Establishment of the inter-retrotransposon amplified polymorphism (IRAP) reaction system and construction of fingerprint in Malus genus.

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 in a reaction volume of 25 μL was as follows:2.5 μL 10×buffer,50 ng template DNA,2.5 mmol·L-1 Mg2+,0.2 mmol·L-1 dNTPs,0.4 μmol·L-1 primer,1 U Taq DNA polymerase;The Optimal amplification program was with the following cycling parameters:94℃ for 2 min;35 cycles of 94℃ for 1 min;specified annealing temperature for 1 min,72℃ for 2 min,final extension at 72℃ for 10 min.The IRAP technique was used to distinguish skin-color and spur-type mutations of the cultivars 'Red Delicious' and 'Fuji' by the constructed fingerprinting.According to to fingerprinting,some bud mutations could be distinguished.In addition,our results suggest that the bud mutations,which have generated new patented varieties of 'Red Delicious' and 'Fuji',appear to derive from retrotransposon insertion or recombination between retrotransposons.