White crappie (Pomoxis annularis) and black crappie (P. nigromaculatus), both members of the Centrarchidae family, are two popular sport fish species in lakes and reservoirs throughout the USA. The Vasa and Nanos-2 genes have been widely used as molecular markers for identifying germ cells in various fish species. However, these two genes have not yet been investigated or reported in crappie species. Identifying and characterizing these genes can have significant implications for germplasm conservation and production of hybrid crappie using innovative hatchery technology, such as xenogenesis.
Conserved sequences of these two genes were identified by comparing available nucleotide sequences from the database of five different fish species: largemouth bass (Micropterus salmoides), catfish (Ictalurus punctatus), tilapia (Oreochromis niloticus), medaka (Oryzias latipes), and zebrafish (Danio rerio). Subsequently, primers were designed based on the conserved regions of these five different fish species to perform quantitative polymerase chain reaction (qPCR) and to determine the expression levels of the target genes in the ovary of crappie species.
The primers designed from conserved regions proved effective for amplifying and identifying the Vasa and Nanos-2 genes in the crappie ovaries. Both target genes were found to be expressed in the ovaries of both crappie species. Gel electrophoresis was conducted to visualize the qPCR products, confirming the successful amplification of Vasa and Nanos-2 genes. The results demonstrated the conserved nature of these genes in both white and black crappies.