ˆ:áë1
=0àáBp½Ž。B¥POPGENE¿‘Á.
‹5

!Y‡Ep

i!Y‡Ep

‡E‹&g
«p

‹  g È ¶ « p

B ¥ NTSYSversion
2.10e;ixyz{,ôLxyÖh×。B¥TFP
GAversion1.30¿‘+B MantelTest;iƒ@
gIj

2 lmY;n
2.1 1+º»
}K}DNAGéh,§ŒISSR½920,
RAPD½96。
:1 犐犛犛犚1+犝犅犆818%8„œ<
犇犖犃KžXžÈ:Z
Fig.1 Amplificationmapof8犛狅狆犺狅狉犪犱犪狏犻犱犻犻
genomeDNAusingISSRprimerUBC818
ò
:M:DNAmarker;1:K};2~8:?P20~140kR
Note:M:DNAmarker;1:control;2~8:20~140kR
:2 犚犃犘犇1+49%8„œ<犇犖犃
KžXžÈ:Z
Fig.2 Amplificationmapof8犛狅狆犺狅狉犪犱犪狏犻犱犻犻
genomeDNAusingprimerRAPD49
ò
:M:DNAmarker;1:K};2~8:?P20~140kR
Note:M:DNAmarker;1:control;2~8:20~140kR
5111
! " # $ !23"
2.2 žÈlm;n
ëP1|ì,ISSRƒæñ183&,L–”
e½9æñ9.1&,Ò+‹g&ƒÁ94


‹5Ä51.37%。ëP2|ì,RAPDƒæñ
37&,L–”e½9æñ6.1&,Ò+‹
g&ƒÁ10,‹5Ä27.03%。
\1 犐犛犛犚;n1+Ÿ ažÈlm
Table1 TheprimeralignmentandamplifiedresultsofISSRanalysis
½9˜™
Primernumber
ºÄ
(5’-3’)
Sequence(5’-3’)
æñ&`p
Tatalbands
‹g&p
Polymorphicbands
‹5
Rateofpolymorphicbands/%
UBC857 AGAGAGAGAGAGAGAKRG 17 12 70.59
UBC818 CACACACACACACACAG 7 2 28.57
UBC850 GTGTGTGTGTGTGTGTYC 9 4 44.44
UBC840 GAGAGAGAGAGAGAGAYT 10 2 20.00
UBC812 GAGAGAGAGAGAGAGAA 7 1 14.29
ISSR04 GAGAGAGAGAGAGAGACT 10 2 20.00
ISSR11 ACACACACACACACACTG 11 8 72.73
ISSR21 GAGAGAGAGAGAGAGATG 7 2 28.57
ISSR25 ACACACACACACACACCT 10 4 40.00
ISSR24 ACACACACACACACACCA 13 7 53.85
ISSR36 GCGTCTCTCTCTCTCTC 8 2 25.00
ISSR47 AGAAGAAGAAGAAGAAGAAGA 7 4 57.14
ISSR02 AGAGAGAGAGAGAGAGCC 12 10 83.33
ISSR05 GAGAGAGAGAGAGAGACC 7 5 71.43
ISSR06 GAGAGAGAGAGAGAGACG 12 9 75.00
ISSR08 GTGTGTGTGTGTGTGTCC 15 14 93.33
ISSR12 CCCTCCCTCCCTCCCT 3 1 33.33
ISSR14 CTTCACTTCACTTCA 7 3 42.86
ISSR10 ACACACACACACACACTA 4 1 25.00
ISSR15 GGAGAGGAGAGGAGA 7 1 14.29
@ÁTotal 183 94 51.37
\2 犚犃犘犇;n1+Ÿ ažÈlm
Table2 TheprimeralignmentandamplifiedresultsofRAPDanalysis
½9˜™
Primernumber
ºÄ
(5’-3’)
Sequence(5’-3’)
æñ&`p
Tatalbands
‹g&p
Polymorphicbands
‹5
Rateofpolymorphicbands/%
RAPD01 CAGGCCCTTC 7 2 28.57
RAPD05 TTCCGAACCC 7 1 14.29
RAPD14 CTACTGCCGT 5 3 60.00
RAPD49 CTGGACGTCA 5 2 40.00
RAPD50 CAAAGCGCTC 8 1 12.50
RAPD65 GGATCGTCGG 5 1 20.00
@ÁTotal 37 10 27.03
2.3 ÷ø†[3a@¡ô;n
2.3.1 ISSRpRAPD”mÜÝnI»¼ ëP3

,ISSR¬±B!Y‡Ep=i!Y‡Ep–*
¦RAPD¬±。Nei’s«pÞ.20ISSR½9=6
RAPD½9æñ]ÕY:B‡EÁ‹&g,WX
Y:KÁ‹&gBħWX
,ISSRBNei’s«pG
0.1851,*¦RAPDBNei’s«p0.0861;ISSRBL–
Shannon«pG0.2773*¦RAPDL–Shannon«p
0.1331。PJISSR¬±|IjŸìB‹g,Ÿâ
»µ;ê‡o‘“BÁÂ@A

\3 犐犛犛犚Y犚犃犘犇@¡÷ø†[3uh./
Table3 ComparisonofgeneticdiversitieslabeledbyISSRandRAPD
±yQ
MarkerType
!Y‡Ep犖犪
Observednumberofaleles
i!Y‡E犖犲
Effectivenumberofaleles
Nei’s‡E‹&g«p犎
Nei’sgenediversity
Shannon’s‹gȶ«p犐
Shannon’sInformationindex
ISSR 1.5137 1.3129 0.1851 0.2773
RAPD 1.2703 1.1374 0.0861 0.1331
6111
!5# Ö!ø!:)*ê‡oÁ‹&gBISSR=RAPDȚz{
2.3.2 ISSRpRAPD”m•”mr"»¼ •8
eUê‡o‘“ISSR¬±B犕犐õ(1.28)5
RAPD¬±(0.20)B6.4[,EJISSR¬±Œ
šìB¬±i5

Ò+
,犐犫avõzÆG0.27=0.12,
ƒDšw
;犈犕犚õzÆG4.7=1.67,0Æ5ÌÆ
2.81[。犈犕犚Do5NXISSR¬± 犕犐õì¦
RAPD¬±B¾DÞE(P4)。
\4 犐犛犛犚a犚犃犘犇@¡ô0./
Table4 Comparisonoflabelingefficiencies
betweenISSRandRAPD
²³Items ISSR RAPD
Y:L–ȶë
Averagebandinformativeness犐犫av
0.27 0.12
i‹®È5Effectivemultiplexratio犈犕犚 4.70 1.67
¬±«p Markerindex犕犐 1.28 0.20
2.4 {<;n
ISSR¬±xyz{,8e^X“BÁƒ„A
p•0.6885~0.8579’“,C×3]ó,Ƀ„A
p<0.832“,â^T8e‘zG7y,EJ
60Coγ*mWX/¨B67Hê‡oB‡Eà。
RAPDxyz{,8e^X“BÁƒ„Ap
•0.8108~0.9730’“,C×4]ó,Ƀ„gA
pG0.94“,|T‘zG5y。
¦ISSR=RAPDB0,1Žl@;ié@^
X
,8e^X“BÁƒ„Ap•0.7108~0.8682
’“

xylmC×5]ó,TISSR=RAPDBé
@xy×8ISSRœÓxy×KÈ|}@A,ÛÆ
‡T‚PAGŠX

:3 8¿œFig.3 Dendrogramof8犛狅狆犺狅狉犪犱犪狏犻犱犻犻groundaccessionsbasedonISSRmarkers
:4 8¿œFig.4 Dendrogramof8犛狅狆犺狅狉犪犱犪狏犻犱犻犻groundaccessionsbasedonRAPDmarkers
  *¥TFPGA¿‘KISSR=RAPD¬±}†IS
SR8RAPDé@¬±BÁƒ„gAp;i Mantel
testƒ@gIj。lmPJ,ISSR=RAPD¬±Bl
mƒ@gW—r
(狉=0.0108),ISSR+RAPD¬±8
ISSR¬±Blm—rƒ@(狉=0.9791,犘<0.05),ISSR
+RAPD¬±8RAPD¬±Blmƒ@gW—r(狉=
0.2141),|?ISSR+RAPD¬±xylm8ISSRƒ
„

ŸA#¦ISSRBxylm。
7111
! " # $ !23"
:5 8¿œFig.5 Dendrogramof8犛狅狆犺狅狉犪犱犪狏犻犱犻犻groundaccessionsbasedonISSRandRAPDmarkers
3 ~Yl
T˜™*¥20ISSR=6RAPD½9z
ÆKUê‡o‘;iHPCRæñ,¿2Y¬±
B@špõ~
,RAPDB½9pëQISSR‹,
ISSR¬±æñBY:Ÿ‹,IjÅB‹gŸ$
%

•Y:Ijâç‚ì¦ RAPD¬±。ISSRz
{IjB‹È¶Ìë

!Y‡Ep

i!Y‡E
p
、Nei’s‡E‹&g«p†Shannonȶ«p–
ì¦RAPD,E£PJISSR½9Kê‡oBÁÂ
‹&gšìB‚ƨ

ˆRAPD½9Kê‡oB
Á‹&gŠkB‚ƨ
。ISSR¬±B 犕犐õ
5RAPDB6.4[,EJISSRŒšìB¬±i
5
。Mantelƒ@gIjPJ,ISSR+RAPD¬±
8ISSR¬±Blm—rƒ@。}‚EJISSR¬
±ŸâIjê‡oBÁÂDo

•Çkg

‹
g

¬±i5!N/ÈRAPD¬±Ÿãä。
ŠØvG
,ISSR¬±È RAPD¬±ŒŸì
Bæñi5=‹g

Cfð!K…†¦ì2
13„XO°Yù;iÁ‹&gz{,lmPJ
ISSRIj‹gimì¦RAPD[9]。¯ˆq˜
§WXB˜™lm

kŸ7!
[10]
•K_}WXñ
Ý\EnyB˜™+@A
,RAPD¬±ÈISSR¬
±|}OóŸG$%BÁÂȶ

T˜B˜™lm
ŸVL¦0Æ
,2Y¬±AÐYDo8ÒӕK
‡Eà;iæñ“½9l@YZBWXÓAB@
A

ë£|?
,60Coγ*m)}ê‡o̔•$%
BÁ‹&g

EJê‡o()*Ì@:HŠkB
ÁÂno
。2Yz7¬±•–S|ž¥¦ê‡oB
Á‹&gz{

wISSR¬±•Çkg、‹g、
¬±i5!N/ÈRAPD¬±Ÿãä。
u>?
[1] €J, lz.89*n•–B˜™;k[J].:9•–;k,
2011,1(2):9097
[2] ÈTU,Ö­Ú,nS£,!.ÔY DNA z7¬±•–†Ò•
•ÖYù´†˜™+Bž¥
[J].:9•–Çw,2013(1):63
67
[3] ©6j,õËV.Co60)*•ìRpPQ=7?‚B7ž[J].
+¬Nñ+w
,2009,31(4):5356
[4] õ$,’Ö,_Œ‘,!.3Yk·7-sÏÐ9ù=!NRà
9ùKÜý=ñ›UVB)*B7ž[J].Nñ+w,2013,21
(3):517525
[5] ;3=,ڂ„,À˜ç,!.ê‡o(犛狅狆犺狅狉犪狏犻犮犻犻犳狅犾犻犪)íž
ËÌÜýB:X+›L
[J].ÜýñÝ()˜™,2005,23(1):
106110
[6] ¶›,;ÄJ,ÖGƒ,!.ê‡oBT{\£•–†U@*¥
0b
[J].+¬":89´†,2003,21(6):2021
[7] õdç,ћ¬,ÒÔ$,!.WX^XNÓKê‡o\=Y7
²@RgB67˜™
[J].É?O,,2014(6):4750
[8] ¶J,$35,fw:,!.úê‡o<+áz˜™[J].+á
1
,2009,31(2):269271
[9] fð,›La,?Ú,!.ìXO°YùÁ‹&gB
RAPD=ISSRz{[J].89Á´†+w,2011,12(6):
860864
[10]kŸ7,yK,¶ÒB,!._}WXñÝ\EnyRAPD=
ISSRz{[J].©4()°+,2009,48(1):2326

%&( , -)
8111