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Genetic Diversity and Genetic Structure Analysis on Natural Populations of Endangered Rhododendron molle G.Don

濒危羊踯躅自然居群的遗传多样性和遗传结构分析



全 文 :书!"#$%&
,2016,36(4):0674-0680
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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Ap

qrabcMNhijst@Au0vgwxyz

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l‚ƒ„
:(1)12X
SSR…$†‡ˆ260`‰Š,z,‹`ŠŒgt‰Š,zŽ{21.667,t‰Š,zŽ(犖犲){5.425,t
h?D‘’“
(犘犐犆){0.900;$”stgShannonhij•Ž(犐){1.768,,zhij•Ž(犎){0.777。(2).
!-–
(JX)gabcdegMN—˜™š›,œžk(ZH)gMNhijst™Ÿ。(3), ¡¢‰Š,z£¤
(IAM)gMNAu¥Ž(犉狊狋){0.142,,z¦(犖犿){1.522;AMOVAApƒ„abcde§—˜(87.71%)Q 
de¨—˜
(12.29%)。(4)MN©ª«¬­ NJApkStructureAp®¯,8`)*ef°A{±Q­e;
Mantel²³´µ,abcMN©ª@¶·©ª¡ƒ¸¹ºj。89®¯,abc™»¼½¶|}{;,¾¿À|}
.!-–
(JX)de,ÁÂÃÄXœžk(ZH)kÅ"ÆÇ(JS)deg|}ÈÉ。rÊËabcÌÍMNhij
kΉ0vAugwxyz

JKL

abc
;SSR;MNhij;ÏÐ#$|}
MNFO$
:Q346.+5   !PQRS:A
犌犲狀犲狋犻犮犇犻狏犲狉狊犻狋狔犪狀犱犌犲狀犲狋犻犮犛狋狉狌犮狋狌狉犲犃狀犪犾狔狊犻狊狅狀犖犪狋狌狉犪犾犘狅狆狌犾犪狋犻狅狀狊狅犳
犈狀犱犪狀犵犲狉犲犱犚犺狅犱狅犱犲狀犱狉狅狀犿狅犾犾犲犌.犇狅狀
CHENGMalong,DAILiangfang,LUOXiangdong,LIUMenglu,ZHOUYi,
CHENGYanqiu,XIEJiankun
(ColegeofLifeScience,JiangxiNormalUniversity,Nanchang330022,China)
犃犫狊狋狉犪犮狋:Inthepresentstudy,12pairsofsimplesequencerepeat(SSR)primerswereusedtoevaluatege
neticdiversityandgeneticstructureof犚犺狅犱狅犱犲狀犱狉狅狀犿狅犾犾犲G.Donineightpopulationsfromsixprov
inces.Theresultsshowedthat:(1)Atotalof260aleleswithanaverage21.667alelesperlocusweream
plifiedfrom193individuals.Averagevaluesofnumberofeffectivealeles(犖犲)andpolymorphisminforma
tioncontent(犘犐犆)were5.425and0.900,respectively.Thisspecieshashighergeneticdiversityandgene
flowamongdifferentpopulations.For犚.犿狅犾犾犲,theShannon’sinformationindex(犐)andNei’sgenediver
sity(犎)were1.768and0.777,respectively.(2)ComprehensiveanalysissuggestedthatJinxi(Jiangxi
Province)populationexhibitsgreatlevelofvariability,whereasthepopulationofZhenghe(FujianProv
ince)exhibitsthelowestlevelofvariability.(3)Thegeneticdifferentiationcoefficient(犉狊狋)basedoninfi
nitealelemodel(IAM)was0.142andthegeneflow(犖犿)was1.522,respectively.Analysisofmolecular
variance(AMOVA)revealedthatthegeneticvariationmainlyoccurredwithinpopulations(accountedfor
87.71%),morethaninterpopulations(accountedfor12.29%).(4)Theresultsofneighborjoining(NJ)a
nalysisbasedongeneticdistanceandstructureanalysisshowedthateightpopulationswereclusteredinto
threegroups.Themanteltestshowedtherewasnosignificantcorrelationbetweengeneticdistanceandge
ographicaldistancein犚.犿狅犾犾犲.Ourresultsindicatedthepriorityshouldbegivento犻狀狊犻狋狌conservation,
naturalpopulationsinJinxi,Zhenghe,andJingshan(HubeiProvince)deservepriorconservation.Thepossi
bleformationofthehighlevelgeneticdiversityandthemoderatediferentiationof犚.犿狅犾犾犲werealsodiscussed.
犓犲狔狑狅狉犱狊:犚犺狅犱狅犱犲狀犱狉狅狀犿狅犾犾犲G.Don;SSR;geneticdiversity;conservationofendangeredplant
  abc(犚.犿狅犾犾犲),ÑÒÓÔÕ、Öa׉,Ø
ÎÔÕ×+abcÙÚÎۏg1`y:”[1],
܏ÌÍgÝÞßà

ØÔÕ×áâ”ãägÉ<
åæ
[23]。
çè

abcéêë썒h”íjî
A

܏É
ð~ñ$wòóôõ

ö
j÷øù÷ú

Íûük­ýþ‰ÿ
[45];
Xû!"
kE$#"$܏%&»g(‚
[6]。
20)*80+,¼-,abc._/Î、/T,
0ð4.1

2.

34

.!kÅ"‰/5Ìh6

!T¶7$8“A9

:; <=h+>g?@
AB_:CgDE

abcg:FGHIJKÉL
M

abcNèA9g¶7k”eŽ“OPQ8

R
ԦST

UVu
”,
W/XNèSY6ðZL

a
bcÏ [gý\]^ÄP

_`ļ|}
[7]。
MNhijApØaßk|}bÏÐ$”g
É<•,
xcdef`$”g¾GCgxhkX
GC—ijknuglhk¾

mx{noÏÐ
#$¿À|}”ekpogñiqr~É<
€
[8]。
stÉuvw
(Simplesequencerepeat,
SSR),xyz4 {|,z}Î,܏†ƒj、Ív
Éuj

Ívš›j‰¿Œ

Ø89efMNhij
g·~Ü
[910]。
{ˀ„ÏÐabc)*deM
NhijkMNlm‚

VWƒ„}-…†´g
12XabcYZ[(SSR)\]Apð)*deg
MNhijkMNlm

89l‚w{abcN:
åæ|}kwjkVWqrgpo~+%€

1 ‡ˆk‰«
1.1 TUVW
Š‹‡ˆBŒ 2014k2015+g4ÎŽ
5Ž,iâB).!/-–‘、’“‘、”•‘,
œ/žk‘

34/-–‘

ÅT/—I‘

Å"
/ÆǑ

2./˜3‘

†8`de,™š193X
`fiâ

‹``fi⨛810m,BŒ»g
iâWœž|zŸ Š‹¡

¢£¤¥¦

§¨-
80℃œ©|z。VW GISŽBŒë]ª«Bi
Œ¶·¬­v

«deg,®¯°6®1。
1.2 犇犖犃XYC犘犆犚Z[
±²³´‰
[11]
‰«~ñabcµ¶,z}
DNA。BW1%·¸¹º»¼½k¾èA¿¿vš
²³DNAiâgÀvkÁv,-20℃Â|zÃW。
15μLgPCR‡Ãf¥ÄÅ7.5μL2×犜犪狇PCR
greenmix("ÆÆUÇ:$Èɏ¢ÊË~),
ÂÌSSR…$(®2)«1.0μL,ddH2O4μL,,
z} DNA1.5μL。d¾ÍÎ{:94 ℃ϗj
3min;94℃—j45s,55℃(ÐA…$56℃)ÑÒ
45s,72℃uj1min,35`ÓG;72℃ÔÕ7min,
™¦4℃|z。‡ÃÖ$¼20bpDNAladderE
{AB“\]

48%%—j¬×ØÙÚº»Î¼
½

ÛÜ¢{1×TBE(pH8.3)。150VÝü¼½
90min,Þßßá[12],4੐á²。
1.3 \]^_`FG
âAB“QøãšfX‰Š,z

]ªÍŸ
gä,ŽQø

åæ

AB“Qø{120bpgÁ™
͟w]{120/120;AB“{120bpk126bpg
ç™ÍŸ]{120/126),͟èé]{“0/0”,êJ
SSR®¤Ž。ëÀ,VW GenALEx6.1ìÎ[13]
ší«MNhij•Ž

VW POPGENE ší
Nei’shij•Ž(犎)、MNAu¥Ž(犉狊狋)_,z¦
(犖犿)[14];ðî,VWGenALEx6.1ìÎÎgAB
a1 678;Table1 Detailedsampleinformationof犚.犿狅犾犾犲populations
de>æ
Populationslocation
i®Ž
Samplesize
¬­v
Longitudeandlatitude
ïð
Altitude/m
34-–Jinzhai,Anhui(JZ) 21 115°47′E,31°11′N 506~706
2.˜3Pan’an,Zhejiang(PA) 24 120°27′E,28°52′N 514~906
.!”• Yongxiu,Jiangxi(YX) 21 115°43′E,29°08′N 16~39
Å"ÆÇJingshan,Hubei(JS) 22 112°50′E,30°59′N 80~164
.!-–Jinxi,Jiangxi(JX) 30 116°45′E,27°50′N 84~100
ÅT—IShaodong,Hu’nan(SD) 33 111°50′E,27°04′N 209~321
.!’“Poyang,Jiangxi(PY) 24 116°57′E,29°16′N 67~104
œžkZhenghe,Fujian(ZH) 18 119°05′E,27°16′N 993~1176
5764… 012,‰:ÏÐabc)*degMNhijkMNlmAp
‰ñ
(AMOVA)òxnfóApMN—˜4abc
de¨

de§gMNAust

ô¦BWPower
MarkerV3.25ìÎX«ŠŒAph?jD‘“
(犘犐犆)•Ž,šíMN©ª(犇)kMNfõv(犌犐);
öW Neighborjoining(NJ)í«m¬­÷,øV
WGenALEx6.1ší¶·©ª,¬Mantel²³,A
pð@MN©ªg¹ºj

™¦BW STRUC
TURE2.3.4ìÎApeflm[15],㚙ùef
}Ž 犓,犓 ñ1~13,10îÉu,BW Structure
Harvesterší△犓 àE{™ù犓 àú“\û[16]。
2 l‚@Ap
2.1 678犛犛犚cd@eB
®89VW12`h?j…$Xabc8`)
*de†193``fnoSSRAp,ü†‡ˆ260
`‰Š,z

t‹`ŠŒ‰Š,zŽ{21.667,
‡Ãýþÿv490~182bp!¨。®89"Wg
12XSSR…$h?jÌ»,‡Ã͟#$,æ÷1
΅$ HD88X—Ief‡Ãl‚"„。Ap®
¯

%ÁŠŒ‡Ãˆg‰Š,zŽñ˜ÌQ

ðΊ
Œ—˜™8gØ HD83,{14`,™hgØ HD34、
HD39k HD64,{26`(®2)。12`ŠŒgh
?jD‘’“
(犘犐犆)40.790~0.941,tà{
0.900,ƒ„abcMN&guçj。«ŠŒgM
NAu¥Ž 犉狊狋àñ˜$̯ƒ,( 0.067~
0.251,tà{0.142,®¯de§—˜Q de
¨—˜

2.2 6789:;<=>?@AB
MNhijApl‚ƒ„

®3),8`abc)
*deÎg‰Š,zŽ{4.087~6.905,t
{5.425,ðΙÍ{-–(JX)de,žk(ZH)d
e™Ÿ
。Nei’s,zhij•Ž(犎)—uP){
0.706~0.820,tà{0.777,8`degMN—
˜
(Nei’s,zhij)*v;ŸJ̀î{žk
(ZH)<ÆÇ(JS)<”•(YX)<’“(PY)<-–
(JZ)<˜3(PA)<—I(SD)<-–(JX)。Shan
non•Ž(犐)tà{1.768(1.493~2.009),ðM
Nhij*v+v@Nei’s,z•Ž(犎)gl‚,
M.20bpDNAladder
÷1 …$ HD88X—Ide‡Ãl‚
Fig.1 AmplificationproductsofShaodongpopulationbyHD88
a2 67812f犛犛犚cd>?@AB
Table2 Geneticdiversityof12SSRlociin犚.犿狅犾犾犲
SSRŠŒ
SSRlocus
‰Š,zŽ
Numberofaleles
(犖犪)
,zhij•Ž
Genediversity
index(犎)
MNAu¥Ž
Coefficientofgene
differentiation
(犉狊狋)
,z¦
Geneflow
(犖犿)
h?D‘“
Polymorphism
information
content(犘犐犆)
HD25 21 0.924 0.067 3.479 0.922
HD26 17 0.883 0.223 0.873 0.877
HD34 26 0.935 0.112 1.975 0.939
HD35 24 0.929 0.095 2.379 0.925
HD37 15 0.812 0.162 1.295 0.790
HD39 26 0.942 0.146 1.450 0.941
HD43 25 0.926 0.172 1.201 0.925
HD55 23 0.919 0.163 1.287 0.917
HD64 26 0.932 0.076 3.040 0.932
HD80 21 0.889 0.251 0.745 0.881
HD83 14 0.864 0.150 1.420 0.853
HD88 22 0.906 0.090 2.537 0.901
t Mean 21.667 0.905 0.142 1.522 0.900
676 ! " # $ % & 36,
®fõ

®3)。-™«MNhij±ŽgApl‚
®¯


(JX)¶7abcef§.’Ìš›gM
N—˜

—I
(SD)、˜3(PA)î!,-–(JZ)、’“
(PY)、”•(YX)、ÆÇ(JS)4`¶7deMNhi
jstdÎ

žk
(ZH)deMNhijst™Ÿ。
2.3 6789:;<=>?DEgh
2.3.1 6789:;<=犃犕犗犞犃FG abc
8`)*deMNAustgAB‰ñApl‚®
¯

®4),abcefg—˜QÐAz4 de§
(87.71%),de¨gMN—˜{12.29%(犘<
0.001)。/l‚@;Nei’s•Žšígef¨Au
¥Ž
(犉狊狋=0.142)²³gl‚fõ。ÁÂ,abc
det,z¦犖犿 {1.522(®2),®¯abcd
e¨wxC0ç12,z34Ö:fo0vg,z


=567ËzMN8—…9gde¨A
u0v

2.3.2 678;?jk`犖犑lOFG 
MN©ª
(犇)kMNfõv(犌犐)Ap®¯(®5),
abc«deNei’sMN©ª_MNfõvgt
àA:{0.1426k0.8662,®¯«de;<º¥
5Ì=

2.˜3
(PA)@ÅT—I(SD)deMN
fõv™Q

{0.9054,ÁÂMN©ª™ø,{
0.0994。.!”•(YX)@œžk(ZH)def
õv™ø

{0.8055,>F¨MN©ª™Q,{
0.2163。¬ Mantel²³´µ,abc«de¨g
MN©ª@¶·©ª¡ƒ¸¹º
(狉=-0.164,犘=
0.248)。
%Áabcde"mg NJ¬­l‚(÷2)
®¯
,8`de;(JS)de{Ⅰ­,-–(JZ)、-–(JX)、žk(ZH)
k’“
(PY)de¬{Ⅱ­,˜3(PA)@—I(SD)2
`de¨MN©ª™ø

º¥™?@

¬{Ⅲ­。
2.3.3 6789:;<=犛狋狉狌犮狋狌狉犲FG , A
¶Bí«gStructurel‚ƒ„,abc«deˆµ
ËfogMNAu

÷3)。△犓 ™Qà"X¾g犓
à{3,®¯abc)*deStructureApg™g
犓 à{3。StructureApl‚ƒ„,abc8`d
ewAî3Q},A:W3”Cá>®„(÷4),w
a3 6789:;<=>?@AB
Table3 Geneticdiversityofnaturalpopulationsof犚.犿狅犾犾犲
de
Population
‰Š,zŽ
Numberof
aleles(犖犪)
‰Š,zŽ
Effectivenumber
ofaleles(犖犲)
Shannon•Ž
Shannon
index(犐)
,zhij•Ž
Genediversity
index(犎)
Do•Ž
Fixation
index(犉)
JZ 7.58 5.061 1.724 0.783 0.415
PA 9.00 6.010 1.893 0.805 0.420
YX 8.83 5.581 1.791 0.770 0.402
JS 7.25 4.720 1.634 0.754 0.330
JX 10.58 6.905 2.009 0.820 0.367
SD 9.25 6.029 1.871 0.806 0.551
PY 7.83 5.004 1.728 0.771 0.449
ZH 6.48 4.087 1.493 0.706 0.188
t Mean 8.35 5.425 1.768 0.777 0.390
  E:de,FÁ®1;ÂÁ。
Note:Populationcodesaresameastable1;Thesameasbelow.
a4 678>?Fmno=pqFG(AMOVA)
Table4 AMOVAofthegeneticdifferentiationlevelin犚.犿狅犾犾犲
—˜>æ
Sourceof
variance
);v
犱犳
‰ñk
Sumof
squares
t‰ñ
Meanof
squares
—˜}A
Variance
component
—˜GAH
Percentageof
variation/%
犘
de¨Amongpopulations 7 278.334 39.762 0.683 12.29 <0.001
de§ Withinpopulations 185 1823.511 9.738 4.869 87.71 <0.001
üšTotal 192 2101.845 5.552 100.00
}¨Amonggroups 2 175.104 87.552 1.153 6.86 <0.001
}§ Withingroups 190 2973.585 15.650 15.650 93.14 <0.001
üšTotal 192 3148.689 16.803 100.00
  E:犘஄5Ý³à—˜QgIH,J`IHØC0KdeÎgi®¬01000îôL*w㗚íêJg。
Note:犘valuesaretheprobabilitiesofhavingamoreextremevariancecomponentthantheobservedvaluesalone.Probabilitieswerecal
culatedby1000randompermutationsofindividualsacrosspopulations.
7764… 012,‰:ÏÐabc)*degMNhijkMNlmAp
a5 犖犲犻’狊>?jk(±M)` >?rst(±M)
Table5 Nei’sgeneticdistance(belowdiagonal)andgeneticidentity(abovediagonal)
de
Population
JZ PA YX JS JX SD PY ZH
JZ - 0.8847 0.8341 0.8572 0.9041 0.8886 0.8703 0.8671
PA 0.1225 - 0.8608 0.8713 0.8969 0.9054 0.8685 0.8521
YX 0.1814 0.1499 - 0.8442 0.8747 0.8559 0.8606 0.8055
JS 0.1541 0.1378 0.1694 - 0.8700 0.8718 0.8593 0.8173
JX 0.1008 0.1088 0.1339 0.1393 - 0.8937 0.8529 0.8529
SD 0.1181 0.0994 0.1556 0.1372 0.1124 - 0.8838 0.8647
PY 0.1389 0.1410 0.1501 0.1516 0.1210 0.1235 - 0.8858
ZH 0.1426 0.1600 0.2163 0.2018 0.1591 0.1454 0.1213 -
÷2 abcef¨NJ¬­Ap
Fig.2 NJanalysison8populationsof犚.犿狅犾犾犲byPowerMarker
÷3 VW△犓XeflmnoAp
Fig.3 Thegroupstructureanalysisonbasing△犓
1~8A:{JZ、PA、YX、JS、JX、SD、PYkZHde;¹ÁgCá,®f`­e。
÷4 WStructureNOg8`abcdeMNlm(犓=3)
Thenumber1to8representedJinzhai,Panan,Yongxiu,Jingshan,Jinxi,Shaodong,Poyangand
Zhenghepopulation.Thesamecolorindicatesthesamepopulation
Fig.4 Estimatedgeneticstructurefor犓=3obtainedwiththeSTRUCTUREprogramfor8populationsof犚.犿狅犾犾犲
¼Pˆ


(JZ)、-–(JX)、žk(ZH)de¬{
f}

˜3
(PA)、—I(SD)、’“(PY)de¬{f
}

”•
(YX)、ÆÇ(JS){f}。nfó5ÌAp
´µ
,StructureApl‚@, MN©ª«g NJ
¬­l‚,®fõ

QR’“
(PY)deè,ðS
7`deg¬­l‚Téfõ。nfóX8deA
{3}noAMOVAAp´µ,abc}¨MN—
˜{6.86%(®4),}¨tAu¥ŽΦ狊狋{0.069
(犘<0.001),UîV¯abc)*de}§—˜Q
 }¨—˜

3 r Ê
MNhijØ$”ÿ…:zk|jnulhg
$W,X

Ø:$hij|}gÉ<}îÐA

f
Y5Z

”

ÏДgMNhijst̟
[17],
:$Wx|jÌÍstgMNhij
[18]。
ab
cØÎabcÙÚÎۏgf`y:”

; ð
ïWßàk[\ÝÞßà

<{?@]î:CDE

876 ! " # $ % & 36,
A9^_Tµý`u

™aOõabcî{ÏÐ
”
[19]。
®89Î
,12XSSR\]Apƒ„abc
܏ÌÍgMNhij
(犘犐犆=0.900,犐=1.768,
犎=0.777),Í ç-VWISSR\]Ap.!a
bcgMNhij
[20](犐=0.5007,犎=0.3342)¼
_ÁÚgbcÔÕ
[21](犐=0.4972,犎=0.3386),d
Ÿ SSR\]Apg66`ÔÕâ”MNhij[22]
(犘犐犆=0.838,犐=2.2293),9ðyz,f‰ewx
89g)*deQø_:?­¤%Á"õ

ff‰
ewxØabc{h+:

gRÌÿ#$

y®A9
P)Ì{xy

ÿ…gdehukÔkSiðjk
ÌhMN—˜

lئ…0vB@_ð:CmDE
ˆµý`u

:ý`u¨%ÿ

MNhij(4n
éoþ

ý`u:CX”egMNlmkMNhijs
tÖ:pQLM

fY5Z

:Cý`uqŸ
deQø

]î$”g¾GCxhÂq

ÁÂ$wx
rz{MN8—k=1sÑ5ide¨Ö:MNA
u

n5qŸ/”eg:zxh

;ç4¦,Ö:M
Ntu
[23]。
v=g89ƒ„

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