Establishment and Optimization of ISSR-PCR Amplification System in SquashChinese Full Text
Zhu Haisheng;Lu Lifang;Wen Qingfang;Lin Yizhang;Crops Research Institute, Fujian Academy of Agricultural Sciences;Vegetable Research Center, Fujian Academy of Agricultural Sciences;College of Horticulture, Fujian Agriculture and Forestry University;
Abstract: In order to obtain the ISSR-PCR reaction system of Squash, We took an optimization experiment with single factor design which was concentrated on five factors of Mg2+, dNTP, Taq DNA polymerase, primer and template DNA by using the squash ‘miben’as the material of filter system to set up a good foundation of analysing the genetic diversity and relationship identification of Squash. The test result showed that the greatest 25 μL ISSR reaction system which contained 2.5 μL 10×Buffer, 2.0 mmol/L Mg2+, 0.3 mmol/L dNTP, 1 U Taq DNA polymerase, 0.48 mmol/L primer, 75 ng template DNA. On this basis, optimized annealing temperature of 12 primers which have been screened, and ultimately determined the optimal annealing temperature of each primer.85 squashes were amplified with primer 891 under the optimized reaction conditions. The test result showed the amplification products which were clear and bright, abundant polymorphism, strong specificity and good repeatability. Accordingly, it was suitable for ISSR analysis of squash.
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- DOI：
10.13271/j.mpb.012.000802
- Series：
- Subject：
- Classification Code：
S642.1
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