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

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

A method for genotyping elite breeding stocks of leaf chicory (Cichorium intybus L.) by assaying mapped microsatellite marker loci

BACKGROUND: Leaf chicory (Cichorium intybus subsp. intybus var. foliosum L.) is a diploid plant species (2n = 18) of the Asteraceae family. The term "chicory" specifies at least two types of cultivated plants: a leafy vegetable, which is highly differentiated with respect to several cultural types, and a root crop, whose current industrial utilization primarily addresses the extraction of inulin or the production of a coffee substitute. The populations grown are generally represented by local varieties (i.e., landraces) with high variation and adaptation to the natural and anthropological environment where they originated, and have been yearly selected and multiplied by farmers. Currently, molecular genetics and biotechnology are widely utilized in marker-assisted breeding programs in this species. In particular, molecular markers are becoming essential tools for developing parental lines with traits of interest and for assessing the specific combining ability of these lines to breed F1 hybrids. RESULTS: The present research deals with the implementation of an efficient method for genotyping elite breeding stocks developed from old landraces of leaf chicory, Radicchio of Chioggia, which are locally dominant in the Veneto region, using 27 microsatellite (SSR) marker loci scattered throughout the linkage groups. Information on the genetic diversity across molecular markers and plant accessions was successfully assessed along with descriptive statistics over all marker loci and inbred lines. Our overall data support an efficient method for assessing a multi-locus genotype of plant individuals and lineages that is useful for the selection of new varieties and the certification of local products derived from Radicchio of Chioggia. CONCLUSIONS: This method proved to be useful for assessing the observed degree of homozygosity of the inbred lines as a measure of their genetic stability; plus it allowed an estimate of the specific combining ability (SCA) between maternal and paternal inbred lines on the basis of their genetic diversity and the predicted degree of heterozygosity of their F1 hybrids. This information could be exploited for planning crosses and predicting plant vigor traits (i.e., heterosis) of experimental F1 hybrids on the basis of the genetic distance and allelic divergence between parental inbred lines. Knowing the parental genotypes would allow us not only to protect newly registered varieties but also to assess the genetic purity and identity of the seed stocks of commercial F1 hybrids, and to certificate the origin of their food derivatives

Pistil transcriptome analysis to disclose genes and gene products related to aposporous apomixis in Hypericum perforatum L.

Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies,which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis),the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell. Despite the importance of apomixis for breeding of crop plants and although much research has been conducted to study this process, the genetic control of apomixis is still not well understood. Hypericum perforatum is becoming an attractive model system for the study of aposporous apomixis. Here we report results from a global gene expression analysis of H. perforatum pistils collected from sexual and aposporous plant accessions for the purpose of identifying genes, biological processes and molecular functions associated with the aposporous apomixis pathway. Across two developmental stages corresponding to the expression of aposporous apomeiosis and parthenogenesis in ovules, a total of 224 and 973 unigenes were found to be significantly up- and down-regulated with a fold change >= 2 in at least one comparison, respectively.Differentially expressed genes were enriched for multiple gene ontology (GO) terms,including cell cycle, DNA metabolic process, and single-organism cellular process. For molecular functions, the highest scores were recorded for GO terms associated withDNA binding, DNA (cytosine-5-)-methyltransferase activity and heterocyclic compound binding. As deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, all genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were analyzed in great detail. Overall, our data suggest that phenotypic expression of apospory is concomitant with the modulation of key genes involved in the sexual reproductive pathway. Furthermore, based on gene annotation and co-expression, we underline a putative role of hormones and key actors playing in the RNA-directed DNA methylation pathway in regulating the developmental changes occurring during aposporous apomixis in H. perforatum

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

Toward a First High-quality Genome Draft for Marker-assisted Breeding in Leaf Chicory, Radicchio (Cichorium intybus L.)

Radicchio (Cichorium intybus subsp. intybus var. foliosum L.) is one of the most important leaf chicories, used mainly as a component for fresh salads. Recently, we sequenced and annotated the first draft of the leaf chicory genome, as we believe it will have an extraordinary impact from both scientific and economic points of view. Indeed, the availability of the first genome sequence for this plant species will provide a powerful tool to be exploited in the identification of markers associated with or genes responsible for relevant agronomic traits, influencing crop productivity and product quality. The plant material used for the sequencing of the leaf chicory genome belongs to the Radicchio of the Chioggia type. Genomic DNA was used for library preparation with the TruSeq DNA Sample Preparation chemistry (Illumina). Sequencing reactions were performed with the Illumina platforms HiSeq and MySeq, and sequence reads were then assembled and annotated. We are confident that our efforts will extend the current knowledge of the genome organization and gene composition of leaf chicory, which is crucial for developing new tools and diagnostic markers useful for our breeding strategies in Radicchio

Large-scale Gene Ontology analysis of plant transcriptome-derived sequences retrieved by AFLP technology

<p>Abstract</p> <p>Background</p> <p>After 10-year-use of AFLP (Amplified Fragment Length Polymorphism) technology for DNA fingerprinting and mRNA profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. AFLP marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cDNA-AFLP for transcriptome profiling and differentially expressed gene cloning. The evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. This may be achieved by means of the Gene Ontology (GO), consisting in three structured vocabularies (<it>i</it>.<it>e</it>. ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. In this paper, the functional annotation of about 8,000 AFLP-derived ESTs retrieved in the NCBI databases was carried out by using GO terminology.</p> <p>Results</p> <p>Descriptive statistics on the type, size and nature of gene sequences obtained by means of AFLP technology were calculated. The gene products associated with mRNA transcripts were then classified according to the three main GO vocabularies. A comparison of the functional content of cDNA-AFLP records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ESTs of Arabidopsis and rice, respectively. On the whole, the statistical parameters adopted for the <it>in silico </it>AFLP-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable GO results. Such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species.</p> <p>Conclusion</p> <p>Reliable GO annotations of AFLP-derived sequences can be gathered through the optimization of the experimental steps and the statistical parameters adopted. The Blast2GO software was shown to represent a comprehensive bioinformatics solution for an annotation-based functional analysis. According to the whole set of GO annotations, the AFLP technology generates thorough information for angiosperm gene products and shares common features across angiosperm species and families. The utility of this technology for structural and functional genomics in plants can be implemented by serial annotation analyses of genome-anchored fragments and organ/tissue-specific repertories of transcriptome-derived fragments.</p

Karyological analysis and DNA barcoding of pompia citron: A first step toward the identification of its relatives

open7Pompia is a citrus fruit endemic of Sardinia, Italy, with an essential oil profile showing outstanding anti-inflammatory and anti-microbic properties. Despite its remarkable pharmaceutical potential, little taxonomic and genetic information is available for this species. We applied flow cytometry and classical cytogenetic techniques to assess the DNA content and to reconstruct the karyotype of several Pompia accessions. Molecular data from plastid DNA barcoding and nuclear DNA sequencing were used to study the genetic distance between Pompia and other citrus species. Flow cytometric estimates of DNA content and somatic chromosome counts suggest that Pompia is a regular diploid Citrus species. DNA polymorphisms of nuclear and chloroplast markers allowed us to investigate the genetic relationships between Pompia accessions and other Citrus species. Based on DNA polymorphism data we propose that Pompia is a very recent interspecific hybrid generated by a cross between C. aurantium (as seed bearer) and C. medica (as pollen donor). Our findings pave the way for further and more specific investigations of local Pompia germplasm resources that may help the preservation and valorisation of this valuable citrus fruit tree.openViglietti G.; Galla G.; Porceddu A.; Barcaccia G.; Curk F.; Luro F.; Scarpa G.M.Viglietti, G.; Galla, G.; Porceddu, A.; Barcaccia, G.; Curk, F.; Luro, F.; Scarpa, G. M