Establishment of a SRAP analysis protocol in Carya cathayensis and a comparison among SRAP，RAPD，ISSR analysis protocols

Key words: 
• cash forestry /
• Carya cathayensis （hickory） /
• sequence-related amplified polymorphism （SRAP） /
• random amplified polymorphic DNA （RAPD） /
• inter-simple sequence repeat （ISSR）

Abstract: In order to have a good molecular marker to reflect inherent genetic characteristics of Linan hickory （Carya cathayensis），the genomic DNA extracted from hickory leaves was used to optimize parameters （constituents） included in a sequence-related amplified polymorphism-polymerase chain reaction （SRAP-PCR） protocol run under the following conditions：pre-denaturing at 94 ℃ for 5 min；5 cycles，each of which denatured at 94 ℃ for 30 s and annealed at 35 ℃ for 30 s with an extension at 72 ℃ for 2 min；30 cycles，each of which denatured at 94 ℃ for 30 s and annealed at 50 ℃ for 30 s with an extension at 72 ℃ for 2 min；and a final extension at 72 ℃ for 8 min. Then，15 pairs of primers out of 100 pairs were screened for SRAP analysis and a comparison was made among SRAP，random amplified polymorphic DNA（RAPD），and inter-simple sequence repeat（ISSR）. The optimized（SRAP-PCR） protocol was as follows：a total volume of 25.00 L containing 1 buffer，0.20 mmolL-1 dNTPs（deoxynucleotide triphosphates），0.20 molL-1 primers，2.00 mmolL-1 Mg2+，33.34 nkat Taq DNA polymerase，and 0.80 mgL-1 genomic DNA（all at a final concentration）. On the average，SRAP，compared to RAPD and ISSR，had the most loci and polymorphic loci amplified by each pair of primers，but SRAP percentages for both polymorphic primer pairs and polymorphic loci were between RAPD and ISSR. The optimized SRAP-PCR reduced the reaction time by half compared with the former protocols. It has been shown that both SRAP and RAPD should be considered when studying hickory.［Ch，6 fig. 4 tab. 28 ref.］