Molecular and phenotypic characterization of a collection of white grain sorghum [Sorghum bicolor (L.) Moench] for temperate climates

AbstractSorghum [Sorghum bicolor (L.) Moench] is a subsistence crop and the main food for populations in arid or semiarid regions and it is appreciated for the production of gluten-free products, forages, raw materials for industrial transformation and packaging. The end-use of different sorghum purposes having various plant or kernel characteristics require specific breeding programs to develop the desired ideotype. Sorghum grains can be classified according to kernel color, tannins and polyphenols content: white, yellow, red, brown, and black. White sorghum is characterized by a low level of total phenolic content and tannins. The advantage of using white sorghum is: increased protein digestibility, nutritional composition and consumer acceptance similar to other cereals. A collection of 117 white grain sorghums was characterized using 10 SSRs and preliminary agronomic observations were made for main traits. SSR analysis revealed from 10 to 33 alleles per locus.Observed heterozygosity was lower than expected according to the reproduction system of sorghum. Phylogenetic analysis revealed 6 main groups of genotypes. Only one group is constituted by genotypes with the same geographical origin (Egypt) while other groups are admixtures of different countries. The principal coordinate analysis revealed good correspondence between genetic profiles and groups evidenced by similar agronomic performances

The Rediscovery of Traditional Maize Agrobiodiversity: A Study Case from Northern Italy

Nowadays, agriculture is under the pressure of climate change and new pathogen outbreaks while farmers are requiring breeders to develop more resistant and resilient genotypes. The genetic base for breeding may be increased through appropriate conservation, description and characterization of local varieties and germplasm collections that have never been used in breeding, and which could be sources of useful alleles. In this framework, the present paper focuses on eight maize landraces of the eastern part of Emilia-Romagna, derived from the Italian maize collection sampled in 1954. Landraces are characterized by a short cycle length and different kernel types—mainly flint-like or an intermediate type of yellow or yellow–orange color—while dent landraces are less represented. Pigmented and white corns are absent even though one landrace (Va213) showed the presence of scattered blue kernels on yellow ears. Ear shape is frequently conical, a trait associated with drought-resistance and common in Italian traditional landraces. Genetic characterization was carried out on 529 individuals by using 10 SSR markers. A total of 68 different alleles, ranging from 4 for markers (phi084 and umc1401) to 11 (phi031) and from 27 (Va217) to 50 (Va211), were evidenced at the individual and population level. AMOVA analysis revealed a small amount (19%) of variability between populations, as supported also by PCoA, with the only exception of Va217, which is different from the others, as evidenced also by phylogenetic analysis. Population structure analysis resulted in the identification of three and four population levels, which are consistent with previous results

Morphological and Genetic Characterization of Local Maize Accessions from Emilia Romagna Region, Italy

Italian maize germplasm is particularly rich in local materials and each region is characterized by the presence of peculiar local varieties deriving from centuries of adaptation, selection and cultivation. While the introduction of hybrids, during the 1950s, led to the disappearing of many of these varieties, some have been maintained in cultivation by farmers, frequently in marginal areas, as a kind of family heritage. Local varieties were identified throughout field surveys carried out in recent years. The discovery of a traditional popcorn variety over the most common flint and semi-flint materials used for production of polenta was interesting. Since these varieties have never been adequately described and reported in scientific literature, this study was aimed to solve this lack of knowledge on recently discovered local maize populations. Characterization represents the first step of a process focused on the preservation and possible exploitation of important genetic resources. Traditional materials are a useful reservoir of genes for adaptation to local conditions and climate changes. Adequate breeding programs can use such germplasm for developing new and more resilient varieties. These local materials have been characterized at the morphological level highlighting plant, ear and kernel differences. Genetic characterization, carried out on 455 individuals by the use of 10 SSR markers, revealed 62 different alleles ranging from four for markers phi127, phi076 and phi084 to nine for marker p-bnlg176. The landraces are well distinguishable at genetic level since 40% of genetic variability is present among accessions. Five landraces are characterized by the presence of private alleles and heterozygosity levels are generally good. These findings support the possibility to correctly preserve local materials through in situ conservation. Phylogenetic analysis evidenced the presence of varietal clusters, the clearest one formed by three red-pigmented accessions. STRUCTURE analysis revealed that five landraces have a well-defined genetic attribution while the remaining two (EMR04-Mais Rosso di Rasora and EMR10-Mais del Principe di Scavolino) are both constituted by two different backgrounds

Development of rapid molecular diagnostics methods for the agro-food sector

L’elaborato è stato strutturato in base alle matrici analizzate. Il primo capitolo è dedicato alla caratterizzazione genetica e conseguente possibilità di utilizzo dei marcatori microsatellitari all’interno di 28 popolazioni locali di mais. L’analisi ha permesso di valutare lo stato di conservazione delle accessioni e realizzare un albero filogenetico per individuare le relazioni esistenti tra le diverse varietà. Su un numero più ridotto di materiali è stata valutata la possibilità di utilizzare i marcatori molecolari per la tracciabilità della filiera maidicola, in particolare farina destinata al consumo umano. La seconda parte mostra un esempio di tracciabilità vegetale nel caso di cacao. L’uso di marcatori molecolari hai ha permesso di identificare un profilo microsatellitare per la cultivar CCN51 (clone) permettendo di distinguerla dalla varietà “National” (popolazione) e, quindi, creare una metodologia per la certificazione delle partite costituite esclusivamente dal clone CCN51. Il terzo capitolo è dedicato alla filiera enologica e vede l’ottimizzazione di metodologie per l’estrazione del DNA da uve, mosto, mosto durante le varie fasi di fermentazione, fino al vino finito sia pre che post imbottigliamento, ottenendo DNA amplificabile lungo tutta la filiera produttiva e utilizzabile per una analisi microsatellitare permettendo di identificare le uve utilizzate con attendibilità diversa nelle varie fasi di lavorazione.The thesis is organized according to the different experiments. The first chapter focus on genetic characterisation of 28 local maize populations. The analysis assessed the conservation status of the accessions and allowed the construction of a phylogenetic tree to identify the relationships existing between the different varieties. The possibility to use a molecular marker based traceability approach was evaluated on a smaller number of maize accession. This analysis focused on maize flour for direct human consumption. The second part is an example of food traceability along the cocoa-chocolate chain. The use of molecular markers allowed the identification of the genetic of the CCN51 cultivar (clone) allowing the discrimination between CCN51 and the 'National' variety (population). The identification and traceability of “pure” CCN51 cocoa batches is possible following this approach. The third chapter focused on wine production chain with the aim of the optimisation of methodologies for DNA extraction from grapes and must during the various winemaking steps until pre and post bottling. The obtained DNA was successfully amplified and used for a microsatellite analysis allowing the identification of the grape varieties used for winemaking process

Comparison of six methods for the recovery of PCR-compatible microbial DNA from an agricultural biogas plant

Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality PCR compatible microbial DNA from an agricultural biogas plant. Within the last two decades, biogas plants have been developed to produce energy from organic wastes and from devoted biomass. The complex biotransformations are performed by a diverse consortium of microorganisms that is an important reserve of genes and enzymatic activities with a huge range of applications in various commercial fields. In this respect, the ability to isolate DNA from a complex matrix is of high importance. Important parameters of the recovered DNA are good yield, purity, and quality. The methods examined showed considerable differences about quantity and quality of the recovered DNA and, usually, it was observed that a higher amount was accompanied by more degradation. DNA purity was determined by its PCR amplificability. Only two methods were able to provide DNA pure enough to be directly amplified. For the rest of the methods, a few intermediate steps such as dilution and/or the addition of polyvinylpyrrolidone were necessary to remove the inhibitors present and to amplify the DNA. Real-time PCR analysis evidenced that, as expected, prokaryotic DNA was much more abundant than eukaryotic DNA, but some methods were more suited to recovering prokaryotic or eukaryotic DNA. The digestion analysis of ribosomal DNA amplicons confirmed the influence of the methods on the final output, allowing the recovery of only a fraction of the present species as determined by sequencing a small prokaryotic and eukaryotic ribosomal library

Morphological and Genetic Characterization of Local Maize Accessions from Emilia Romagna Region, Italy

Italian maize germplasm is particularly rich in local materials and each region is characterized by the presence of peculiar local varieties deriving from centuries of adaptation, selection and cultivation. While the introduction of hybrids, during the 1950s, led to the disappearing of many of these varieties, some have been maintained in cultivation by farmers, frequently in marginal areas, as a kind of family heritage. Local varieties were identified throughout field surveys carried out in recent years. The discovery of a traditional popcorn variety over the most common flint and semi-flint materials used for production of polenta was interesting. Since these varieties have never been adequately described and reported in scientific literature, this study was aimed to solve this lack of knowledge on recently discovered local maize populations. Characterization represents the first step of a process focused on the preservation and possible exploitation of important genetic resources. Traditional materials are a useful reservoir of genes for adaptation to local conditions and climate changes. Adequate breeding programs can use such germplasm for developing new and more resilient varieties. These local materials have been characterized at the morphological level highlighting plant, ear and kernel differences. Genetic characterization, carried out on 455 individuals by the use of 10 SSR markers, revealed 62 different alleles ranging from four for markers phi127, phi076 and phi084 to nine for marker p-bnlg176. The landraces are well distinguishable at genetic level since 40% of genetic variability is present among accessions. Five landraces are characterized by the presence of private alleles and heterozygosity levels are generally good. These findings support the possibility to correctly preserve local materials through in situ conservation. Phylogenetic analysis evidenced the presence of varietal clusters, the clearest one formed by three red-pigmented accessions. STRUCTURE analysis revealed that five landraces have a well-defined genetic attribution while the remaining two (EMR04-Mais Rosso di Rasora and EMR10-Mais del Principe di Scavolino) are both constituted by two different backgrounds

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-b-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

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-gradeDNAfrom 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