华扁穗草ISSR-PCR反应体系优化及引物筛选

Optimization of ISSR-PCR System and Primer Screening for Blysmus sinocompressus

利用正交试验设计方法,对影响华扁穗草ISSR-PCR反应中Mg2+、Taq DNA聚合酶、dNTP、引物和DNA模板等5个因素进行优化筛选,以期建立最佳反应条件。同时,筛选扩增条带清晰稳定的ISSR引物,经退火温度试验得到各个引物的最佳退火温度。结果表明:华扁穗草20μl ISSR-PCR最佳反应体系包括1.80 mmol/L Mg2+、0.80U Taq DNA聚合酶、0.100mmol/L dNTP、0.6μmol/L引物和20ng DNA模板;筛选出扩增条带清晰稳定的10条引物。体系验证和引物筛选试验表明,其在华扁穗草不同个体中能够扩增出条带清晰、稳定性好的条带,可用于后续华扁穗草遗传多样性分析,为华扁穗草野生资源保护和优良牧草种质资源选育提供理论依据。

To optimize the ISSR-PCR system for Blysmus sinocompressus,an orthogonal design was selected at four levels of five factors（Mg2＋,Taq DNA polymerase,dNTP,primer and DNA template）.Then,based on the optimal ISSR-PCR amplification system,ISSR primers with clear bands and the annealing temperatures were selected.As a result,the optimal PCR mixture（20μL）contained 1.80 mmol/L Mg2＋,0.80 U Taq DNA polymerase,0.100mmol/L dNTP,0.6mol/L primer and 20 ng template DNA.Ten primers were screened with clear and consistent bands.With the optimal system of ISSR-PCR and primer screening,clear and steady bands were obtained in different individuals of Blysmus sinocompressus.The establishment of ISSR-PCR system could favor the genetic diversity of Blysmus sinocompressus pasture germplasm with molecular marker techniques.It was useful for the conservation of wild resource of Blysmus sinocompressus and excellent forage germplasm breeding.