: A, ; B, 3 ; C, 2 ; D, , AFLP Vos (1995) AFLP,, : E2F: 5 2CTC GTA GAC TGC GTA CC23 ; E2R: 3 2CAT CTG ACG CAT GG

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1 2009, 36 (3) : Acta Horticulturae Sinica AFL P 13, 13, 1, 1, 1, 23 3 ( 1, ; 2, ) : 2, AFLP, 64, 43, , bp, : ; AFLP; : S : A : X (2009) O ptim iza tion of AFL P System for Fuji and Iden tif ica tion Early2ma tur ing Sport from Changfu 2 L I Yuan2jun 13, TANG Mei2ling 13, YU Q ing 1, L IU Mei2ying 1, SONG Lai2qing 1, and HAN Zhen2hai 23 3 ( 1 Fruit Institute, Yantai A gricultural Science A cadem y, Yantai, Shandong , China; 2 Institute of Horticultural P lants, China A gricultural U niversity, B eijing , China) Abstract: U sing AFLP to compare the early2maturing sport and its mother p lant in Changfu 2. Sixty2 four selective p rim er pairs were adop ted in AFLP analysis. detected by 43 pairs of AFLP p rim ers. Two thousand and seven hundred clear bands were There were 7 obvious bands difference between early2m aturing sport and mother p lant under 4 pair p rimers. The molecular weight range of the polymorphic fragm ents were mainly bp. The results indicated that there were some molecular2level differentiation between Changfu 2 and its early2maturing sport. Key words: app le; AFLP; bud mutation The app lication of AFLP in the research of app le bud mutation was successful.,,,, RAPD AFLP ISSR MSAP (, 2003;, 2005;, 2006;, 2007;, 2007), AFLP (Vos et al., 1995) AFLP , 3, 2, AFLP, AFLP, AFLP 2, DNA, AFLP : ; : : ( ) ; 863 (2006AA10Z1B6) Author for correspondence ( E2mail: rschan@cau1edu1cn)

2 : A, ; B, 3 ; C, 2 ; D, , AFLP Vos (1995) AFLP,, : E2F: 5 2CTC GTA GAC TGC GTA CC23 ; E2R: 3 2CAT CTG ACG CAT GGT TAA25 ; M 2F: 5 2GAC GAT GAG TCC TGA G23 ; : M2R: 3 2TAC TCA GGA CTC AT25 ; E20: 5 2 GAC TGC GTA CCA ATT CA23 ; M 2C: 5 2GAT GAG TCC TGA GTA ACC23 ; 3, 1 1 Table 1 Tested pr im er pa irs and their sequence in the study Primer Sequence Primer Sequence E1 E2 E3 E4 E5 E6 E7 E8 5 2GACTGCGTACCAATTCAGC23 M1 5 2ATGAGTCCTGAGTAACAA23 5 2GACTGCGTACCAATTCAGG23 M2 5 2GATGAGTCCTGAGTAACAC23 5 2GACTGCGTACCAATTCAAG23 M3 5 2GATGAGTCCTGAGTAACAG23 5 2GACTGCGTACCAATTCACT23 M4 5 2GATGAGTCCTGAGTAACAT23 5 2GACTGCGTACCAATTCACA23 M5 5 2GATGAGTCCTGAGTAACTG23 5 2GACTGCGTACCAATTCACC23 M6 5 2GATGAGTCCTGAGTAACTA23 5 2GACTGCGTACCAATTCACG23 M7 5 2GATGAGTCCTGAGTAACTC23 5 2GACTGCGTACCAATTCAAC23 M8 5 2GATGAGTCCTGAGTAACTT AFLP AFLP 3 SDS (, 2001 ) CTAB DNA, / 3, 012% RNase, m in, 100 ng L - 1 EcoR M se 40 L DNA 500 ng, M se I EcoR I 3 U 2 Y/T Buffer, 37 6 h, m in 10 L Vos ( 1995) PCR 1 /2, 95 5 m in, 6 L 6 % 2 h, DNA AgNO 3, m in AFL P DNA CTAB SDS SDS, DNA, ( ), CTAB DNA ( ), A 260 /A , AFLP

3 : AFLP 329 1 CTAB SD S D NA 1 5: CTAB DNA; 6 12: SDS DNA F ig.

extracted by CTAB method; 6-12: The DNA extracted by SDS method.

2 2 The selective am plify ing result w ith tem pla tes d iluted d ifferen tly

3 3 : AFLP CTAB SD S D NA 1 5: CTAB DNA; 6 12: SDS DNA F ig. 1 The apple D NA extracted by m od if ied CTAB and SD S m ethod 1-5: The DNA extracted by CTAB method; 6-12: The DNA extracted by SDS method. 10 2, 10 ; 20 30, F ig. 2 2 The selective am plify ing result w ith tem pla tes d iluted d ifferen tly from pre2am plified product 30 m in 10 m in 3, 20 m in 30 m in,, 10 m in, 10 m in 6,,,,, F ig. 3 3 The electrophoresis result of d ifferen t AgNO 3 2sta ined tim e

330 36 212 AFL P 64, 2 43, 2 700 AFLP, 62 ( E2ACT/M 2NNN E2 AAG/M 2NNN 4 ) ; 16,, ; 5 2 AFL P Table 2 AFL P fragm en t num ber am plif ied by d ifferen t prim er com b ina tion s Primer M2CAA M2CAC

4 AFL P 64, 2 43, AFLP, 62 ( E2ACT/M 2NNN E2 AAG/M 2NNN 4 ) ; 16,, ; 5 2 AFL P Table 2 AFL P fragm en t num ber am plif ied by d ifferen t prim er com b ina tion s Primer M2CAA M2CAC M2CAG M2CAT M2CTG M2CTA M2CTC M2CTT E2AGC E2AGG (2) E2AAG (1) E2ACT E2ACA (3) 73 (1) E2ACC E2ACG E2AAC :,, ; 3 ; Note: The fragment was too large to number for p rimer combination with ; No product with primer combination with 3 ; Numbers in bracket show the specific segment number found by electrophoresis observation. 4 E2AAG /M 2NNN ( 1 8) E2ACT /M 2NNN ( 9 16) F ig. 4 D NA band pa tters am plif ied by pr im er com b ina tion E2AAG ( 1-8) and E2ACT ( 9-16) w ith M 2NNN 213 AFL P 4 4 ( 5 ),, 214 AFL P 43 4, 7, bp, 4, 3 ( 6 3 ) 215, 2, 2 693, 7, Nei L i (1979), , , 0126%

3 : AFLP 331 5 4 4 1. ; 2. 3 ; 3. 2 ; 4. 1 F ig. 5 The electrophoresis result am plied by four pr im er com b ina tion s in sports m uta tion 1. Mutation from mother p lant; 2.

3 Table 3 Polym orph ic fragm en t by sport and contract Primer combination Molecular weight of polymorphic fragment Sport E2AGG/M2CTT 350 700 E2AAG/M2CAG 500 E2ACA /M2CTG 600

5 3 : AFLP ; 2. 3 ; 3. 2 ; 4. 1 F ig. 5 The electrophoresis result am plied by four pr im er com b ina tion s in sports m uta tion 1. Mutation from mother p lant; 2. Mutation from grafting for three years; 3. Mutation from grafting for two years; 4. Mutation from grafting for one year. 3 Table 3 Polym orph ic fragm en t by sport and contract Primer combination Molecular weight of polymorphic fragment Sport E2AGG/M2CTT E2AAG/M2CAG 500 E2ACA /M2CTG E2ACA /M2CTA Mother p lant 6 4 1: ; 2: F ig. 6 PAGE electrophoresis of 4 pa irs of AFL P prim er com binations in sport and con tract 1: Mother p lant; 2: Sport. AFLP,, AFLP AFLP (, 2000;, 2001;, 2006), 3 AFLP, CTAB DNA, DNA,

6 332 36,, AFLP, AFLP, 64, 4,, 7,, DNA 4, 2,, AFLP SCAR,,, References Huang Yu2hui, Chen Tao, W u Xiao2wei, Yang Rong2zhong, L i Yang2rui AFLP analysis of sugarcane mutants. Southwest China Journal of Agricultural Sciences, 20 (4) : ( in Chinese),,,, AFLP., 20 (4) : Nei M, L iw H Mathematicalmodel for studying genetic variation in term s of restriction endonucleases. Proceedings of the NationalAcade2 my of Sciences, USA, 76, N ing Yun2ye, Xiong Q ing2e, ZengW ei2guang, Xiao Guang2rong Studies red flesh sport from Red Sun kiwifruit using RAPD marker. Acta Horticulturae Sinica, 30 (5) : ( in Chinese),,, RAPD., 30 (5) : Shi L i2xue, Sha Guang2li, W ang B in, Sun Zhong2xu Establishment and op tim ization of AFLP technology system used to mark characteris2 tics of app le. Shandong Agricultural Science, (2) : ( in Chinese),,, AFLP., (2) : Vos P, Hogers R, B leekerm, ReijansM, van de Lee T, HornesM, Frijters A, Pot J, Peleman J, KuiperM, Zabean M AFLP: A new technique for DNA fingerp rinting. Nucleic Acids Res, 23 (21) : W ang Cai2hong, W ang Q ian, Dai Hong2yi, Jia Jian2hang, W ang B in, Shu Huai2rui DNA temp late making and technology system building for AFLP analysis of apple genome. Journal of Shandong Agricultural University: Natural Science, 32 (2) : ( in Chinese),,,,, AFLP DNA. :, 32 (2) : Xiao Xuan, Sun M in, W ang Xin2yan, Q iao A i2m in, Jiang Bo Identification of large fruit and sweet osmanthus flavour sport from Shi xia longan using RAPD marker. Acta Horticulturae Sinica, 32 (4) : ( in Chinese),,,, RAPD., 32 ( 4) : Xie Ji2rong, L iang Guo2lu, L i Shu2fa, Cheng Zai2quan, Tang Kai2xue, Huang Xing2qi Identification of p ink sp rout mutation from Gold Island rose. Northern Horticulture, (11) : ( in Chinese),,,,, , (11) : Zeng Bai2quan, Gan L in, Xiong Xing2yao, Deng Xiu2xin, Deng Zi2niu Analysis of B ingtang O rangeπs good bud mutation by AFLP. Acta Agriculturae Universitatis Jiangxiensis, 28 (7) : ( in Chinese),,,, AFLP., 28 (7) : Zhu Jun, W ang Tao, L i Guang2chen, Zhao Yu2jun, ZhangW en, Zhou A i2qin EstablishmentAFLP system for app le. Journal of China Ag2 ricultural University, 5 (3) : ( in Chinese),,,,, AFLP., 5 (3) :

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L

AFLP % 1. 1 DNA 5 RAPD DNA 0. 1g % ~ ph 6 10 DNA 9 40μl TE SSR STS 20ng /L 2012 13 2 201-206 Journal of Plant Genetic Resources / 071001 41 SSR 21 SSR 41 52 2 ~ 4 2. 5 Shannon 0. 21 ~ 1. 40 0. 73 5. 54 3 7 9 13. 16% ~ 186. 49% 5. 48 6 11 SSR Genetic Diversity of Peanut Arachis