First attempt to validate multiplex PCR with sex markers SSM4 and ALLWSex2 in long-term stored blood samples of eastern North American shortnose sturgeon (Acipenser brevirostrum).

Sex-specific information is crucial for sturgeon culture, conservation, and fisheries management. However, identifying the sex of sturgeon is difficult, especially for immature individuals. Two recent studies identified two female-specific loci (AllWSex2 and SSM4) that are conserved among many Acipenserid species, but they have not been validated for all species within this family. The objectives of this study were to 1) determine whether SSM4 can be used to sex shortnose sturgeon; 2) develop and test a multiplex PCR technique using both ALLWSex2 and SSM4 for sexing shortnose sturgeon; 3) determine if long-term storage of blood samples can be used to sex shortnose sturgeon; and 4) test the effect of storage temperature on DNA degradation. DNA was extracted from frozen RBC samples from 36 fish which had previously been sexed. A multiplex PCR was set up using three pairs of primers: AllWSex2 and SSM4, as female-specific loci, and mtDNA as an internal control which were all run on a 2 % agarose gel. AllWSex2 and SSM4 allowed for perfect discrimination of sex. While there was DNA degradation, as a result of long-term storage and temperature, the signal was still strong enough after 8 years of cold storage to delineate sex. This suggests that researchers now have the ability to reexamine archived/frozen samples to determine sex of their fish.