毛叶山桐子性别相关ISSR分子标记的筛选与分析

Screening and analysis of sex-related ISSR molecular marker in Idesia polycarpa Maxim. var. vestita Diels

摘要:

为筛选与毛叶山桐子性别相关的分子标记，本研究优化了毛叶山桐子ISSR-PCR反应体系，并利用优化后的体系逐一筛选100条ISSR引物，然后通过聚丙烯酰胺凝胶电泳进行多态性检测．实验确立出最佳的25 μL反应体系为：55 ng DNA模板，1 U Taq酶，2 μL dNTPs（2.5 mmol/L），2 μL引物（10 μmol/L），2.5 μL 10 ×Taq Buffer（含Mg2+），超纯水补足总25 μL．利用该体系逐一筛选ISSR引物，在多次重复后只有UBC841引物扩增出的差异性条带可以稳定存在．PAGE电泳结果显示，UBC841扩增产生一条差异性条带，大小在250-300bp之间，存在于毛叶山桐子雌株中，但后期的测序发现该条带是由大小相近的条带组成的混合条带．虽然如此，引物UBC841仍可以作为毛叶山桐子性别相关的分子标记．

Abstract:

In order to screen the sex-related ISSR molecular marker in Idesia polycarpa Maxim. var. vestita Diels，the inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) system was established and optimized．And we found the best ISSR-PCR reaction system with total volume of 25μL was 55 ng DNA template， 1U Taq polymerase，2μL dNTPs tendency（2.5 mmol/L)，2μL primers (10μmol/L)，2.5μL 10 x Taq Buffer (including magnesium ion)and then adding ultrapure water to make up total 25μL．With this system we discovered only one ISSR primer——UBC841，which could amplify one stable female-special fragment，with size of 250-300bp．By sequencing the female-special fragment，we found the fragment was made up of many fragments with similar sizes，which indicated the female-special fragment showed by PAGE  electrophoresis was a mixed bands．Even so，the primer UBC841 still can be used for sex-related markers to identify the gender of Idesia polycarpa Maxim. var. vestita Diels．