Matteo Busconi

DNA extraction from olive oil and its use in the identification of the production cultivar

Abstract DNA recovery from food samples might be of great importance when the raw material used in the production process has to be traced. We were interested in verifying the presence of nucleic acids in extra virgin olive oil in order to determine the cultivar of origin of the olives used for the production. A reliable DNA extraction method for extra virgin olive oil has been defined, as far as both quantity and quality are concerned, and the possibility of using this DNA for fingerprinting the original cultivar has been demonstrated. DNA extraction was tested on four monovariety oils, plus four commercial extra virgin olive oils. The DNA in the extracted solution was of chloroplast and nuclear origin since we were able to amplify cloned cultivar RAPD and AFLP fragments homologous to nuclear DNA of other species. It has also been shown that DNA purified from oil can be used for AFLP analysis and that the profile of the DNA purified from a monovariety oil corresponds to the profile of the DNA purified from the leaves of the same cultivar.

Spread of herbicide-resistant weedy rice (red rice, Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy.

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide-resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F2 or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.

Development of SCAR Markers for Germplasm Characterisation in Olive Tree (Olea europea L.)

A set of 14 SCAR markers were developed starting from RAPD, AFLP and SAMPL analysis of several olive germplasm accessions. Eight RAPD, two AFLP and four SAMPL fragments were converted into dominant and codominant SCARs by cloning and sequencing the selected fragments. The markers obtained were evaluated on forty different olive cultivars from different Italian production areas (mainly from Liguria). The combined use of these SCARs made possible to univocally identify 26 cultivars while the remaining 14 will require the development of further markers since most of them are placed in a main group containing six genetically similar cultivars (among which Frantoio and Taggiasca) and four minor groups containing two cultivars each. A total of 31 different haplotypes were identified and the analysis of several individual plants indicated no intra-cultivar variability. Considering the SCAR polymorphism two alleles were scored for each markers with the only exception of markers IGPS3 and IGPS4 showing 4 alleles with 7 recognised groups and 5 alleles with 4 groups, respectively. Though less polymorphic in comparison with other markers like SSRs, the developed SCARs proved useful in genotype identification. In addition, they could potentially be used for breeding applications and forensic analysis.

Evaluation of biodiversity of lactic acid bacteria microbiota in the calf intestinal tracts

Amplified fragment length polymorphism (AFLPs) were used to analyse the naturally occurring flora of lactic acid bacteria (LAB) in gastrointestinal tracts of two healthy 65-day-old calves. More than 1,000 of presumptive LAB were collected and cultured from the gastrointestinal tracts and, among the isolated colonies, a total of 311 strains were analysed and separated into eight clusters based on AFLP banding patterns. To precisely determine the species inside the clusters, partial sequences of fragments of the 16S ribosomal DNA gene were determined, and sequence homology searches were conducted through GenBank on few strains per cluster. The most representative genera of LAB were Lactobacillus (169 isolates, 54% of total) and Streptococcus (99 isolates, 32% of total), while the most frequent species was identified as L. mucosae with 86 different isolates (51% of the Lactobacillus spp. and 28% of the total). This report gives a first characterization of LAB strain biodiversity recovered directly from calf intestine and is the first account of the presence of the L. mucosae species in calves. Moreover it demonstrates that the AFLP is a robust and useful technique for characterizing the strain level of LAB microflora.

AFLP and MS-AFLP Analysis of the Variation within Saffron Crocus (Crocus sativus L.) Germplasm

The presence and extent of genetic variation in saffron crocus are still debated, as testified by several contradictory articles providing contrasting results about the monomorphism or less of the species. Remarkably, phenotypic variations have been frequently observed in the field, such variations are usually unstable and can change from one growing season to another. Considering that gene expression can be influenced both by genetic and epigenetic changes, epigenetics could be a plausible cause of the alternative phenotypes. In order to obtain new insights into this issue, we carried out a molecular marker analysis of 112 accessions from the World Saffron and Crocus Collection. The accessions were grown for at least three years in the same open field conditions. The same samples were analysed using Amplified Fragment Length Polymorphism (AFLP) and Methyl Sensitive AFLP in order to search for variation at the genetic (DNA sequence) and epigenetic (cytosine methylation) level. While the genetic variability was low (4.23% polymorphic peaks and twelve (12) effective different genotypes), the methyl sensitive analysis showed the presence of high epigenetic variability (33.57% polymorphic peaks and twenty eight (28) different effective epigenotypes). The pattern obtained by Factorial Correspondence Analysis of AFLP and, in particular, of MS-AFLP data was consistent with the geographical provenance of the accessions. Very interestingly, by focusing on Spanish accessions, it was observed that the distribution of the accessions in the Factorial Correspondence Analysis is not random but tends to reflect the geographical origin. Two clearly defined clusters grouping accessions from the West (Toledo and Ciudad Real) and accessions from the East (Cuenca and Teruel) were clearly recognised.

Evidence of a sirtuin gene family in grapevine (Vitis vinifera L.)

Sirtuins are known as regulators of age-dependent gene transcription and chromatin modification in yeast and in animals, but information about their occurrence and role in plants is scarce. Sirtuin-like sequences were amplified using two highly degenerate primers designed comparing sirtuin sequences of seven different plant species, and characterised at the sequence level in Vitis vinifera L. cv. Barbera clone R4. The data were confirmed and analysed by searching the published database from two independent grape genome projects with the obtained sequences. Two putative sirtuin genes containing sirtuin-conserved domains were found on chromosome 7 and on chromosome 19, suggesting the occurrence of sirtuins in grapevine.

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.

Bacterial ecology of PDO Coppa and Pancetta Piacentina at the end of ripening and after MAP storage of sliced product.

The objective of this study was to evaluate the microbiota of two typical Italian PDO delicatessens Coppa and Pancetta Piacentina, produced in Piacenza area (Italy). Classical and molecular approaches were employed, in order to acquire knowledge on their bacterial ecology and its evolution after slicing and MAP storing; thus, the biodiversity of characteristic bacterial community, already present or introduced during such procedures, was studied in both full ripened and sliced samples from two producers (A and B) of the PDO district, packaged under MAP and stored at 2 and 8 °C for 30 days. The microbiota of the two kinds of Italian delicatessen demonstrated peculiar differences, particularly regarding the staphylococci and lactic acid bacteria (LAB) ratio. Moreover, some species within these two groups appeared to be linked to the kind of product: Leuconostoc, Lactobacillus versmoldensis and Staphylococcus saprophyticus were found only in Pancetta while Lactobacillus pentosus, Staphylococcus equorum, Staphylococcus xylosus, Staphylococcus sciuri and Macrococcus caseolyticus occurred only in Coppa. Also, both delicatessens from producer A were richer in LAB compared to those of producer B and the opposite applied for staphylococci. Interestingly, Tetragenococcus halophilus was detectable in all the samples and its presence in the sausage environment has been reported only for Capocollo. Storage did not substantially modify the microbiota composition, the only changes being the relative abundance of same sequences; S. xylosus was prevalent before slicing process and S. equorum at the end of MAP storage at both 2 °C and 8 °C. Concerning microbial contamination during the slicing process, our results suggest that the adopted procedures assure high hygienic quality standard of these typical products, with exception of a contamination by Psychrobacter psychrophilus in Coppa B. The possible origin of species rarely or never reported in the sausage environment and detected in this study is discussed.

Wine Resveratrol: From the Ground Up

The ability of the grapevine to activate defense mechanisms against some pathogens has been shown to be linked to the synthesis of resveratrol and other stilbenes by the plant (inducible viniferins). Metabolized viniferins may also be produced or modified by extracellular enzymes released by the pathogen in an attempt to eliminate undesirable toxic compounds. Because of the important properties of resveratrol, there is increasing interest in producing wines with higher contents of this compound and a higher nutritional value. Many biotic and abiotic elicitors can trigger the resveratrol synthesis in the berries, and some examples are reported. Under the same elicitation pressure, viticultural and enological factors can substantially affect the resveratrol concentration in the wine. The production of high resveratrol-containing grapes and wines relies on quality-oriented viticulture (suitable terroirs and sustainable cultural practices) and winemaking technologies that avoid degradation of the compound. In general, the oenological practices commonly used to stabilize wine after fermentation do not affect resveratrol concentration, which shows considerable stability. Finally the paper reports on two sirtuin genes (SIRT) expressed in grapevine leaves and berries and the role of resveratrol on the deacetylation activity of the encoded enzymes.

Gene flow from transgenic rice to red rice (Oryza sativa L.) in the field

In this study, we simulate a transgenic rice crop highly infested with red rice to examine transgene transfer from a transgenic line (A2504) resistant to glufosinate ammonium to cohabitant red rice. The red rice was sown along with the transgenic line at the highest density found in naturally infested crops in the region. Agricultural practices similar to those used to control red rice infestation in northern Italy rice fields were used to reproduce the local rice production system. During the first 2 years, the field was treated with herbicide at the appropriate time; in the first year the dosage of herbicide was three times the recommended amount. In this first year, detectable red rice plants that escaped herbicide treatment were manually removed. Nevertheless, two herbicide-resistant hybrid plants (named 101 and 104) were identified in the experimental field during the second year of cultivation. Phenotypic and molecular characterisation suggests the hybrid nature of these two plants, deriving from crossing events involving A2504, respectively, with red rice (plant 101) and the buffer cultivar Gladio (plant 104). The progeny of two subsequent generations of the two plants were examined and the presence of the transgene detected, indicating stable transfer of the transgene across generations. In conclusion, despite control methods, red rice progeny tolerant to the herbicide can be expected following use of transgenic rice and, consequently, difficulties in controlling this weed with chemicals will emerge in a relatively short time.