中华白海豚SRAP分子标记多态性的初步研究

首次将SRAP(sequence-related amplified polymorphism)方法引入中华白海豚(Sousa chinensis)多态性研究中,利用高分辨率的毛细管凝胶电泳分析了模板DNA、Mg2+、dNTP、引物、Taq DNA聚合酶等因素对SRAP-PCR扩增结果的影响.确定了me1-em5为扩增中华白海豚基因组DNA的最佳引物对.并进一步确立了适合中华白海豚的SRAP最佳反应体系:25 mm3体系中,模板DNA浓度为0.8 ng/mm3,Mg2+浓度为1.0 mmol/dm3,dNTP浓度为0.1 mmol/dm3,引物浓度为0.2μmol/dm3,Taq DNA聚合酶浓度为0.04 U/mm3.利用该反应体系对10个白海豚样品进行SRAP分析的结果表明,不同样品间DNA谱带清晰、多态性丰富.证实该体系稳定可靠,可以用于中华白海豚的分子标记研究.

Study on the SRAP marker polymorphism of Chinese white dolphin (Sousa chinensis)

Sequence related amplified polymorphism（SRAP） is a new molecular marker system based on PCR,with the advantages of simplicity,stability,high reproducibility and high co-dominance.In this paper,SRAP is first used to study the genetic diversity of Chinese white dolphin（Sousa chinensis）.All factors,including template DNA,Mg2＋,dNTP,primer pairs,and Taq DNA polymerase,which affect the SRAP-PCR reactions,are optimized using the capillary electrophoresis.Our results reveal that the best SRAP primer set for Chinese white dolphin genomic DNA is me1（5＇-TGAGTCCAAACCGGATA-3＇） and em5（5＇-GACTGCGTACGAATTAAC-3＇）.And the optimal reaction system is template DNA 0.8 ng/mm3,Mg2＋ 1.0 mmol/dm3,dNTP 0.1 mmol/dm3,primer 0.2 μmol/dm3,Taq DNA polymerase 0.04 U/mm3 in a 25 mm3 reaction.Then,we evaluate this SARP system using 10 samples from different Chinese white dolphin.Results show that polymorphism among samples are abundant and clear,which indicate that this system can be used as a suitable and reliable method for the molecular marker research of Chinese white dolphin.