Optim ization of SRAP - PCR System in Pepper Using Orthogonal Design and Selection of Primers
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1 2010, 32 (3) : Acta Agriculturae Universitatis J iangxiensis http: / /xuebao. jxau. edu. cn E - mail: ndxb7775@ sina. com SRAP - PCR 1, 2, 1, 13, 1, 3 (1.,330200; ; 3.,330200) 2., : DNA,L 16 (4 5 ), SRAP 5( Taq DNA Mg 2 + dntps DNA),,SRAP - PCR : Taq DNA U Mg mmol/l dntps 0. 2 mmol/l0. 8 mol/ldna 50 ng, 10 L 3,,198 SRAP 35 SRAP :; SRAP; ; ; : S641. 3: A : (2010) Optim ization of SRAP - PCR System in Pepper Using Orthogonal Design and Selection of Primers ZHOU Kun2hua 1, 2, FANG Rong 1, CHEN Xue2jun 13, M IAO Nan2sheng 1, WANG W u2liang 3 (1. Vegetable and Flower Institute, J iangxi Academy of Agricultural Sciences, Nanchang , China; 2. The Key Laboratory of O il Crop, J iangxi Academy of Agricultural Sciences, Nanchang , China; 3. J iangxi Academy of Agricultural Sciences, Nanchang , China) Abstract: The L 16 (4 5 ) othogonal design was used to op tim ize SRAP - PCR system in pepper (Capsicum spp. ) with five key factors( Taq DNA polymerase,mg 2 +, dntps, primerand template DNA, respectively). The results showed that the optimized SRAP - PCR system for pepper was: U Taq DNA polymerase, 0. 6 mmol/l Mg 2 +, 0. 2 mmol/l dntps, 0. 8 mol/l p rimer, 50 ng temp late DNA in a total of 10 L reaction solution. The op tim ized SRAP - PCR system was tested on three pepper germp lasm s and was steady and reliable. 35 p rimer com binations were selected w ith abundant polymorphism from 198 p rim er combinations. The op tim ized SRAP - PCR system and polymorphism p rimer com binations could be app lied to research on molecular genet2 ics in pepper. Key words: pepper; SRAP; orthogonal design; system op tim ization; selection of p rim ers : : : ( ) : ( ),,,, ; E - mail: zhoukun hua6080@163. com; 3 :,,, E - mail: @163. com
2 596 SRAP( sequence - related amp lified polymorphism, ) L i Quiros [ 1 ] PCR (polymerase chain reaction, ) GC ( ) AT ( ),PCR,,,,, [ 2-7 ] (Capsicum spp. ), C,,,, [ 8 ], AFLP RAPD ISSR [ 9-12 ], SRAP [ ],SRAP,SRAP10 L, SRAP,SRAPSRAP,, B 9431 (C. annuum. var. longum ) H108 (C. B 9431 H108, frutescens) SRAP - PCR Taq DNA Mg 2 + dntps Buffer Fermantas, (marker) 100 bp DNA laddertakara SRAP,Me8 ( 5 - TGAGTCCAAACCGGCAC - 3 )Em21 ( 5 - GACTGCG2 TACGAATTCAA - 3 ), 1. 2 DNA CTAB [ 15 ] DNA Uvm ini DNA, 50 ng/ L, SRAP - PCR,L 16 (4 5 ) [ 16 ], SRAPTaq Mg 2 + dntps DNA 5 16, 2 ( 12), 10 L 1 PCR Tab. 1 The designa tion of factors and the ir levels in PCR reaction Factors (10 L) Levels ( final concentration of 10 L reaction system) Taq /U Taq polymerase Mg 2 + / (mmol L - 1 ) dntps/ (mmol L - 1 ) / ( mol L - 1 ) Primer DNA /ng Temp late DNA PCR PCR EppendorfPCR,: 94 5 m in; 94 1 m in, 35 1 m in, 72 1 m in, 5 ; 94 1 m in, 50 1 m in, 72 1 m in, 35 ; m in, 4 PCR 60 g/l (7 mol/l), 1 TBE, 70 w 1. 5 h [ 17 ],
3 3 :SRAP - PCR PCR [ L 16 ( 4 5 ) ] Tab. 2 The table of orthogona l design of PCR[ L 16 ( 4 5 ) ] Treat Taq /U Taq polymerase / Mg 2 + / (mmol L - 1 ) dntps/ (mmol L - 1 ) / ( mol L - 1 ) Primer DNA /ng Temp late DNA [ 18 ], 16, 2 [ 19 ] K i k i,r, k i Excel, SRAP,198 SRAP PCR, PCR 16, Taq DNA Mg 2 + dntps DNA 5, ( 1) 15 16,,,,, ; 13 I II,M: 100 bp DNA ladder; 1 16: ,, From left to right were repeat I and II. M: 100bp DNA ladder; 1 16: 1 16, ( 3) treats in table 2. R ( 4), 1 ( 400) 5, dntps R Fig. 1 Nucleus sup raop ticus( 400)
4 598, 7. 0,dNTPs;DNA R, 1. 9, DNA PCR: dntps >> Taq >Mg 2 + >DNA 3 PCR Tab. 3 The score result of PCR products Repeat I II Average Tab. 4 In tu itive result of the orthogona l design Parameter Taq Taq polymerase Mg 2 + dntps Primer DNA Temp late DNA K K K K k k k k R PCR Taq 2, Taq U,, U 1 U, Taq, ; Taq,,,,, Taq U 2 Taq Fig. 2 Relationship between quantity of Taq DNA polymerase and mean of results 3 Mg 2 + Fig. 3 Relationship between quantity of Mg 2 + concentration and mean of results Mg 2 + 3,Mg mmol/l 1. 5 mmol/l 2, Mg mmol/l,, 2. 5 mmol/l, Mg 2 + Taq
5 3 :SRAP - PCR dntps Fig. 4 Relationship between quantity of dntps concentration and mean of results, dntps, Mg 2 +,Mg 2 + Taq,, Mg mmol/l dntps 4,dNTPs,dNTPs 0. 1 mmol/l, 0. 2 mmol/l, 0. 3 mmol/l, ; dntps 0. 4 mmol/l, dntpspcr,,, dntps 0. 2 mmol/l 5 Fig. 5 Relationship between quantity of p rimer concentration and mean of results 6 DNA Fig. 6 Relationship between quantity of temp late DNA and mean of result ,0. 2 mol/l 0. 4 mol/l,, 0. 6 mol/l 0. 8 mol/l, PCR,, ;,0. 8 mol/l DNA 6, DNA ng,, DNA 50 ng,,,dna, DNA, ;, dntps,, M 100 bp DNA ladder; 1: B 9431 H108; 2:, H108;3: B 9431,DNA 50 ng M: 100 bp DNA ladder; lane 1: B 9431 H108; lane 2: H108; 2. 3 lane 3: B 9431, SRAP PCR : Taq U, Mg 2 + Fig. 7 The verification results of 8 p rimer combinations 2 mmol/l, dntps 0. 1 mmol/l,0. 8 mol/l, based on the op tim ized SRAP - PCR system
6 600 DNA 50 ng,10 L 8 3 DNA, :, ( 7),, DNA SRAP DNA 198 SRAP,,,28 70,183,92. 42% ; (5 ) 35,17. 68% 3 SRAP DNA, RAPD ISSR, PCR SRAP, [ 20 ], SRAP,,SRAP,, SRAP, PCR Taq Mg 2 + dntps, DNA 5, dntps,dna, [ 21 ] [ 22 ] [ 23 ] [ 24 ], Mg 2 + SRAP,,,, SRAP - PCR: Taq U,Mg mmol/l, dntps 0. 2 mmol/l, 0. 8 mol/l,dna 50 ng, 10 L [ 25 ] [ 26 ] 10 L SRAP,, Taq,Mg 2 + dntpsdna [ 14 ] 25 L, Taq, 10 L 25 L,, 10 L, DNA SRAP : [ 1 ]L i G, Quiros C F. Sequence - related amp lified polymorphisim ( SRAP), a new marker system based on a simp le PCR reac2 tion: its app lication to mapp ing and gene tagging in B rassica[ J ]. Theor App l Genet, 2001, 103: [ 2 ],,,. SRAP [ J ]. (C ) :, 2004, 36 (16) : [ 3 ]L in Z, Zhang X, N ie Y, et al. Construction of a genetic linkage map for cotton based on SRAP[ J ]. Chinese Science Bulle2 tin, 2003, 98 (19) : [ 4 ],,,. SRAP [ J ]., 2005, 15 ( 2) : [ 5 ],,,. SRAP[ J ]., 2007, 23 (4) : [ 6 ] FerriolM, Pico B, Nuez F. Genetic diversity of a germp lasm collection of Cucurbita pepo using SRAP and AFLP markers[ J ]. Theor App l Genet, 2003, 107: [ 7 ],,,. SRAP F 2 [ J ]., 2004, 31 (6) : [ 8 ],,,. (Capsicum spp. )[ J ]., 2004, 16 (3) : [ 9 ] Paran I, Aftergoot E, Shifriss C. Variation in Capsicum annuum revealed by RAPD and AFLP markers[ J ]. Euphytica, 1998, 99: [ 10 ] Sergio L, A lberto A, Luciana Q, et al. RAPD and AFLP assessment of genetic variation in a landrace of pepper (Capsicum annuum L. ) grown in North W est Italy[ J ]. Genetic Resources and Crop Evolution, 2003, 50: ( 632 )
7 632 [ 2 ],,,. [ J ]., 2005, 27 (5) : [ 3 ],. [ J ]., 2003, 12 (4) : [ 4 ]. [ J ]., 2004, 23 (2) : [ 5 ]Moreno D A, V illora G, Ruiz J M, et al. Growth conditions, elemental accumulation and induced physiological changes in Chinese[ J ]. Chemosphere, 2003, 52 (6) : [ 6 ],. Cd Hg Pb Cu [ J ]., 2007, 16 (4) : [ 7 ],,,. [J ]., 2008, 27 (2) : [ 8 ],,,. [J ]., 2006, 37 (6) : [ 9 ],,,. [ J ]., 2004, 19 (6) : [ 10 ],,,. Cd Pb [ J ]., 2005, 24 (2) : [ 11 ],,. [J ]., 2003, 18 (4) : [ 12 ]A llan D L, JarrellW M. Proton and copper absorp tion by maize and soybean root cell - walls[ J ]. Plant Physiology, 1989, 89 (3) : [ 13 ]ChoudharyM, Bailey L D, Grant C A, et al. Effect of Zn on the concentration of Cd and Zn in p lant tissue of two durum - wheat lines[ J ]. Canadian Journal of Plant Science, 1995, 75 (2) : [ 14 ]B ranquinho C, B rown D H, Catarino F. The cellular location of Cu in lichens and its effects on membrane integrity and chlorophyll fluorescence[ J ]. Environ - mental and Experimental Botany, 1997, 38 ( 2) : [ 15 ],,. [J ]. :, 2005, 26 (5) : 79-81, 85. [ 16 ],,,. [ J ]., 2007, 23 (4) : [ 17 ],. [ J ]. :, 2006, 27 (5) : ( 600 ) [ 11 ],,,. 5 RAPD[J ]., 2006, 33 (4) : [ 12 ],,,. RAPD ISSR[ J ]., 2007, 27 (4) : [ 13 ],,,. [J ]., 2006, 26 (12) : [ 14 ],. SRAP - PCR [ J ]., 2004, 2 (5) : [ 15 ]Murry H G, Thompon W F. Rap id isolation of higher weight DNA [ J ]. Nucleic Acid Res, 1980, 8: [ 16 ]. [M ]. :, 2000: 383. [ 17 ],,,. DNA[ J ]., 2002, 24 (3) : [ 18 ],,,. PCR[ J ]., 1998, 23 (4) : [ 19 ],,,. ISSR - PCR [ J ]., 2006, 42 (6) : [ 20 ],,,. SRAP [ J ]., 2006, 4 (5) : [ 21 ],,,. SRAP - PCR [J ]., 2009, 18 (3) : [ 22 ],. SRAP - PCR[ J ]., 2008, 47 (12) : [ 23 ],,,. SRAP - PCR [J ]., 2007, 16 (4) : 1-6. [ 24 ],,,. SRAP [ J ]. :, 2009, 26 (1) : [ 25 ],,,. SRAP[ J ]., 2007, 22 (4) : 112. [ 26 ],,,. SRAP - CR [ J ]., 2009, 5 (4) :
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