Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers

In an attempt to develop a technique for the identification of grape cultivars in commercial wines, a method for the extraction of DNA from must and experimental wines was adapted and optimal PCR conditions for the amplification of this DNA were established. DNA was analyzed during the fermentation process for six cultivars (Chardonnay, Clairette blanche, Grenache noir, Merlot, Muscat blanc à petits grains, and Syrah). The extractions were performed on solid parts in suspension as well as on the aqueous fraction. Expected profiles for these cultivars were obtained with DNA extracted from the solid parts during all of the fermentation process and for the wine. The analysis of DNA extracted from aqueous fractions was less reproducible, and microsatellite amplifications were obtained only in the case of Clairette blanche, Merlot, and Syrah wines. Results demonstrate that the purification process is adequate for the analysis but that the DNA concentration represents the main limiting factor. Technical improvements of the method are discussed.     

Development of Expressed Sequence Tag (EST)-based Cleaved Amplified Polymorphic Sequence (CAPS) markers of tea plant and their application to cultivar identification

To develop cleaved amplified polymorphic sequence (CAPS) markers for cultivar identification of the tea leaf, 5 primer pairs designed on the basis of genes that encode proteins related to nitrogen assimilation and 26 primer pairs based on expressed sequence tag (EST) sequences of the root of tea plant were screened. From combinations of primer pair and restriction enzyme that showed polymorphism among tea plants, 16 markers were selected and applied to DNA fingerprinting of Japanese tea cultivars. Sixty-three cultivars, except for a bud sport (Kiraka) and its original cultivar (Yabukita) and a pair that was the progeny of the same crossing parent (Harumoegi and Sakimidori), were distinguished from one another. By combining the 16 markers with previously developed CAPS markers and observing the physical appearance, 67 cultivars were distinguishable. The cultivars involve approximately 95% of total tea cultivating area in Japan; therefore, about 95% of tea leaves produced in Japan can be authenticated by labeling their cultivars.     

Application of random amplified polymorphic DNA markers to evaluate intraspecific genetic variation in the Elymus alaskanus complex (Poaceae)

Random amplified polymorphic DNA (RAPD) markers were used to evaluatethe levels and patterns of genetic diversity in ten Elymusalaskanus populations, which were collected from Canada, USA,Greenland and Russia. Ten arbitrarily chosen decamer primers were used in thisstudy. The results revealed high levels of variation. The mean number of allelesper locus (Ap) was 1.5, ranging from 1.4 to 1.6, the meanpercent of polymorphic loci (Pp) was 49.5%, ranging from35.1% to 64.9%, and the mean gene diversity (Hep) was0.162, varying from 0.142 to 0.262. The total variation wasH _T = 0.403. When partitioned(G _ST), 60% of the total variation was foundamong the populations. Although the genetic diversity values obtained with RAPDsare much higher than for allozymes, they are similar regarding how the geneticvariation is distributed among populations. In addition, a similar geneticpattern of population differentiation, where populations from Greenland andthe USA (violaceus and latiglumis)were clearly separated from the others (hyperarcticus,komarovii and sajanensis), wasrevealed by both the cluster and principal coordinates analyses.     

Genetic fingerprinting of grape plant (Vitis vinifera) using random amplified polymorphic DNA (RAPD) analysis and dynamic size-sieving capillary electrophoresis

Dynamic size-sieving capillary electrophoresis with laser-induced fluorescence detection (DSCE-LIF) was combined with random amplified polymorphic DNA (RAPD) analysis to demonstrate the feasibility of the genetic analysis of grape plant varieties and clones within a variety. Parameters of the genomic DNA extraction process, as well as those of the RAPD analysis, were optimized specifically for this application. Polymorphic DNA fragments were generated for four different grape plant varieties including Cabernet Franc, Cabernet Sauvignon, Merlot, and Chardonnay. Relative to slab gel electrophoresis (SGE) with ethidium bromide staining, DSCE-LIF provided superior separation efficiency and detection limits in the analysis of DNA polymorphic bands. Optimal DSCE-LIF analyses were achieved using a 10-fold RAPD sample dilution, hydrodynamic sample injection, and 100 ng/mL of YO-PRO-1 DNA intercalator in the dynamic size-sieving buffer solution. In addition, the reproducibility of both the DSCE-LIF and RAPD analyses were demonstrated.     

Genetic variation of Ethiopian and Eritrean sorghum (Sorghum bicolor (L.) Moench) germplasm assessed by random amplified polymorphic DNA (RAPD)

The extent and patterns of distribution of genetic variation among 80 sorghum (Sorghum bicolor (L.) Moench) germplasm accessions from Ethiopia and Eritrea were investigated using RAPD with 20 oligonucleotide primers. The primers generated a total of 147 polymorphic bands across the 80 accessions with a mean of 7.35 bands per primer. Estimation of the extent of variation by the Shannon-Weaver diversity index revealed an intermediate level of overall variation (H = 53), although the levels varied among regions of origin of the accessions. Partitioning of the total variation revealed considerable variation (77%) within the region of origin of the accessions and the remainder (23%) among regions of origin. Similarly, a large portion (94%) of the total variation was found within the adaptation zones compared to among the adaptation zones (6%). The results suggest a weak differentiation of the sorghum material both on regional and agro-ecological bases, which could be ascribed to the high rate of outcrossing in cultivated sorghum and its free natural hybridization with its wild and weedy relatives, as well as to seed movement by humans. The average genetic dissimilarity was found to be 36% among the 80 accessions and 13% among the 15 regions of origin. Cluster analysis failed to group accessions of the same region or the same adaptation zone, which further confirmed the weak differentiation of the material studied. The clustering pattern of the regions of origin was broadly concordant with previous clustering patterns obtained using morphological characters, in which regions with broad agro-climatic conditions were grouped together.     

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Summary Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.     

Analysis of genetic diversity in Indian taro [Colocasia esculenta (L.) Schott] using random amplified polymorphic DNA (RAPD) markers

Taro [Colocasia esculenta (L.) Schott] germplasm accessions collected from different parts of India were subjected to RAPD (Random Amplified Polymorphic DNA) analysis to assess the genetic diversity prevalent and also to test the genetic basis of morphotypic classification. Thirteen random decamer primers out of the 22 tested were used to analyse 32 taro accessions belonging to 28 morphotypes. Three out of these thirteen primers analysed showed 100 per cent polymorphism. Per cent polymorphism varied from 60 to 100 among the polymorphic primers. High genetic diversity was revealed as the similarity coefficient values ranged from 0.50 to 0.98. No two accessions analysed in the present study showed a similarity coefficient value of one thereby indicating their distinctness and presence of high genetic diversity in Indian taro germplasm. Dendrogram obtained from UPGMA analysis grouped 32 accessions in four clusters and three accessions were placed as outliers. Clustering pattern did not show any strict relationship with geographical distribution, morphotype classification and genotypic diversity. Further, accessions classified, as belonging to the same morphotypic group did not always cluster together. Presence of a very close genepool of the wild, weedy and cultivated forms with extreme levels of phenotypic and genotypic variation is suggested as the reason for high genetic diversity reported. Usefulness of DNA markers such as RAPD in characterising and assessing the genetic diversity in Indian taro germplasm is hereby demonstrated.     

Regional origin assignment of red wines from Valencia (Spain) by (2)H NMR and (13)C IRMS stable isotope analysis of fermentative ethanol.

The use of the stable hydrogen and carbon isotope ratios of fermentative ethanol as suitable environmental fingerprints for the regional origin identification of red wines from Valencia (Spain) has been explored. Monovarietal Vitis vinifera L. cvs. Bobal, Tempranillo, and Monastrell wines have been investigated by (2)H NMR and (13)C IRMS for the natural ranges of site-specific (2)H/(1)H ratios and global delta(13)C values of ethanol over three vintage years. Statistically significant interregional and interannual (2)H and (13)C abundance differences have been noticed, which are interpreted in terms of environmental and ecophysiological factors of isotope content variation. Multivariate discriminant analysis is shown to provide a convenient means for integration of the classifying information, high discriminating abilities being demonstrated for the (2)H and (13)C fingerprints of ethanol. Reasonable differentiation results are achieved at a microregional scale in terms of geographic provenance and even grapevine genotypic features.     

Detection of SNPs in fish DNA: application of the fluorogenic ribonuclease protection (FRIP) assay for the authentication of food contents

The fluorogenic ribonuclease protection (FRIP) assay was used to detect single nucleotide polymorphisms (SNPs) in commercially produced fish products. By using fluorescence resonance energy transfer (FRET) between fluorophore and quencher labeled probes, the species-specific cleavage of sample RNA was detected by measuring the fluorescence intensity during the FRIP assay. We were able to discriminate raw and thermally processed eel and tuna species using the FRIP-based SNP detection method. Furthermore, the intensity of fluorescence was correlated with the mutant/wild-type ratio. These results suggest that the FRIP assay is a useful method for the in situ confirmation of labels of fishery foods during food production.     

Genetic variation in captive population of chinese alligator, Alligator sinensis, revealed by random amplified polymorphic DNA (RAPD)

Chinese alligator (Alligator sinensis) is an endemic species in China. The likely extinction of it in the wild has been recognised. To prevent this species becoming extinct, the Anhui Research Centre of Chinese Alligator Reproduction (ARCCAR) was established in Xuanzhou, Anhui Province in 1979, where has been established the largest captive population of Chinese alligator (XZSP) in the world. Another farm (CXSP) was established by villagers in Changxin, Zhejiang Province. The results of an investigation of the two captive subpopulation structures by genetic analysis are presented in this paper. We examined the genetic variation in the two captive subpopulations using RAPDs. Thirty-one random primers were selected among 199 random primers screened. A total of 193 reproducible RAPD fragments were scored among 43 individuals, of which 21 (10.88%) were polymorphic. The genetic distances between 43 individuals ranged from 0 to 0.0376 with average of 0.0104±0.0055 S.E. The genetic similarity in CXSP (0.9948±0.0029 S.E.) was higher than that in XZSP (0.9894±0.0055 S.E.). The founder effect is a possible explanation for very low genetic variation in CXSP. Analysis of the RAPD data showed that the mean phenotypic band frequencies of each polymorphic loci was 0.6656±0.3730 S.E. The lowest phenotypic band frequency (0.0233) was found in four of those polymorphic loci. There was no genetic difference between the two subpopulations (D_ij=0.0009). According to the dendrogram and the distribution of polymorphic fragments in two subpopulations, CXSP originated genetically from XZSP. This paper summarises a preliminary research on genetic structure in populations of Chinese alligator. Although there is higher genetic similary (0.9896±0.0055 S.E.) in captive population of A. sinensis, we did not determine whether or not loss of genetic variation had occurred in relation to a wild control population. The data of malformed offspring will be collected carefully, and wild samples be added to set up a control population in future study.     

Varietal differentiation of red wines in the Valencian region (Spain)

Ninety-one young varietal wines from the Valencian community (Spain), made from Cabernet Sauvignon, Tempranillo, Monastrell, and Bobal grapes, were tested on the basis of 33 variables: 9 conventional parameters, 10 alcohols and polyols, and 14 esters. Discriminant analysis was used to identify and explain the differences among samples, as well as to determine whether it is possible or not to differentiate among varieties. This differentiation (100% of the samples) has been possible due to the new discriminant analysis based on only 11 main variables: total acidity, cis-3-hexenol, methanol, glycerol, 2,3-butanediol, isobutyric alcohol, 1-pentanol, acetaldehyde, ethyl propionate, ethyl decanoato, and gamma-butyrolactone, which allow differentiating 100% of the 1994 vintage and 97% of the 1995 vintage.     

Toward the determination of the geographical origin of Emmental(er) cheese via high resolution MAS NMR: a preliminary investigation

We tested the potential of High-Resolution MAS NMR spectroscopy to study 20 samples of Emmental cheeses from 7 different geographical regions. Principal component analysis (PCA) and discriminant analysis (DA) were used to analyze the data set of 1H HRMAS NMR spectra and succeeded in grouping the studied samples according to their geographical origins.     

Characteristic phenolic composition of single-cultivar red wines of the Canary Islands (Spain)

The detailed phenolic composition of five single-cultivar (Baboso Negro, Listán Negro, Negramoll, Tintilla, and Vijariego Negro) young and aged (vintages 2005-2009) red wines of the Canary Islands has been determined by HPLC-DAD-ESI-MS(n). Despite the total monomeric anthocyanin content decreasing for older wines in each set of single-cultivar wines, the corresponding anthocyanin profiles remained almost unchanged. Although all wine anthocyanin profiles were dominated by malvidin 3-glucoside, their differentiation by grape cultivar was possible, with the exception of Listán Negro. In contrast, the total content of non-anthocyanin phenolics did not appreciably change within vintages but polymerization, hydrolysis, and isomerization reactions greatly modified the phenolic profiles. Aglycone-type flavonol profiles offered the best results for differentiation of the wines according to grape cultivar (Listán Negro and Negramoll; Baboso Negro and Vijariego Negro; and Tintilla). Within flavan-3-ols, the B-ring trihydroxylated monomers ((-)-epigallocatechin and (-)-gallocatechin) and also (-)-epicatechin provided additional cultivar differentiation. Hydroxycinnamic acid derivatives and stilbene profiles were very heterogeneous with regard to both grape cultivar and vintage and did not significantly contribute to wine differentiation, even when structure-type profiles were obtained, with the exception of Tintilla, which always appeared as the most different single-cultivar wines. Finally, most Canary Islands wines showed characteristic high contents of stilbenes, especially trans-resveratrol.     

Genetic diversity of <Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass. (Asteraceae) from Ethiopia as revealed by random amplified polymorphic DNA (RAPD)

Genetic diversity of 70 populations of niger (Guizotia abyssinica) representing all its growing regions in Ethiopia was investigated using random amplified polymorphic DNA (RAPD) to reveal the extent of its populations genetic diversity. Ninety-seven percent of the loci studied was revealed to be polymorphic for the whole data set. The within population diversity estimated by Shannon diversity index and Nei gene diversity estimates was revealed to be 0.395 and 0.158, respectively. The extent of genetic variation of populations from major niger producing regions was significantly lower than that of populations from other regions; however, it is distributed regardless of altitude of growth. Genetic differentiation between populations was estimated with Shannon index as G^′ _ST (0.432), Nei’s G _ST (0.242) and AMOVA based F _ST (0.350) and appears to be equivalent to the average values calculated from various RAPD based studies on outcrossing species. Higher proportion of the variation detected by AMOVA resided within populations (64.58%) relative to the amount of variation among populations (35.42%). UPGMA cluster analysis showed that most of the populations were clustered according to their region of origin. However, some populations were genetically distant from the majority and seem to have unique genetic properties. It is concluded that the crop has a wide genetic basis that may be used for the improvement of the species through conventional breeding and/or marker assisted selection. Collection of germplasm from areas not yet covered and/or underrepresented is the opportunity to broaden the genetic basis of genebank collection.     

New evidence on the origin of mangosteen (Garcinia mangostana L.) based on morphology and ITS sequence

Mangosteen (Garcinia mangostana L.), known as one of the most desirable tropical fruits of Southeast Asia, has been considered as an obligate agamospermous hybrid, thought to have arisen from two wild species, G. celebica L. (syn. G. hombroniana Pierre) and G. malaccensis Hook. f. However, this putative origin was based on a misidentification of G. malaccensis, which was confused for G. penangiana Pierre. Intensive field studies and molecular investigations based on internal transcribed spacer (ITS) sequence data of 22 samples were conducted, which included six samples of true G. malaccensis. Morphological observation shows that mangosteen highly resembles G. malaccensis, particularly in its vegetative and fruit characters, even sharing similar taste of ripe fruits. ITS data revealed that mangosteen shared more than 99 % of its sequence with G. malaccensis with a few accessions identical with wild populations in Peninsular Malaysia. Phylogenetic analysis revealed that clades of mangosteen are paraphyletic per se, but monophyletic if both mangosteen and G. malaccensis are grouped together. This show that mangosteen and G. malaccensis are so closely related that they should be combined together as one species. I propose two theories on the origin of mangosteen, first, that it is a hybrid of different varieties of G. malaccensis, and second, that it may be a product of multiple, superior selections from different populations of female trees of G. malaccensis originating in Peninsular Malaysia.     

One-step triplex-polymerase chain reaction assay for the authentication of yellowfin (Thunnus albacares), bigeye (Thunnus obesus), and skipjack (Katsuwonus pelamis) tuna DNA from fresh, frozen, and canned tuna samples

A one-step triplex-polymerase chain reaction (PCR)-based assay was developed to discriminate between three tuna species, Thunnus albacares, Thunnus obesus, and Katsuwonus pelamis, even in highly processed food samples such as canned or cooked tuna. Diagnostic nucleotides were identified by direct sequencing and alignment of part of the mitochondrial cytochrome b gene of 30 authenticated exemplars, which allowed us to evaluate intraspecific variation and the genetic distance between three tuna species. The assay relies on a one-step triplex-PCR reaction in which in a single tube species-specific amplification products are generated only in the presence of the correct template nucleic acid and the species of origin of the DNA is indicated by the distinctive size of the PCR product. The identification of tuna species can be performed with a good accuracy, low cost, and with potential automation for large-scale high-throughput screenings in small in-house laboratories.     

Characterization of the mean degree of polymerization of proanthocyanidins in red wines using liquid chromatography-mass spectrometry (LC-MS)

An HPLC-MS method for the characterization of proanthocyanidins (PA) has been refined. Further application to red wines provided interesting conclusions about the composition of the flavanol fraction and PA extractability during winemaking. The yield in PA extraction increases with the length of the postfermentative maceration (PFM), as well as the mean degree of polymerization (mDP) of wine flavanols. In early winemaking events mostly monomers to trimers are extracted from grape solids, whereas PFM is required for the significant extraction of higher oligomers. Nevertheless, at the end of a regular process of elaboration the mDP is not very high and does not usually exceed a value of 2.3, dimers and trimers being the predominant flavanols in red wines. With regard to groups of compounds, gallocatechins and prodelphinidins (located in the skins) are extracted rapidly in the first stages of the winemaking. On the contrary, long postfermentative macerations are required for the extraction of galloyled derivatives from the seeds. PA extractability is also dependent on the grape variety used for winemaking. Thus, wines made with Graciano grapes were found to require a longer process of PFM than those made from Tempranillo grapes to obtain similar yields in the extraction of flavanols.