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Authentication of honey based on a DNA method to differentiate Apis mellifera subspecies: Application to Sicilian honey bee (A. m. siciliana) and Iberian honey bee (A. m. iberiensis) honeys
No full textHoney contains intrinsic markers that can be used to identify its origin. In this study, we used the honey as source of honey bee DNA and developed a test to detect the entomological origin of the honey by identifying the Apis mellifera subspecies using an informative mitochondrial DNA region. We then applied this method for the authentication of A. m. siciliana and A. m. iberiensis honeys. DNA was extracted from 60 honey samples produced in several Italian regions (including Sicily and close minor islands), Portugal, Spain and other countries. PCR primers were designed to amplify a fragment of 85 bp (A. mellifera C lineage; highly frequent in A. m. ligustica, the Italian honey bee that is frequently implicated in human introductions worldwide), or 138 bp (M lineage; characteristic of A. m. mellifera) or 152 bp (A lineage; of the honey bee subspecies of African origin). All sampled Sicilian honeys (that were from A. m. siciliana) showed only the fragment of 152 bp, confirming its expected origin. All honeys from A. m. iberiensis showed only the fragment of 152 bp or only the fragment of 138 bp or both, in agreement with the hybrid origin of Iberian honey bee populations. All other analyzed honeys showed the fragments of 85 bp or 85 + 138 bp, suggesting that they were produced from other subspecies. This authentication system could be a useful tool to support conservation genetic programs that rely on marketing links between honey bee genetic resources and the honey they produce
Honey Environmental DNA Can Be Used to Detect and Monitor Honey Bee Pests: Development of Methods Useful to Identify Aethina tumida and Galleria mellonella Infestations
Full text linkEnvironmental DNA (eDNA) contained in honey derives from the organisms that directly and indirectly have been involved in the production process of this matrix and that have played a role in the hive ecosystems where the honey has been produced. In this study we set up PCR-based assays to detect the presence of DNA traces left in the honey by two damaging honey bee pests: the small hive beetle (Aethina tumida) and the greater wax moth (Galleria mellonella). DNA was extracted from 82 honey samples produced in Italy and amplified using two specific primer pairs that target the mitochondrial gene cytochrome oxidase I (COI) of A. tumida and two specific primer pairs that target the same gene in G. mellonella. The limit of detection was tested using sequential dilutions of the pest DNA. Only one honey sample produced in Calabria was positive for A. tumida whereas about 66% of all samples were positively amplified for G. mellonella. The use of honey eDNA could be important to establish early and effective measures to contain at the local (e.g., apiary) or regional scales these two damaging pests and, particularly for the small hive beetle, to prevent its widespread diffusion
Genotyping-by-sequencing of honey derived environmental DNA can retrieve information on the Apis mellifera subspecie.
No full textHoney contains environmental DNA (eDNA) traces derived from all organisms that directly or indirectly contributed to its production or that have been part of the production niche and environment from which this matrix is obtained. We recently demonstrated that honey constitutes an easily accessible source of Apis mellifera DNA useful to retrieve population genetic information. We also recently demonstrated that honey bee mitochondrial DNA (mtDNA) specific lineages detected in the honey can be used to authenticate the entomological origin of the honey. In this study we analysed honey DNA and integrated honey bee mtDNA information with nuclear genome polymorphisms to set up an improved tool that can detect the honey bee subspecies using these two genome levels. To this aim, we designed and tested a genotyping by sequencing (GBS) assay to analyse 121 single nucleotide polymorphisms (SNPs) of A. mellifera nuclear genome using eDNA extracted from honey. Results were integrated with information derived from previous studies and whole genome resequencing datasets. Genomic analyses were obtained for 61 specimens (honey samples and honey bees) collected in a few Italian regions (Emilia-Romagna, Liguria and Sicily) and that included: (i) individual honey bees of the subspecies A. m. ligustica, A. m. mellifera and A. m. siciliana; (ii) groups of pooled DNA samples from more than 30 A. m. ligustica workers belonging to the same colonies from which honey samples (see below) have been collected; (iii) honey samples obtained from 32 single hives; (iv) undifferentiated honey samples produced from A. m. ligustica and A. m. siciliana. The GBS runs produced more than 53 million reads that were used to obtain genotype information of the selected bi-allelic SNPs. Allele frequency estimation combined with several multidimensional scaling approaches were able to 25th Congress of Animal Science and Production Associationcorrectly assign the honey to the honey bee subspecies that produced it with high correlations between samples and runs. Overall, results obtained from GBS demonstrated the possibility to use A. mellifera nuclear genome variability to authenticate the entomological origin of the honey by detecting the honey bee subspecies.
Acknowledgements
This study was supported by Regione Emilia Romagna – BEE-RER3
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A genotyping by sequencing approach can disclose Apis mellifera population genomic information contained in honey environmental DNA
Full text linkAwareness has been raised over the last years on the genetic integrity of autochthonous honey bee subspecies. Genomic tools available in Apis mellifera can make it possible to measure this information by targeting individual honey bee DNA. Honey contains DNA traces from all organisms that contributed or were involved in its production steps, including the honey bees of the colony. In this study, we designed and tested a genotyping by sequencing (GBS) assay to analyse single nucleotide polymorphisms (SNPs) of A. mellifera nuclear genome using environmental DNA extracted from honey. A total of 121 SNPs (97 SNPs informative for honey bee subspecies identification and 24 SNPs associated with relevant traits of the colonies) were used in the assay to genotype honey DNA, which derives from thousands of honey bees. Results were integrated with information derived from previous studies and whole genome resequencing datasets. This GBS method is highly reliable in estimating honey bee SNP allele frequencies of the whole colony from which the honey derived. This assay can be used to identify the honey bee subspecies of the colony that produced the honey and, in turn, to authenticate the entomological origin of the honey
Entomological signatures in honey: an environmental DNA metabarcoding approach can disclose information on plant-sucking insects in agricultural and forest landscapes
Full text linkHoneydew produced from the excretion of plant-sucking insects (order Hemiptera) is a carbohydrate-rich material that is foraged by honey bees to integrate their diets. In this study, we used DNA extracted from honey as a source of environmental DNA to disclose its entomological signature determined by honeydew producing Hemiptera that was recovered not only from honeydew honey but also from blossom honey. We designed PCR primers that amplified a fragment of mitochondrial cytochrome c oxidase subunit 1(COI) gene of Hemiptera species using DNA isolated from unifloral, polyfloral and honeydew honeys. Ion Torrent next generation sequencing metabarcoding data analysis assigned Hemiptera species using a customized bioinformatic pipeline. The forest honeydew honeys reported the presence of high abundance of Cinara pectinatae DNA, confirming their silver fir forest origin. In all other honeys, most of the sequenced reads were from the planthopper Metcalfa pruinosa for which it was possible to evaluate the frequency of different mitotypes. Aphids of other species were identified from honeys of different geographical and botanical origins. This unique entomological signature derived by environmental DNA contained in honey opens new applications for honey authentication and to disclose and monitor the ecology of plant-sucking insects in agricultural and forest landscapes
An over-time distribution map of Apis mellifera mitotypes in Italy constructed using an environmental DNA approach.
No full textFor the role that Apis mellifera plays in the agroecological systems, a growing interest across Europe is emerging to preserve the genetic integrity of honey bee populations and subspecies. ..
Application of next generation semiconductor based sequencing to detect the botanical composition of monofloral, polyfloral and honeydew honey
No full textHoney is one of the most frauded food products. Therefore, it is important to develop new analytical systems useful for its authentication. Honey contains intrinsic markers that can be used to identify and monitor its origin, including plant DNA mainly derived by pollen. In this study, we applied a next generation sequencing approach for honey authentication by detecting the prevalent botanical contribution and botanical composition of honeys of different origin. DNA was isolated from nine honeys (six monofloral honeys produced in Italy, two polyfloral honeys produced in East Europe and Chile respectively, and one honeydew honey) and PCR amplified for a chloroplast trnL barcoding fragment. Obtained amplicons were sequenced using the Ion Torrent sequencing platform. Sequence data was interpreted using a customized bioinformatic pipeline that used a reference plant sequence dataset derived by more than 150,000 entries. A total of 254 botanical groups were identified from the nine analysed samples, ranging from 37 groups in orange tree blossom honey to 74 in eucalyptus tree blossom honey. The prevalent expected botanical origin was confirmed in five out of six monofloral honeys. The plant signature of the labelled lime tree blossom honey did not confirm the expected botanical prevalence. The most represented botanical group in the honeydew honey was Castanea. The botanical composition of monofloral and polyfloral honey samples was useful to infer their geographical origin. The metabarcoding based system applied in this study captured the botanical signature of all analysed honey samples and provided information useful for their authentication
Distribution of the Main <i>Apis mellifera</i> Mitochondrial DNA Lineages in Italy Assessed Using an Environmental DNA Approach
Full text linkGrowing interest has been emerging on the need to monitor the genetic integrity of the European Apis mellifera subspecies that could be threatened by the human-mediated dispersion of non-native populations and lines. Mitochondrial DNA (mtDNA) lineages can provide useful information for this purpose. In this study, we took advantage of the environmental DNA (eDNA) contained in the honey, which can be analyzed to detect the main groups of mitotypes of the honey bees that produced it. In this study, we applied this eDNA to produce a distribution map all over the Italian peninsula and the two major islands (Sicily and Sardinia) of the following three honey bee mtDNA lineages: A, C and M. A total of 607 georeferenced honey samples, produced in all Italian regions, was analyzed to detect these lineages. The A lineage was widespread in Sicily, as expected, considering that A. m. siciliana carries the African lineage. Surprisingly, this lineage was also reported in about 14% of all other samples produced in almost all continental regions, and in Sardinia. The applied method obtained an updated distribution map of honey bee mtDNA lineages that could be useful to design policies for the conservation of Italian honey bee genetic resources
Application of next generation semiconductor based sequencing for species identification in dairy products
Full text linkIn this study, we applied a next generation sequencing (NGS) technology (Ion Torrent) for species identification based on three mitochondrial DNA (mtDNA) regions amplified on DNA extracted from dairy products. Sequencing reads derived from three libraries, obtained from artificial DNA pools or from pooled amplicons, were used to test the method. Then, sequencing results from five libraries obtained from two mixed goat and cow milk samples, one buffalo mozzarella cheese, one goat crescenza cheese and one artisanal cured ricotta cheese, were able to detect all expected species in addition to undeclared species in a few of them. Mining generated reads it was possible to identify different dairy species mitotypes and the presence of human DNA that could constitute a potential marker to monitor the hygienic level of dairy products. Overall results demonstrated the usefulness of NGS for species identification in food products and its possible application for food authentication
Exploiting phenotype diversity in a local animal genetic resource: identification of a single nucleotide polymorphism associated with the tail shape phenotype in the autochthonous Casertana pig breed
Full text linkCasertana is a local pig breed mainly raised in Central-South regions of Italy. Pigs of this breed are considered the descendants of the ancient Neapolitan population that largely influenced the constitution of the modern commercial pigs. The pigs of this breed are usually curly-tailed, like several other domestic pig populations. However, Casertana population shows some variability for this trait, including animals having straight tail as observed in wild boars. In this study, we run, for the first time, a genome wide association study (GWAS) comparing the curly tailed (no. = 53) and straight tailed (no. = 19) Casertana pigs to identify genomic regions associated with the tail shape phenotype in Sus scrofa. All animals were genotyped with the Illumina PorcineSNP60 BeadChip v.2. GEMMA software was used in the GWAS for which we were able to correct for stratification in the analysed cohort. A single nucleotide polymorphism (rs81439488), located on porcine chromosome 12, was significantly associated with the investigated trait. This marker is close to the SRY-box 9 (SOX9) gene that encodes for a transcription factor that is required during sequential steps of the chondrocyte differentiation pathway, notochord maintenance and skeletogenesis. As the shape of the tail could be important in relation to the problem of tail biting in pigs, the obtained results might open new perspectives for defining selection programs answering indirectly animal welfare issues. This work demonstrated that autochthonous animal genetic resources might be used to disclose genetic factors affecting peculiar traits by exploiting segregating phenotypes and genetic variability
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