萝卜IRAP技术体系建立与品种指纹图谱构建

Establishment of inter-retrotransposon amplified polymorphism( IRAP )reaction system and construction of cultivar fingerprint in radish( Raphanus sativus L. )

为建立萝卜逆转座子间扩增多态性(IRAP)技术体系,基于萝卜Ty1-copia类逆转座子逆转录酶的保守序列设计引物,对IRAP-PCR反应主要因素进行分析。建立的萝卜IRAP标记技术体系(20μl)为:20 ng基因组DNA模板,1×PCR buffer,0.25 mmol/L d NTPs,2.0 mmol/L Mg2+,0.4μmol/L引物,1 U Taq DNA聚合酶。将所建立的IRAP标记技术体系应用于14个萝卜品种指纹图谱分析,结果显示,筛选出的2个特异引物Rs Ty1F5和Rs Ty1F10在14份萝卜材料中共扩增得到了16个多态性条带,可以将14份萝卜材料完全区分开,每份种质都有独特的指纹图谱,表明IRAP技术可以有效地应用于萝卜种质鉴定和指纹图谱的构建。

Based on the conserved domain of Ty1-copia-like retrotransposon reverse transcriptase of radish,inter-retrotransposon amplified polymorphism（ IRAP） primers were designed. Several important reaction factors of IRAP were studied to establish and optimize IRAP marker system in radish. The IRAP-PCR was performed in a optimized 20-μl reaction mixture containing 20 ng DNA,1 × PCR buffer,0. 25 mmol / L d NTPs,2. 0 mmol / L Mg2 ＋,0. 4 μmol/L primer and 1 U Taq DNA polymerase. DNA fingerprint maps of 14 radish genotypes were constructed with optimized IRAP molecular marker system. A total of 16 polymorphic bands were generated with two primers,Rs Ty1F5 and Rs Ty1F10. The 14 radish genotypes could be absolutely distinguished with the two IRAP primers. Each radish genotype had a unique fingerprint,suggesting that IRAP marker was practical and effective in radish cultivar identification and fingerprint construction.