Multiplex PCR-based genotyping of mitochondrial DNA from two Gnathopogon species found in the Lake Biwa system: Its development and application to field-collected eggs from the South Basin

The endemic Lake Biwa species Gnathopogon caerulescens has become severely endangered since the 1990s due to deterioration of its spawning habitat, much of which was in the South Basin of the lake. To aid in the conservation of the species, a rapid molecular method was devised for discriminating it from a sympatric congener, Gnathopogon elongatus, widely distributed in western Japan. Three allele-specific primers were developed for the mitochondrial cytochrome b gene regions of three lineages of the two species: Cytb465hon_c for G. caerulescens, and Cytb315tam_e1 and Cytb246tam_e2 for the E1 and E2 lineages, respectively, of G. elongatus. Using the same reverse primer (Cytb522R), the specific primers for G. caerulescens and the E1 and E2 lineages of G. elongatus were designed to amplify ca. 80-, 230-, and 310-bp fragments, respectively. Multiplex PCR reactions including these four primers produced species/lineage-specific fragments. Although false fragments sometimes appeared, they did not affect discrimination between the two species. To demonstrate the efficacy of this method, field-collected eggs were analyzed, a total 1,238 eggs being collected from 37 egg populations on submerged roots of Salix trees (the spawning substrate preferred by G. caerulescens) on the coast of the South Basin between 18 April and 16 May 2019. Analysis of 298 eggs (approximately equal samples from each population) using a published DNA method resulted in 249 being successfully sorted into the genera Gnathopogon (206 eggs), Carassius (33), and Cyprinus (10). Subsequent analysis of the Gnathopogon eggs with our method detected 185 G. caerulescens and three G. elongatus (E1) eggs, 18 being unidentified. G. caerulescens eggs were included in 32 of the 37 egg populations from throughout the South Basin, except the southernmost region, indicating that the geographic distribution of spawning sites of the species is rapidly recovering to pre-1960s levels (based on literature estimates).