A Multistep DNA-Based Methodology for Accurate Authentication of Sturgeon Species

Abstract

The sturgeons (order Acipenseriformes) are caviar producers and some of the most valuable fish species worldwide. Due to different reasons, wild populations are now at the brink of extinction. The high demand for caviar has led to the development of aquaculture for restocking and caviar production. Since the caviar from different species has different prices depending on the quality and attempts of commercial fraud based on species substitution have been found, correct species identification is more than necessary. We report a new multistep methodology for an accurate species identification based on both nuclear and mitochondrial markers. Our test integrates data from the analysis of microsatellites (Afu19, Afu34, Afu39, Afu54, Aox27, AoxD234, AnacC11 and AnacE4), nuclear gene markers (RPS7, vimentin and rhodopsin) and mtDNA barcoding to give a reliable molecular diagnostic for five sturgeon species (Huso huso, Acipenser stellatus, Acipenser ruthenus, Acipenser gueldenstaedtii and Acipenser baerii). In addition to species identification, our methodology allows the identification of bester, sterbe and best beluga hybrids, but also the identification of hybrids of unknown origin. This methodology has a good potential to contribute to the conservation of highly threatened sturgeon populations and also to the traceability of their products.

Species Identification of Caviar Based on Multiple DNA Barcoding

“…The comparison and analysis of the data using the NCBI and BOLD databases showed that the similarity of each sample with the database was ≥98%, and the matching degree was high. The primers (set I) and self-designed COI gene primers (set II) achieved the same results of species identification, but neither could distinguish between A. baerii , A. gueldenstaedtii , A. naccari , A. persicus , and A. sinensis (Samples S1, S7, S11, S14, S19), which was consistent with the results of the previous reports [ 20 , 23 ]. In contrast, D-loop gene primer (set III) completely identified the sturgeon maternal information of all caviar samples, including A. baerii , A. gueldenstaedtii , A. schrenckii , H. dauricus, and H. huso ; with the majority of samples being identified as H. dauricus (52.4%) ( Table 2 ).…”

“…This study used plant specific DNA barcodes ( tRNA-Leu ) [ 32 ] and fish universal primers ( COI ) [ 24 ] for identifying 21 batches of caviar, which ruled out the possibility of counterfeit caviar with non-sturgeon and plant-derived ingredients at the first step. Although COI -based DNA barcoding were suitable for detecting a wide range of animal species, several studies have shown limitations in the identification of sturgeon relatives [ 20 , 23 ] and we further confirmed this view. However, we successfully identified the maternal source of all caviar using primer set III based on the D-loop gene.…”

“…It is necessary for regulatory authorities to develop standardized molecular technology to identify caviar. In the past decade, researchers have used multiple microsatellites and SNP-sites for the identification of hybrid sturgeon and have combined them with DNA barcoding to achieve identification of parental information for the hybrid sturgeon [ 20 , 34 ]. In this study, the mitochondrial DNA was first selected for preliminary identification of caviar and, despite only being able to identify the maternal source, it was effectively applied to exclude the presence of heterologous substitutions and to assist the other methods to identify hybrids.…”

“…Since DeSalle and Birstein et al [ 13 , 14 ] first proposed that DNA molecular methods could identify sturgeon species and caviar authenticity, many relevant studies have reported that some techniques have been used to identify sturgeon purebreds, crossbreds and their caviar, such as polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), amplified fragment length polymorphism (AFLP), microsatellite marker, and DNA barcoding [ 15 , 16 , 17 , 18 , 19 , 20 ]. DNA barcoding has become a widely and commonly used technology in the identification of fish species, playing an important role in the identification of maternal species in sturgeon, and various mitochondrial markers are well-established for identifying sturgeon and caviar.…”