Figure 1.
Pearls of Pinctada margaritifera, P. maxima and P. radiata.
A) Natural pearls (P. radiata): radiography of a necklace and a cross-section of a pearl showing the three layers: the periostracum rich in organic material (OM) (inner layer), the prismatic layer (middle layer), and the aragonitic nacre or mother of pearl layer (outer layer). B) Beadless (without a nucleus) cultured pearls also called ‘Keshi’ (P. maxima): radiography of a necklace and a cross-section showing the nacreous layer around an inner cavity filled with OM. C) Beaded cultured pearls: radiography of a necklace with P. margaritifera pearls and cross section of an Akoya pearl showing the nacreous layer around an internal nucleus and an OM “pocket” on the right (Photos and radiographies A–C: H.A. Hänni). D) Necklaces with P. margaritifera pearls (lower row left), P. radiata pearls (upper row) and P. maxima pearls (lower row right). The inset shows the historic natural pearl “the Peregrina” which was found in the 16th century. This pearl and its necklace were sold for $11.8 million at a Christie's auction in December 2011 in New York. The PCR-RFLP method described here could provide scientific validation of the provenance of historic pearls (Photos: Swiss Gemmological Institute SSEF). E) Scanning electron microscope side-view image of aragonite tablets of the nacreous layer of a P. margaritifera pearl (Photo: Marcel Düggelin, ZMB, Basel University).
Figure 2.
Schematic representation of the experimental procedures used for DNA extraction and PCR amplicon analysis.
In methods A and B pearls were broken open using forceps to expose the internal organic material and nacre (mother of pearl). In method C samples were obtained by drilling a 1-mm diameter hole through the pearls and the hole was enlarged internally using a 0.9 mm drill head.
Table 1.
DNA profiles of pearl samples from Pinctada margaritifera (PMR), P. maxima (PMX) and P. radiata (PR) based on four different molecular markers.
Table 2.
Sequencing success rate associated with different molecular markers from pearl DNA extracts of Pinctada margaritifera, P. maxima and P. radiata using methods A, B and C (Fig. 2).
Figure 3.
A PCR-RFLP assay of the ITS2 region applied to pearls from Pinctada margaritifera, P. maxima and P. radiata.
(A) PCR products of 575 bp (P. margaritifera), 571 bp (P. maxima) and 590–91 bp (P. radiata) obtained with ITS2 universal primers (5.8S-F and 28S-R) and (B) RFLP patterns of ITS2 amplicons (from A) obtained after digestion with RsaI. MW: molecular weight size marker, 100-bp DNA ladder; lanes 1–3: P. maxima (PMX) pearls; lane 4: P. margaritifera (PMR) pearl; lanes 5–10: P. radiata (PR) pearls; lanes 11–16: P. margaritifera pearls; lane 17: PCR negative control; lanes 18 and 19: P. radiata and P. margaritifera positive controls. Note: The P. maxima positive control is shown in Figure 4.
Figure 4.
Blind PCR-RFLP assay with eighteen pearls of unknown identity.
(A) PCR products of 575 bp (Pinctada margaritifera), 571 bp (P. maxima) and 590–91 bp (P. radiata) obtained with ITS2 universal primers (5.8S-F and 28S-R) and (B) of 335 bp obtained with 28S-R and the P. margaritifera specific primer ITS2-Marg-F. (C) RFLP patterns of ITS2 gene fragments (from A) obtained after digestion with RsaI. MW: molecular weight size marker, 100 bp DNA ladder; lanes 1–18: pearl isolates; lanes 19–20: DNA extraction negative controls; lane 21: PCR negative control; lanes 22–23: P. radiata and P. margaritifera positive controls; lanes 24–26: P. radiata, P. margaritifera and P. maxima positive controls showing ITS2 PCR products (upper gel) and ITS2-RFLP patterns (lower gel).
Figure 5.
Examples of pearls of Pinctada margaritifera, P. maxima and P. radiata used in this study before and after micro-drilling.
We used a drill head attached to a Dremel Workstation to produce pearl powder used for DNA extraction. Recovered pearl powder (nacre and organic material) can be seen in the Petri dish. P. margaritifera (PMR), P. maxima (PMX) and P. radiata (PR).
Table 3.
ITS2 profiles of pearls from Pinctada margaritifera (PMR), P. maxima (PMX) and P. radiata (PR) using a practically non-destructive method (Fig. 2C).