ALLELIC DIVERSIFICATION, SYNTENIC ALIGNMENT AND EXPRESSION PATTERNS OF NANOS2 IN POLYPLOID GIBEL CARP

Germline stem cells (GSCs) have the self-renewal ability to continuously give rise to gametes throughout their life. Owing to its unique reproductive and sex determination modes, polyploid gibel carp (Carassius gibelio) has become a promising model organism for reproductive and developmental genetics in vertebrates. Although several germ cell marker genes, such as vasa, dazl and dnd, have been studied in gibel carp, GSC genes has not been identified. Here we identified two divergent nanos2 homeologs (named Cgnanos2a and Cgnanos2b), each of which has three alleles. Phylogenetic and synteny analyses indicated that two rounds of polyploidization occurred during allo-autohexaploid gibel carp evolution. Cgnanos2a and Cgnanos2b were most expressed in testis, followed by ovary. They also highly expressed in ovary at 25 dph, and then decreased sharply. The expression levels of Cgnanos2a in testis and ovaries from 25 dph to 60 dph were higher than those of Cgnanos2b (P<0.05), indicating significant expression bias between Cgnanos2a and Cgnanos2b. Finally, Cgnanos2a and Cgnanos2b specifically expressed in a subset of small germ cells with a diameter of <20 μm in the ovary, and abundant Cgnanos2a transcripts were detected in isolated cells or in doublets of testis. Considering these characteristics, Cgnanos2a and Cgnanos2b might be the markers of gibel carp GSCs. In addition, their transcripts were also detected in spermatogonia and spermatocytes. Therefore, we described the development of early ovarian cysts with nanos2 positive cells, which will help to isolate gibel carp GSCs. The sequence characteristics, evolution and expression patterns of two divergent nanos2 genes in polyploid gibel carp provide a paradigm for the evolution of duplicated genes in recurrent polyploidy vertebrates.