SRAP分析体系的优化及在枇杷种质资源研究上的应用

以me7(5’-TGAGTCCAAACCGGTCC-3’)和em7(5’-GACTGCGTACGAATTCAA-3’)正反向引物组合对西班牙枇杷品种Javierin进行了SRAP分析体系的优化,结果表明,在25μL反应体系中,5种主要成分的适宜浓度或用量分别是:dNTPs0.3mmol/L,Mg2+2.5mmol/L,TaqDNA聚合酶1.0U,引物0.3μmol/L,模板DNA20ng,优化的扩增程序为:94℃预变性5min,94℃变性1min,35℃复性1min,72℃延伸1min30s,5个循环;之后94℃变性1min,50℃复性1min,72℃延伸1min30s,35个循环;最后72℃下延伸10min。并将该优化的体系在来自中国、西班牙、日本、意大利和美国的46份枇杷种质资源上进行了SRAP扩增的初步应用,经琼脂糖和聚丙烯酰胺凝胶电泳均获得了清晰、重复性好的SRAP指纹图谱。

Optimization of SRAP analysis and its application in germplasm research of Loquat(Eriobotrya japonica)

An optimal system of SRAP analysis for Spanish loquat cultivar Javierin (Eriobotrya japonica Lindl. cv. Javierin) was established with the primer combination of me7 (5’-TGAGTCCAAACCGGTCC-3’) and em7 (5’-GACTGCGTACGAATTCAA-3’). The results showed that the optimum concentrations of 5 important ingredients were: dNTPs 0.3 mmol/L, Mg2+ 2.5 mmol/L, Taq DNA polymerase 1 U , primers 0.3 μmol/L and template DNA 20 ng. The optimized program was pre-denaturing at 94 ℃ for 5 min, then denaturing at 94 ℃ for 1 min, annealing at 35 ℃ for 1 min and extending at 72 ℃ for 90 s for the first five cycles; followed by another 35 cycles with annealing temperature at 50 ℃, and finally terminated by post-extending at 72 ℃ for 10 min. With this system, 46 accessions of Eriobotrya from China, Spanish, Japan, Italy and USA were preliminarily analyzed and the protocol resulted in clear and robust SRAP patterns after electrophoresis on agarose and polyacrylamide gels.