Epigenetic Variability Among Saffron Crocus (Crocus sativus L.) Accessions Characterized by Different Phenotypes.

This work represents the first epigenomic study carried out on saffron crocus. Five accessions of saffron, showing differences in tepal pigmentation, yield of saffron and flowering time, were analyzed at the epigenetic level by applying a methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach. Five accession-specific hypomethylomes plus a reference hypomethylome, generated by combining the sequence data from the single accessions, were obtained. Assembled sequences were annotated against existing online databases. In the absence of the Crocus genome, the rice genome was mainly used as the reference as it is the best annotated genome among monocot plants. Comparison of the hypomethylomes revealed many differentially methylated regions, confirming the high epigenetic variability present among saffron accessions, including sequences encoding for proteins that could be good candidates to explain the accessions' alternative phenotypes. In particular, transcription factors involved in flowering process (MADS-box and TFL) and for the production of pigments (MYB) were detected. Finally, by comparing the generated sequences of the different accessions, a high number of SNPs, likely having arisen as a consequence of the prolonged vegetative propagation, were detected, demonstrating surprisingly high genetic variability. Gene ontology (GO) was performed to map and visualize sequence polymorphisms located within the GOs and to compare their distributions among different accessions. As well as suggesting the possible existence of alternative phenotypes with a genetic basis, a clear difference in polymorphic GO is present among accessions based on their geographic origin, supporting a possible signature of selection in the Indian accession with respect to the Spanish ones.

Epigenetic Barcodes for Detection of Adulterated Plants and Plant-Derived Products.

In this chapter, we report a possible alternative use of epigenetics by applying methylation-sensitive amplified fragment length polymorphisms (MS-AFLP) to saffron traceability. Saffron is the most expensive plant-derived product in the world and one of the most frequently adulterated. One of the most frequent adulteration is by adding to saffron stigmas different parts of the saffron flower itself to increase volumes. While DNA is the same in all the parts of the plant, the epigenetic state can vary according to the organ and/or tissue of origin, making it possible to discriminate the stigmas from the other parts of saffron flower. In the subsequent method, the protocol to carry out a MS-AFLP analysis of saffron DNA methylation patterns is described.

Epigenetic stability in Saffron (Crocus sativus L.) accessions during four consecutive years of cultivation and vegetative propagation under open field conditions.

Saffron (Crocus sativus L.) is a sterile species that is vegetatively propagated in the field, year by year, via the production of new corms. While Saffron's genetic variability is extremely low, phenotypic variation is frequently observed in the field and epigenetics could be a possible origin of these alternative phenotypes. Present day knowledge on Saffron epigenetics is very low or absent. In the present paper, to deepen existing knowledge, we focused on the epigenetic differences and stability among 17 Saffron accessions, of different geographic origin, during four consecutive years of vegetative propagation under open field conditions. Before the analysis, the selected accessions have been cultivated in the same field for at least three consecutive years. Despite the low genetic variability and the prolonged co-cultivation in the same environment, Methylation-Sensitive Amplified Fragment Length Polymorphism (MS-AFLP) analysis revealed a very high epigenetic difference among accessions, making it possible to discriminate them based on the epigenetic profiles. During the four years of the study, a little variation has been observed within accessions following different patterns, slightly modifying the accession epigenotypes but not enough to even them to a more uniform profile. These results confirm that, under natural conditions, Saffron epigenotypes are highly stable, supporting a role for epigenetics in phenotypic variability.

Phenolic Profiling for Traceability of Vanilla ×tahitensis.

Vanilla is a flavoring recovered from the cured beans of the orchid genus Vanilla. Vanilla ×tahitensis is traditionally cultivated on the islands of French Polynesia, where vanilla vines were first introduced during the nineteenth century and, since the 1960s, have been introduced to other Pacific countries such as Papua New Guinea (PNG), cultivated and sold as "Tahitian vanilla," although both sensory properties and aspect are different. From an economic point of view, it is important to ensure V. ×tahitensis traceability and to guarantee that the marketed product is part of the future protected designation of the origin "Tahitian vanilla" (PDO), currently in progress in French Polynesia. The application of metabolomics, allowing the detection and simultaneous analysis of hundreds or thousands of metabolites from different matrices, has recently gained high interest in food traceability. Here, metabolomics analysis of phenolic compounds profiles was successfully applied for the first time to V. ×tahitensis to deepen our knowledge of vanilla metabolome, focusing on phenolics compounds, for traceability purposes. Phenolics were screened through a quadrupole-time-of-flight mass spectrometer coupled to a UHPLC liquid chromatography system, and 260 different compounds were clearly evidenced and subjected to different statistical analysis in order to enable the discrimination of the samples based on their origin. Eighty-eight and twenty three compounds, with a prevalence of flavonoids, resulted to be highly discriminant through ANOVA and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) respectively. Volcano plot analysis and pairwise comparisons were carried out to determine those compounds, mainly responsible for the differences among samples as a consequence of either origin or cultivar. The samples from PNG were clearly different from the Tahitian samples that were further divided in two different groups based on the different phenolic patterns. Among the 260 compounds, metabolomics analysis enabled the detection of previously unreported phenolics in vanilla (such as flavonoids, lignans, stilbenes and other polyphenols).

Genetic and Epigenetic Approaches for the Possible Detection of Adulteration and Auto-Adulteration in Saffron (Crocus sativus L.) Spice.

Saffron (Crocus sativus L.) is very expensive and, because of this, often subject to adulteration. Modern genetic fingerprinting techniques are an alternative low cost technology to the existing chemical techniques, which are used to control the purity of food products. Buddleja officinalis Maxim, Gardenia jasminoides Ellis, Curcuma longa L., Carthamus tinctorius L. and Calendula officinalis L. are among the most frequently-used adulterants in saffron spice. Three commercial kits were compared concerning the ability to recover PCR-grade DNA from saffron, truly adulterated samples and possible adulterants, with a clear difference among them, mainly with the processed samples. Only one of the three kits was able to obtain amplifiable DNA from almost all of the samples, with the exception of extracts. On the recovered DNA, new markers were developed based on the sequence of the plastid genes matK and rbcL. These primers, mainly those developed on matK, were able to recognize saffron and the adulterant species and also in mixtures with very low percentages of adulterant. Finally, considering that the addition of different parts of saffron flowers is one of the most widespread adulterations, by analyzing the DNA of the different parts of the flower (styles, stamens and tepals) at the genetic and epigenetic level, we succeeded in finding differences between the three tissues that can be further evaluated for a possible detection of the kind of fraud.

Direct amplification of new cellulase genes from woodland soil purified DNA.

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-ß-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.