梅花品种资源叶绿体基因组SSR研究
»29 9 12007 M1 Ø v Ð Ð JOURNALOFBEIJINGFORESTRYUNIVERSITYVol.29, Supp.1Jan., 2007là ° ù2006-11-16htp Á[ “SE1 SÐÁù[ “300070641、SE S/ZÏù[ “2005004。»BT g ö,p V,¬ q。ö1ùîZ_ó ±þ÷ÐâÕ。ÈÅ010-82376017-601 EmailL ¹¶100083Ø vÐ10|Q。¼½ ÖÕ÷ s8yFSSRùî吕英民1, 2 殷 婧1 杨 果1 张启翔1,21Ø vÐó Ðý 2SE½ñýñ/ ùîÏK1¾ùî æ¨ ñÊ¥B 1õ¤¥ s8DNAcpDNASSRþNTCP9 948z ¼½ ¥yFDNA,É s8DNASSRS k。[48ñ ¼½ ÖÕ¥cpSSR·Õ ¹,¨NTSYSpc2.11 Èq9ØNeis1972L. Ö“ 、e Ä ¥“ SM、Jaccard Ä ¥“ Dice Ä ¥“ ,isYÉ Ë、 ½s。V¥s ¿cpDNAsÄ£ U¥ ¼½ ÖÕW¥“dÄ1“。1oM ¼½,cpSSR,·Õm öL Ying-min1, 2;YINJing1;YANGGuo1;ZHANGQi-xiang1, 2.cpSSRanalysisofPrunusmumeculti-varresources.JournalofBeijingForestryUniversity200729 Supp.148-53[Ch, 22 ref.]1 ColegeofLandscapeArchitecture, BeijingForestryUniversity, 100083, P, R.China;2 ChinaNationalCenterforFlowerEngineeringTechnique, Beijing, 100083, P, R.China.Forty-eightcpDNAfingerprintsofPrunusmumecultivarswerebuiltupwiththeprimerNTCP9 fromtobaccocpDNAbyusingthecpSSRtechnique.BasedoncpSSRfingerprintsdataof48accessionsofP.mumecultivars, thecomplexityofrelationshipandoriginationofP.mumecultivarswereanalyzedbyusingNTSYSpc2.11toclusterandarangementanalysisofNeis1972geneticdistance, SMcoefi-cient, JaccardcoeficientandDicecoeficient.Keywords Prunusmume, cpSSR, DNAfingerprint®¿ s8DNAchloroplastDNA, cpDNAµÚ¥M o,yN ¼½PrunusmumecpDNAÐ ð±þ¥cpDNA µ]÷。 Ù ð±þ  ¾ Pinus、Ö ÜZeamaysϵZ 륡[ 1-15] , ¼½Ï,Ãnµ1cpDNASSRs¥¡。Ð5cpDNASSR¥ ÿ ÂDNASSR¥ ÿ [ 16] , V_©] 、Õ、 ÖÕcpDNA¥µslM, uY Tø1“Í¥yZ ëµ µ¥ª ]。ùî ñÊB 1õ¤Nicoti-anatabacum¥cpDNASSRþ 948z ¼½ ¥yFDNA,ÉcpDNASSRS k、。V¥s ¿cpDNAsÄ£ U¥ ¼½ ÖÕW¥“dÄ1“,[F ¼ ÖÕ÷¥ùî、 ¹ æ¨,[ ¼½ÕÉ÷ ¹¥s0Sùî Ø ,ÉBy ¼L. õ ím ö、s0S£ùâÕ、1y¥çÊÐ X H©ùîýTµBç¥ Ø I L¨N´。1 材料与方法1.1 ¼½ ÖÕ k cpSSR k“ ÖnV1 1 2Ø ¼½ öóq ¼½Ï 48ñ ¼½ ÖÕ。1.2 kÐsZE1.2.1 试验仪器及试剂 k î P¨¥N ®ÏS j SÐý v½ñ î ÖÉ î L i4。þ¤h1Ø Øpù½ 3þµK ³、Z 3ý 3þýñ/ µK ³。 M ½EcoRI MseI、T4 õ¤ ½1 g 3þ/ ³。Taq ½1¿ Å 3þýñ ³、ØåS 3þ/ µK3 © ³。 ÖA TAØåS 3þ/ µK3 © ³。9 1 g ö© ¼½ ÖÕ÷ s8yFSSRùî49 ðÄÐ k4SÁsB。V1 ¼½cpSSR k“ Ö¨VTABLE1 SerialnumberofP.mumesamplesforcpSSRI| ÖÕ I| ÖÕ I| ÖÕ 1 lÖÜ 17 e£ 33 Öî¥2 m öâ 18 Be£ 34 x½äõ¥3 v } sl 19 ëÀ 35 äõ sl¥4 sl 20 õ*â½ 36 5¥5 * sl 21 v½ ¼37 Ö;¥6 õK 22 B Ä 38 ¥7 ϯK¥ 23 õv £ 39 C Ê¥8 }´ 24  ® 40 ª9 A 25 2Ø*£ 41 ÖÕ10 *x 26 FÜKl 42 ¥ Â11 v 27 U 43 ½ Â12 äõ ûî 28 x 44 1¥13 2ë é 29 ¥ 45 K Å ¦14 T 30 Ö 46 ö¥15 x¥ 31 Ø }ÖÜ 47 Æ 3¥16 Å¥ 32 Ö7 ¼48 l£É¶1.2.2 引物筛选 k¨13 ÷¿Ö Ü、õ¤、 ¾ s8DNAcpSSRþ[ 3, 17-19] , 9 Û |¥5z ¼½ ¥yFDNAÉsþ ñÊ, ñʲTϵ8_© ÿ,]61.54,KÔʨ 9Kz¥B 1õ¤¥ s8DNASSRþNTCP9 ¼½yFDNAÉ s8DNASSRS k。õ¤NTCP9þ½ 5′-CTTCCAAGCTAACGATGC-3′5′-CTGTCCTATCCATTAGACAATG-3′1.2.3 试验方法及试验数据的记录分析方法¨F.JamesRohlf2001NTSYSpc2.11 Èq9 ØM »“ ÐL. Ö“ 。L. Ö“ dij¨Neis1972 Ö“ 。Ä ¥“ ¨SM“ e Ä ¥“ SSMad abcdJ“ Jaccard“ SJa abcDice“ SC2a 2abcTÏ,aV U ñOTUI ¥¹“1”¥ñ ;bV UBñOTU¹“1”, 6BñOTU¹“0”¥ñ;cV UBñOTU“0”, 6BñOTU“1”¥ñ;dV U ñOTU¥¹“0”¥ñ 。[SAHNClusteringÉdF î Ø Ü EUPGMA Ës,9 ØM »“ r,i¨MX-COMPMatrixcomparisonÏ¥COPHprogramñ½É2»Manteltest_,9 Ø“ [ 20] ˲Tñ¥M1“ rij。2 结果与分析2.1 ¼½ ÖÕcpSSR·Õm ö Ës s8DNA¥±ÏsöV¥£ U¼½ ÖÕW¥ Tø1“。Ü ñÊ P¨¥þ 915HSSR ÿ{, c Ë Â/。m1 þNTCP9·ÕmFIGURE1 cpSSRfingerprintofprimercombinationsNTCP92.2 ¼½ ÖÕcpDNASSRM » Ë®cpSSRsþNTCP9¤¥·Õm önm1 c²TÉ Ës,¤ m。1¨Neis1972L. Ö“ ¤¥ Ë Âm2 î U。2¨e Ä ¥“ SM¤¥ ˲T Âm3 î U。3¨Jaccard Ä ¥“ ¤¥ Ë Âm4î U。4¨Dice Ä ¥“ ¤¥ Ë Âm5î U。51 4Õ Ës²T Âm6 î U。50 Ø v Ð Ð »29 m2 ¨Neis1972L. Ö“ ¤¥ Ë FIGURE2 cpSSRdendrogramofNeis1972geneticdistancecoeficientsm3 ¨SM Ä ¥“ ¥ Ë FIGURE3 DendrogramofsimplematchingcoeficientsSMm4 ¨Jaccard Ä ¥“ ¤¥ ËmFIGURE4 DendrogramofJaccardcoeficientsm5 ¨Dice Ä ¥“ ¤¥ ËmFIGURE5 DendrogramofDicecoeficients9 1 g ö© ¼½ ÖÕ÷ s8yFSSRùî51 m6 ˲T1 mFIGURE6 MXCOMPmatrixcomparisonofdendrograms52 Ø v Ð Ð »29 2.3 ¼½ ÖÕcpDNASSR·Õ Ës2.3.1 相关系数 2维比较9 Ø4Õ Ë¥M1“ V[ AòÕ Ë“ Ð Ë ¥ z; Ö“ ¤¥“ Ë ²TÐM »“ Dice¥ Ë dÈÚ,M1“ rij¹-0.984 44;ÐJ¤¥ Ë ¥M1“ rij¹-0.951 74;ÐSM¤¥ Ë ¥M1“ rijK®,9r -0.923 36,t“ rij¥ ´ûÚ¿0.9,ñûrdÈz¥Y。2-wayManteltest²TV ü ˲T zQ c¥cpSSR·Õ f , V[ AòM1“ I³ ˲TdÈM »。2.3.2 树状图聚类情况ʨ¥scpSSRþNTCP9_©¥48ñ ¼½ ¡ ÖÕ“ Ö¥cpDNA ˹14ñ, Ï13ñ Ë ÿ,] î_© Ë ¥92.86。sY¨Neis1972L. Ö“ 、DiceÄ ¥“ 、Jaccard“ SM Ä ¥“ ¤¥ ˲TÉ1 , V[C Ù ò“ ¥´ÐM],¨Neis“ 、D“ 、J“ ¤M]¥ ˲T,SM“ ¤¥ ˲TM »,Ç {µ],vÁ|48z ¼½ ks¹ BF»BFv } sl。»Fe£、Be£、ëÀ、B Ä、v½ ¼、õ*â、õv £、 sl、õK、 A、ϯK¥、 }´,ö1¹ ¼、 ÒÁ ¼½。» ØFÖ7 ¼、 2Ø*£、 Ø }ÖÜ、lÖÜ、m öâ、¥、Ö 、x、*x、 2ë é、 T、ÖÕ、ª、 K Å ¦、¥ Â、½ Â,ö1¹ÖÜ、x ¼ Ù ¼ Ë。» ¹FFÜKl、 U、Å¥、 ö¥、ÖÍ¥、x¥、x½äõ¥、¥、 släõ¥、 C Ê¥、Ö;¥、5¥、Æ 3¥,ö1¹¥ ¼ Ë。»ôF §、* sl、v、äõûî、l£É¶。» BF 1¥。ÐAFLP ˲TM1 VC;µM],9µ]。K üA¥]¿cpSSRS¥cpDNA¥L.M » üAÚ¿AFLPS¥DNA¥L.M »,µ ÖÕ¥M »“ r1。iV üt ¼½ ¡ ÖÕ¥ s8yü M],7 ·¾sþ 9Hq/, s8yF¥M » Ú,9ü ª ]÷ Ú,MºW¥µs9¸ ðsþHq/kY。î ðy V ¼½¥ s8yF ÖÕ £ Ü µ Ú¥]÷,cpDNA¥L. p。3 遗传相似系数分析与讨论ô 48z ¼½ cpSSR ÿ{9 Ø L.M »“ ,²T V©, k¥48ñ ¼½ ÖÕWL.M »“ ¥M¹0.400~ 1.000, Ü L.M»“ ¹0.806。 Ï, ¼ ¼½ ÖÕWL.M »“ ¥M¹0.533~1.000, Ü L.M »“ ¹0.824; sl ¼½ ÖÕWL.M »“¥M¹0.533~0.667, Ü L.M »“ ¹0.600; ÒÁ ¼½ ÖÕWL.M »“ ¥M¹0.800~1.000, Ü L.M »“ ¹0.889;x ¼½ ÖÕWL.M »“ ¥M¹0.867~1.000, Ü L.M »“ ¹0.933;ÖÜ ¼½ÖÕWL.M »“ ¥M¹0.733~1.000, ÜL.M »“ ¹0.880;ë é ¼½ ÖÕWL.M »“ ¥M¹0.667~ 1.000, Ü L.M»“ ¹0.773;¥ ¼ Ë ÖÕWL.M »“¥M¹0.733~1.000, Ü L.M »“ ¹0.926; ¼ Ù ¼ Ë ¼½ ÖÕWL.M »“ ¥M¹0.800 ~ 1.000, Ü L.M »“ ¹0.907。 1¥、v } sl¥ s8DNAÐ ð ¼½ ÖÕµv¥µs,V ü ñ ÖÕMsÄ *, ð S¥ ÖÕ。 ¼ ¼½ ÖÕ¥M »“ MKv,V üñ Ë¥ ¼½ ÖÕ ÷ *,ÉÄ å N É。ô 48ñ ¼½ ¡ ÖÕ¥cpDNASSRs, V[ A ¼½ ¡ ÖÕW Ù iBç¥cpDNAMs, L.1“ ¯ Í, ª ü s8DNA¥ oç,Q ¼½ ¡ ÖÕWy ÔN¥Ä。®þNTCP9_©µ¥ ¼½ ¡ ÖÕcpDNAL.M »r 1.000, è  A、õv £、v½ ¼ÐB Ä©, ñ Ì V LµL.M »¥ß, s8DNA ¥µs¾þ/ z¥_©。 VnÇYVBÕsþ ÉcpSSRùî±þ ÖÕW¥ Tø1“ ¥。[ ²TsV ü, ¼½ ¡ ÖÕW¥cpDNAµ Ú¥L.M »,9iBç¥L.µs9 1 g ö© ¼½ ÖÕ÷ s8yFSSRùî53 sÄ。 ˲TCµ¥s Ë“d,¯ iBç¥ f , ª ü ¼½ ¡ ÖÕ¥L.ß1 ¯,w© ÉÄ、 ¡âVñÏ, û Ú¥L.ç¥] H,µBç¥L.Ms,®¿ s8DNA ç¥ TL., V £U ÖÕW÷* ùBt¥ Tø1“。 6,cpSSR¥ÖÕkY ï ÂAFLP, V4 s8y TL.,¹ùî ¼½ ÖÕ¥ Tø1“4 ¥± I。Á YË 2ØÏ ö 3 ¼½ ö¥ B óÚý, 2Ø jvÐó\Ðý\¿ ö¬ q,óq öÏS ¼½ùîÏ ° i öÚý、foö ©k §Úý“ ¼½“ Ö HóÍ¥ £ fúù¦û。 I Ó D[ 1] JURGEF.Geneticflexibilityofplantchloroplast[J].Nature,1999, 398115-116.[ 2] WOLFEKH, LIWH,SHARPPM.Ratesofnucleotidesubstitu-tionvarygreatlyamongplantmitochondrial, chloroplast, andnu-clearDNAs[J].ProcNatlAcadSciUSA, 1987, 84249 054-9 058.[ 3] CHENGYJ, GUOWW, DENGXX.cpSSRAnewtooltoan-alyzechlorplastgenomeofcitrussomatichybrids[J].ActaBotani-caSinica, 2003, 458906-909.[ 4] PLOMIONC, LEPROVOSTG, POTD, etal.Polencontamina-tioninamaritimepinepolycrossseedorchardandcertificationofimprovedseedsusingchloroplastmicrosatelites[J].CanadianJournalofForestResearch, 2001, 31101 815-1 816.[ 5] VIARDF, EL-KASSBYYA, RITLANDK.DiversityandgeneticstructureinpopulationsofPseudotsugamenziesiPinaceaeatchloroplastmicrosateliteloci[J].Genome, 2001, 443336-344.[ 6] FERRISFCR,KINGRA,HEWITTGM,etal.ChloroplastDNArecognizesthreerefugialsourcesofEuropeanoaksandsuggestsin-dependenteasternandwesternimmigrationstoKnland[J].Hered-ity, 1998, 805584-593.[ 7] ISODAK, SHIRAISHIS, WATANABES, etal.Molecularevi-denceofnaturalhybridizationbetween, AbiesveitdhiandA.homolepisPinaceaerevealedbychlorplast, mitochondrialandnuclearDNAmarkers[J].MolEcol, 2000, 9121 965-1 974.[ 8] JASONWL,MICHAELJH, SCOTTAJ, etal.Cytogenomicanalysesrevealthestructuralplasticityofthechloroplastgenomeinhigherplants[J].PlantCel, 2001, 13245-254.[ 9] KATOS,YAMAGACHH, SHIMAOTOY, etal.ThechloroplastgenomesofazukibeananditscloserelativesAdeletionmutationfoundinweedazukibean[J].Hereditas, 2000, 132143-48.[10] ROSAA, FRANCOISL, MARIAT, etal.Chloroplastmicrosatel-litepolymorphismsinVitisspecies.[J].Genome, 2002, 4561 142-1 149.[11] RICHARDGO,JEFFREYDP.ChloroplastDNAsystematicsAreviewofsanddataanalysis[J].AmerJBot, 1994, 811 205-1 224.[12] SCOTTNS, POSSINGHAMJV.ChangesinchloroplastDNAlev-elsduringgrowthofspinachleaves[J].JExpBot, 1983,341 756-1 767.[13]¢ç1, ÙTÝ,Û¡Ó. ¦ ÖÕSSRs¥ùî[J].óq±þÐùî, 2003, 215444-448.[14] ´¿,f ,ô,ñ¹ ¦.SSRs0S¥ 7/ ùîÉZ[J]. 2 jÐ, 2002, 154106-109.[15]ñ W½, gZ,,©. s8DNA Ë ¥RFLP¥sÛÚB“dÐùîÏ¥¨[J].±þs ËÐ, 2000, 38297-110.[16]çu¬, 3, p. õ k õ 8¥ s8±Ïs[J]. SÐ, 2000, 36346-51.[17]f,â FÉ,Û ù ¥. ° ¾ 8¥ s8SSRs[J].L., 2004, 264486-490.[18] Ê ¨7. æ¨SSRkç £Õ0B¥ùî[J].Õ0,2003123-25.[19] ®{ ù, d b,· .. 2l 、ö7 3l ôYl s8DNAKÅ M ½m ö¥ùî[J].L., 1995, 1764-6.[20]· XÐ. 3þ Ð[M].Ø SÐñ, 1999.[21]ç .É.ÏS ¼½ ÖÕm½[M].ØÏS ñ, 1989.[22]¢î .±þs0Sð ØÐZE[M].É ìµ 2 SÐ/ ñ, 2003.责任编辑 李 慧