Development of novel microsatellite marker panel in threatened tetraploid mahseer, Tor tor (Hamilton 1822) for insights into its genetic diversity and population structure

Tor tor (Hamilton 1922), a natural polyploid fish species, is commonly known as the red-fin or deep bodied mahseer and is the type species of the Tor genus. It was assessed as ‘Near Threatened’ in the IUCN Red List, due to rapidly declining populations in wild and recently reassessed as Data Deficient. To generate the genetic structure information in this species, co-dominant markers face polyploidy specific hitches in genotyping, such as auto/tetrasomy, double reduction and missing dosage information. In the present study, a total of 25 polymorphic microsatellite loci was developed through long- read single molecule, real time (SMRT) sequencing technology, to genotype 139 T. tor samples from six natural riverine populations of India, to determine intra-specific genetic variation in this species. Upon sequencing 1.5 kb SMRT bell library, the clustering and assembly of high-quality CCS reads resulted into 9746 contigs and 2129 contained repeat sequences. The majority of repeats were dinucleotide (67.8%), followed by trinucleotide (21.4%) and tetranucleotide (10.7%). Out a total of 118 SSR loci primers designed, 25 loci showed polymorphism. The microsatellite genotype analyses with these loci indicated T. tor as an auto-tetraploid, with tetrasomy segregation and occurrence of double reduction. The microsatellite loci identified were highly polymorphic (average PIC 0.6687).. The genetic diversity parameters, observed and expected heterozygosities ranged from 0.1268 to 0.5047 and from 0.4989 to 0.9212, respectively, whereas inbreeding (FIS) values from 0.2334 to 0.8379. The average ρ statistics value of 0.0739 (p = 0.00) indicated the presence of genetic structure in the natural population, and along with pair-wise ρ statistics and DAPC analysis pointed to the occurrence of three genetic stocks. The identified microsatellite loci were found to be useful in generating information on genetic diversity and fine scale population structure of tetraploid T. tor, which would be valuable for conservation and management of this threatened species.