Massively parallel sequencing and capillary electrophoresis of a novel panel of falcon STRs: Concordance with minisatellite DNA profiles from historical wildlife crime

Abstract

Birds of prey have suffered persecution for centuries through trapping, shooting, poisoning and theft from the wild to meet the demand from egg collectors and falconers; they were also amongst the earliest beneficiaries of DNA testing in wildlife forensics. Here we report the identification and characterisation of 14 novel tetramer, pentamer and hexamer short tandem repeat (STR) markers which can be typed either by capillary electrophoresis or massively parallel sequencing (MPS) and apply them to historical casework samples involving 49 peregrine falcons, 30 of which were claimed to be the captively bred offspring of nine pairs. The birds were initially tested in 1994 with a multilocus DNA fingerprinting probe, a sex test and eight single-locus minisatellite probes (SLPs) demonstrating that 23 birds were unrelated to the claimed parents. The multilocus and SLP approaches were highly discriminating but extremely time consuming and required microgram quantities of high molecular weight DNA and the use of radioisotopes. The STR markers displayed between 2 and 21 alleles per locus (mean = 7.6), lengths between 140 and 360 bp, and heterozygosities from 0.4 to 0.93. They produced wholly concordant conclusions with similar discrimination power but in a fraction of the time using a hundred-fold less DNA and with standard forensic equipment. Furthermore, eleven of these STRs were amplified in a single reaction and typed using MPS on the Illumina MiSeq platform revealing eight additional alleles (three with variant repeat structures and five solely due to flanking SNPs) across four loci. This approach gave a random match probability of < 1E-9, and a parental pair false inclusion probability of < 1E-5, with a further ten-fold reduction in the amount of DNA required (~3 ng) and the potential to analyse mixed samples. These STRs will be of value in monitoring wild populations of these key indicator species as well as for testing captive breeding claims and establishing a database of captive raptors. They have the potential to resolve complex cases involving trace, mixed and degraded samples from raptor persecution casework representing a significant advance over the previously applied methods.

Keywords: DNA forensics; Massively parallel sequencing; Peregrine; Raptor persecution; STR multiplex; Wildlife laundering.

Graphical abstract

Fig. 1

Determining relatedness using all 22 SLP and STR markers. (A) Figure shows pairwise comparisons between each of the 49 peregrines. Above the diagonal, increasing numbers of loci at which no alleles are shared are indicated on a scale from 0 to 22 (green to red). Below the diagonal are the coefficients of relatedness (r) as determined by ML-Relate; likely first-degree relatives are shown by blue shades, these are clustered along the diagonal and grouped by heavy black boxes. The first two boxes from left to right (and top to bottom) correspond to the adults and confirmed offspring of breeding pair BP1 (two adults (M,F), six offspring (O)) and BP11 (two adults, one offspring) with at least one shared allele at every locus. The following seven boxes enclose inferred full sibships (Sib1–7). The remaining 20 individuals appear unrelated to all others. The 31 individuals marked (U) were used to estimate allele frequencies. (B) The number of loci showing no shared alleles between confirmed parent/offspring (dark shades), inferred full sibling (mid shades) and more distant relationships (light shades) are shown in the histograms for the eight minisatellite SLPs (blue), 14 CE STRs (red) and all markers combined (grey). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Genetic incompatibilities between young and claimed breeding pairs. Number of mismatching loci in pairwise comparisons between the young birds and both claimed (dark shades) and all other breeding pairs (light shades) with eight minisatellite SLPs (blue), 14 CE STRs (red) and all markers combined (grey). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Additional variation revealed by MPS. (A) Graph showing the number of alleles detected by CE (pale grey bars) and MPS (dark grey bars) for each of the 11 STRs. (B) Schematic allele structures are shown for Fpeμ56_1 together with their CE lengths indicated on the left and their frequencies amongst the 31 unrelated birds on the right. Most variation is seen within a single GATA[n] repeat array (pink) (n = 13–18 copies), but additional tetranucleotide repeats upstream of this can also vary in sequence and/or number, as can the nucleotide at the 27th position downstream of the terminal GAGA repeat which can be either a G as in the published genome sequence (red), or an A (dark or light blue). Pie charts to the right indicate the frequencies of the different allele structures with the colour of the slice corresponding to the status of this flanking SNP. Two birds typed by CE as length homozygotes were identified by MPS as heterozygotes carrying isometric alleles (black star). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)