Primer design of D-loop region for wild population genetics of Rusa timorensis in Indonesia

Abstract

Abstract Rusa timorensis (Javan deer) is endemic wildlife in Indonesia and is estimated at less than 10.000 individuals with continuously declining populations due to habitat loss and illegal hunting in the wild. This declining low population indicates a greater risk of extinction. Unfortunately, the genetic information of the wild Javan deer population for conservation management strategies still lacks data due to challenging sampling in the wild. Most recent studies were analysing the breeding populations outside Indonesia. Here, we propose the primer design of the D-loop genetic marker to determine the genetic population of wild Javan deer. We used metadata analysis of genetic sequences and new samples from five wild populations to design the specific primer of the D-loop region of the wild Javan deer in Indonesia. We used software, i.e.., Primer3 to design the primers, BLAST for specificity and Oligo Analyzer™ Tool for efficiency of the primer. The Annealing temperature optimisation started with pre-denaturation at 94 °C followed by 35 cycles of denaturation at 95°C; 51-56°C annealing for each one degree’s different per PCR treatment; and 72°C extensions. We successfully designed a specific primer (RL-3.1a) to amplify 235 bp of the D-loop region at 52°C annealing’s temperature.