Efficiency of DNA Mini-Barcoding to Assess Mislabeling in Commercial Fish Products in Italy: An Overview of the Last Decade

The problem of fish traceability in processed products is still an important issue in food safety. Major attention is nowadays dedicated to consumer health and prevention of possible frauds regulated by national and international laws. For this reason, a technical approach is fundamental in revealing mislabeling at different levels. In particular, the use of genetic markers has been standardized and DNA barcoding is considered the gold-standard strategy to examine and prevent species substitution. Considering the richness of available DNA databases, it is nowadays possible to rapidly reach a reliable taxonomy at the species level. Among different approaches, an innovative method based on DNA mini barcoding has recently been proposed at an international level. Starting from this evidence, we herein illustrate an investigation dealing with the evolution of this topic in Italy over the last decade. The molecular analysis of 71 commercial fish samples based on mini-COI sequencing with two different primer sets reached an amplification success rate of 87.3 and 97.2%. The investigation revealed four major frauds (5.8%) and four minor ones (5.8%). Results highlighted a decrease in incorrect labeling in Italy from 32% to 11.6% over the last decade, although a recurrent involvement of “endangered” species sensu IUCN was still observed.

Weathered Antlers: A Valuable Source of DNA Useful for Conservation Purposes for Cervids

Increasingly, conservation genetics pinpoint the use of biological matrices collected without stressing wildlife. Cervid’s antlers seem to fit with this need. We verified the amplification success rate from DNA obtained from red deer antlers collected in the State Nature Reserve of Bosco della Mesola, Northern Italy and its use for conservation purposes. Here occurs the only native red deer population of peninsular Italy, recently recognized as a distinct subspecies (Cervus elaphus italicus). Four antlers stored at room temperature for four years and four samples highly degraded by environmental conditions were analyzed using a multimarker approach. We utilized a simple, inexpensive method to extract DNA from drilled antlers powder. This study confirms that weathered antlers can be a suitable source of DNA also in Mediterranean climate characterized by strong seasonal fluctuations, and not only in dry climates. Our results pointed out that only burr drilling yielded good quality amplifiable DNA. Antlers can be used in particular for molecular genetic studies on rare or threatened species of cervids as providing an efficient and cost-effective non-invasive sampling.

DNA Mini-Barcodes, a Potential Weapon for Conservation and Combating Illegal Trade of Pangolin

Background Smuggling and illegal trade of pangolins and their scales has drastically reduced the wild population of pangolins. Accurate species identification is currently in urgent need as a powerful weapon for combating pangolin smuggling and trade and conserving the already endangered pangolin species. Aim of the study To develop an efficient method based on DNA mini-barcodes for accurate pangolin species identification and authentication of processed pangolin scales against the non-target species. Materials and methods The primers for amplifying the DNA mini-barcodes were designed based on cytochrome C oxidase subunit I (COI) gene fragments. The mini-barcodes were compared with the two universal barcodes (COI and Cytb) for performance in pangolin species identification by calculating the Kimura-2-parameter (K2P) distance, assessing the clustering dendrogram, and analyzing the BLAST similarity and barcoding gap. The accuracy of the three barcodes was also compared for authentication of pangolin scales against non-target species. Results Comparison of the three barcodes showed that the mini-barcode form COI had the highest amplification success rate (100%) and high variable sites (40.0%), with the ratio of mean inter- to intraspecific distance ratio was 25 and a distinct DNA barcoding gap. In the neighbor-joining (NJ) tree constructed based on the mini-barcode regions, each species of the pangolin family formed an obvious clade respectively, and the clades were all separated from those of the non-target species, indicating that the genetic information in the mini-barcode was sufficient for species identification. Conclusion The DNA mini-barcodes based on COI gene fragments provide an effective and accurate method for identification of pangolin species and authentication of pangolin scale products.

Short-read DNA Sequencing Yields Microsatellite Markers for Rheum

Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.

An optimised protocol for barcoding museum collections of decapod crustaceans: a case-study for a 10 - 40-years-old collection

The sequencing of the crustacean collection of the MNHN, Paris, constitutes a promising yet very challenging barcoding project. For the collection’s crustacean specimens preserved in ethanol, some of which were collected up to 40 years ago, the conventional COI barcoding procedure of amplification with Folmer primers failed for more than half of the specimens (58%, n = 1920). We hypothesised that this failure may have been due to incompatible mismatches between the crustaceans targeted and the Folmer primer sequences and/or the amount of degradation of the DNA extracted from museum specimens. The comparison of the Folmer primers against the COI sequences from GenBank complete decapod mitochondrial genomes revealed that the annealing regions were, in fact, rather conserved, suggesting that the amplification failures were due more likely to the low quality of the DNA isolated. Using an alignment of all available decapod sequences we designed two internal primers in the middle of the barcoding COI region and also selected two additional external primers to be used as alternative to the standard Folmer primers. Using a two-overlapping-fragments amplification strategy and different primer combinations, our new protocol significantly increased the amplification success rate of the collection material from 42% with the Folmer primers to 84%, recovering an additional 364 complete barcodes and 443 minibarcodes (i.e. fragments of less than 400 base pairs), and expanding the species coverage from 254 to 397 barcoded crustaceans.

Assessment of SSR Marker Transfer from the Cultivated Strawberry to Diploid Strawberry Species: Functionality, Linkage Group Assignment, and Use in Diversity Analysis

The cultivated strawberry, Fragaria ×ananassa Duchesne ex Rozier, originated via hybridization between octoploids F. chiloensis (L.) Mill. and F. virginiana Mill. These three octoploid species are thought to share a putative genome composition of AAA`A'BBB`B'. Diploid F. vesca L., is considered to have donated the A genome. Current attention to the development of a diploid model system for strawberry genomics warrants the assessment of simple sequence repeat (SSR) marker transferability between the octoploid and diploid species in Fragaria L. In the present study, 23 SSR primer pairs derived from F. ×ananassa `Earliglow' by genomic library screening were evaluated for their utility in six diploid Fragaria species, including eight representatives of F. vesca, four of F. viridis Weston, and one each of F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandshurica Staudt, F. iinumae Makino, and F. nilgerrensis Schltdl. ex J. Gay. SSR primer pair functionality, as measured by amplification success rate (= 100% - failure rate) in each species, was ranked (from highest to lowest) as follows: F. vesca (98.4%) > F. iinumae (93.8%) = F. nubicola (93.8%) > F. mandshurica (87.5%) > F. nilgerrensis (75%) > F. viridis (73.4%). The extent to which these octoploid-derived SSR primer pairs generated markers that could be added to the F. vesca linkage map also was assessed. Of the 13 F. ×ananassa SSR markers that segregated codominantly in the F. vesca mapping population, 11 were assigned to linkage groups based upon close linkages to previously mapped loci. These markers were distributed over six of the seven F. vesca linkage groups, and can serve as anchor loci defining these six groups for purposes of comparative mapping between F. vesca and F. ×ananassa.