RSAP标记技术在紫菜遗传多样性检测及种质鉴定中的应用

The Application of RSAP Marker Technique in Diversity Detection and Germplasms Identification of Porphyra

限制性位点扩增多态性（restriction site amplification polymorphism，RSAP）是根据基因组内普遍存在的酶切位点来设计特殊的引物，通过简单的梯度PCR反应来产生多态性。本研究首次将RSAP标记技术成功应用于紫菜遗传多样性检测和种质鉴定。利用所建立的适合于紫菜RSAP分析的PCR反应体系和反应条件，使用30对引物对15个紫菜系DNA进行了PCR扩增，经过3次重复验证，有12对引物能够扩增出清晰且稳定的条带。这12对引物共扩增出413条带，其中408条为多态性条带，多态性比例96％，平均每对引物产生34条多态性条带，片段大小在50-500bp之间。利用这413条带进行聚类分析，产生了这15个紫菜系的进化树。15个紫菜系在0.69相似系数水平上分为两大类：一类包括坛紫菜；另一类包括条斑紫菜和少精紫菜。选2对引物R1／R6和R3／R4所扩增的10条稳定且重复性好的条带，构建了这15个紫菜系的RSAP-DNA指纹图谱。在该指纹图谱中，每个紫菜系都有其独一无二的指纹模式，能够很容易地与其它紫菜系相区分。

Restriction site amplified polymorphism （RSAP） requires just a simple polymerase chain reaction （PCR） to generate polymorphic markers around restrictive site. An RSAP analytic system was set up and successfully used in Porphyra in this study. Fifteen Porphyra lines were screened by the RSAP analytic system with 30 primer-pairs, twelve of which produced stable and reproducible amplification patterns in three repeated experiments. The 12 primer-pairs produced 413 amplified fragments, 408 of which （96 % ） were polymorphic, with an average of 34 polymorphic fragments for each primer-pair, ranging in size from 50 bp to 500 bp. The 413 fragments were scored one by one and then used to develop a dendrogram of these Porphyra lines. These Porphyra lines were divided into two major groups at the 0.69 similarity level, one group contained only P. haitanensis lines and the other group contained P. yezoensis and P. oligospermatangia lines. From the 408 polymorphic fragments, ten amplified by two primer-pairs R1/R6 and R3/R4, were used to develop the RSAP- DNA fingerprints of the 15 Porphyra lines. In the DNA fingerprints, each of the 15 Porphyra lines has its u- nique binary code and can be easily distinguished from each other.