Optim ization of SRAP - PCR System in Pepper Using Orthogonal Design and Selection of Primers

Transcription

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

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