Preliminary Screening Molecular Markers Linked to Non-Acid/Acid Fruit Traits in Peach

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1 2004,37(12): Scientia Agricultura Sinica F 1 69 RAPD AFLP BSA / 301 RAPD S % 128 AFLP 18 3 E-ACT/M-CAT E-TA/M-CTC E-AT/M-CTA / cm D/d 2 AF TA-CTC RAPD AFLP BSA AF AT-CTA D/d Preliminary Screening Molecular Markers Linked to Non-Acid/Acid Fruit Traits in Peach WU Jun 1,3, SHU Huai-rui 1, ZHANG Kai-chun 2, ZHANG Xue-ning 2, JIANG Li-jie 2 1 College of Horticulture Shandong Agricultural University Tai an ; 2 Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, Beijing ; 3 College of Horticulture Nanjing Agricultural University, Nanjing Abstract In this research molecular markers linked to non-acid /acid trait in peach fruit were detected with RAPD and AFLP technique coupled with BSA method in 69 F 1 population hybrid from the intra-specific cross between peach cultivars Jingyu and Flavortop. Of 301 random primers, only the pirmer S 332 produced a polymorphic band linked to non-acid fruit trait, high recombination ratio of specific fragment was 28.9%. Among 128 pairs of AFLP primer combination, 18 pairs produced polymorphism between two bulks. The result of individual amplification showed that only three of them amplified specific fragments linked to fruit non-acid/acid trait. The specific fragments derived from primers combination E-ACT/M-CAT, E-TA/M-CTC, and E-AT/M-CTA, the linkage distance were 16.2, 1.47, and 2.99 cm, respectively. The D/d gene linkage groups was established, and two nearest markers AF TA-CTC and AF AT-CTA located in two sides of D/d locus, respectively. Key words: Peach; Non-acid/acid; RAPD; AFLP; BSA Yoshiba Monet non-acid F 1 69 RAPD randomly [1] Dirlewanger [2, 3] amplified polymorphic DNA AFLP (1975-) Tel: baafs.net.cn zhangkaichun@

2 12 / 1893 (amplified fragment length polymorphism ) bulked segregation analysis, BSA 36 1 min 30 s 72 2 min 39 / TAE 4 5V cm -1 EB 1994 [Prunus persica (L.) Batsch cv. Jingyu] [Prunus persica (L.)Batsch var. nectarina Maxim cv. Flavortop] 69 F 1 T 4 DNA 1U ìl ìl 25 ìl / 6% 10 Mapmaker/Exp3.0 [6] 10 ml 20 ml Kosambi [7] NaOH 3 40 mmol L -1 [2,3] 3 mmol L -1 V 1 N 10 = V 2 V 2 ml V 1 NaOH ml N NaOH mol L -1 CTAB [4] DNA RNaseA DNA 50 ng ìl -1 / BSA [5] 6 DNA DNA 1.0 ìl, 10 ìl, 25 mmol L -1 MgCl ìl PCR mmol L -1 dntp 1.5 ìl, 7.5 ng ìl ìl, DNA 5 U ìl ìl 1.0% Life technologies AFLP core reagent kit (cat.no: ) 12.5 ìl DNA 50ng ìl -1 5 ul EcoR I/ Mse I (1.25U ìl -1 ) 1.0 ul 5 Reaction Buffer 2.5ìl 12.5 ìl DNA 12 ìl 2 NaOH(0.1 mol L -1 ) centimorgan cm ph min = =3.84, / D/d / Fig.1 The distribution of fruit titratable acid in the progeny 15 ìl PTC-100 / RAPD PCR s s 301 9

3 % 2 = =3.84, F % / S 332 ( D/d 46.9 cm TCAACGGGAC) RAPD bp M. Marker( Lambda DNA/ EcoR I+Hind III Marker); 1-6. Acid individuals; Non-acid individuals; The specific fragment indicated by an arrow Fig.2 Amplification of polymorphic primer S 332 in the progeny / 4 A 128 AFLP / d AdaD adad Adad adad AFLP DNA % 2 = ( =3.84, ) F Mapmaker/Exp 3.0 D/d 1.47 cm E-AT/M-CTA 200 bp 69 B 5 18 AFLP 3 / 4 d / 2.90% 2 = =3.84, A D/d E-TA/M-CTC 2.99 cm 140 bp E-ACT/M-CAT A (3) C

4 12 / bp 6 4 A B D CDcd cdcd CDcd cdcd 14.39% 2 =3.71 ( =3.84, Mapmaker D/d 16.2 cm Mapmaker/Exp3.0 D AFLP AF TA-CTC AF AT-CTA AF ACT/CAT RAPD. E-ACT/M-CAT. E-TA/M-CTC. E-AT/M-CTA Primers combination E-ACT/M-CAT;. Primers combination E-TA/M-CTC;.Primers combination E-AT/M-CTA. 1.DNA pool of non-acid trait; 2. DNA pool of acid trait; 3. Jingyu ; 4. Flavortop Fig. 4 Amplification of specific fragments in parents and two DNA bulks A The specific fragment A indicat by an arrow. 1. DNA pool of non-acid trait; 2. DNA pool of acid trait; 3-13.non-acid individuals; acid individuals.m. Marker ( pbr322 DNA/Msp markers) DNA Fig. 3 Segregation of specific fragment A amplified by E-TA/M-CTC in the progeny S 332 (7) 2 AF TA-CTC AF AT-CTA D/d 2 AF AT-CTA AFLP AFLP [8] RFLP restriction fragment length polymorphism

5 B The specific fragment B indicated by an arrow. 1. DNA pool of non-acid trait; 2. DNA pool of acid trait; 3-13.non-acid individuals; acid C The specific fragment C indicated by an arrow. 1. DNA pool of non-acid trait;2. DNA pool of acid trait; non-acid individuals; acid individuals. M. Marker ( pbr322 DNA/Msp markers) individuals. M. Marker ( pbr322 DNA/Msp markers) Fig.5 Segregation of specific fragment B amplified by E-AT/M-CTA in the progeny RAPD AFLP 301 RAPD 3 AFLP S 332 RAPD RFLP AFLP D AFLP 10 RAPD [9] RAPD DNA Fang [14] BSA [10~12] Fig.6 Segregation of specific fragment C amplified by E-ACT/M-CAT in the progeny RAPD (pummelo 2240) Acitric RAPD [13, 14]

6 12 / Wang [15] 2 D/d Fig. 7 Linkage group of D/d gene SCAR sequence characterized amplified regions 1.0 cm / References / [1] Dirlewanger E, Pronier V, Parvery C, Rothan C, Guye A, Monet R. Dirlewanger [2] Genetic linkage map of peach [Prunus persica (L.) Batsh] using morphological and molecular markers. Theoretical and Applied Genetics,1998,97: ph [2] Dirlewanger E, Moing A, Pronier V, Svanellla L, Guye A, Monet R. D Detection of QTLs controlling peach fruit acidity and sweetness. Acta Horticulturae, 1998,465: [3] Dirlewanger E, Moing A, Rothan C, Svanellla L, Pronier V, Guye A, / Plomion C, Monet R. Mapping QTLs controlling fruit quality in peach [Prunus persica (L.) Batsh]. Theoretical and Applied Genetics, Dirlewanger [3] D 1999,(98): D [4] Maria T D, Roberta Q, and Ignazio V. A peach linkage map 2 RAPD 6 3 cm intergrating RFLPs, SSRs, RAPDs, and morphological markers. BSA AFLP Genome, 2001, 44: D [5] Michemor R W, Paran I, Kesseli R V. Identification of markers linked D to disease resistance gene by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using AFLP segregating populations. Proceedings of the National Academy of Sciences of the USA, 1991,88: F 2 [6] Lincoln S E, Daly M J, Lander E S. Constructing Genetic Linkage AFLP SCAR Maps with Mapmaker/Exp Version 3.0: A Tutorial and Reference Manual. A Whitehead Institute for Biomedical Research Technical Report (3rd. edition). January,1993.

7 [7].,1999,7(2): , 1996,29(2):8-16. Mo H D. Advances in quantitative genetics QTL mapping and its applification. Scientia Agriculture Sinica, 1996, 29(2): 8-16.(in Chinese) [8],,,,, Henry T Nguyen,. AFLP.,2000,16 (1): Li C Y, Zheng H G, Weng M L, Jia J H, Mou T M, Nguyen H T, Wang B. AFLP analysis of photoperiod-sensitive genic male sterile( PGMS) rice mutant lines. Chinese Journal of Biotechnology, 2000,16 (1): (in Chinese) [9],. AFLP. Rikkerink E, Roche P, Ryder C, Sansavini S, Schmidt H, Tartarini S,, 1997,17(1):6-11. Yin D M, Huang M R. AFLP molecular marker and its appliciation in the breeding of plant. Progress in Biotechnology, 1997,17(1):6-11. (in Chinese) [10] Warburton M L, Becerra-Velasquez V L, Goffreda J C, Bliss F A. Utility of RAPD markers in identifying genetic linkages to genes of economic interest in peach. Theoretical and Applied Genetics, 1996,93: [11],,,,,. (Malus) Dw RAPD. Zhang K C, Bi X Y, Li R Q, Jin S X, Wu L P, Yin S P. RAPD markers linked to dominant Dw gene in Malus. Journal of Agriculture Biotechnology, 1999,7(2): (in Chinese) [12],,. RAPD.,2000,27(5): Wang Y J, Wang X P, Zhou P, Zheng X Q. A RAPD marker linked to Anthracnose resistant gene of wild grape native to China. Acta Horticulturae Sinica, 2000,27(5): (in Chinese) [13] Maliepaard C, Alston F H, Arkei G ven, Brown L M, Chevreau E, Dunemann F, Evans K M, Gardiner S, Guilford P, Heusden A W, Janse J, Laurens F, Lynn J R, Manganaris A G, Nijs A P M, Periam N, Verhaegh J J, Vrielink-van Ginkel M, King G J. Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers. Theoretical and Applied Genetics,1998,97: [14] Fang D Q, Federici C T, Roose M L. Development of molecular markers linked to a gene controlling fruit acidity in citrus. Genome, 1997,40: [15] Wang Q, Zhang K, Qu X, Jia J, Shi J, Jin D, Wang B. Construction and characterization of a bacterial artificial chromosome library of peach. Theoretical and Applied Genetics,2000,103:

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