Distinguishing Forest and Savanna African Elephants Using Short Nuclear DNA Sequences

Abstract

A more complete description of African elephant phylogeography would require a method that distinguishes forest and savanna elephants using DNA from low-quality samples. Although mitochondrial DNA is often the marker of choice for species identification, the unusual cytonuclear patterns in African elephants make nuclear markers more reliable. We therefore designed and utilized genetic markers for short nuclear DNA regions that contain fixed nucleotide differences between forest and savanna elephants. We used M13 forward and reverse sequences to increase the total length of PCR amplicons and to improve the quality of sequences for the target DNA. We successfully sequenced fragments of nuclear genes from dung samples of known savanna and forest elephants in the Democratic Republic of Congo, Ethiopia, and Namibia. Elephants at previously unexamined locations were found to have nucleotide character states consistent with their status as savanna or forest elephants. Using these and results from previous studies, we estimated that the short-amplicon nuclear markers could distinguish forest from savanna African elephants with more than 99% accuracy. Nuclear genotyping of museum, dung, or ivory samples will provide better-informed conservation management of Africa's elephants.

Single‐nucleotide polymorphism discovery and panel characterization in the African forest elephant

“…SNPs in two African savannah elephant samples and BLAST alignment of these alleles to the published L. africana assembly found only two markers to be polymorphic (data not shown), which is consistent with the species separation (Ishida et al, 2011).…”

“…However, the genetic structure of forest elephant populations in Central Africa is expected to be weak (Johnson, 2008) due to relatively high mobility of individuals, suggesting that with some further testing on populations outside of Gabon, these markers may have wider use for individual ID across the species range. In contrast, preliminary testing of our 107 SNPs in two African savannah elephant samples and BLAST alignment of these alleles to the published L. africana assembly found only two markers to be polymorphic (data not shown), which is consistent with the species separation (Ishida et al., 2011). …”

Sequence variation of koala retrovirus transmembrane protein p15E among koalas from different geographic regions

“…PCR amplicons were treated with Exonuclease I (USB Corporation, Cleveland, OH) and shrimp alkaline phosphate (USB Corporation) to remove excess primers and unincorporated dNTPs (Hanke and Wink, 1994). Sanger sequencing was conducted as previously described (Ishida et al, 2011) and used the ABI 3730XL capillary sequencer at the UIUC Core DNA Sequencing Facility. Sequencher 5.1 (Gene Codes Corp.) was used to examine, edit and concatenate the sequences.…”

Evidence for selection at HIV host susceptibility genes in a West Central African human population

“…Amplified fragments were examined on a 1% ethidium bromide stained agarose gel, and purified with Exonuclease I (Life Technologies) and shrimp alkaline phosphatase (Affymetrix Corporation, Santa Clara CA) to remove primers and unincorporated dNTPs prior to sequencing. In some cases (listed in Additional file 1: Table S7), the M13 forward (TGTAAAACGACGGCCAGT) or the M13 reverse sequence (CAGGAAACAGCTATGAC) was added to the 5’ end of PCR primers, to permit the use of M13 forward or reverse primer in sequencing reactions [83]. Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) with 0.12 μM of primer (PCR and sequencing primers are listed in Additional file 1: Table S7), and the ABI 3730XL capillary sequencer at the University of Illinois Core DNA Sequencing Facility.…”