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Microsatellite Primer Screening and Population Genetic Diversity of Bletilla striata (Thunb.) Rchb. f.

白芨SSR引物筛选及群体遗传多样性研究



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LIJun1,YANGHeng1,ZHOUTianhua1,2
(1ColegeofBiologicalScienceandEngineering,ShaanxiSciTechUniversity,HanzhongShaanxi723001,China;2Qinling
BashanMountainsBioresourcesComprehensiveDevelopmentColaborativeInnovationCenter,Hanzhong,Shaanxi723001,China)
犃犫狊狋狉犪犮狋:犅犾犲狋犻犾犾犪狊狋狉犻犪狋犪(Thunb.)Rchb.f.isanendangeredplantinChinawithimportantmedicinalval
ue.Inthisstudy,thegenomicsequencelibraryandmicrosatelitelibraryof犅.狊狋狉犻犪狋犪wassetupbasedon
Iluminasequencing.SSRprimersweredesignedandcarriedouttotesttheirpolymorphismwith80indi
vidualsfrom4wildpopulations.Fourrelatedspeciesof犅.狊狋狉犻犪狋犪wereusedtoinvestigatedtheircross
taxatransferability.Inaddition,geneticdiversityandgeneticdifferentiationofthespecieswerealsoinves
tigated.Theresultsshowedthat:(1)Microsatelitesequenceswereabundanceinthegenomeof犅.狊狋狉犻犪
狋犪,17841lociweredetectedintotal.100pairofprimersweredesigned,20ofthemperformedhighpoly
morphismafterPCRandpolyacrylamidegelelectrophoresis,thealelesrangedfrom2to6withaverageof
3.85perlocus.Thecrosstaxatransferabilitywasalsoexaminedin4relatedspecies,mostof20lociwere
provedamplifiableinthesespecies.(2)Thestudyrevealedthatthegeneticdiversityof犅.狊狋狉犻犪狋犪was
high(犖a=3.85,犐=1.07,犎=0.6147),geneticdifferentiationwasprominentamongpopulations(犌st=
0.43).Thegeneflowamongpopulationswaslimited(犖m=0.8676),theadjacentpopulationsperformed
tightgeneticrelationshipasrevealedbyclusteranalysesofpopulations.Thereasonsforgeneticconstruc
tionandconservationalstrategiesofthespecieswerealsodiscussedinthisstudy.
犓犲狔狑狅狉犱狊:犅犾犲狋犻犾犾犪狊狋狉犻犪狋犪(Thunb.)Rchb.f.;microsatelite;geneticdiversity;population;primerscreening
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²³«r20‹ij‚ƒ…¢´ÀÁ°±› ‹rDNA(À1);ø©…°±{r¹­,|ºL k2~6†,ýJ«:D^_k0.3500~0.7000,¨ ¯«:D^_k0.4685~0.7653,[rAMOVA X —±« ,犌stk0.43,À¥Ã57%Œª“)vˆ‰ á , Ã43%Œª“)vˆ‰¼,ˆ‰¼¨PdºÁ r™šXœ 。MantelTestw £(¤4)¤¥,i jȉ»¼r™šÎÏeo®‘rÍFÎϺÁ r†®ÑÑÒ (狉=0.2943,犘 <0.01)。 2.4 ,~犛犛犚N]oLK| tMNŒËÌX— , z{ UPGMA ËÌá ( À2)ºÃ,4†ij‡6ȉP™š–Ã¥ºr© ª , ÍFÎϒrLGȉ€ZBȉŒœÑÒÄ ’ , ¦YXȉ€oÄ3†È‰ŒœÑÒÄÅ。æ tGenAlEx6.3MNŒÈ‰T®ÑX—, £ ( À3)e UPGMAËÌX—r £DðfY,4† ij‡6ȉDðX; 。 `4 45€e;犖犲犻狊=>‚ƒ(ßÆÇ)„ =>…†‡ ( –ÆÇ ) K| Table4 Neisgeneticdistance(belowdiagonal) andgeneticidentity(abovediagonal) ȉ Population LG ZB HY YX LG  0.7234 0.6744 0.6810 ZB 0.3238  0.6507 0.6469 HY 0.3939 0.4297  0.5348 YX 0.3841 0.4356 0.6258  `3 45e;,ˆ‰:=>KŠ‹h Table3 Theparametersofgeneticdifferentiationandtheestimatesofgeneflowamongpopulationsof犅.狊狋狉犻犪狋犪 ȉ Population ½¾¹­,|º Numberofaleles(犖a) Ô¹­,|º Effectivenumberofaleles(犖e) Shannonº Shannonindex(犐) ,|Ž›Âº Genediversityindex(犎) ZB 2.4500 2.0318 0.7434 0.5010 LG 2.4000 2.0398 0.7508 0.5029 HY 2.8500 1.7816 0.6926 0.4120 YX 2.4500 1.9991 0.7386 0.4929 ˆ‰Â½Populationlevel 2.5375 1.9631 0.734 0.4772 $ˆÂ½Specieslevel 3.85 2.7125 1.0780 0.6147 È : ȉòÉ>¤1;ß> Note:PopulationcodesaresameasinTable1;Thesameasbelow M.PUC18;1~20.HYȉ†Š À1 …$Bjssr14‹éêg8(HY)ȉrSSR°± 1~20.SamplesofHYpopulation Fig.1 SSRamplificationproductsgeneratedbytheprimerpairsBjssr14forHY(Sichuanhanyuan)population 74317¨              M N,¹:ijSSR…$²³Œ‰Š™šŽ›./ À2 ,vNeis™šÎÏrij)*ȉr UPGMAËÌá Fig.2 UPGMAdendrogramof犅.狊狋狉犻犪狋犪populations usingNeisunbiasedgeneticdistances À3 ,vGDrij)*ȉT®ÑX— Fig.3 PCAanalysisof犅.狊狋狉犻犪狋犪populations usinggeneticdistances 2.5 67z{Axy [r²³±q20‹¢´µ¶°±«ŽZ¨ ©rij‚ƒ…$ , ætø©…$‹s*r4† ij’“ˆ ( aij 、 Üij 、 (öij€)Õ ) ›Ö Œ°± , htˑ’“”C#–˜‹°± £Œ w 。  £¤¥ , aij 、 Üij€(öijˆ‰ A ,20‹…$¾¢w«¹­,|(¤5),üÊr 40A30w«¬Ù,|•,²³«r …$ûPij›#$A[t , PÔ+oěAw Ãf¶r[t 。 3 Ÿ ù Ö./[r‹ij4†‡6ȉrˑ’“ª C#–˜Žÿ , ²³«20‹µ¶¸,ŽZ\9 ­®¨©rSSR…$。‹80ïij‡6ˆ$78 rŽÿ £X—¤¥ ,20‹…$ªK±77†¹­ Œª® , ½¾·‹…$3.85†,¤¥²³«r‚ ƒ…$ŽZ¨© 。 ŽZ\9"ó (PIC)^ _k `5 20Œ67Œ445bcd]z{Axy(n=10) Table5 Universaldetectionof20pairsofprimersamong4犅.狊狋狉犻犪狋犪closelyrelatedspecies …$­® SSRlocus w«r¹­,|No.ofalelesdetected aij犅犾犲狋犻犾犾犪狊犻狀犲狀狊犻狊 Üij犅犾犲狋犻犾犾犪犳狅狉犿狅狊犪狀犪 (öij犅犾犲狋犻犾犾犪狅犮犺狉犪犮犲犪 )Õ犛狆犻狉犪狀狋犺犲狊狊犻狀犲狀狊犻狊 Bjssr01 2 1 1 - Bjssr02 1 1 1 1 Bjssr03 2 1 1 - Bjssr04 1 1 1 1 Bjssr05 1 1 2 - Bjssr06 1 1 1 - Bjssr07 2 2 1 1 Bjssr09 1 1 1 - Bjssr11 2 1 1 - Bjssr13 1 1 1 - Bjssr14 1 2 1 1 Bjssr15 1 1 1 - Bjssr16 1 1 1 1 Bjssr17 1 1 1 1 Bjssr18 1 1 1 1 Bjssr19 1 1 2 - Bjssr20 1 1 1 1 Bjssr22 1 1 1 - Bjssr23 1 2 1 1 Bjssr24 1 1 1 1 È :n.·ˆ‰†Šº;1.ýwqÆ:Y;2.ýwq«:Y Note:n.Numberofindividuals;1.Observedhomozygote;2.Observedheterozygote 8431 ! " # $ % &                   36_ 0.3572~0.7229,½¾@k0.5479,À¥Ö./ z{rSSR¶·ŠÒ‹v4†ij‡6ȉ™š Ž›rX—ËXÔ 。 |kq¸xŸ€e" SSR­®r­ÌxŸP$ˆ¼Ðﰏ,:[ Úf†$ˆr‚ƒ…$P>+§>›ä^͒$ ˆ¼ÐÃWt [16]。 ôŁ‚ƒhtTrf†Î üÓfρ‚ƒ­®PÐf$ˆAXϔwx , Ñ ¾ûÒtv’“ˆrX— [17]。 Ö./„0²³r ijSSR…$¢´P4†ij’“ˆª40 AÀÁ°± , À¥SSR…$[tQ¸。|•,² ³«rijSSR…$¢´Òtvij‰Š™š% X—r./ , >mw¢´tvij›oĒ“$ˆ r‰Š™š%./ 。 ø‹vhtSSRXY¶·‹ ðl^_ij‡6ˆ$78rX—eëì , ôü˜ +%Ó¦ 。 )*ÔA , 6$:¤¼«“r™šŽ› , oP Ö$–¾ÓDNAr©ª。|•[rXYÕµ“X —$ˆä‰Š™šŽ›ÍkÖv , wÍk×Ø 。 ÑÖvX—™š$$Öw , ÓX—6$r,|¬¦ Ò¤¼¬ , Ù¶˜uv|ÚÛs}ï¶Ù“r-Ü , w§y›‹}Z€}U“8r›Ý , ašbr—ü Í×Øû¼ 。 ѧޢ5X®’$ˆ , ¦«û wC¬ßk®àr>ˆ§>Œˆ 、 Œ¬ 、 ‹Ò 、 œ% ¬á‡†Š»¼rDNA r⁌ª。Ö./ht SSRXY¶·Iy,<¼ijP$ˆÂ½–,ýJ «:D^_k0.3500~0.7000,¨ ¯«:D^_ k0.4685~0.7653,ŽZ\9"ó^_k 0.3572~0.7229,[r AMOVA X—ºÃ,à 57%Œª“)vˆ‰á,Ã43%Œª“)vˆ‰ ¼ , À¥ijP$ˆÂ½–r™šŽ›ÒÚ¨© 。 Ã./¤¥ , PÍFXè–yãr#$ÐÃÄ 2r™šŽ› , ®„ , Xè^_ã`r$ˆÐÃã År™šŽ› [1820]。 ijP$ˆÂ½–ÐÃÄ År™šŽ› , û¢eij›#$r6Óä€) *XèÃÑ 。 ij›#$dŽXèPØÙ100~ 3200mrÚÛÜÝÞß,àáÞ、âãÕäåæç A 。 u›#$›vå8aÄær$ˆÌÊ , Xè5 ÉÄ` , Péê 、 ëh 、 ! 、 ì" 、 íî 、 çè¹Ír 45¾ÃXè , ”«Ž(¾Ãd˜žˆ# 。 f†$ ˆ‹uvŒœrW‘¢éTUý…vu$ˆÓêРè©r™šŽ› , |• , ³*¢vƒkrDst Œ)*Œër›ÝbYijr)*78nò , b Óu›#$ìír,|îÉæoÐè©r™šŒ ª , æo¢´ç¸Í6¨ß“ 。 ij (犅犾犲狋犻犾犾犪狊狋狉犻犪狋犪Rchb.f.)ÓAlñ~¯ õ#$ , wÓqUrst#$ , ‡678ïð , ñk ¯õ 。 ijPƒGôõ¬NßíòóDçô , íò Òºß2 , õö’’C‹ijr}•õ–./Ď , Pf¶fD–÷„˜kl±×e78pørùú , b|s}û͏ýür›Ý¦ýÀr‡6ijeô õijijìZrþÿ , bY˜ƒ!‹‡6ijr rDst , n–Ô+#rˆY"
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k~ij›#78r¯õ 、 ht€ëì ¶˜Q¸,- 。 ‹Ž!M : [1] (ó),* +,,Y-,¹.§>.Ú½߱3ŒCO2/ Å9:]t‹a0à126óŒoXr›Ý
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