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

Key words: 
• cash forestry /
• Torreya grandis ‘Merrillii’ /
• inter simple sequence repeats （ISSR）-polymerase chain reaction（PCR） /
• primer screening

Abstract: 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.］