Characterization of Microsatellite Markers for Paternity Analysis in Captive Loggerhead Turtles (Caretta caretta)

In order to avoid inbreeding, it is important to verify parentage of animals kept in zoos and aquariums. However, determination of parentage is difficult in the case that one female had opportunities of matings by a number of males. In this situation, DNA parentage testing provides reliable results. Microsatellite markers are cost-effective tools for parentage testing and are widely used. In the Port of Nagoya Public Aquarium, Nagoya, Japan, one male (Individual ID: CcW_07) and one female (Individual ID: Cc97_11) of loggerhead turtle (Caretta caretta) had been kept in the same tank since 28 March 2019 in order to breed in captivity. However, mating behavior of CcW_07 could not be observed. Therefore, on 9 May 2020, another male (Individual ID: Cc95_25) instead of CcW_07 was kept together with Cc97_11. However, mating behavior of Cc95_25 could not be confirmed as was the case with CcW_07. After that, Cc97_11 laid eggs on 28 May (Clutch No.1), 14 June (Clutch No.2), and 30 June (Clutch No.3), 2020. Since female turtle has an ability to store sperm within an oviduct over a long period, paternity of these offsprings was unclear in this situation. Therefore, in this study, we applied previously developed microsatellite markers for their paternity examination. Blood samples were collected from two candidate fathers (CcW_07 and Cc95_25), mother (Cc97_11), and 40 offsprings from three clutches. DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Fourteen microsatellite markers were used for genotyping (Table 1). Based on the degree of polymorphism, these markers were selected from a previous study of loggerhead turtle [1]. The forward primer for each marker was labelled with fluorescent dye. PCR was conducted in 13 μ l reaction volumes containing 12 ng DNA, 1 × GeneAmp PCR Buffer (Applied Biosystems, Foster City, CA, USA), 0.2 mM of each dNTP (Applied Biosystems), 6.25 pmol of each primer, and 0.3 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems). Thermal cycling conditions consisted of: initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. Capillary electrophoresis of PCR products was conducted using an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems). Fragment size was determined based on the GeneScan 500 LIZ Size Standard (Applied Biosystems) using GeneMapper software 6 (Applied Biosystems). The number of alleles, observed heterozygosity (HO), unbiased expected * Corresponding author:Ryo TADANO (E-mail: tadano@gifu-u.ac.jp) ABSTRACT This study evaluates the utility of previously developed microsatellite markers in a paternity test of captive loggerhead turtles (Caretta caretta). Two candidate males, one female, and 40 offsprings from three distinct clutches were genotyped for 14 markers. The number of alleles and observed heterozygosity per locus ranged from three to five and 0.488 to 1.000, respectively. Based on these multi-locus genotypes, paternity of all offsprings could be successfully determined. The results of this study indicate that these microsatellite markers provide reliable information for DNA parentage testing of loggerhead turtles.