广西植物Issue(6)：899-904,6.DOI:10.11931/guihaia.gxzw201405042
多用途树种无患子 ISSR-PCR 体系建立与检测
Detection and establishment of ISSR-PCR system for multipurpose tree species Sapindus mukorossi
摘要
Abstract
Sapindus mukorossi is a traditional and important virescencetree species in the south part of China with rich saponins in peel and rich oil in seed.This tree species is one of the newly-developed woody oil species that were approved by the State Forestry Administration of China.For optimizing ISSR-PCR reaction system of S .mukorossi , based on KOD FX High-fidelity DNA Polymerase Kit,orthogonal design experiments were conducted.The main fac-tors affecting ISSR-PCR amplification,such as suitable concentration of primer,dNTPs,KOD FX DNA polymer-ase,DNA template and 2×PCR buffer for KOD FX were studied.Furthermore,the annealing temperature was op-timized on the base of the above tests.An ideally ISSR-PCR reaction system was established,namely 25 μL reaction system containing primer 0.5 μmol.L-1 ,dNTPs 0.05 mmol.L-1 ,KOD FX DNA polymerase 0.06 U.μL-1 、DNA tem-plate 1.0 ng.μL-1 and Mg2+ 1.0 mmol.L-1 .The optimal PCR amplification program was:The profile of ISSR-PCR was an initial denaturation step for 2 min 94 ℃,followed by 35 cycles of 30 s at 98 ℃ for denaturation,30 s at 48.6 ℃ for annealing,90 s at 68 ℃ for extension,finally extension at 68 ℃ for 7 min and holding the samples at 4℃.This optimized ISSR-PCR reaction system would provide the basis for the analysis of genetic diversity,genetic structure,germplasm innovation and molecular assisted selection in S .mukorossi .关键词
无患子/ISSR/PCR 反应体系/正交设计Key words
Sapindus mukorossi/ISSR/PCR reaction system/orthogonal design experiments分类
农业科技引用本文复制引用
刁松锋,邵文豪,姜景民,栾启福..多用途树种无患子 ISSR-PCR 体系建立与检测[J].广西植物,2015,(6):899-904,6.基金项目
国家林业公益性科研专项(201404104,200804032) （201404104,200804032）
浙江省重大科技专项重点农业项目(2011C12015)。 （2011C12015）
