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Genetic Diversity Analysis of Tea Germplasm in Qiannan Prefecture by SSR Markers

黔南60份茶树种质资源遗传多样性的SSR分析



全 文 :书!"#$%&
,2016,36(6):1117-1124
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E F:efg:;hNLMR0]^ijklmn,` aSSRopqrst,u:;60vLM]^wxyDNA
jklmnoz

{|}B
:15u~$€lPn,‚ƒlPn„o…e98.64%。15uSSR~$†‡ˆ‰
147Š‹ŒŽ‚ƒQ73.7786ŠŽ‚ƒ,‘’Š~$‡ˆ9.8Š‹ŒŽ‚ƒ,4.9186ŠŽ‚
ƒ
。15Š+aŽ5†“N280R‚ƒ”,‘’ŠŽ518.7R‚ƒ”。jklPV•1–—?˜e0.1239~
0.9268,‘0.5725,‘‹Œ™š›、‘œ™š›Q‘ShannonsV•žŸo e0.4700、0.6023、
1.4644。¡¢£oz¤,60v¥¦§jk¨©ªŸ«0.2051~0.8636¬§,­‘jk¨©ªŸ0.4775e®
¯

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犌犲狀犲狋犻犮犇犻狏犲狉狊犻狋狔犃狀犪犾狔狊犻狊狅犳犜犲犪犌犲狉犿狆犾犪狊犿犻狀犙犻犪狀狀犪狀
犘狉犲犳犲犮狋狌狉犲犫狔犛犛犚犕犪狉犽犲狉狊
ZHANGMingze,YAOYuxian,CHENShijun
(ColegeofBioscienceandAgronomy,QiannanNormalUniversityforNationalities,Duyun,Guizhou558000,China)
犃犫狊狋狉犪犮狋:Inthisstudy,fifteenSSRprimerpairswithpolymorphismwereusedtoassessgeneticdiversity
andrelationshipof60wildteagermplasmsfromQiannanprefecture.Theresultsshowedthatthepercent
ageofpolymorphicbands(犘犘犅)was98.64%.Atotalof147observedalelesand73.7786effectiveal
lelesweregenerated,withameanof9.8and4.9186perlocus.Totaly280genotypesweredetectedinal
materials,withameanof18.7foreachpolymorphismprimerpairs.Thepolymorphisminformationcon
tentvariedfrom0.1239to0.9268,withanaverageof0.5725.Theaverageobservedandexpectedhet
erozygositieswere0.4700and0.6023,respectively.TheaverageShannon’sinformationindexwas1.464
4.Thesimilaritycoefficientamong60teagermplasmswas0.2051to0.8636.Whenthesimilaritycoeffi
cientwas0.4775,eightmajorgroupsweregeneratedfromaltheaccessionstestedbyUPGMAclustering
analysis.Theresultsshowedthattherewerenosignificantcorrelationbetweengeneticandgeographicdis
tanceamongtheteagermplasms,andsomeindividualsinthesamepopulationdistributedindifferent
groupsofthecluster.Theseresultssuggestedthatthematerialsusedintheexperimentpossessedabroad
geneticvariation,showingahighlevelofgeneticpolymorphismamongteagermplasmsrevealedbySSR
markers.
犓犲狔狑狅狉犱狊:tea;germplasmresources;SSRmolecularmark;geneticdiversity;Qiannanprefecture
  (¿ÔLM]^oÕ?˜Ö,jklmnÒ
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ח”LM]^Íl
[1]。
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Table1 Codeandoriginofteaaccessionsusedinthisstudy
ef
No.
¶^
Origin
ef
No.
¶^
Origin
ef
No.
¶^
Origin
Y1 cdgh Mochong,Duyun Y21 cdiBGujiang,Duyun W41 jc´kShuixiang,Shandu
Y2 cdBlJiangzhou,Duyun Y22 ÚmnmGoushan,Dushan W42 opqrZhucang,Wengan
Y3 ú@sYunwu,Guiding Y23 ú@sYunwu,Guiding W43 ‘t+(Tongzhou,Pingtang
Y4 cdgh Mochong,Duyun Y24 cdiBGujiang,Duyun W44 ‘t+(Tongzhou,Pingtang
Y5 cduv Huanghe,Duyun Y25 cdwxBaimang,Duyun W45 Úm1mYingshan,Dushan
Y6 cdymTuanshan,Duyun Y26 cdBlJiangzhou,Duyun W46 z{|} Mazhi,Longli
Y7 ÚmnmGoushan,Dushan Y27 cdBlJiangzhou,Duyun W47 ‘t+(Tongzhou,Pingtang
Y8 cd~QFenghe,Duyun Y28 cdgh Mochong,Duyun W48 z{|} Mazhi,Longli
Y9 cdBlJiangzhou,Duyun Y29 cdgh Mochong,Duyun W49 ú@sYunwu,Guiding
Y10 cdgh Mochong,Duyun Y30 ú@sYunwu,Guiding W50 ú@sYunwu,Guiding
Y11 cdBlJiangzhou,Duyun Y31 cdBlJiangzhou,Duyun W51 ‘tÎtDatang,Pingtang
Y12 ÚmnmGoushan,Dushan Y32 cdiBGujiang,Duyun W52 €mFengshan,Fuquan
Y13 ú@sYunwu,Guiding W33 ‚´ƒ„Ningwang,Huishui W53 ‘t+(Tongzhou,Pingtang
Y14 cdgh Mochong,Duyun W34 Úm1mYingshan,Dushan W54 ú@sYunwu,Guiding
Y15 cdgh Mochong,Duyun W35 jc´kShuixiang,Shandu W55 Úm1mYingshan,Dushan
Y16 cdwxBaimang,Duyun W36 ‚´ƒ„Ningwang,Huishui W56 Úm1mYingshan,Dushan
Y17 cdiBGujiang,Duyun W37 z{|} Mazhi,Longli W57 ‘t+(Tongzhou,Pingtang
Y18 cdgh Mochong,Duyun W38 …†‡ˆNajiang,Luodian W58 …†‡ˆNajiang,Luodian
Y19 ú@sYunwu,Guiding W39 ‚´ƒ„Ningwang,Huishui W59 ú@sYunwu,Guiding
Y20 cdiBGujiang,Duyun W40 ‚´ƒ„Ningwang,Huishui W60 ú@sYunwu,Guiding
8111 ! " # $ % &                   36‰
ª,
eLM/R

™!caâã2Š‹Œ

1 ¥¦_Ôô
1.1 RSTU
L“é:;k*ðŽ“

JKL“i¨
ďQst‘Íl

ɒ“”•L–¨Ä
—O˜™Æš

›¿J‘ìL–uœ”ꪏ
ͺiyž

ƒ
$ɒ“L
–웿J‘2Š¢g

£¤O웿J
‘¥ÇG¡

†G¡:;¿¦œ”hN”LMR0
]^60Šêª(}1)。mꧨ©ª«»¬­ë

,¯
ÂÝÞ-80℃。
1.2 X Y
1.2.1 犛犛犚Z[ ‚ƒODNA2«Ga°wi
CTAB[13]ô,DNA01Q±›a0.8%²³´µ¶
·¸¹Œ

aŸ ~$eSSRº»~$,$"¼½
¾¿ÀN$stÁÂÃÄŚå

~$ÆÇòÈ
Kaundun[14]、ɂʍ[15]、öaË[13]、ÌBÍ[16]
iÎÏ

JÊâãlPnÍ,i40u~$,­¥¦
Y1、Y7、Y14、W36、W54‚ƒODNAWÐÑ,wx
lPnÒâ

_Óâãy15u~$(}2)。
1.2.2 犘犆犚Z[ PCR‡ˆÔJýª(25μL)e:
40ng/μLÐÑDNA(2.0μL)、~$(É0.5μL)、
10mmol·L-1dNTPs(0.5μL)、10×PCRÔJÕ

(2.5μL)、2U犜犪狇DNA ¢š×(0.5μL)、
ddH2O18.5(μL)。‡ˆØÆe:94 ℃ ٖn
5min;94℃–n30s,51℃Ú60℃¸ÛÜ30s,
72℃ÝÞ30s,35ŠßØ;ßØ{फ72℃ÝÞ
10min。“$4℃¯Â。á8e:GeneAmpPCR
System9600(PerkinElmer,USA)。‡ˆ“$Ga
â㗷¸

ä庻¹Œ

«96æµmÑi’Š
æÊo ç1μLèáiPCR“$,8.5μLéêë
Q0.03μLROX500op1ìq,Áí,95℃–n5
min,` a DNA ŒÆá ABI3730(AppliedBiosys
tems,FosterCity,USA)wxä庻¹Œ。
1.3 \]^_`CD
`aGeneMapperv4.0îÞï¡·¸{|Ÿ
Œ

4ú‡ˆðñۛ

‚ƒ”Ÿ

‹ŒŽ‚ƒŸ
(犖a)、Ž‚ƒŸ(犖e)、‹Œ™š›(犎o)、œ
™š›
(犎e)、ShannonV•lmnžŸ(犐)òó
¯

¥¦¸Ç¶^¿§jk»¼›
(geneticidenti
ty)QjkÀÁ(geneticdistance),­ì犉*ó1Ê
i犉it、犉is、犉staPopGeneVer.1.32îÞoz,
ôŒîÞõOöI

±*óiDNAðñۛ÷ø
eA、B†€nopqr‚ƒ”ŸŒ,³Ê,犉it
žóù‰i‚ƒ”iúûü…_Oýœü…«)
²ýÊiþÁ؛
,犉isž‚ƒ”iúûü…_
Oýœü…«ÿ²ý

!²§"$R§

ÊiþÁ
Q2 abcd
Table2 InformationofSSRmarkers
~$
Primer
"#f
Accession
No.
45‚Æ
Repeatmotif
~$ÆÇPrimersequence(5′→3′)
þ$Forward Ô$Reverse
ÛÜ%›
Annealing
temperature
/℃
“$Îø
Product
size/bp
QNSSR01 CV69972 (TTTTTA)3 GTCAAGAAAGCTCAAGGC AAGACCCATACAAAAGATACT 51 137~157
QNSSR02 CV067063 (TC)15 GGAGCATTGAAGCGAGAAAT ACGCTTCGAGTACTCCCTGA 55 165~199
QNSSR04 CV011305 (GGAAA)11 TAGCTCGCACACAACACCAC TCCAACGACACACTCTCTGC 58 170~204
QNSSR05 CV014525 (GTGGA)5 ATCCACCGTATGATGCTT TGTCTTGTGACCAAATTGAC 51 264~276
QNSSR06 FF682718 (CT)15 AAAGAGGAGAGGCGAGGACAG TTCAGGATACGCTTTGATGCC 58 85~136
QNSSR07 DN976198 (AAGAA)3 GCAGAAAACCCTGTCAAT ATCACCACCCCACCATAC 51 123~175
QNSSR08 FE942905 (CCGCCA)3 TGCCCACACCACGAATACGAC GAAGATGGTTGCGAATGGCT 57 115~135
QNSSR09 AJ621796 (CA)12 GCATCATTCCACCACTCACC GTCATCAAACCAGTGGCTCA 57 146~184
QNSSR11 AJ621798 (TG)13 CACATTGTGGCGTGTTATTAATTT ACATTGGCTATCTCTCATCATGG 56 285~287
QNSSR12 AJ621786 (GT)16 GAATCAGGACATTATAGGAATTAA GGCCGAATGTTGTCTTTTGT 53 171~237
QNSSR15 (ATG)10 GCGTCGTCCCTTCTTTCTAA GGGCAGCCATAACCACTACT 57 139~177
QNSSR16 (TTC)10 CAGGGTTGCAAGAAGTACCG ATCAACCGTATGGGCAAAAG 55 108~158
QNSSR18 (AG)14 GGGAGAACCAACCCAGTCTAT CCCAATCCGCTGTAGTAGGA 58 133~159
QNSSR21 CV013826 (CTC)5 ATGAGAAGGAGGACGATG CATTTATGGACCTGTTCG 51 122~124
QNSSR23 CV011305 (TG)8(AG)10 TGGATTCCACCCAGAGTCC CCACCGACTCGATGACATAA 57 130~174
91116œ              ABC,::;60vLMR0]^jklmniSSRoz
؛
,犉st}ÿ²ý¬§ijkoá؛。ôŒ
Ä&犖m=0.25×(1-犉st)/犉stóùԂƒ(
ʛ
(犖m)。lPV•)1(犘犐犆,polymorphism
informationcontent)aPowermarker3.25îÞo
z

lPnÝ*ŸìlPnÝ*„o…
(犘犘犅,
percentageofpolymorphicbands)` aExcel*ó。
¥¦Šý§jk¨©ªŸ

jkÀÁaîÞ NT
SYS2.10eoz,ôŒîÞöI,òȧ+,[17]
iÔô

±¸ÇۛiDNAðñ-e»ŠŽ‚
ƒ

‰.RŽ‚ƒ;/¯
“1”,¸ «;/¯“0”,
]012ŸŒ34

opª*M#ZGa5ç67
uùt‘ô
(UPGMA)¢£ozÔô\],«î
ÞÊi Matrixcomp.plotu¢£{|Q¨©ªŸ
34¬§i¨Änwxoz

Ga TFPGA wx
Mantel¹Œ,Œú¥¦¶^¿¿OÀÁ_¥¦¶^
¿jkÀÁi¨ÄĪ

2 {|_oz
2.1 犛犛犚abNeVTU=>?@ACD
`aÒâ‰i15uSSR~$,‡ˆ:;¸Ç
¿OØÙi60vhNLMR0]^DNA,{|€


ºÆmaʇˆ‰[*
(85~287bp),·¾Ñ
Í6i45nQlPn

jklmnoz{|

}3)}B,15uSSR~$†¹Œ8147Š‹Œ
Ž‚ƒ

Ž‚ƒŸ1
(犖a)–áe2~23Š,‘
Ž‚ƒŸe9.8Š,³ÊlPn‚ƒ145Š,9:
‡ˆŽ‚ƒŸi98.64%。’u SSR ~$‘
Ž‚ƒŸe4.9186。†¹Œ8280Š‚ƒ
”

‚ƒ”Ÿ–áe2~45Š,‘‚ƒ”Ÿ18.7
Š

‹Œ™š›
(犎o)–;«0.0000~0.8500¬
§

œ™š›
(犎e)–;«0.1282~0.9388¬
§

‘‹<™š›
(0.4700)øï‘œ™š›
(0.6023)。lPV•)1(犘犐犆)–á?˜ÍÎ,_
ø¯e0.1239,_ίe0.9268,‘0.5725;
ShannonV•žŸ(犐)–;«0.3099~2.8622¬
§

‘1.4644。¸ÇiŸŒžq}B15uLM
SSR~$™=:;60vhNLM]^jk
lmn

³Ê5ŠSSR~$(QNSSR02、QNSSR04、
QNSSR06、QNSSR18、QNSSR23)i犐≥1.5、犘犐犆
≥0.8ì 犖a≥10,e_i~$。
ŸŒ}B

:;60vhNLM]^яÒÓi
jklmn

Ž‚ƒŸ_Ž‚ƒ–—ü…
¨Ä>

Ž‚ƒŸ_ShannonsV•žŸi
–á?ŽÎý»¼

Ž‚ƒŸÍÎiSSRŽ
Q3 犛犛犚abZ[fgV?hAcd
Table3 ResultsandthepolymorphisminformationofSSRprimers
~$
Primer
‚ƒ”Ÿ
No.of
genotypes
‹ŒŽ
‚ƒŸ
犖a
lPnÝ*Ÿ
No.of
polymorphic
bands
lPnÝ*
„o…犘犘犅/%
Ž
‚ƒŸ
犖e
‹Œ™š›
犎o
œ™š›
犎e
lPnV•
)1
犘犐犆
Shannon
žŸ犐
QNSSR01 4 4 3 75.00 1.7337 0.5667 0.4268 0.3582 0.7174
QNSSR02 29 16 16 100.00 9.1837 0.4333 0.8986 0.8814 2.4256
QNSSR04 33 14 14 100.00 8.4606 0.8167 0.8892 0.8711 2.3343
QNSSR05 7 5 5 100.00 1.3213 0.0833 0.2452 0.2346 0.5587
QNSSR06 45 23 23 100.00 14.4869 0.6667 0.9388 0.9268 2.8622
QNSSR07 6 5 5 100.00 1.2746 0.1500 0.2172 0.2087 0.5049
QNSSR08 4 4 3 75.00 1.1456 0.1333 0.1282 0.1239 0.3099
QNSSR09 21 13 13 100.00 4.1190 0.4500 0.7636 0.7350 1.8695
QNSSR11 2 2 2 100.00 1.8967 0.0000 0.4768 0.3610 0.6657
QNSSR12 13 10 10 100.00 1.6412 0.3333 0.3940 0.3802 0.9723
QNSSR15 24 10 10 100.00 5.5771 0.7167 0.8276 0.7982 1.9148
QNSSR16 16 9 9 100.00 4.1119 0.7500 0.7632 0.7188 1.5922
QNSSR18 33 12 12 100.00 8.1630 0.8333 0.8849 0.8657 2.2564
QNSSR21 3 2 2 100.00 1.3846 0.2667 0.2801 0.2392 0.4506
QNSSR23 40 18 18 100.00 9.2784 0.8500 0.8997 0.8844 2.5319
‘ Mean 18.7 9.8 9.7 - 4.9186 0.4700 0.6023 0.5725 1.4644
šóTotal 280 147 145 98.64 73.7786 - - - -
0211 ! " # $ % &                   36‰
5³ShannonsV•žŸ@ÍÎ,ShannonsV•ž
ŸNŽ‚ƒŸA™Ô²ýijklmnØ
›

°WeA6iíBžq

2.2 ijWCkCD
u¥¦i¸Ç¶^¿

cd

ú

Úm

‚´

jc

z{

…†

op

‘t



§jkoáwx
oz

{|

}4)}B,Ç»¶^¿ì‚ƒlmn
(犉is)‘¯e-0.0408,lŸŽ5}.™šýþ
,
;¸
Ƕ^¿§‚ƒlmn
(犉it)‘¯e
0.1936,}B:;hNLM]^:ý™šp¸C。
¸Ç¶^¿§jko᪟
(犉st)e0.2252,}B
22.55%ijko᪟«復¸Ç¶^¿§。
¥¦¸Ç¶^¿§‚ƒ(
(犖m)‘¯e0.8601,
‚ƒ(Íø

DB¸Ç¶^¿§‚ƒ!(؛ÍE

2.3 ijWlmno=>pqCD
u¥¦i¸Ç¶^¿

cd

ú

Úm

‚´

jc

z{

…†

op

‘t



§jk»¼›Q
jkÀÁwxoz{|

}5)}B,ɶ^¿§j
k»¼›?˜e0.6430~0.9439,jkÀÁ?˜
e0.0577~0.4416。cd_úijk»¼›_
Q4 TUWCk
Table4 Thegeneticdifferentiationamongorigins
~$
Primer
Ç»¶^¿ì‚ƒlmn
犉is
¸Ç¶^¿§‚ƒlmn
犉it
¸Ç¶^¿§jko᪟
犉st
‚ƒ(
犖m
QNSSR01 -0.5176 -0.3533 0.1083 2.0583
QNSSR02 0.2479 0.4866 0.3174 0.5378
QNSSR04 -0.1930 0.1451 0.2834 0.6321
QNSSR05 0.5722 0.6131 0.0956 2.3655
QNSSR06 -0.0743 0.1418 0.2011 0.9930
QNSSR07 0.0419 0.1883 0.1528 1.3857
QNSSR08 -0.1883 -0.0670 0.1021 2.1986
QNSSR09 0.1776 0.3798 0.2459 0.7668
QNSSR11 1.0000 1.0000 0.2912 0.6085
QNSSR12 -0.2341 0.1243 0.2904 0.6109
QNSSR15 -0.0576 0.1668 0.2122 0.9281
QNSSR16 -0.1761 0.0741 0.2127 0.9253
QNSSR18 -0.1773 0.0370 0.1820 1.1234
QNSSR21 -0.2840 0.0893 0.2907 0.6100
QNSSR23 -0.3045 -0.0564 0.1902 1.0644
‘ Mean -0.0408 0.1936 0.2252 0.8601
Q5 TUWlmn(uF¢­µ)o=>pq(uF¢­G)
Table5 MatrixofNeisgeneticidentity(abovediagonal)andgeneticdistance(belowdiagonal)among10origins
¶^
Origin
cd
Duyun
ú
Guiding
Úm
Dushan
‚´
Huishui
jc
Sandu
z{
Longli
…†
Luodian
‘t
Pingtang
op
Wengan

Fuquan
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