铲鲟微卫星引物对中华鲟的适用性研究
APPLICABILITY OF MICROSATELLITE PRIMERS DEVELOPED FROM SHOVELNOSE STURGEON IN CHINESE STURGEON
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摘要: 将21对铲鲟(Scaphirhynchus platorynchus Rafinesque)的微卫星引物在中华鲟(Acipenser sinensis Gray)基因组DNA上进行PCR扩增,14对(约占67%)引物得到了扩增产物,其中有10对(约占4%)表现为多态性,但只有2对(9.5%)引物Spl-100和Spl-16的多态性较高,且带型清晰,可直接作为分子标记应用于相关的研究.此外,对具有特异性扩增,但有Stutter band现象的4对引物的部分可分离PCR产物进行了回收和测序,并对其中的3对引物的序列进行了重新设计,最后得到两对可应用于中华鲟的引物As-100和Spl-170b.研究结果表明,对相近种的微卫星引物进行优化设计来获得一个物种的微卫星引物是一条简捷有效的途径.Abstract: Twenty one pairs of microsatellite primers developed from shovelnose sturgeon (Scaphirhynchus platorynchus) were tested on Chinese sturgeon (Acipenser sinensis). About 67% (14 loci) of the S.platorynchus primers amplified successfully with 48% (10 loci) being polymorphic. However, only 9 5% (2 loci) of these primers (Spl-100 Spl-168) produced clear and sharp products without stutter bands. Several amplified fragments produced by four pairs of the rest primers, which generated stutter bands in Chinese sturgeon, were sequenced. One of these four loci (Spl-123) was proved without repeat motif. Then, the other three, Spl-106,Spl-163, and Spl-70α, were redesigned with Primer premier 5.0 software and renamed as Spl-106a, Spl-163a, and Spl-170b. Further amplifications using these primers in Chinese sturgeon showed that Spl 106a and Spl 170b could give clear and polymorphic bands without any stutter, but Spl 163a did not get any bands. Moreover, motif sequences obtained by both Spl-106 and Spl-106a in Chinese sturgeon are the same but differ to those in shovelnose sturgeon. It seems that the redesigned primer Spl 106a could be a species specific primer for Chinese sturgeon, and we finally renamed it as As 100. This result indicates that it is viable to obtain species specific microsatellite primers by redesigning from the primers of related species.
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