Cross-amplification testsof ungulate primers in the endangered Neotropical pampas deer (Ozotocerosbezoarticus)

Departamento de Genética – IIBCE Unidad Asociada de Facultad de Ciencias,Montevideo, Uruguay

Conflicts: At the beginningof the 20th century, the pampas deer (Ozotoceros bezoarticus) hadlarge populations that occupied a wide range of open habitats throughout SouthAmerica. However, today a few small isolated populations remain, and the pampasdeer is considered one of the most endangered neotropical species of deer(González et al., 1998). The fast development of cost-efficient tools tomeasure genetic diversity in populations of endangered species such as thepampas deer is needed for conservation management efforts.

ABSTRACT. In cross-species amplificationtests of 15 ungulate primers in pampas deer, five were retained to form a smallpanel of highly polymorphic loci that could be used to efficiently screenpopulations of this endangered species. The polymerase chain reactions wereperformed incorporating the universal fluorescent labeled M13 (-21) primer. In69 pampas deer, average allelic diversity was 15, expected heterozygosity was0.869 and the mean polymorphic information content value was 0.847. Paternityexclusion probabilities over loci were NE-1P = 0.01336 and NE-2P = 0.00135, andcombined non-exclusion probability of identity was P_(ID) =3 × 10^-8.

Key words: Bovidae, Cervidae, Cross-speciesamplification, Microsatellite, Pampas deer

INTRODUCTION

At the beginning of the20th century, the pampas deer (Ozotoceros bezoarticus) had largepopulations that occupied a wide range of open habitats throughout South America. However, today a few small isolatedpopulations remain, and the pampas deer is considered one of the mostendangered neotropical species of deer (González et al., 1998). The fastdevelopment of cost-efficient tools to measure genetic diversity in populationsof endangered species such as the pampas deer is needed for conservationmanagement efforts.

Microsatellite markers areuseful in assessing population structure and trends, patterns of dispersal,social organization, levels of inbreeding, population relationships, and inplanning translocations (Beja-Pereira et al., 2004).

Although cloning is themost accurate technique for developing species-specific primers, the time andeffort required are substantial (Galan et al., 2003). For this reason, severalstudies have taken advantage of the relatively highly conserved microsatelliteflanking regions of domestic species. A large number of polymorphic loci havebeen reported and these have been applied in wild populations ofphylogenetically closely related species (Galan et al., 2003; Vial et al.,2003; Maudet et al., 2004).

The objectives of thepresent study were: i) to test in pampas deer 15 microsatellite loci isolatedfrom cattle, sheep and goat, that have been demonstrated to be polymorphicacross other Cervids (Table 1), and ii) to characterize a small set of highlypolymorphic microsatellite loci across six pampas deer populations.

MATERIAL AND METHODS

DNA was isolated fromtissue samples according to González et al. (1998). Primers were first testedin three pampas deer samples. A single polymerase chain reaction per locus wasperformed with the universal fluorescent labeled M13 primer(5’[6-FAM]TGTAAAACGGCCAGT-3’)following the procedure described by Schuelke (2000). We used a reaction volumeof 20 μL, containing 50ng DNA, 5 pmol of specific forward primer with its M13 (-21) sequence, 20 pmolof the universal M13 (-21) primer, 20 pmol of specific reverse primer, 1X Taqbuffer with KCl, 3.25 mmol MgCl₂, 0.5 μg/μL BSA, 2 mMdNTPs, and 1.25 unit Taq polymerase (Fermentas).

The thermo-cyclingconditions included two stages; during the first 29 cycles, the forward primerwas incorporated. This stage consisted of denaturation at 94°C for 30 s, annealing at 56°C (for all of our specificprimers) for 45 s, and extension at 72°C for 45 s. This was followed by 9 cycleswhere the universal M13
(-21) primer was incorporated in the annealing step at 53°C, and a final extensionphase of 10 min at 72°C.To confirm amplification, 5 μL of each product was electrophoresed on a 2% agarose gel (Invitrogen)for 180 min. Afterward, 1 μL polymerase chain reaction product was added to 9 μL formamide/ROX solution (AppliedBiosystems), electrophoresed and detected on an ABI PRISM 3100 Genetic Analyser(Applied Biosystems). Migration was performed in a 22-cm length capillary arrayusing POP4 polymer (Applied Biosystems) with the following parameters: 15 kV,100 amp, and 11 mW at 60°Cfor 35 min. Fragment size analysis was performed using the GeneScan^®and Genotyper^® software (Applied Biosystems).