Optimization of an ISSR-PCR system and primer screening for Torreya grandis ‘Merrillii’
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摘要: 以香榧Torreya grandis Merrillii基因组DNA为材料，对影响简单序列重复区间扩增?鄄聚合酶链式反应（ISSR-PCR）的Taq酶用量、Mg2+浓度、模板DNA用量、磷酸碱基脱氧核苷酸（dNTPs）浓度和引物浓度等进行了优化试验。优化后的香榧ISSR-PCR扩增体系为：总容积20 L，包括30 ng模板DNA，16.67 nkat Taq酶，0.350 molL -1引物，1.625 mmolL-1 Mg2+，0.250 mmolL-1 dNTPs，10 PCR buffer 2 L。PCR扩增程序：94 ℃变性5.0 min；35个循环：94 ℃变性30 s，退火45 s，退火温度视引物而定，72 ℃延伸1.5 min；最后72 ℃延伸7.0 min，4 ℃保温。在此基础上，从77个ISSR引物中筛选出34个适合香榧ISSR分析的引物，且通过梯度PCR确定了各自最适的退火温度。研究结果为香榧遗传图谱构建打下了基础。图4表1参12Abstract: With genomic DNA of Torreya grandisMerrillii，an optimization experiment was performed in terms of Taq DNA polymerase，Mg2+，DNA template，deoxynucleotide triphosphates（dNTP）s，and primer concentration. The optimized system was as follows：a total polymerase chain reaction （PCR） volume of 20 L included 30 ng DNA，16.67 nkat Taq polymerase，0.350 molL-1 primer，1.625 mmolL-1 Mg2+，0.250 mmolL-1 dNTPs，and 2 L 10 PCR buffer. Then，a PCR amplification program was established with denaturing at 94 C for 5.0 min followed by 35 cycles of denaturing at 94 C for 30 s，annealing for 45 s （annealing temperatures varied with primers） with extension at 72 C for 1.5 min，extension at 72 C for 7 min，and finally maintained at 4 C. Based on this optimized system，34 inter simple sequence repeat （ISSR） primers suitable for the species were screened from 77 primers with gradient PCR being used to propose the optimal annealing temperature. This experimental result has laid a foundation for genetic map construction in T. grandis Merrillii. ［Ch，4 fig. 1 tab. 12 ref.］
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2010.01.014
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