Development of multiplex microsatellite panel to assess the parentage verification in and differentiation degree of reindeer populations ( Rangifer tarandus)

Reindeer ( Rangifer tarandus), the only member of the genus Rangifer, is one of the most interesting object to investigate genetic diversity. One of the technique of studying the genetic structure of populations and parentage identification is to create panels of STR (short tandem repeats) markers. The aim of the current study was the development of multiplex panel of STR markers and assessment of its application to assign the parents and to study biodiversity of Russian reindeer populations. As a biological material for research we used tissue samples (part of ear’s lobes) of reindeer of Even (EVN, n = 44), Evenk (EVK, n = 44), Nenets ( n = 45) breeds and Tyva population (TUV, n = 35). DNA extraction was performed using Nexttec columns (Germany) according to the manufacturer’s instructions. Polymorphism of nine STR markers (NVHRT76, RT9, NVHRT24, RT30, RT1, RT6, RT27, NVHRT21 and RT7) was determined by own procedures using ABI 3130xl DNA analyzer («Applied Biosystems», USA). Statistical analysis was performed in MS Excel 2007 with the plugin GenAIEx v. 6.5, software MSA 4.05, Phylip, v. 3.5c, Treev32 and Structure, v. 2.3.4. The studied populations of reindeer were characterized by relatively high levels of genetic diversity. The average number of alleles per locus was 6.11±0.56 in TUV, 6.67±0.50 in NEN, 8.00±0.76 in EVN and 8.89±0.65 in EVK. The smallest effective number of alleles per locus was detected in TUV (3.37±0.47), the maximal value was in EVK (4.89±0.46 alleles per locus), and EVN and NEN occupied an intermediate position (4.42±0.53 and 3.90±0.38, respectively). The number of alleles in single loci ranged from four in NVHRT21 and NVHRT24 for TUV to twelve in RT7 for EVK and RT1 for EVN. The probability of matching genotypes (PI) for the nine loci ranged from 1.84×10 -9 in NEN to 5.9×10 -11 in EVK, showing the high power of the proposed marker panel for parentage identification. The calculation of the mean values of similarity coefficient Q in the ith cluster with the most probable number of clusters such as k = 3 and k = 4 (Q i/k) revealed high heterogeneity of genetic structure of studied populations. The highest degree of genetic differentiation was shown for TUV (Q 2/3 = 0.899±0.034, Q 3/4 = 0.883±0.035) and for NEN (Q 3/3 = 0.885±0.031, Q 4/4 = 0.813±0.038). The EVN and EVK population were close to each other, and a clear clustering between them was not observed. An estimation of R st (AMOVA) showed that 11.4 % of the total molecular variability was caused by differences between populations, and 88.6 % was due to individual differences between animals (p st showed similar trends. TUV population was the most distinct comparing to other populations (D Nei = 0,283-0,502, F st = 0,299-0,452), while it was the most differ from NEN and the closest to EVN. The minimal genetic differences were observed between EVN and EVK (D Nei = 0,068, F st = 0,032). The results show high functional power of the developed STR panel to identify the parentage and to study biodiversity in Russian reindeer populations.