香榧ISSR-PCR扩增条件的优化和引物筛选

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 μL，包括30 ng模板DNA，16.67 nkat Taq酶，0.350 μmol·L -1引物，1.625 mmol·L^-1 Mg²⁺，0.250 mmol·L^-1 dNTPs，10 × PCR buffer 2 μL。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1参12

Optimization of an ISSR-PCR system and primer screening for Torreya grandis 'Merrillii'

With genomic DNA of Torreya grandis ' Merrillii', an optimization experiment was performed in terms of Taq DNA polymerase, Mg~(2+), DNA template, deoxynucleotide triphosphates (dNTP)s, and primer concentration. The optimized system was as follows: a total polymerase chain reaction (PCR) volume of 20 μL included 30 ng DNA, 16.67 nkat Taq polymerase, 0.350 μmol·L~(-1) primer, 1.625 mmol·L~(-1) Mg~(2+), 0.250 mmol·L~(-1) dNTPs, and 2 μL 10 × PCR buffer. Then, a PCR amplification program was established with denaturing at 94℃ for 5.0 min followed by 35 cycles of denaturing at 94℃ for 30 s, annealing for 45 s (annealing temperatures varied with primers) with extension at 72 ℃ for 1.5 min, extension at 72℃ for 7 min, and finally maintained at 4℃. 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 pro-pose the optimal annealing temperature. This experimental result has laid a foundation for genetic map con-struction in T. grandis' Merrillii.