苹果IRAP反应体系的建立和指纹图谱构建

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

利用已获得的苹果Ty1-copia类逆转座子LTRs建立了苹果逆转座子间扩增多态性(IRAP)技术体系,对影响IRAP-PCR扩增结果的若干因子进行了研究。优化的反应体系包含:2.5μL10×buffer,50 ng模板DNA,2.5mmol.L-1Mg2+,0.2 mmol.L-1dNTPs,0.4μmol.L-1引物,1 UTaqDNA聚合酶,反应体积为25μL;优化的PCR扩增程序为:94℃2 min;94℃1 min,退火1 min,72℃2 min,循环35次;72℃延伸10 min;退火温度根据引物温度设定。该IRAP体系分别用于‘元帅’和‘富士’的短枝和着色芽变指纹图谱的分析,构建的指纹图谱可以作为芽变鉴定的依据,表明这些突变可能系逆转座子转座插入或逆转座子间重组所致。

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.