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,2016,36(7):1351-1356
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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c
,
vwxyzt1upq{|
(犘犘犅)}96.02%,~(犐)0.4889,Neist
u(犎)0.3324。
hiwxz
,
t1upq{|
(犘犘犅)33.51%,~(犐)0.1943,Neist
u(犎)
0.1332。zt
u(犎T)0.3239,cjt
u(犎s)}0.1332。AMOVAhiS
W
,
hi犖m 0.3492。hizNeis0.1481~0.4385。UPGMA,
0.327,7chi,9U ¡、¢£、¤¥¦§¨hi©ª,«¬®£T
hi©ª
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犌犲狀犲狋犻犮犇犻狏犲狉狊犻狋狔狅犳犘犪狆犺犻狅狆犲犱犻犾狌犿犿犻犮狉犪狀狋犺狌犿犇犲狋犲犮狋犲犱犫狔犐犛犛犚犇犪狋犪
LIZongyan1,GUANMingyuan1,LIJing1,LIMingyang2
(1LandscapeandArchitectureFaculty,SouthwestForestryUniversity,Kunming650224,China;2HorticultureandLandscape
Colege,SouthwestUniversity,Chongqing400715,China)
犃犫狊狋狉犪犮狋:犘犪狆犺犻狅狆犲犱犻犾狌犿犿犻犮狉犪狀狋犺狌犿hasthehighornamentalvalueknownasslipperorchids,whichwas
restrictedlydistributedinkarstlimestoneareasofsoutheastYunnan,southwestGuizhou,northernand
westernGuanxiProvince.Thisstudyfocusedonthegeneticdiversityof7populationsfromSoutheast
YunnandetectedbyISSRmarkers.101bandsweregeneratedfrom160individualsby10primers,among
which97bandsbelongedtopolymorphicandeachprimeramplified10.1bands.Therewasahighergenetic
diversityindexatspecieslevel(犘犘犅=96.02%;犐=0.4889;犎=0.3324),butalowergeneticdiversity
indexatpopulationlevel(犘犘犅=33.51%;犐=0.1943;犎= 0.1332).Totalgenediversityreached
0.3239andgenediversitywithinpopulationsattainedto0.1332.Neisgeneticdifferetationamongpopu
lations(犌st)was0.1481-0.4385.Geneflow(犖m)was0.3492.Theseindicatedthattherewasahigher
populationdifferentiationamongpopulations.BasedontheUPGMAclusterdendrogram,thesevenpopu
lationswereclassfiedintotwogroupsatgeneticdistanceof0.327.FirstgroupincludedGulinqing,Magu,
Tianba,JiahanqingandYangliujing;SecondgroupwasmadeupbyDouzuiandXiaobazipopulations.The
clusterresultindicatedthattherewasnosignificantcorrelationbetweengeneticdistanceandgeographical
distance.
犓犲狔狑狅狉犱狊:犘犪狆犺犻狅狆犲犱犻犾狌犿 犿犻犮狉犪狀狋犺狌犿;geneticdiversity;geneticdifferentiation;geneticdistance;
bloodrelationship
defg(犘犪狆犺犻狅狆犲犱犻犾狌犿犿犻犮狉犪狀狋犺狌犿)S¼
(½(
、
¾¿!("À
、
Á!!ÀzÂÃqÄ
ÅÆÇÈ
[12],
rÉÊË*zÌÍÎÏ
,
Ð20ÑÒ
80ÓÔÕÖ×Ø,vÙÚÛWÜÝÞß,ÝCITES
à9:HI#$
,
áâãä
。
Nåæçè
,
éêëìíîïdefgðñ
、
ò0$%
、
óvt
uôJK
[39]。
def
gëìõö÷øùúò
[3],
Ðûü1ý
,
de
fgþ|ÿÕ|Ë!
,
"#Cò$vTÿÕ|
%W&}32.4%[4];rÉÊË*zÌÍ.u,d
efg³tX\()*vz+,
,
-¸Pz.
/01 犘.犿犪犵犻犮Lantern(defg × 23f
g
)[5];
defg#451t
,
ç6789ËW
,
-
ç/
、
ç:
、
s;<;zS°Ër=67
*
[6];
;>ô
[7]
S?4cdefghit
u,
@[
:
r$vwxzt
u*hiwx
,
Ê20.3% zt
uØÐhizS,hi
Swx*7c(hizSRAPA
z
[8];
B9CzDй¯JK@[
:
defg
ijs89DyzS¼EFGü
,
H
I@Öo·¸zJ¹¯
,
ËWI@Öo·
¸zK¹¯
[9]。
ISSR(intersimplesequencerepeat) LM
N2OPàÈklt1DNA ,þQRNMNS
T
[10],
ÐU5×Ø
,
hit
uV
S?s·ïWoz,-
[1113]。
X½(zÄÅÆ
´ÈY&Z[fgz\Ûs]^s]
,
_[`
IdefgÞabc%WzÈd
,
Þ_ISSRef
bg´Èhit
uBhS?
,
rHIij
µkõAÉÊÖþlm
。
1 nopq
1.1 PQ%R
rJKnoÞastuÇ
、
¯vwx3y
7chi,zhi{|}Þ
~,¶u
cj
,
zcN4
2m ×y,7chin
160c
r(@2),
HsqþQ
-70℃!H。
1.2 STUVW犘犆犚XY
Þ_z1.5×CTABqidefg
©DNA,DNA0.8%$,
æ30ng/μL-20℃H_。
þQs_zISSRm$[W{ÂW
%
(UBC)íÕ6z77cm$,Dss10bZ
¡kl
、
¢0pq£¤¥¦
、
t1u˧zm$©
,zPCRkl±¨
j°
:2.0mmol/L MgCl2、dNTPs0.40mmol/
L、©ªÙÜ«¬ 5.0ng/μL、1.25 U/20μL
犜犪狇DNA<、m$ÙÜ«¬0.35μmol/L。IS
SRPCRkl®P:94℃¯8u5min,94℃8u
45s,48~52℃°±45s,72℃²³90s,35c´
µ
,72℃²³10min,4℃H。kl¢$2.0%
。
1.3 Z[\]
pq¶·Éʹ¸¹º|zkl»¼
,
{
½0/1¾¿,¨ _POPGENE1.32ÀÁ[14,15]Bh
t1Â3ÃS|
(犘犘犅)、ÌôÂ(犖a)、Ê
ÄôÂ
(犖e)、Neist
u(犎)、
ShannonMÅ(犐)、ijzt
u(犎T)、
ijIt
u
(犎s)、ijzS°
(犌st)、Neis(犇)(犖m)Æ。Ç
ANeis,^ _ NTSYSpc1.21[16]ÀÁb
hiBh UPGMAhiº»¯°S?。ST
89zpÈS?
(AMOVA)_ GenALEXÀÁ
Æ
[17]。
2 ³S?
2.1 STUV^_
Þ_,zISSRPCRkl±¨j°,b77c
ISSRm$klÊ¢$zÊ42c,DsÉso10c
kl ¡uË*
、
t1u˧pq£¤zm$B
hhit
uS?
(
@1)。m$klpq%t
`1 abST5XYc_
Table1 Amplifiedresultsofthedifferentprimers
m$PÊ
Primercode
Pà
Sequence
(5′→3′)
klpq
No.of
bands
t1upq
No.of
polymorphic
bands
t1uÃS|
Percentageof
polymorphic
bands/%
UBC814 (CT)8A 9 9 100
UBC825 (AC)8T 10 9 90
UBC831 (AT)8YA 8 7 87.5
UBC834 (AG)8YT 10 10 100
UBC848 (CA)8RG 10 9 90.0
UBC855 (AC)8YT 12 12 100
UBC856 (AC)8YA 12 12 100
UBC862 (AGC)5 10 10 100
UBC840 (GA)8YT 12 12 100
UBC849 (GT)8YA 8 7 87.5
Total 101 97 96.04
2531 ! " # $ % & 36Ë
`2 ,4犐犛犛犚Z[567897d:;5<=>?@eZ
Table2 Geneticdiversityindicesofsevengeographicalpopulationsof犘.犿犻犮狉犪狀狋犺狌犿
hi
Population
r
Sample
ÌôÂ
犖a
ÊÄôÂ
犖e
Neis
t
u 犎
Shannons
犐
t1u
ÃS|犘犘犅/%
¯ ¡ MGMG 22 1.3663 1.2539 0.1428 0.2094 36.63
¯¤¥ MGJHQ 19 1.2871 1.1801 0.1020 0.1516 28.71
¯U MGGLQ 25 1.4059 1.2682 0.1519 0.2241 40.59
uǬ WSDZ 22 1.3168 1.2383 0.1332 0.1928 31.68
uǦ§¨ WSYLJ 19 1.2871 1.2267 0.1220 0.1750 28.71
uÇ®£T WSXBZ 21 1.3465 1.2556 0.1430 0.2078 34.65
vwx¢£ MLPTB 32 1.3366 1.2474 0.1374 0.1996 33.66
hiPopulation 1.3352 1.2386 0.1332 0.1943 33.51
$vSpecies 160 1.9604 1.5529 0.3244 0.4889 96.04
Ì
:犖a.ÌôÂ;犖e.ÊÄôÂ;犎.Neist¬;犐.Shannons;犘犘犅.t1uÃS|
Note:犖a.Observednumberofaleles;犖e.Effectivenumberofaleles;犎.Neigenediversity;犐.Shannonsinformationindex;犘犘犅.
Thepercentageofpolymorphicbands
`3 67897d:;5犖犲犻狊<=fgh(bÍÎyp)W<=ij(bÍÎýp)
Table3 Neisgeneticidentity(abovediagonal)andgeneticdistance(belowdiagonal)of犘.犿犻犮狉犪狀狋犺狌犿
hi
Population
¯U
MGGLQ
¯¤¥
MGJHQ
¯ ¡
MGMG
uǬ
WSDZ
uǦ§¨
WSYLJ
uÇ®£T
WSXBZ
vwx¢£
MLPTB
¯U MGGLQ 0.6778 0.8046 0.7460 0.7072 0.6768 0.7045
¯¤¥ MGJHQ 0.3889 0.7758 0.7279 0.8623 0.7199 0.7845
¯ ¡ MGMG 0.2174 0.2539 0.7843 0.7452 0.6876 0.7343
uǬ WSDZ 0.2931 0.3176 0.2429 0.7400 0.7970 0.7847
uǦ§¨ WSYLJ 0.3464 0.1481 0.2940 0.3012 0.6450 0.7945
uÇ®£T WSXBZ 0.3903 0.3287 0.3746 0.2269 0.4385 0.7080
vwx¢£ MLPTB 0.3503 0.2427 0.3089 0.2424 0.2301 0.3453
12p,m$855、856840,tm$klpq
8~10p,Ê6cm$klt1ÃS|}100%。
2.2 67895<=>?@
10cm$nklo101cpq(@2),rst
1upq97c,t1pq{|}96.04%,zcm
$xÏkl¢010.1cpq。U ¡hi
t1uÃS|Ë*
,
SÐ40.59%36.63%,¤
¥¦§¨hizt1uÃS|%®
,
ÑÊ
28.71%。7 chizÊÄôÂ(犖e)
1.1801~1.2682Ò;Neist¬(犎)8Ó
0.1020~0.1519,Shannon(犐)Ï0.1516~
0.2241。®£T¢£hizt
u¹ÈÔ
W
,
¤¥hiIzt
uË!
(犘犘犅=
28.71%;犎=0.1020;犐=0.1516)。Uhi
zt
uwx%*
,Shannon (犐)
0.2241。hiwxzShannon Neis
t¬0.19430.1332,$vwxzSÐ
0.48890.3244。zt
u(犎T)
0.3239,hiIzt
u(犎s)0.1332。
2.3 :;k<=ijlmnEo
õA·zhizNeis(@3),Õ
hiz®£T¦§¨z%W
,
0.4385,Ö¤¥¦§¨hiÊ%®
,
0.1481,7 chi x Ï
0.2992。UPGMA(×1),}
0.327,7chi,9cS©
ª
,
9SU ¡hi©ª
,
«S
¢£
、
¤¥¦§¨hi©ª
,
«¬®
£Thi©ª
,
Øþïhi³´µ
¶·¸¹¯u
。
2.4 6789:;k5<=Jp
ijS°[[ÙÜt
uz8
9ØÛz`
[18],
Sz*!@[hiz8
9Swx
[1920]。犌st、犘犺犻犘犜 [Ô¸ÀÁÆ
35317Ú ;<=,ô:ISSRzdefghit
uJK
×1 defg7chizº»¯°
Fig.1 Dendrogramanalysisshowingrelationships
amongsevenpopulations
zS
。
ÛÜ
,7chizS°(犌st)}
0.5888,ïhiË*zSwx。
AMOVAÊ39.60%zST89ØÛh
i
,
Ý9Þ*ßàUô&áz
[21],
®
³犌stzSwx,³犘犺犻犘犜ÏÕ¹¸。Öhi
xÏ
(犖m)Ñ0.3492。
râ
,
¸ 9 ´ È h i z S °
(犘犺犻犘犜)¹ÈËW,uÇ®£T³¬z犘犺犻犘犜
Ï0.304,¬³¦§¨z0.430,®£T³¦
§¨zS°}0.492,xÏS°
}0.409;Ö ¯ ¡UÑ0.319,U
³¤¥z犘犺犻犘犜 0.472, ¡³¤¥Ò
0.381。¢£hi³rã6chi犘犺犻犘犜 Ï
0.338~0.413Ò,³¬S%®,³U
%W
。7chiS%®z¦§¨
¤¥
,
}0.278;%Wz¦§¨®£T,}
0.492。7chixÏ犘犺犻犘犜 Ï0.392。hi
犘犺犻犘犜 ϱ²ozS®¬³ Neis
¯°[9äz
。
å榧¨¬hiÕ»h
i
,
çhizxÏS°èË*
,
é[
¹¸´ÈÕhi_Ë*zS
,
±
²ïhiSwx³´µ¶[·¹
¯u
。
3 ê k
3.1 6789:;5<=>?@
9ëì
,
Á¼ví{î¼vÉÊË*z
t
u
,
FG#$ÊË!zt
u
,
t
u
ï!z#$ïFG
[2223]。
çïØïtzJK@[
,
FG#$ðÊñ*zt
u
。
Þ_ISSRef
bòóô
(犌犾犲犺狀犻犪犾犻狋狋狅狉犪犾犻狊)、õ:öç÷(犆犪犿犲犾
犾犻犪狆狌犫犻狆犲狋犪犾犪)FGvt
uwxz·
,
vhiwxíHøË*zt
u
,
hi
SðÔ*
[2425]。
Þ_ISSRefg4FGvzJK@[,
ùÄú
(犇犲狀犱狉狅犫犻狌犿犳犻犿犫狉犻犪狋狌犿)vwxz
t
uÔ!
,
çhiwxzt
uË!
[26]。
r78b7cdefghizISSR@Ö
o$vwxzt
uË*
(犘犘犅=96.04%,犎
=0.3244,犐=0.4889),hiwxzt
uÏ
*yûv
(犘犘犅=33.51%,犎=0.1332,犐=
0.1943)。$vzt
u(犎T)0.3239,
hiIzt
uÑ0.1332。ük³
SRAPA;>zýCk[9äz[89],þ
defgvwxt
uÿ*hiwx
,
ç³
ßàUô&ázË*wxzhit
u
(犘犘犅
=80.28%,犎=0.2847,犐=0.4236)ÊËWo
!
[21]。
åæFG$zt
u³ñt"ʯ
,
-
D#ÜÁ¼vz$vsSÖØ
、
S¼$%
、
0&p
Á
、
ðó°¶ô
。Hamrick[22]ìhi89W
®zëì("õâðó°¶
、
S¼$%0
)*
,
Á¼
、
9,ö+$,vTz$v--ÉÊ
Ë*zt
u
。
defgZHøvË*wxz
t
u³r9,
、
./0r
、
vT,.u
、
S
¼0&zt
12¹¯
。
3.2 6789:;5<=Jp
9ëì
,犘犺犻犘犜S30~0.05Ò
é[hiSñ4
,
30.05~0.15Òé[h
isô®¬S
,
30.15~0.25Òé[
hiSW
,
3犘犺犻犘犜 W0.25@[hiS
Y W
[27]。7 c d e f g h i z S
(犘犺犻犘犜)xÏwx}0.392,5Sé[defgh
iSW
,
³ AMOVA Õ¹¸,!
Neisz犌st(0.5888),6Õ¹¸´È78Ä
úhi犌st(0.5637),ÿ*defg¾9
,:Èz犌stwx[21]。defgzISSRST
`a
,^
_Ô¸zS?pq
(犘犺犻犘犜,AMOVA);
JK@[
:
9,iz
xÏS°犌st0.23,=,i犌st
0.19,Ð,i犌st0.59[28]。defg>9ç
ò#$
,
hizS·¸
。
$vzðó°¶[(hiwxyzS
wxz2ì"
,Avise[29]ì4?hZcz+
$CòövT¹ºz#$hiÒzS·
4531 ! " # $ % & 36Ë
¸。
defgëì÷ò
[3],
r?hÊ@
,
ëì[BhhiIíç#4ÒzCò
,
Aª
89ëìØÛhiI
,
Öhizçò[BC
Ê@z
;
DO
,
defgvTÿÕ.uÉÊEF
z0&st.3
,
äGä$v´µHuS¼
,
Aª
hiz´µËÿ
。
9ëì
,´
µIJm
\Õ,
,
ÖÕ,ðñpK·m\Õ,L°M!i
jzt
u
,¸
ðJM!ij,
,
NijzS
[3032]。
hi%JKs
,
ÇA 犖m zW®OS*(≥1.0)、s(0.250~
0.99)、!(0.0~0.249)Pcô:wx[33]。3{ü
`QOS
,
üâJKzdefghiR
!wx
,
Ñ0.3492,¹¸zISSRefz
(
、
¾¿Á!,:ÈdzdefgzðÑ
Ê0.7201[21]。9ëÖS,stN_Ô[·,T
ìÊñUÜzV&ÊÄ´WâijS
[34],
&XHÔ[AªdefghiS·
¸zëì"
。
ÊïØïtzJKØ[
,
89ëì[s
t
、
W8
、
Y8ô¢0
,
ÖDZ
Ô9¡¢089
[35]。
[ü¹ñÕzijþ
\]ðz+
,
EÊczstN_ðZ
Ê%O
[35]
ì³´µÒ^_
·¸¹¯ul`aY8vizSs\a
Ô&bczN_
。
"def2zdefghi
s
,
hicjëìõg¶u¢0
,
GähisÊ
ÄcjÜýM5ª®hi
,
häWÕ0
Y8z}|
;
üâji¬
、
®£T¦§¨hi
®hi
,
WjðlïhizS
。
DO
,
"de®zhis
,
íç#4UcjÜkU
,
äAªhiIçòwxýMþ|M!
,
hi
It
uwxýM898®
,
Öhi
zvTÊ@ðNhizS
。
qr!L
:
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æ){
[J].X\%&,2004,31(4):540542.
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[J].ÃqÂÃq#$%&,2010,18(2):459468.
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