Establishment Of The Inter-retrotransposon Amplified Polymorphism (IRAP) Marker System And Identification Of Apple Bud Mutation Clones

Apple(Malus domestica Borka) is one of the most important fruit crops in the world, by long evolution, the gentic variability within each species is very abundant. In this study, mutants of cultivar'Red Delicious'and'Fuji'are test targets,and we carried out some works: establishment of the IRAP-PCR reaction system in Malus genus;construction of fingerprint in cultivar'Red Delicious'and'Fuji', primary sport identifyication;a special fragment was generated with ARL5 primer, two primers were designed based on the sequence to transform IRAP marker into SCAR marker,the IRAP marker was successfully converted into SCAR marker; cloning the flanking sequences adiacent to the special fragment using inverse PCR method,it lays a foundation of study on mechanism of mutation and genome function.1. Several main factors influencing the IRAP-PCR reaction were analyzed in order to establish the inter-retrotransposon amplified polymorphism (IRAP) molecular marker system in Malus genus. The primers were selected from a pool of primers designed from apple Ty1-copia like retrotransposon long terminal repeat (LTR) sequences. The optimal system was in a reaction volume of 25μL including: 2.5μL 10×buffer, 50 ng template DNA, 2 mmo1-L^-1 Mg²⁺, 0.2 mmo1-L^-1 dNTPs, 0.4μmo1-L^-1 primer, 1 U TaqDNA polymerase; The optimal amplification program was with the following cycling parameters: 94°C for 2 min; 35 cycles of 94°C for 1 min; specified annealing temperature for 1 min, 72°C for 2 min, final extension at 72°c for 10 min. The IRAP technique was used in the present study to distinguish skin-color and spur-type mutations of the cultivars'Red Delicious'and'Fuji'by the constructed fingerprinting. According to fingerprinting, some bud mutations could be distinguished.2. Fifteen Red Delicious bud sport clones and sixteen Fuji bud sport clones were distinguished by IRAP molecular marker based on primers designed with Ty1-copia-like retrotransposons LTRs. The primers amplified polymorphic, clear bands and could differentiate the Red Delicious bud sport clones and Fuji bud sport clones. The phylogenetic relationship among 15 Red Delicious bud sport clones and 16 Fuji bud sport clones based on SAHN analysis showed that Red Delicious bud sport clones could be grouped into 4 families using similarity 0.76 as the standard. The first group was composed of 10 bud sport clones, which were Red Delicious, Jujiadian spur starking, Starspur Ultra Red Delicious, Miller Spur Delicious, Hardispur Delicious, Starking, Richard, Well spur Delicious, Early Red Delicious and Chinese Marshal 1. The second group comprised Meiguihong, Oregon spur Delicious and Show Red. Starkrimson and Hardi Bolaite were as a single group respectively. 16 Fuji sport clones could be grouped into 5 families based on similarity 0.74. The first group contained 10 sport clones, which were Nagafu 3, Shengfangfu 1, Nagafu 6, Qunfu 1, Yanfu 1, Yujingâ…¡, Yanshouâ… , Fujiâ… , Hongwangjiang and Nagafu 7. The second group comprised Fuji and Akifu 5. The third included Akifu 1 and Akifu 2. Changyeâ… and Zhaitengâ…¡were as a single group respectively. The results showed that the insertion orientations of retrotransposons were independent in apple genome. Transpositional insertion and recombination between retrotransposons were two of important mechanisms inducing apple somatic cell variation. The primers developed in the experiment provided useful tools for identification of apple bud sport clones, which laid the foundations for the further study of the mechanisms of apple bud mutations.3. A special fragment generated with the primer ARL5 in'Meiguihong', the spurtype mutant of the cultivar'Red Delicious', was sequenced. The IRAP marker was successfully converted into SCAR marker. The optimal system of SCAR marker was in a reaction volume of 25μL including: 50 ng template DNA, 2.5μL 10×buffer, 3 mmo1-L^-1 Mg²⁺, 0.2 mmo1-L^-1 dNTPs, 0.2μmo1-L^-1 primer, 1 U TaqDNA polymerase. The SCAR marker will be useful for spur-type character selection and marker-assisted selection in apple breeding programs.4. Inverse PCR technique is a method by which flanking sequences adjacent to known DNA fragments can be cloned. 2μg template DNA were extracted and digested with restriction enzymes Hindâ…¢in 50μL. The optimal system of ligation was in a reaction volume of 50μL including: 0.8μg digested DNA, 5μL10×T4 ligation buffer, 5 U T4DNA ligation enzyme. Digested DNA was ligated overnight at 16°C. The optimal system of IPCR marker was in a reaction volume of 50μL including: 5μL 10×LA PCR buffer(Mg²⁺ plus), 40 ng template DNA, 0.2 mmo1-L^-1 dNTPs, 0.4μmo1-L^-1 primer IP1 and IP2, 2.5 U TaKaRa LA TaqDNA polymerase; The optimal amplification program was as follows, 94°C for 4 min; 35 cycles of 94°C for 1 min; 60°C for 1 min, 72°C for 4 min, final extension at 72°C for 10 min. Flanking sequence amplified by inverse PCR technique was 1.5 kb.