Developing a molecular identification assay of old landraces for the genetic authentication of typical agro-food products: The case study of the barley 'Agordino'

The orzo Agordino is a very old local variety of domesticated barley (Hordeum vulgare ssp. distichum L.) that is native to the Agordo District, Province of Belluno, and is widespread in the Veneto Region, Italy. Seeds of this landrace are widely used for the preparation of very famous dishes of the dolomitic culinary tradition such as barley soup, bakery products and local beer. Understanding the genetic diversity and identity of the Agordino barley landrace is a key step to establish conservation and valorisation strategies of this local variety and also to provide molecular traceability tools useful to ascertain the authenticity of its derivatives. The gene pool of the Agordino barley landrace was reconstructed using 60 phenotypically representative individual plants and its genotypic relationships with commercial varieties were investigated using 21 pure lines widely cultivated in the Veneto Region. For genomic DNA analysis, following an initial screening of 14 mapped microsatellite (SSR) loci, seven discriminant markers were selected on the basis of their genomic position across linkage groups and polymorphic marker alleles per locus. The genetic identity of the local barley landrace was determined by analysing all SSR markers in a single multi-locus PCR assay. Extent of genotypic variation within the Agordino barley landrace and the genotypic differentiation between the landrace individuals and the commercial varieties was determined. Then, as few as four highly informative SSR loci were selected and used to develop a molecular traceability system exploitable to verify the genetic authenticity of food products deriving from the Agordino landrace. This genetic authentication assay was validated using both DNA pools from individual Agordino barley plants and DNA samples from Agordino barley food products. On the whole, our data support the usefulness and robustness of this DNA-based diagnostic tool for the orzo Agordino identification, which could be rapidly and efficiently exploited to guarantee the authenticity of local varieties and the typicality of food products

Current advances in genomics and breeding of leaf chicory (Cichorium intybus L.)

This review gives an overview of agricultural topics on a non-model species, in other words, leaf chicory. Often classified as a minor crop, \u201cRadicchio\u201d, the Italian name of leaf chicory, is assuming a very important role at both a local and national level, as it characterizes a high proportion of the agricultural income of suited areas. Botanical classification along the genus Cichorium is reported and a detailed description of the most important cultivated biotypes typical of northern Italy is presented. A special consideration is reserved to breeding aspects, from molecular marker-assisted selection to the implementation of the first genome draft and leaf transcriptomes. Sexual barriers, for example, self-incompatibility or male-sterility, are described in great detail with the aim to be utilized for breeding purposes. The main aspects of seed production are also critically presented. In conclusion, the present work is a sort of handbook to better understand this orphan crop and it is mainly directed to breeders and seed producers dealing with leaf chicory

DNA barcoding as a molecular tool to track down mislabeling and food piracy

DNA barcoding is a molecular technology that allows the identification of any biological species by amplifying, sequencing and querying the information from genic and/or intergenic standardized target regions belonging to the extranuclear genomes. Although these sequences represent a small fraction of the total DNA of a cell, both chloroplast and mitochondrial barcodes chosen for identifying plant and animal species, respectively, have shown sufficient nucleotide diversity to assess the taxonomic identity of the vast majority of organisms used in agriculture. Consequently, cpDNA and mtDNA barcoding protocols are being used more and more in the food industry and food supply chains for food labeling, not only to support food safety but also to uncover food piracy in freshly commercialized and technologically processed products. Since the extranuclear genomes are present in many copies within each cell, this technology is being more easily exploited to recover information even in degraded samples or transformed materials deriving from crop varieties and livestock species. The strong standardization that characterizes protocols used worldwide for DNA barcoding makes this technology particularly suitable for routine analyses required by agencies to safeguard food safety and quality. Here we conduct a critical review of the potentials of DNA barcoding for food labeling along with the main findings in the area of food piracy, with particular reference to agrifood and livestock foodstuffs

cpDNA barcoding by combined End-Point and Real-Time PCR analyses to identify and quantify the main contaminants of oregano (Origanum vulgare L.) in commercial batches

Oregano (Origanum vulgare L.) is a flowering plant that belongs to the mint family (Lamiaceae). It is used as a culinary herb and is often commercialized as a fine powder or a mixture of small fragments of dried leaves, which makes morphological recognition difficult. Like other commercial preparations of drugs and spices, the contamination of oregano mixtures with vegetable matter of lower quality, or the use of generic misleading names, are frequent and stress the need to develop a molecular traceability system to easily, quickly, and cheaply unveil these scams. The DNA-based analytical approach known as cpDNA barcoding is particularly suited for fraud identification in crop plant species (fresh products and food derivatives), and it represents a promising traceability tool as an alternative or complement to traditional detection methods. In the present study, we used a combined approach based on both qualitative and quantitative cpDNA barcoding with end-point and real-time polymerase chain reaction (PCR) analyses to assess the type and degree of contamination in commercial batches of common oregano. In a preliminary qualitative screening, we amplified, cloned, and sequenced a number of universal trnH-psbA- and trnL-barcoded regions, to identify the main contaminants in the samples under investigation. On the basis of these findings, we then developed and validated a species-specific and sequence-targeted method of testing for the quantitative assessment of contaminants, using trnL gene intron assays. Surprisingly, the results obtained in our case study indicated an almost total absence of O. vulgare in the commercial batches analyzed, but a high presence of group I contaminants (Satureja pilosa Velen.), and a moderate presence of group II contaminants (Cistus lanidifer L./Cistus albidus)

Le nuove vie del miglioramento genetico delle piante agroalimentari: dalle tecnologie di breeding cisgenico a quelle di editing genomico.

Advances in genome sequencing have led to the rapid development of new biotechnologies for the genetic improvement of crop plants. Among the new biotechnological tools, the most promising are cisgenic breeding and genomic editing, which have great potentials for the intensification of sustainable agriculture systems and the implementation of food security, in response to the growing population and the climate change that we are witnessing. Contrary to what happens with transgenesis, the plant genetically modified by these new technologies has the peculiarity of not having exogenous coding DNA and therefore it is not distinguishable from plants improved using traditional plant breeding schemes. Our goal is to explain how these new bio- technologies work, highlighting the potential applications and drawbacks, compared to traditional plant breeding techniques

Critical Aspects on the Use of Microsatellite Markers for Assessing Genetic Identity of Crop Plant Varieties and Authenticity of their Food Derivatives

A total of 90 original articles concerning the varietal characterization and identification by means of SSR analysis of the five most economically relevant crops in Italy (i.e., Olea europaea L., Solanum lycopersicum L., Vitis vinifera L., Triticum spp. and Malus × domestica Borkh.) have been selected and reviewed. Since the genetic traceability of processed products may result more complex, wine and olive oil have been considered too. Specifically, this chapter deals with three main aspects: (i) the criteria adopted for the selection of the most appropriate number, type, and distribution of SSR marker loci to be employed for varietal genotyping, (ii) the use of genetic statistics and parameters for the evaluation of the discriminant ability and applicability of SSR marker loci, and (iii) how to make different experimental works on the same species that are standardized, reliable, and comparable. What emerges from the studies reviewed here is a lack of wider consensus among the authors regarding the strategy to design and to adopt for genotyping plant varieties with SSR markers. This finding highlights the urgent need to establish a common procedure, especially for characterizing and preserving landraces, and for supporting its rediscovery and valorization locally

De novo sequencing of the Hypericum perforatum L. flower transcriptome to identify potential genes that are related to plant reproduction sensu lato

Background: St. John's wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Recently gained biological information has shown that this species is also an attractive model system for the study of a naturally occurring form of asexual reproduction called apomixis, which allows cloning plants through seeds. In aposporic gametogenesis, one or multiple somatic cells belonging to the ovule nucellus change their fate by dividing mitotically and developing functionally unreduced embryo sacs by mimicking sexual gametogenesis. Although the introduction of apomixis into agronomically important crops could have revolutionary implications for plant breeding, the genetic control of this mechanism of seed formation is still not well understood for most of the model species investigated so far. We used Roche 454 technology to sequence the entire H. perforatum flower transcriptome of whole flower buds and single flower verticils collected from obligately sexual and unrelated highly or facultatively apomictic genotypes, which enabled us to identify RNAs that are likely exclusive to flower organs (i.e., sepals, petals, stamens and carpels) or reproductive strategies (i.e., sexual vs. apomictic). Results: Here we sequenced and annotated the flower transcriptome of H. perforatum with particular reference to reproductive organs and processes. In particular, in our study we characterized approximately 37,000 transcripts found expressed in male and/or female reproductive organs, including tissues or cells of sexual and apomictic flower buds. Ontological annotation was applied to identify major biological processes and molecular functions involved in flower development and plant reproduction. Starting from this dataset, we were able to recover and annotate a large number of transcripts related to meiosis, gametophyte/gamete formation, and embryogenesis, as well as genes that are exclusively or preferentially expressed in sexual or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. Conclusions: Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity of the flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding of the molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum

Uncovering the sources of DNA found on the Turin Shroud

The Turin Shroud is traditionally considered to be the burial cloth in which the body of Jesus Christ was wrapped after his death approximately 2000 years ago. Here, we report the main findings from the analysis of genomic DNA extracted from dust particles vacuumed from parts of the body image and the lateral edge used for radiocarbon dating. Several plant taxa native to the Mediterranean area were identified as well as species with a primary center of origin in Asia, the Middle East or the Americas but introduced in a historical interval later than the Medieval period. Regarding human mitogenome lineages, our analyses detected sequences from multiple subjects of different ethnic origins, which clustered into a number of Western Eurasian haplogroups, including some known to be typical of Western Europe, the Near East, the Arabian Peninsula and the Indian sub-continent. Such diversity does not exclude a Medieval origin in Europe but it would be also compatible with the historic path followed by the Turin Shroud during its presumed journey from the Near East. Furthermore, the results raise the possibility of an Indian manufacture of the linen cloth

Venetian local corn (Zea mays L.) germplasm: Disclosing the genetic anatomy of old landraces suited for typical cornmeal mush production.

Due to growing concern for the genetic erosion of local varieties, four of the main corn landraces historically grown in Veneto (Italy) \u2014 Sponcio, Marano, Biancoperla and Rosso Piave \u2014 were characterized in this work. A total of 197 phenotypically representative plants collected from field populations were genotyped at 10 SSR marker loci, which were regularly distributed across the 10 genetic linkage groups and were previously characterized for high polymorphism information content (PIC), on average equal to 0.5. The population structure analysis based on this marker set revealed that 144 individuals could be assigned with strong ancestry association (>90%) to four distinct clusters, corresponding to the landraces used in this study. The remaining 53 individuals, mainly from Sponcio and Marano, showed admixed ancestry. Among all possible pairwise comparisons of individual plants, these two landraces exhibited the highest mean genetic similarity (approximately 67%), as graphically confirmed through ordination analyses based on PCoA centroids and UPGMA trees. Our findings support the hypothesis of direct gene flow between Sponcio and Marano, likely promoted by the geographical proximity of these two landraces and their overlapping cultivation areas. Conversely, consistent with its production mainly confined to the eastern area of the region, Rosso Piave scored the lowest genetic similarity (<59%) to the other three landraces and firmly grouped (with average membership of 89%) in a separate cluster, forming a molecularly distinguishable gene pool. The elite inbred B73 used as tester line scored very low estimates of genetic similarity (on average <45%) with all the landraces. Finally, although Biancoperla was represented at K = 4 by a single subgroup with individual memberships higher than 80% in almost all cases (57 of 62), when analyzed with an additional level of population structure for K = 6, it appeared to be entirely (100%) constituted by individuals with admixed ancestry. This suggests that the current population could be the result of repeated hybridization events between the two accessions currently bred in Veneto. The genetic characterization of these heritage landraces should prove very useful for monitoring and preventing further genetic erosion and genetic introgression, thus preserving their gene pools, phenotypic identities and qualitative traits for the future

The Molecular Determination of Hybridity and Homozygosity Estimates in Breeding Populations of Lettuce (Lactuca sativa L.)

The development of new varieties of horticultural crops bene\ufb01ts from the integration of conventional and molecular marker-assisted breeding schemes in order to combine phenotyping and genotyping information. In this study, a selected panel of 16 microsatellite markers were used in di\ufb00erent steps of a breeding programme of lettuce (Lactuca sativa L., 2 n = 18). Molecular markers were \ufb01rst used to genotype 71 putative parental lines and to plan 89 controlled crosses designed to maximiserecombinationpotentials. Theresulting871progenyplantswerethenmolecularlyscreened, and their marker allele pro\ufb01les were compared with the pro\ufb01les expected based on the parental lines. Theaveragecross-pollinationsuccessratewas68\ub133%,so602F1hybridswerecompletelyidenti\ufb01ed. Unexpected genotypes were detected in 5% of cases, consistent with this species\u2019 spontaneous out-pollination rate. Finally, in a later step of the breeding programme, 47 di\ufb00erent F3 progenies, selected by phenotyping for a number of morphological descriptors, were characterised in terms of their observed homozygosity and within-population genetic uniformity and stability. Ten of these populations had a median homozygosity above 90% and a median genetic similarity above 95% and are, therefore, particularly suitable for pre-commercial trials. In conclusion, this study shows the synergistic e\ufb00ects and advantages of conventional and molecular methods of selection applied in di\ufb00erent steps of a breeding programme aimed at developing new varieties of lettuce