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Analysis of Genetic Diversity of Germplasm Resources of Lycium barbarum L.Revealed by SRAP in Xinjiang of China

新疆枸杞种质资源遗传多样性的SRAP分析



全 文 :书!"#$%&
,2016,36(4):0681-0687
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D E:pqSRAPrstuOPv34jk[\>]wxyz-{|}~€.0H,34jk[\>]r
‚ƒ„…†[‡ˆm‰Š‹

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:(1)‘’12vSRAP“$”•–‘310—˜;,™nyš-˜;264
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yš-›œž84.61%,“$yš-RŸ ¡(犘犐犆)¢£¤¥0.76~0.93,ž0.83;¦§¨˜<©
(犖犪)、ª«¨˜<©¬(Ne)、Neis<©yz-­¬(犎)、ShannonRŸ­¬(犐)ž®r¯1.8461、1.3869、
0.2280ƒ0.3520。(2)°‚r±²³,ˆ´µ¶·’wx¸¹¬0.5903~0.9032,Dwx¸¹-¬
0.70ƒ0.76º,»¼30½z¾r¯r2_‚ƒ5—¿‚;JÀtr±Œ8ƒ°‚Œ8<ÁÂÃ。0H²³,3
4jk[\>]wxyz-ÄÅÆ

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jk
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(SRAP);°‚r±;JÀtr±;wxyz-
IJBK$
:Q346+.5 !LMNO:A
犃狀犪犾狔狊犻狊狅犳犌犲狀犲狋犻犮犇犻狏犲狉狊犻狋狔狅犳犌犲狉犿狆犾犪狊犿犚犲狊狅狌狉犮犲狊狅犳犔狔犮犻狌犿
犫犪狉犫犪狉狌犿犔.犚犲狏犲犪犾犲犱犫狔犛犚犃犘犻狀犡犻狀犼犻犪狀犵狅犳犆犺犻狀犪
ZHAMeiqin1,ZHAOYulin2,LIJiang1,ZHUYu1,LUOShuping1,HUYue1
(1.ColegeofForestryScienceandHorticulture,XinjiangAgriculturalUniversity,Urumqi830052,China;2.ManagementCen
terofJingheCountyWolfberry,Jinghe,Xinjiang833300,China)
犃犫狊狋狉犪犮狋:SRAPmarkerswereusedtostudythegeneticdiversityandphylogeneticrelationshipforpro
vidindatheoreticalbasisforidentificationandcrossbreedingofgeneticresourcesof犔狔犮犻狌犿犫犪狉犫犪狉狌犿L.in
XinjiangofChina.Theresultsare:(1)atotalof310lociwereproducedby12pairsofSRAPprimers,and
polymorphiclociwere264whichaccountedfor84.61%inthetotalamplifiedfragments.Thepolymor
phisminformationcontent(犘犐犆)valuesofthesemarkersvariedfrom0.76to0.93withanaverageof0.
83.Meanvaluesofobservednumberofaleles(犖犪),effectivenumberofaleles(犖犲),Nei’sgenediversity
(犎)andShannon’sinformationindex(犐)were1.8461,1.3869,0.2280and0.3520.(2)Rangeofgenet
icsimilarity(犌犛)was0.5903to0.9032among30wolfberrysamples.Clusteranalysisshowedthatsam
plescouldbedividedinto2groupsand5subgroupsaccordinglyatthe犌犛of0.70and0.76.Theprincipal
coordinateanalysisandclusteringresultsarebasicalyconsistent.Researchshowsthegeneticdiversityof
germplasmresourcesof犔.犫犪狉犫犪狉狌犿L.inXinjiangwasrelativelyabundant.Thegeneticdifferencebe
tweenwildspeciesandcultivars(lines)wasrelativelylarge,showadistantrelationship,andthegenetic
differenceamongcultivars(lines)wasrelativelysmal,showacloserelationship.
犓犲狔狑狅狉犱狊:犔狔犮犻狌犿犫犪狉犫犪狉狌犿L.;sequencerelatedamplifiedpolymorphism(SRAP);clusteranalysis;prin
cipalcoordinatesanalysis;geneticdiversity;
  jk(犔狔犮犻狌犿犫犪狉犫犪狉狌犿 L.)ÔÕ/(Solanace
ae)jkÖ(犔狔犮犻狌犿L.)’Â[y×IØÙÚN[1]。
jkSÂ[ÛÜÝ]#$

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Table1 Numberandnameofthematerials
¥¦Code ²³Names ¥¦Code ²³Names ¥¦Code ²³Names         
1 gk1¦Jingqi1 11 1021 21 ´k7¦ Ningqi7
2 gk3¦Jingqi3 12 1101 22 ´p1¦ Ningcai1
3 gk4¦Jingqi4 13 1102 23 _µÙjk Hempleafwolfberry
4 gk5¦Jingqi5 14 1103 24 ¶k1¦ Mengqi1
5 gk7¦Jingqi7 15 1104 25 ·¸jk犔狔犮犻狌犿犮狔犾犻狀犱狉犻犮狌犿
6 gk8¦Jingqi8 16 1202 26 o¹jk Whitethornwolfberry
7 1012 17 1407 27 Ⱥ8jk Wildredfruitwolferry
8 1016 18 0901 28 Ȼ8jk Wildblackfruitwolfberry
9 1017 19 ´k1¦ Ningqi1 29 ÈI¼½w8 Wildlilaccolorfruitwolfberry
10 1018 20 ´k5¦ Ningqi5 30 ÈI¾8jk Wildyelowfruitwolferry
286 ! " # $ % &                   36¿
T2 PQ犛犚犃犘YZ[Table2 SRAPprimersequencesusedintheexperiment
¥¦Code
ÒÓSequence(5′→3′)
Primersequence(5′→3′)
¥¦Code
ÒÓSequence(5′→3′)
Primersequence(5′→3′)
me1 5′TGAGTCCAAACCGGATA3′ em1 5′GACTGCGTACGAATTAAA3′
me2 5′TGAGTCCAAACCGGAGC3′ em2 5′GACTGCGTACGAATTAAC3′
me4 5′TGAGTCCAAACCGGACC3′ em3 5′GACTGCGTACGAATTAAG3′
me5 5′TGAGTCCAAACCGGAAG3′ em5 5′GACTGCGTACGAATTACA3′
me6 5′TGAGTCCAAACCGGTAA3′ em6 5′GACTGCGTACGAATTACC3′
me7 5′TGAGTCCAAACCGGACG3′ em8 5′GACTGCGTACGAATTACT3′
em9 5′GACTGCGTACGAATTAGA3′
em10 5′GACTGCGTACGAATTAGC3′
1.2 ] ^
1.2.1 ,_` 犇犖犃 Aabcde –qÀF
CTABƒ‡Áˆ´µ¶<©;DNA[21],qgÂÃ
oȧDNAÅ%,1%ÆÇÈÉ©Êˌ§™Š
Ì-ƒÍ%
,EBvw(,É©`΂¦ÏÐÑÒ


qddH2O¼DNAÅ%ÔÕÖ50ng/μL(


™°-40℃ØÙn±q。
1.2.2 YZfg ›qSRAPڅۓ$ÜÝLi
¨+$ÒÓ

²2),Þ7ßIyI$yàª
X+á&`
,SRAPڅۓ$â12 ,ãã;&
`144—“$;&,PCR…b¸~´ä10×Buffer

 Mg2+)、犜犪狇DNA°&z、dNTPs、100bpDNA
markerƒddH2Ožå5æçI£/O("è)ªX


Lˆ´µ¶néÁ6š--ËÌÄ_’jk
DNABê5—v144vSRAP“$€.,ëì
‘Dí+îï

ðj-、
yš-ŃSRAP
“$qó›ªDNAz¾•–。
1.2.3 犘犆犚hi PCR…bDBioRadPCRÄ7
€.

D25μLSRAP-PCR…bxn, 10×
Buffer(  Mg2+ )2.5μL,dNTP(10 mmol/L)
0.5μL,DNABê(50ng/μL)1μL,犜犪狇DNA°
&z
(2.5U/μL)0.4μL,څ“$(10μmol/L)â
0.5μL,:ddH2O Ö25μL。PCR …bàҁ:
94℃ñ¢-5min,94℃¢-1min,35℃[-
1min,72℃òó1min…b5—ôõ,94℃¢-
1min,50℃[-1min,72℃òó1min…b35—
ôõ
,72℃òó7min,4℃™°。
1.2.4 犘犆犚hijZAde PCR•–ú$`
2μL6×LoadingBufferö÷(,D6%ø¢-°ù
úûüÉ©7ÊËr.

Ê8ýþÿ1×TBE,
200V!Ê"ÊËí50min,Êˌ#(,$vƒv
wÒ

D%&7¦Ïr± ÷ÐݸuÓ
,´
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(j3—:[。
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®

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“1”,5÷u“0”,f/+,
01¬234

pqPIC_CALCVersion0.6â5
6yšRŸ ¡
(犘犐犆)®;7qPOPGENE1.32
â56•–ú$’yš˜;¬

yš-8rœ
(犘犘犅)、¦Ï¨˜<©¬(犖犪)、ª«¨˜<©¬
(犖犲)、Neis<©yz-­¬(犎)ƒShannonRŸ
­¬
(犐)¨ wxyz-ܬ;pqNTSYSpc2.1eâ
56"Ýjk[\·’Jaccard¸¹-¬
(犌犛),-ø:9`;:v6Pžƒ(UPGMA)€
.°‚r±

Q/TreeplotB;I`|}~°‚
c

ë(

ç‹wx¸¹¬€.JÀtr±{<=
>?@·cƒ+?kA;cIŒ“°‚Œ8

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2.1 犛犚犃犘YZhiopBC
pq6š--ËÌÄ_’jkDNABê€.
“$

L144vSRAP“$n‘•– ÷


ðjÇyš-ŃSRAP“$12v,r¯
v30½jkz¾€.SRAPPCR•–,•–˜;
’rs¡JKrîD100~1500bpû·,™n
SRAP“$ me4em8’•–cdc1。v12—
SRAPyš-“$;&D›ª´µ7•–’˜;€
.‚r±

˜;‚Œ8²3。12vSRAPy
š-“$”Œ§‘DNA˜;310—,Þªyš-
˜;264—,Bv“$Œ§’˜;¬14~34—"
¨

ž25.83—,yš˜;¬11~34—"¨,
3864C           @AB,¨ :34jk[\>]wxyz-’SRAPr±
M.DL1500;¥¦1~30ݲ1
c1 “$;&me4-em8vjk›ª´µ’SRAP•–
No.1-30arethesameasTable1
Fig.1 SRAPamplificationof犔狔犮犻狌犿犫犪狉犫犪狉狌犿L.germplasmbyprimercombinationme4em8
T3 PQ67<=>?@Aqr
Table3 Thestatisticsofgeneticdiversityof犔.犫犪狉犫犪狉狌犿
“$
Primer
•–D÷¬
Totalbands
yš- ÷
Polymorphic
bands
yš8rœ
Polymorphic
rate
犘犘犅E%
yš-RŸ ¡
Polymorphism
information
content(犘犐犆)
¦§¨˜<©¬
Observednumber
ofaleles
(犖犪)
ª«¨˜<©¬
Effective
numberof
aleles(犖犲)
Neis<©yz-
Neisgene
diversity(犎)
F^­¬
Shannons
information
index(犐)
me2em6 27 27 100.00 0.79 2.0000 1.4550 0.2603 0.3963
me4em2 27 16 59.26 0.88 1.5926 1.3346 0.1898 0.2834
me4em3 23 16 69.57 0.85 1.6957 1.3196 0.1944 0.3005
me4em6 25 17 68.00 0.93 1.6800 1.3698 0.2093 0.3121
me4em8 23 21 91.30 0.82 1.9130 1.4328 0.2504 0.3829
me4em10 33 27 81.28 0.86 1.8128 1.3733 0.2178 0.3440
me5em5 30 24 80.00 0.78 1.8000 1.2056 0.1340 0.2245
me7em10 29 28 96.55 0.77 1.9655 1.4522 0.2613 0.3996
me2em1 34 34 100.00 0.78 2.0000 1.3609 0.2377 0.3874
me2em9 14 11 78.57 0.88 1.7857 1.4860 0.2754 0.4160
me1em5 18 17 94.44 0.76 1.9444 1.3242 0.1996 0.3195
me6em5 27 26 96.30 0.86 1.9630 1.5285 0.3055 0.4578
ž Mean 25.83 22 84.61 0.83 1.8461 1.3869 0.2280 0.3520
ž22—。"ݓ$;&•–yš-8rœ°
DÄ_ËÌ

L 59.26% ~100% "¨,ž
84.61%。™n,me2em6ƒ me2em1“$;&y
š-8rœë$

žGH100%;me4em2“$;&
yš-8rœëI

59.26%。12v“$Œ§˜
;’yšRŸ ¡
(犘犐犆)®¢£¤¥D0.76~
0.93û·,ž0.83。J³_ñr“$;&•–
yš-ÄÅÆ
,SRAPtuK&ó34jk[\>
]wxyz-0H

ÞªÄ$’yš-Œ§ê

2.2 <=>?@BC
pqPOPGENE1.32â5v30½jkz¾•
–’310—˜;€.r±,L?¬‹²³:¦§¨˜

(犖犪)Mó1.5926~2.0000,ž1.8461;
ª«¨˜<©
(犖犲)Mó1.2056~1.5285,ž
1.3869;Nei′s<©yz-­¬(犎)Mó0.1340~
0.3055,ž0.2280;ShannonRŸ­¬(犐)M
ó0.2245~0.4578,ž0.3520。NV0HŒ
8²³

34jk[\>]wxËÌÄ_

wxyz
-LÄ$

2.3 <=st@BC
pq NTsyspc2.10eâ56SRAP“$ú
I’›ª˜;’wx¸¹¬
(犌犛),30½jkz
¾ã㷒wx¸¹¬Mó0.5903~0.9032û
·

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(犌犛)®0.7361;wx¸¹¬ëВÔ

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ƒ
‘´
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²³|}~ëÎ

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‘1101’
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J
,30½jkz¾·wx¸¹¬ÄI,°DÄ$
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wx¸¹-¬Geneticsimilaritycoefficient
1~30µ¶¥¦Ý²1
c2 30½jk[\’UPGMA°‚r±c
No.1-30arethesamematerialsasTable1
Fig.2 UPGMAdendrogramfor30resourcesof
犔.犫犪狉犫犪狉狌犿L.basedonSRAPdata

gk5¦’、0901、1016、1017ƒ‘´ k7¦’,™n
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A.Threedimensionalgraph;B.Planargraph;No.1-30arethesamematerialsasTable1
Fig.3 PrincipalcoordinatesanalysisofLyciumbararumL.genotypesfromSRAPmarkers
5864C           @AB,¨ :34jk[\>]wxyz-’SRAPr±
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