New multiplex PCR assays for estimating genetic diversity in European grayling ( <i>Thymallus thymallus</i> ) by polymorphism of microsatellite DNA

Abstract The European grayling ( Thymallus thymallus L.) is a vulnerable freshwater fish that plays an important role in the conservation of the biodiversity of European ichthyofauna and is a key species in recreational fishing. Multiplex PCR is a technique used to estimate genetic diversity that saves both time and money. This paper presents three multiplex PCR assays for amplifying 11 microsatellite markers from European grayling DNA. The steps necessary for constructing multiplex assays and verifying genotyping results are described. The compositions of the PCR mixture and the characteristics of the PCR thermal profile recommended for amplification in multiplex mode, and protocols used in genotyping them by using automatic DNA sequences are also described. Consequently, our method enables a near four-fold reduction in time and costs of the PCR stage of studies on genetic variation. Moreover, the next step of analysis (measuring the length of DNA fragments) can be done faster and easier without decreasing the precision or quality of genotyping results. This method was tested by assessing the genetic variation in a grayling stock kept under aquaculture conditions, and it has proved useful in the management of the genetic variation of this species.