基于正交试验设计优化三疣梭子蟹SRAP-PCR反应体系

相关序列扩增多态性（sequence related amplified polymorphism,SRAP）是一种多态性高的新型分子标记,其扩增结果稳定,容易操作和引物通用性高,在遗传多样性分析、种质鉴定和遗传图谱构建等方面的广泛应用逐渐从植物转移到水产动物中。为了建立三疣梭子蟹（Portunus trituberculatus）SRAP技术体系,文章利用正交设计L16（45）对三疣梭子蟹SRAP-PCR反应体系的5因素TaqDNA聚合酶、镁离子（Mg2＋）、模板、三磷酸脱氧核苷（dNTPs）和引物]在4个水平上进行优化试验,结果得出各因素水平变化对PCR反应的影响从大到小依次为模板（纯度1.75~1.90,质量浓度10~70ng.μL-1）,Mg2＋（1.60~2.00mmol.L-1）,dNTPs（0.10~0.40mmol.L-1）,TaqDNA聚合酶（0.50~2.00U）和引物（0.10~0.40μmol.L-1）;筛选出各反应因素的最佳水平,建立三疣梭子蟹SRAP-PCR反应的最佳体系（25μL）为TaqDNA聚合酶0.50U,Mg2＋2.20mmol.L-1,模板DNA10ng,dNTPs0.20mmol.L-1,引物0.20μmol.L-1。这一优化体系可望在三疣梭子蟹遗传多样性和性别连锁标记等研究中应用。

Optimization of SRAP-PCR system for Portunus trituberculatus based on orthogonal experimental design

SRAP is a novel molecular marker with high polymorphism.It is reliable in amplification,easy to operate and has a high universality in primer pairs.SRAP technique has been widely used for studies from plants to aquatic animals in the fields of genetic diversity,germplasm identification,genetic map construction and so on.Based on an orthogonal experimental design,we optimized the five factors of SRAP-PCR amplification system for Portunus trituberculatus（TaqDNA polymerase,Mg2＋,template,dNTPs and primer）from four levels.The results show that the effects of these factors on PCR reaction from strong to weak is as follows：template（purity 1.75~1.90,concentration 10~70 ng·μL-1）,Mg2＋（1.60~2.00 mmol·L-1）,dNTPs（0.10~0.40 mmol·L-1）,TaqDNA polymerase（0.50~2.00 U）and primer（0.10~0.40 μmol·L-1）.The optimal SRAP-PCR reaction system（25 μL）can be summarized as follows：0.50 U TaqDNA polymerase,2.20 mmol·L-1 Mg2＋,10 ng DNA template,0.20 mmol·L-1 dNTPs and 0.20 μmol·L-1 primer.This optimized system may be helpful in the studies of genetic diversity and sex-linked markers for P.trituberculatus.