山核桃SRAP体系的建立及与RAPD和ISSR标记的比较

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

以山核桃Carya cathayensis基因组DNA为模板,对聚合酶链式反应(PCR)体系各组分进行了梯度实验,优化出条带清晰、重复性好的相关序列扩增多态性聚合酶链式反应(SRAP-PCR)扩增体系,并筛选了引物。该体系(25.00μL)为:1×缓冲液0.20 mmol.L-1,脱氧核糖核苷酸(dNTPs),0.20μmol.L-1引物,2.00 mmol.L-1镁离子(Mg2+),33.34 nkat Taq DNA聚合酶,0.80 mg.L-1基因组DNA(以上均为终浓度)。反应条件为94℃预变性5 min;94℃变性30 s,35℃退火30 s,72℃延伸2 min,5个循环;94℃变性30 s,50℃退火30 s,72℃延伸2 min,30个循环;72℃延伸8 min,4℃保存,反应时间比其他体系缩短了一半。从100对引物中筛选出了适用于山核桃的引物15对。在山核桃中,随机扩增多态性DNA(RAPD),简单序列重复区间(ISSR),SRAP等3种标记,以SRAP标记每对引物扩增的位点数及每对引物扩增的多态性位点数为多,但SRAP多态性引物的比例、多态性位点比例居于另2种标记之间。在山核桃研究中可以考虑使用SRAP及RAPD标记。

In order to have a good molecular marker to reflect inherent genetic characteristics of Lin＇an 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 μL containing 1 × buffer,0.20 mmol·L-1 dNTPs（deoxynucleotide triphosphates）,0.20 μmol·L-1 primers,2.00 mmol·L-1 Mg2＋,33.34 nkat Taq DNA polymerase,and 0.80 mg·L-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.]