Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
书书书
!"#$%&
,2020,40(6):0969-0977犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
犱狅犻:10.7606/j.issn.10004025.2020.06.0969 http://xbzwxb.alljournal.net
!"#$
:20200209;%&"!'#$
:20200509
()*+
:'!()*+%,-./01
(2019JM476);!23+40156
(2020KJWL21)
,-./
:7 8
(1983-),9,:;
,<=
,>?@A#$BCDE%./
。Email:jinglee211@163.com
FGHI
:JKK
,:;
,LMN
,O;./DP=
,>?@A#$QRD$SH./
。Email:missilexll@163.com
(0犐犛犛犚123456789:;
<=>?@1A
! "
1,#$%
2,&'(
1,)*+
1,,--
1
(1!2TU%V D$QWXYZ%V
,#$RD$[\./]^_`a
,!2710065;2!23bc4defgh
,!2
710061)
B C
:!ijk
(犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿)lmnopqrstuvwxk+#$
,yz{|g}~���x�
�#$
。�./�uISSRBC��4d
,��)g}!���x7�!ijk��������������
B�
,|g}�D!ijk����x����U ¡¢
。�£¤¥
:(1)@100¦ISSR§$g¨©ª«12¦�
E�p
、]¬�x§$
,̈®¯«136�°^
,�E°^±B²
(犘犘犅)³100%,́ µNeis,¶���·¸
(犎e)
sShannonG¹·¸
(犐)Bº|0.3186s0.4843,��»xNeis��¼½¾0.0333~0.1701¿»
,Neis��
ÀÁ¾0.8435~0.9673¿»
,́µ��BÃĸ
(犌st)s,¶Å
(犖m)Bº|0.2229s1.7430。(2),ÆUP
GMAÇsÈÉÇx��ÄÊËÌ�£Í¤¥«ÎÏ��Q'!��¿»Ð¾��BÃ
。´µÑ!ijk��»
��BÃQ�UÒ½¿»ÀÓ�Ô¤Õ
。./Ö|
,!ijk��¾BC×ØÑmnÙÚx�����
,ÛÎÏ
��Q'!��yÜDÝ��BÃ
;ISSRBC��ÞuÆ!ijk�������
、�������»��BÃ
ßàáx./
。
DEF
:!ijk
;�����
;����
;ISSR
GH1IJ
:Q346+.5;Q789 KL3MN
:A
犌犲狀犲狋犻犮犇犻狏犲狉狊犻狋狔狅犳犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿
犘狅狆狌犾犪狋犻狅狀狊犚犲狏犲犪犾犲犱犫狔犐犛犛犚犃狀犪犾狔狊犻狊
LIJing1,ZHANGXiaofei2,CHENJueyi
1,WULinqin1,XULingling
1
(1KeyLaboratoryofPlantmicrobeCollaboration Research,SchoolofBiologicalandEnvironmentalEngineering,Xian
University,Xian710065,China;2XianAgriculturalTechnologyExtensionCentre,Xian710061,China)
犃犫狊狋狉犪犮狋:犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿isauniqueorchidspecieswithhighornamentalandmedicinalvalue.As
oneendangeredspecies,itiscurrentlylistedasanationalprotectedplant.Inthisstudy,geneticstructure
andgeneticdiversityof7犆.狋犻犫犲狋犻犮狌犿populationscollectedinwesternChinawasanalyzedusingISSRmo
lecularmarkers.Thisstudycanprovideareferenceforspeciesconservationinnaturalhabitatsof犆.狋犻犫犲狋犻
犮狌犿.Theresultsshowed:(1)12polymorphicISSRmarkerswithstrongrepeatabilitywerechosenfrom
100ISSRprimers.Atotalof136bandswereidentified,andthepercentageofpolymorphicbands(犘犘犅)
was100%.Neisgenediversity(犎e)andShannonsinformationindex(犐)were0.3186and0.4843,re
spectively.TheNeisgeneticdistancerangedfrom0.0333to0.1701andgeneticsimilarityrangedfrom
0.8435to0.9673amongpopulations.Theoverallgenedifferentiationcoefficient(犌st)was0.2229and
theestimatedvalueofgeneflowwas1.7430.(2)TheUPGMAandNeighborJoiningclusteringanalysis
bothindicatedcleardifferentiationbetweenpopulationsfromSichuanandShaanxi.Theoverallcorrelation
betweengeneticdistanceandgeographicaldistancewasnotsignificant.Thisstudyindicatedhighgenetic
diversityin犆.狋犻犫犲狋犻犮狌犿populationsandgreatdivergencebetweenSichuanandShaanxipopulationsonmo
lecularlevel.ISSRmarkerscouldbeusedforinvestigatingthegeneticstructure,diversityandgeneticdif
ferentiationof犆.狋犻犫犲狋犻犮狌犿populations.
犓犲狔狑狅狉犱狊:犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿;geneticdiversity;geneticstructure;ISSR
k+
(Orchidaceae)H|zC#$xâã+Ä
,
fäBåÆ��æ�DEÄÊ
,mnçpx$��
��
。k+#$xèéêë�ìpÂíÃ
,�îI
pÂïâÃ
,ÛðQíñxòóôõöñx¨DÓ
Ä
,lzC#$g÷|�ÃxÌ�¿â
[12]。øùk
+#$�ÌÙÚ
,úûÆüýíxDXþÿsD!
"í#
,�$%&�'()*+Bå
,¶,-¿ü.
#$�
,/012DX3ÃQ45x67
[3]。
!ijk
(犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿)Äk+j
k8�9D:;#$
,<DÆ=>2300~4600m
p?@A:Bg'CDé�xEFG
,>?BåÆ
g}!H��
,IJ!i
、KH
、ÎÏß�
[4],¾'!
LM?�NnOPBå
[5]。\�¸jk8#$â
�
,!ijkQôRí
,üSTíÃõUìôÁ
“V
W
”,mnçpxqrvwstuvw
。ûÆXÌ�
jk#YxZ�[
,\¿]F^_
、ZÂ`aßb
c!b
,!ijkcádeDXfgxh]67
,�
Djk��¸ijkGl
,mny�dop
[6]。û
Æ6qjk8#$xXYrssètuоoãv
w
,��Djk����x��yx¾yz
。IJ
!ijk¾{x��jk8#$yz|})*��
~�
(IUCN){ � � � � � � � $ �
(https://
www.iucnredlist.org/)。
��l�{xB��
,ûBå¾âñ�»�
�gx\��µ�õ
,��x���������
���«üDÐ��s���Ãx{¾��
,Nl
�ñ$�ùU��
、�ñ�t��x]?¡¢
[78]。
6qÓÆjk8#$x��BC��%àáynâ
�./&�
。��ß
[9]�10�jk8#$xITS
�{��ÝÄÊ�tB�
,�10�jkB|3�
;
���ß
[10]FZISSRB���� ?�Ô\Q�
jk8#$mn-ÙÚx�����
,ÄÊËÌQ
ôE%BÌ�£,;â¡
;Qianß
[11]./��g
}g¢�Båx£¤jk
(犆.犼犪狆狅狀犻犮狌犿)¥ ¾
��×ئl$�×ØÑ�����Ío§
,̈Û
��»��BÃZÂ-p
;Pandeyß
[12]©uRª
P��./�£«�Bå¾"¬x®,jk
(犆.
犽犲狀狋狌犮犽犻犲狀狊犲)��x��������BÃZÂÍ
-§
。Liß
[13]¯�!ijk°�Ý20�mn�E
�xRªP��°^
,̈Û±�RªP��ã�¸
N�uÆjk8x²Qjk
(犆.犳犾犪狏狌犿)s¥³j
k
(犆.犫犪狉犱狅犾狆犺犻犪狀狌犿)́ �µ¶�
。Guoß
[14]�
u2��·µ,¶¸¹rps16strnLFH|BC
��
,¾g}KH
、ÎÏß�Båx9�!ijk�
U��g¨®¯º7��»¼
,̈����»��
BÃ�¤
,ú½(�«�¤x¾Ä�UBå¿À
。
ISSR(intersimplesequencerepeat)ÁÂ�]
¬�»�{
,lâ5,ÆRªP��°�xBC�
�4d
,ÃÄÝRAPD、AFLPsSSR4dx~^
,
mn���E�p
、��G¹iÙÚ
、ÅÄÆÇÈ�
�ÉÊ
、(¤�����
,ÛË`]¬�
,ÌHÂ
�
,õ;§Íßí^
,l./D$µ¶�»ÄÊ�Ã
��{��ÎÏxUÐBC��
,fä�uÆ#$
����Ññ��������ßÒ*x./
g
[15]。ÓÔu½ÕISSR���uÆ!ijk��
��%àáx./&�
。
;./©uISSRBC��4d��Ö)g}
ÎÏs'!x7��D!ijk��x��ÎÏ�
�B�
,×Øü�����×Ø�����
,$�Ô
\��»x��BÃZÂQ,¶Å×Ø
,ÙÚ�$
�xWX�������
,@����%xÛÂ
×ØP¡�jkád��x{¾sܾ¶Ý
,|�
�g}�D!ijk�����«À�x�tÞß
s����
。
1 àásàÇ
1.1 OPQR
�Ë!ijk�;Æ2015~20179Bº�Æ
ÎÏ
、'!p=>?�
,JKK:;Ññ
。�â�Ö
�G¹ãÕ«1。ä����ÖÔåÆ10Y#$
x�¸�æ
(�Öçèéåi�¸�æ
,øiêë�
#YìíD�îõÔïðñ
),�ÖìòÁ`��ó
079 ! " # $ % & 40ô
S1 6789:;OP?TUVWX
Table1 Collectioninformationof犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿populationsinthisstudy
��õö
Populationcode
�Ö�^
Location=>
Altitude/m��¸
Samplesize�U÷�
Geocoordinate
�Öç»
Collectiondate(yearmonth)
SP ÎÏøùúSongpanCounty,Sichuan 3190~3365 64 32.75°N,103.82°E 201506
KD ÎÏûñ KangdingCounty,Sichuan 2891~2945 23 30.00°N,101.94°E 201607
XJ ÎÏ%üú XiaojinCounty,Sichuan 3494~3720 26 31.15°N,102.76°E 201706
PW ÎÏØýúPingwuCounty,Sichuan 3385~3410 13 32.58°N,104.62°E 201706
GZ ÎÏþÿú GanziCounty,Sichuan 3780~3826 10 31.53°N,99.74°E 201607
TB '!! ú TaibaiCounty,Shannxi 3066~3219 15 33.98°N,107.75°E 201706
MX '!yú MeixianCounty,Shannxi 3210~3274 12 34.02°N,107.97°E 201706
"g#Ð
,$ì%&_`a'ÐÆ-80℃()*u
。
1.2 Y Z
1.2.1 犇犖犃[\ $�Y!ijk�¸�æ�;
|�°
,�u#$,¶� DNA�éË+"
(Tian
gen)�é,¶�DNA,$1%,-./012®¯
DNA�i
,̈u NanoDropRi34
/5 ®¯6
(Thermo)̄ ñDNAx7Â�8Â
,®¯Ä¿ì�
ÐÆ-20℃*u
。
1.2.2 犐犛犛犚]^_`a犘犆犚bc 9¢\:ã
;<=>ã%?åx100¦ISSRFu§$�{
(§$@A801~900)��§$Äõ
(Ñ=DYD$
YZnB?C
)。@7�!ijk��gBºD$
EªFâ�DNA�;�100¦ISSR§$��©
ª
,̈@DNAGH7Â
、§$3IJÂ�PCRK
W¸ßL¸��PCR��µÄx~Ã
。÷Mªé
12¦�E�p
、öñ�
、NO¦%PQxISSR§
$
(«2)uÆ�Ëjk��ISSRPCRNO��
。
ISSRPCR��µÄ
(20μL)IC
:2×Taq
PCRMasterMix10μL(Tiangen),ISSR§$1μL
(10mmol/L),DNA GH1μL�ddH2O8μL。
PCRNOZ�
:95℃�Î�3min;95℃30s,49~
59℃3I1min(DISSR§$3IJÂãÕ«2),
72℃RS1.5min,̈ 35�KW
,÷ì72℃RS10
min。���Tì
,é8μLPCRÜ$Æ1.5%,-
./ 0 � � 1 2 ® ¯
,1 2 ® ¯ � £ ¾ UVP
BiodocIT220/0õUÄÊgqV¨WX�Y
。
1.3 defgh1A
NOÜ$¾12ZÑxäâ¦%[H1�BC
��
,\/0g\â°]ÑxDNA¦%xn¥�
�Ê0
,n%x�|1,¥%x�|0,�^0/1_�
¸¢`a
。¾bñ�Ë!ijk��ÍcÆ Har
dyWeinbergØdxq�G
,0eD5��L¸
。
�uPopgene1.32fgBº0e�E°^±B²
(犘犘犅)、q¯ß°,¶¸
(犖a)、nhß°,¶¸
(犖e)、Neis�����·¸
(犎e)、ShannonG¹·
¸
(犐)、́ �µ,¶���
(犎t)、��{,¶���
(犎s)、��»��B÷¸
(犌st)、Neis��¼½
(犇)。Arlequin35fg�� AMOVA BCàiB
�
[16]。�u MEGA6fgBº,Æ UPGMA(un
weightedpairgroupmethodusingarithmeticalav
erages)ÇsÈÉÇ
(NeighborJoining)���¿»
��ËÌB�
,�^ÄÊËÌj
[17]。9¢D�Ö�
klÂ0e«õ���»x�U¼½
,̈FZGe
nepop ¾ m Y m
(http://www.genepop.curtin.
edu.au/genepop_op6.html)��õ���»x�
�¼½Q�U¼½¿»xÀÓ�B�
[18],$®`�
�»x��BÃZÂlnQ�UÒ½¤ÕÀÓ
[19]。
2 �£QB�
2.1 犐犛犛犚bcij
�u12¦©ªopxISSR§$�7���
¨163Y!ijk�;��PCRNO
,k12®¯
Q¦%Ê0
,̈®¯º136�DNA¦%
,ØÍä�
§$NO«11.3¦
,NO¸¹ã%;100 ~
2000bp¿»
,üg§$810、811、834、847¾!i
jk�B�Ë�;gxISSRPCRNO12Z¾q
Z1o¥
。o®¯«x¦%Ímn�E�
,́�µ
�E�¦%=²|100%(«2),r�;./goª
ISSR§$¾!ijkgÍmnopx�E�
,Û�
Ë!ijk��mnÙÚx�����
,�µ»�
�iÏ-ã
。
2.2 6789:;<=>?@1A
�!ijk7��D���������B
�
,́�µNeis,¶���·¸
(犎e)|0.3186,
1796s 7 8
,ß
:,ÆISSRBC��x!ijk�������B�
ShannonG¹·¸
(犐)|0.4843。¾Ô\jk��
g®¯ºx�E�°^¸��E�°^±B²Ín
iÏ
,ügÎÏøù��
(SP)±´5L¸Í|÷
p
,Bº|129�s94.85%,t'!yú��
(MX)x�E�°^¸÷å
,�E�°^±B²÷
§
,Bº|84�s61.76%。7���xq¯ß°
,¶¸
(犖a)¾1.6176~1.9485¿»
,nhß°,
¶¸
(犖e)¾1.3586~1.5007¿»
。!ijk7
���xNeis,¶���ã%«�|
:øù��
(SP)>ûñ��
(KD)> Øý��
(PW)> !
��
(TB)> yú��
(MX)> %ü��
(XJ)> þ
ÿ��
(GZ),̧ wuv¾0.2958~0.2125¿»
;
tShannonG¹·¸«�|
:øù��
(SP)>û
ñ��
(KD)> Øý��
(PW)> ! ��
(TB)>
%ü��
(XJ)> yú��
(MX)> þÿ��
(GZ),̧ wuv¾0.4482~0.3204¿»
(«3)。
2.3 6789:;<=1k1A
�!ijk7���»x��ÀÁÂ���¼
½��0e
,�£«�7���x Neis��ÀÁ
¾0.8435~0.9673¿»
,Neis��¼½
(犇)¾
0.0333~0.1701¿»
,ügÎÏþÿ��
(GZ)Q
'!yú��
(MX)¿»��ÀÁÂ÷§
,��¼
½÷ã
;tÎÏ%ü��
(XJ)QØý��
(PW)¿
»x��ÀÁÂ÷p
,��¼½÷%
(«4)。�µ
��BÃB�¤¥
,7�!ijk��´,¶���
·¸
(犎t)|0.3097±0.0230,��{,¶���
A.1~182�|ÎÏûñ
(KD)��1~18A�µ
;B、C.1~222�|ÎÏøù
(SP)��1~22A�µ
;D.1~182�|ÎÏ%ü
(XJ)��1~18A�µ
;M.DL2000(¦%û%ºã¡w|
:100bp,250bp,500bp,750bp,1000bp,2000bp)
Z1 §$810(A)、811(B)、834(C)、847(D)¾�B!ijk_`�;gISSRPCRNO�£
A.1-18.SichuanKangding(KD)populationsampleNo.1-18;B,C.1-22.SichuanSongpan(SP)populationsample
No.1-22;D.1-18.SichuanXiaojin(XJ)populationsampleNo.1-18;M.DL2000(bandsizesfromsmalltolargeare100bp,
250bp,500bp,750bp,1000bpand2000bp)
Fig.1 ISSRPCRfingerprintsofpart犆.狋犻犫犲狋犻犮狌犿samplesusingprimer810(A),811(B),834(C)and847(D)
279 ! " # $ % & 40ô
S2 犐犛犛犚]^WXabcij
Table2 InformationofISSRprimersandamplificationresults
§$�A
Primercode§$�{
Primersequence(5′→3′)3IJÂ
Tm/℃
´¦%¸
Totalnumberofbands
�E�¦%¸
Numberofpolymorphicbands
�E�¦%=x
Percentageofpolymorphicbands(犘犘犅)/%
810 GAGAGAGAGAGAGAGAT 53 16 16 100
811 GAGAGAGAGAGAGAGAC 54 14 14 100
834 AGAGAGAGAGAGAGAGYT 53 15 15 100
835 AGAGAGAGAGAGAGAGYC 51 15 15 100
836 AGAGAGAGAGAGAGAGYA 51 11 11 100
847 CACACACACACACACARC 54 9 9 100
855 ACACACACACACACACYT 55 9 9 100
857 ACACACACACACACACYG 54 13 13 100
876 GATAGATAGACAGACA 49 9 9 100
881 GGGTGGGGTGGGGTG 54 10 10 100
887 DVDTCTCTCTCTCTCTC 59 5 5 100
895 GTTGGTAGCTCTTGATC 55 10 10 100
´0Total 136 136 100
S3 67897l:;犐犛犛犚<=>?@fg
Table3 Thegeneticdiversitystatisticsof7populationsof犆.狋犻犫犲狋犻犮狌犿
��
Population
q¯ß°,¶¸
Observednumberofalleles(犖a)
nhß°,¶¸
Effectivenumberofalleles(犖e)
Neis,¶���
Neisgenediversity(犎e)
ShannonG¹·¸
Shannonsinformationindex(犐)
�E�°^¸
Numberofpolymorphicloci
�E�°^±B²
Percentageofpolymorphicloci/%
SP 1.9485 1.5007 0.2958 0.4482 129 94.85
KD 1.8309 1.4236 0.2566 0.3931 113 83.09
XJ 1.7941 1.3586 0.2207 0.3433 108 79.41
PW 1.7574 1.4026 0.2401 0.3651 103 75.74
GZ 1.6324 1.3608 0.2125 0.3204 86 63.24
TB 1.7132 1.4024 0.2336 0.3512 97 71.32
MX 1.6176 1.3950 0.2253 0.3331 84 61.76
ØÍw Average 1.7563 1.4062 0.2407 0.3649 102.86 75.63
´�µTotal 2.0000 1.5343 0.3186 0.4843 136 100
·¸
(犎s)|0.2407±0.0148,́ �µx��BÃ
ĸ
(犌st)|0.2229,,¶Å
(犖m)|1.7430。û
,ÞÕ
,!ijk��»´µ��BÃZÂ-p
(0.15<犌st<0.25),��»,¶Å-y
。
û«5Þ$z«
,!ijk7��Ë��¿»
xài�B|0.6133,o{ài=²|25.58%;
��{�xài�B|1.7842,o{ài=²|
74.42%,ÛiÏÍ|¤Õ×Ø
(犘 <0.001)。r�
!ijk�;»x��BÃ>?|)Æ��{�x
�µ¿»
,üw|)Æ��¿»
。
2.4 6789:;mI1A
Z2¤¥
,,Æ UPGMAÇ�^xÄÊËÌj
g
,ÎÏ%ü��
(XJ)、Øý��
(PW)sþÿ��
(GZ)Ë|â�B}
,ÎÏøù
(SP)sûñ��
(KD)
Ë|~â�B}
;±5�ÎÏ���Ö|âã}
;~
âã}�û'!! ��
(TB)syú��
(MX)́
����õ
(Z2,A)。,ÆÈÉÇ�^xÄÊËÌ
j
,«�|ÎÏ%ü��
(XJ)、þÿ��
(GZ)sØ
ý��
(PW)�Ë|â�B}
,*ì�QÎÏûñ
��
(KD)、øù��
(SP)¡w�Äõâ�ãB}
,
~âã}�\�û'!! ��syú��o�õ
(Z2,B)。û,ÞÕ
,�*,Æ��¼½�éÔ\
xËÌàÇ
,opºx��»ÄÊËÌj����
Ô\
,ú´�ËÌ�£Í¤¥ÎÏ��Q'!��
¿»yÜDÝâñ×Øx��BÃ
。,Ü
,�eÇ
Ô\Ü
,Ô\ËÌàÇpºxËÌ����xiÏ
3796s 7 8
,ß
:,ÆISSRBC��x!ijk�������B�
S4 67897l:;n5犖犲犻狊<=opqa<=rs
Table4 Neisgeneticidentityandgeneticdistanceamong7populationsof犆.狋犻犫犲狋犻犮狌犿
��Population SP KD XJ PW GZ TB MX
SP — 0.9052 0.8966 0.9011 0.8783 0.8843 0.8767
KD 0.0996 — 0.9196 0.9063 0.8747 0.8777 0.8597
XJ 0.1091 0.0838 — 0.9673 0.9515 0.8905 0.8595
PW 0.1041 0.0984 0.0333 — 0.9272 0.8756 0.8537
GZ 0.1298 0.1339 0.0497 0.0756 — 0.8672 0.8435
TB 0.1230 0.1304 0.1159 0.1328 0.1425 — 0.9621
MX 0.1316 0.1512 0.1514 0.1581 0.1701 0.0387 —
«�
:�ÑÛ|��ÀÁÂ
,�GÛ|��¼½
Note:Geneticidentityisabovethediagonal,whilegeneticdistanceisbelowthediagonal
S5 67897l:;犐犛犛犚34512Yt1A
Table5 Molecularvarianceanalysis(AMOVA)in7populationsof犆.狋犻犫犲狋犻犮狌犿basedonISSRmarkers
ÎÏ|�
Sourceofvariation)ûÂ
df
Øàs
Sumofsquares
ài�B
Variancecomponents
ài=²
Percentageofvariation/%
犘w
犘value
��» Amongpopulations 6 71.291 0.6133 25.58 <0.001
��{ Withinpopulations 158 183.266 1.7842 74.42 <0.001
´ÎÏ Total 163 245.757 2.3975 100
�ñĸFixationindex 犉st=0.2356
SP.ÎÏøù��
;KD.ÎÏûñ��
;XJ.ÎÏ%ü��
;
PW.ÎÏØý��
;GZ.ÎÏþÿ��
;TB.'!! ��
;
MX.'!yú��
Z2 ,ÆUPGMAÇ
(A)sÈÉÇ
(B)�^x!ijk
7���ÄÊËÌj
SP.SichuanSongpanpopulation;KD.SichuanKangdingpopulation;
XJ.SichuanXiaojinpopulation;PW.SichuanPingwupopulation;
GZ.SichuanGanzipopulation;TB.ShannxiTaibaipopulation;
MX.ShannxiMeixianpopulation
Fig.2 Phylogeneticclusteringof7populationsof
犆.狋犻犫犲狋犻犮狌犿basedonUPGMA(A)
andNeighborJoining(B)method
Z3 !ijk7�����¼½Q�U¼½
»ÀÓ�B�
Fig.3 Correlationanalysisbetweengeneticdistanceand
geographicaldistanceof7populationsof犆.狋犻犫犲狋犻犮狌犿
Þ�NQD��{������xiÏnÓ
。
2.5 6789:;<=rshuvrsnoD@
1A
|Ý®¯!ijk��»x��BÃZÂln
Q�UÒ½ÀÓ
,�!ijkõ���»x��¼
½Q�U¼½¿»xÀÓ��� Mantel®`
(Z
3),pº��¼½Q�U¼½xm�&�àZ狔=
479 ! " # $ % & 40ô
1.11×10-4狓+0.07,ÀÓĸ犚|0.6481(犘 =
0.086>0.05,1000w$E��
),«�!ijk
��»��¼½Q�U¼½¿»ÀÓ�Ô¤Õ
,�
�»x,¶�žâñZÂÑ2ºÝ�U¼½xB
�
,ú!ijk��»ÜDx��Bý(�«�
�x�UBåG+
。
3 Ø
#$������s�����./
,Þ$�
���¾Ý���#$¾;���Ã�"x,-Ñ
���#$x�D������
。;./�uIS
SR��4d
,¾BC×ØÑ�Bå¾g}ÎÏs
'!´(x7��D!ijk�������Q�
�����Ý®¯
,̈���»��Ý��BÃQ
,¶ÅB�
。¾!ijkg¨©ªpº12�ISSR
�E�°^
,Û�E�¦%±B²Íp³100%,r
�ISSRBC��4dÞH|â�phx./�
¹
,�uÆ!ijk����%./Ò*
。
��xD�WX
、Båuv
、��ã%sè
éà+ßÍ����x����s�����ÜD
ðñ
,�tðñ�$�xWX���s�Ã��
。
!ijk��B�|p=>?�
,Fðmn-
öñxDE��
,ÛBå�=>�ÂxiÏ�jk
��ÝÔ\xD��»
,¾)*ìEG!ijkð
(�«%��)*+Bå
[20]。U ѱ�BåG
+ð¶��%tP¡���Î
,¶�»Ò½tP¡
ß°,¶¾��{$E�ñ
、��»ÜD��BÃ
。
±��ôy¾!ijkâ�µ¶�8x��%./
gæípºÝ _
:g}g¢�x£¤jk
(犆.犼犪
狆狅狀犻犮狌犿)��{�����§t��»��BÃ
×Øp
[11];¬}"�®,jk
(犆.犽犲狀狋狌犮犽犻犲狀狊犲)
�����×ا
,��BäÕ
,Û��¼½Q�
UÒ½¤ÕÀÓ
[12];¡!Båxk+#$犗犲犮犲狅
犮犾犪犱犲狊犿犪犮狌犾犪狋犪��{�����×ا
,��»
mn¤Õx��BÃ
,ü./IÖ|)QNî
、��
�Î
、Ô\xªF¢�
、å¸�µñéßÍÞ�lð
ñ�����s����x¶Ý
[21]。£kX{x
jk
(犆.犮犪犾犮犲狅犾狌狊)��������BÃZÂÍ
8Ægß×Ø
,��»x��¼½Q�UÒ½¤Õ
ÀÓ
,Û��x�����×ØQ��ã%¤ÕÀ
Ó
[22]。BåƤ}x£¤jk��{s��»Í
½��о��ÎÏ
,r�£¤jk��¾���
�Ã"Ñ¥�Æ��¦§À\x¨©xª«�
�
[23]。;./op��xD5��L¸�£Í«
�!ijk��{Ímn-p×Øx�����
,
e¯BåÆg}!�x!ijk¾�Ã�Zµs½
k�Z
“¬h�
”,Ûª«��x�����½1
2ãá®45
,¾�Ãg��WXÎÃÜDÝ-Ù
Úx�����
。
����·��g��ÎÏBåxç�¿À
,
¯°
、±Î
、ªF
、���Î
、Ò½ßÍlðñ���
���x¶Ý
。,¶Ål��»����²�Ãx
>?eb�
,,¶Å�ÙÚ��{x�����
,t
³��»��BÃxZ´y
。U ÑÖ|
,µ
犖m>4ç
,��»,¶�ÅÀ�¶B
,,¶Å�·
¸����ÎxHut³Ô\��³ºÍ�Ã
,¹
º��»��BÃxÜD
[24]。;./�£«�
,7
�!ijk��»´µ,¶Å犖m=1.7430<4,
«�!ijk��»x,¶ÅÔ»$¹ºû���
Χ¥x����BÃ
。7���»x��¼½-
ã
(0.0333~0.1701),NQ!ijk��»,¶
Å×Ø-§nÓ
。“{ètĸ
”犉N¼|
“µ�Ä
¸
”,犉Ê0igx犉stðu|¯i>&�»x��
BÃZÂ
。Fð�犉stwBõ4�½¾�»
,BºQ
�µ»��BÃZÂp§xÓÄ��|
:µ犉st|0
~0.05ç
,Ö|�µ»��BÃo%
;犉st|0.05~
0.15ç
,Ö|�µ»Ð¾gßZÂx��BÃ
;犉st
|0.15~0.25ç
,�µ»��BÃ-ã
;t犉st>
0.25ç,�Ö|�µ»noãx��BÃ
[25]。,¶
BÃĸ犌stQ犉stÓÄ¿À
,µâ�°^Án2�
ß°,¶ç
,_IÀß
;µn��ß°,¶ç
,犌stß
Æonß°,¶x犉stx\ÂØÍw
。¶,�ÆIS
SR��tÃ
,犌stÞQ犉stw��Ì=¨$,ÄÅ�
�»��BÃZÂ
。;./g!ijk��»Ð¾
-ã��BÃ
(犌st=0.2229),±Q��»��¼
½-ã
、�µ»,¶Å-yxB��£â¡
。
!ijk8Æ)�Æs$�
,ú¾)*¦gG
ÇÈ¡É�îÊËÌ�õÍ�_
。¾)*}!ij
kxnh�îÎÏIJ3�ÐÑÑÒ
,Á犅狅犿犫狌狊
犾犲狆犻犱狌狊,�ÓÐÑ犅.犾狌犮狅狉狌犿s犅.犺狔狆狀狅狉狌犿[26],
ûÆÐÑÑÒx!buv-%
,e¯nBxQîÅ
lîõ!ijk��»ÜD��BÃxª¶¿â
。
�¸k+#$x�CR%Û�ÔÕÖ
,¾)*ìE
G�¸�CèN×nØYÙv
,Ánçå¸�Cn
E�Ú¼½�Û¨�¾ü.��{õÍñé
[2728],
¶,!ijk�C$À�N×|>Þ�lü��»
ÜD-ã��BÃx~â�ª¶
。~Ü
,;./�
��Ë!ijk��»x��Bý(�«�¤x
5796s 7 8
,ß
:,ÆISSRBC��x!ijk�������B�
�UÒ½BåG+
,e¯üª¶Þ�Q�jk$�
��*¥�
、�"Ñ�Dx�C$EN×Ag$�
XÌ!bnÓ
。
Guoß
[14]©u2��·µDNA¸¹rps16s
trnLF�{H|BC��
,�Bå¾g}KH
、Î
Ïs!ix9�!ijk��x����s���
����Ý®¯sB�
,ü�£��!ijk��
»Ð¾p×Øx��BÃ
,Û��»��¼½Q�
UÒ½Ômn¤ÕÀÓ�
,Q;./� ÀÅ
。t
ü./�£Ö|!ijkx��{������×
ا
,Q;./�£Ôâ¡
。;./�uISSRB
C��
,���Ë7�!ijk��{mnÙÚx
�����
。./â�$�'��x����Q�
����
,ªFÔ\̼xBC��ÜÜ�îõB
��£xiÏ
[29]。�·µBC��mn�Ý�Ø
Ä��xí^
,FðÔ�D]�
[30],Û�·µ,¶
�|�»µ
,Q3,¶�À=
,�Æ��x�·µ,
¶�xnh�µ-%
,ü��ÎÏ/Þ02º�µ
��h�xðñ
[3132]。��C#$tÃ
,QîxB
½s�Cx×ålßñ�µ����x]?¶Ý
,
¾#$°QìNî¨ôõ�CxZZg
,#$x�
�í#çãZÂ2ºÝà;xðñ
。¶,
,3,¶
BC���/\ÄÊ���«�������xÙ
ÚÂ
。;./o�uxISSRBC��(ÆÇÈ+
��
,ã�|¤���
,����E�ÙÚ
[15],¶,
¾./�µ�����ç
,����À=Æ�·µ
BC��mn/pxáâÂ
。
;./�£«�
,�Ë!ijk��x���
��-ÙÚ
,t��»��BÃZÂ-p
,��»,
¶Å-y
。e¯!ijk¾�s�ÃZZgÜDÝ
ÙÚx�����
,tûÆX|ãi�[P¡�D
��¸ijkGlxAg�D绨ÔãÚ
,³p
!ijk�����¾âñZÂÑ#çäp$�
å
。*tûÆæ�X|¶Ý
,qZ�[
、D�WX
z°�|çè§^ß
,\k�D!ijkDXx
é¸Ã
,&�ÉGêëì%
,�WX�¾x���´
y
,ád��xíî
[3335],,Æ;./�£
,¾,^
ïÃÄ�é��Þß|\ð�!ijk�D��x
��
:(1)¾!ijkxÔ\Bå��øÞ��xñ
Ö�DjkÔ\��x�C
,ò\ó9â¥
,̂ò!
ijk�Có
,$����Djkx�����
;
(2)�Æ��ÉG-%
、�����-§x!ijk
���éô���
,ûÆ!ijkx)*�_²è
n9.57%~13.8%[36],
FcùUXõÞ¾Qs��
XYNî
,�pNîs�_²
,̈\ðµ�#$��
öÇ�Â
,¹º÷�÷[�|
;(3)ªF�ø!ij
kDÐxDX��XYÛ��#
,̂òùx��
,$
O\��¸i
;(4)Þ©u!ijk�C
、�¸ß�
��æsú4d./
,sú!ijk_DûuÆ�
�¬ü
;(5)FZ�ý�þ�Mt
,\ðX��WX
�#$��x��ÿ!
,��!ijkDÐxªD
X
。Ôì./�n"�â#Nã���*
,\ã�
�i
,̈©u��BC��À�ÄxàÇ
,�g}!
ijk�D��x������������/\
$áÄÊxB�sÙv
,|!ijk#$��x�
���U ¡¢
。
wxKL
:
[1] FAYMF,CHASEM W.Orchidbiology:fromLinnaeusvia
Darwintothe21stcentury[J].犃狀狀犪犾狊狅犳犅狅狋犪狀狔,2009,104
(3):359364.
[2] SWARTSND,DIXONK W.Perspectivesonorchidconser
vationinbotanicgardens[J].犜狉犲狀犱狊犻狀犘犾犪狀狋犛犮犻犲狀犮犲,2009,
14(11):590598.
[3] FAYMF,PAILLERT,DIXONKW.Orchidconservation:
makingthelinks[J].犃狀狀犪犾狊狅犳犅狅狋犪狀狔,2015,116(3):
377379.
[4] %&'
,(j)
,*+�
,ß.jk8#$./��
[J].,"
bc+%
,2012,51(9):17331735.
CHENLF,LIUSY,JIANGPD,犲狋犪犾.Researchprogress
on犆狔狆狉犻狆犲犱犻狌犿 Linn.[J].犎狌犫犲犻犃犵狉犻犮狌犾狋狌狉犪犾犛犮犻犲狀犮犲狊,
2012,51(9):17331735.
[5] -Ø.
,J/ý
,0 1
,ß.'!k+3�ù�Y#$
[J].
!"F%V%&
,2005,20(3):6061.
YANGPH,XUZ W,FENG N,犲狋犪犾.Threenewrecord
speciesofOrchidaceaeinShaanxi[J].犑狅狌狉狀犪犾狅犳犖狅狉狋犺狑犲狊狋
犉狅狉犲狊狋狉狔犝狀犻狏犲狉狊犻狋狔,2005,20(3):6061.
[6] ZHENGBQ,ZOULH,LIK,犲狋犪犾.Photosynthetic,mor
phological,andreproductivevariationsin犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻
犮狌犿inrelationtodifferentlightregimesinasubalpineforest
[J].犘犔狅犛犗狀犲,2017,12(7):e0181274.DOI:10.1371/jour
nal.pone.0181274.
[7] BAO W Q,WUYUNTN,LITZ,犲狋犪犾.Geneticdiversity
andpopulationstructureof犘狉狌狀狌狊犿犻狉犪 (Koehne)fromthe
TibetplateauinChinaandrecommendedconservationstrate
gies[J].犘犔狅犛犗狀犲,2017,12(11):e0188685.DOI:10.1371/
journal.pone.0188685.
[8] CHENC,LURS,ZHUSS,犲狋犪犾.Populationstructureand
historicaldemographyof犇犻狆狋犲狉狅狀犻犪犱狔犲狉犻犪狀犪 (Sapindaceae),
anextremelynarrowpalaeoendemicplantfromChina:implica
tionsforconservationinabiodiversityhotspot[J].犎犲狉犲犱犻狋狔,
2017,119(2):95106.
[9] � �
,7JØ
,Ù 2
,ß.10�jk8#$rDNAITS�{
679 ! " # $ % & 40ô
x34QB�
[J].5*ã%%&
(U%6
),2012,39(6):
689695.
JIANGM,LIWP,ZHOUJ,犲狋犪犾.Cloningandanalysisof
rDNAITSsequencesfromten犆狔狆狉犻狆犲犱犻狌犿plants[J].犑狅狌狉
狀犪犾狅犳犣犺犲犼犻犪狀犵犝狀犻狏犲狉狊犻狋狔(ScienceEdition),2012,39(6):
689695.
[10] ���
,%&'
,(j)
,ß.� ?�Ô\Q�jk8#
$�����xISSRB�
[J].!"bF+4ã%%&
()
*+%6
),2014,42(9):137143.
SUNYY,CHENLF,LIUSY,犲狋犪犾.ISSRanalysisfor
geneticdiversityof犆狔狆狉犻狆犲犱犻狌犿inChangbaiMountainarea
[J].犑狅狌狉狀犪犾狅犳 犖狅狉狋犺狑犲狊狋犃牔犉 犝狀犻狏犲狉狊犻狋狔 (Nat.Sci.
Ed.),2014,42(9):137143.
[11] QIANX,LIQJ,犲狋犪犾.Conservationgeneticsofanendan
geredlady'sslipperorchid:犆狔狆狉犻狆犲犱犻狌犿犼犪狆狅狀犻犮狌犿inChina
[J].犐狀狋犲狉狀犪狋犻狅狀犪犾犑狅狌狉狀犪犾狅犳犕狅犾犲犮狌犾犪狉犛犮犻犲狀犮犲狊,2014,15
(7):1157811596.
[12] PANDEY M,RICHARDSM,SHARMAJ.Microsatellite
basedgeneticdiversitypatternsindisjunctpopulationsofa
rareorchid[J].犌犲狀犲狋犻犮犪,2015,143(6):693704.
[13] LIJ,LUO YB,XU LL.Developmentofmicrosatellite
markersfor犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿 (Orchidaceae)andtheir
applicabilitytotworelatedspecies[J].犃狆狆犾犻犮犪狋犻狅狀狊犻狀犘犾犪狀狋
犛犮犻犲狀犮犲狊,2017,5(12):1700084.
[14] GUOJL,CAO WJ,LIZM,犲狋犪犾.Conservationimplica
tionsofpopulationgeneticstructureinathreatenedorchid
犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿[J].犘犾犪狀狋犇犻狏犲狉狊犻狋狔,2019,41(1):
1318.
[15] Ò^£.ISSRBC���ü¾#$��%./gx�u
[J].��
,2002,24(5):613616.
WANGJB.ISSRmarkersandtheirapplicationsinplantge
netics[J].犎犲狉犲犱犻狋犪狊(Beijing),2002,24(5):613616.
[16] EXCOFFIERL,LISCHERHEL.Arlequinsuitever3.5:a
newseriesofprogramstoperformpopulationgeneticsanaly
sesunderLinuxandWindows[J].犕狅犾犲犮狌犾犪狉犈犮狅犾狅犵狔犚犲
狊狅狌狉犮犲狊,2010,10(3):564567.
[17] TAMURA K,STECHERG,PETERSOND,犲狋犪犾.MEGA6:
molecularevolutionarygeneticsanalysisversion6.0[J].犕狅
犾犲犮狌犾犪狉犅犻狅犾狅犵狔犪狀犱犈狏狅犾狌狋犻狅狀,2013,30(12):27252729.
[18] ROUSSETF.Geneticdifferentiationandestimationofgene
flowfromFstatisticsunderisolationbydistance[J].犌犲狀犲狋
犻犮狊,1997,145(4):12191228.
[19] DINIZFILHOJAF,SOAREST N,LIMAJS,犲狋犪犾.
Manteltestinpopulationgenetics[J].犌犲狀犲狋犻犮狊犪狀犱犕狅犾犲犮狌
犾犪狉犅犻狅犾狅犵狔,2013,36(4):475485.
[20] CHUNGMY,LOPEZPUJOLJ,MAKIM,犲狋犪犾.Genetic
diversityinthecommonterrestrialorchid犗狉犲狅狉犮犺犻狊狆犪狋犲狀狊
anditsrarecongener犗狉犲狅狉犮犺犻狊犮狅狉犲犪狀犪:inferenceofspecies
evolutionaryhistoryandimplicationsforconservation[J].
犑狅狌狉狀犪犾狅犳犎犲狉犲犱犻狋狔,2012,103(5):692702.
[21] UENOS,RODRIGUESJF,ALVESPEREIRAA,犲狋犪犾.
Geneticvariabilitywithinandamongpopulationsofaninva
sive,exoticorchid[J].犃狅犅犘犾犪狀狋狊,2015,7:plv077.
[22] BRZOSKOE,WR?BLEWSKAA,TAAAJI,犲狋犪犾.Ge
neticdiversityof犆狔狆狉犻狆犲犱犻狌犿犮犪犾犮犲狅犾狌狊inPoland[J].犘犾犪狀狋
犛狔狊狋犲犿犪狋犻犮狊犪狀犱犈狏狅犾狌狋犻狅狀,2011,295(14):8396.
[23] CHUNGJM,PARKK W,PARKCS,犲狋犪犾.Contrasting
levelsofgeneticdiversitybetweenthehistoricallyrareorchid
犆狔狆狉犻狆犲犱犻狌犿犼犪狆狅狀犻犮狌犿andthehistoricallycommonorchid
犆狔狆狉犻狆犲犱犻狌犿 犿犪犮狉犪狀狋犺狅狊inSouth Korea[J].犅狅狋犪狀犻犮犪犾
犑狅狌狉狀犪犾狅犳狋犺犲犔犻狀狀犲犪狀犛狅犮犻犲狋狔,2009,160(2):119129.
[24] WHITLOCKMC,MCCAULEYDE.Indirectmeasuresof
geneflowandmigration:FST≠1/(4Nm+1)[J].犎犲狉犲犱犻狋狔,
1999,82(2):117125.
[25] ROUSSETF.Geneticdifferentiationandestimationofgene
flowfromFstatisticsunderisolationbydistance[J].犌犲狀犲狋
犻犮狊,1997,145(4):12191228.
[26] LIP,LUOYB,BERNHARDTP,犲狋犪犾.Deceptivepollina
tionoftheladysslipper犆狔狆狉犻狆犲犱犻狌犿狋犻犫犲狋犻犮狌犿 (Orchidace
ae)[J].犘犾犪狀狋犛狔狊狋犲犿犪狋犻犮狊犪狀犱犈狏狅犾狌狋犻狅狀,2006,262(12):
5363.
[27] MACHONN,BARDINP,犲狋犪犾.Relationshipbetweenge
neticstructureandseedandpollendispersalintheendangered
orchid犛狆犻狉犪狀狋犺犲狊狊狆犻狉犪犾犻狊[J].犖犲狑犘犺狔狋狅犾狅犵犻狊狋,2003,157
(3):677687.
[28] JACQUEMYNH,BRYSR,犲狋犪犾.Aspatiallyexplicitanaly
sisofseedlingrecruitmentintheterrestrialorchid犗狉犮犺犻狊
狆狌狉狆狌狉犲犪[J].犖犲狑犘犺狔狋狅犾狅犵犻狊狋,2007,176(2):448459.
[29] WUCA,CAMPBELLDR.Cytoplasmicandnuclearmarkers
revealcontrastingpatternsofspatialgeneticstructureina
natural犐狆狅犿狅狆狊犻狊hybridzone[J].犕狅犾犲犮狌犾犪狉犈犮狅犾狅犵狔,
2005,14(3):781792.
[30] BIRKYC W.Uniparentalinheritanceofmitochondrialand
chloroplastgenes:mechanismsandevolution[J].犘狉狅犮犲犲犱犻狀犵狊狅犳
狋犺犲犖犪狋犻狅狀犪犾犃犮犪犱犲犿狔狅犳犛犮犻犲狀犮犲狊狅犳狋犺犲犝狀犻狋犲犱犛狋犪狋犲狊狅犳
犃犿犲狉犻犮犪,1995,92(25):1133111338.
[31] BIRKYC WJr,MARUYAMA T,FUERSTP.Anap
proachtopopulationandevolutionarygenetictheoryfor
genesinmitochondriaandchloroplasts,andsomeresults[J].
犌犲狀犲狋犻犮狊,1983,103(3):513527.
[32] PETITRJ,DUMINILJ,FINESCHIS,犲狋犪犾.INVITED
REVIEW:Comparativeorganizationofchloroplast,mito
chondrialandnucleardiversityinplantpopulations[J].犕狅
犾犲犮狌犾犪狉犈犮狅犾狅犵狔,2004,14(3):689701.
[33] VOLISS.HowtoconservethreatenedChineseplantspecies
withextremelysmallpopulations?[J].犘犾犪狀狋犇犻狏犲狉狊犻狋狔,
2016,38(1):4552.
[34] VOLISS.Speciestargetedplantconservation:timeforcon
ceptualintegration[J].犐狊狉犪犲犾犑狅狌狉狀犪犾狅犳犘犾犪狀狋犛犮犻犲狀犮犲狊,
2015,63(4):232249.
[35] VOLISS.SecuringafutureforChinasplantbiodiversity
throughanintegratedconservationapproach[J].犘犾犪狀狋犇犻
狏犲狉狊犻狋狔,2018,40(3):91105.
[36] 78á
,7 +
,9:¢
,ß.jk8#$x°Qs�_b
E
[J].DE%&
,2010,30(12):31823187.
ZHENGGL,LIP,TAIYD,犲狋犪犾.Floweringandfruitset
dynamicsin犆狔狆狉犻狆犲犱犻狌犿[J].犃犮狋犪犈犮狅犾狅犵犻犮犪犛犻狀犻犮犪,2010,
30(12):31823187.
(!"
:#$%
)
7796s 7 8
,ß
:,ÆISSRBC��x!ijk�������B�