Ioannis V Ganopoulos

Abstract:

Abstract: Legumes and common bean (Phaseolus vulgaris L.), in particular, are important crops worldwide, consumed either as dried seeds or fresh fruits. Correct identification of common bean varieties is important, in order to ensure food quality, safety and authenticity for consumers. Recently, DNA based methods, including molecular markers like microsatellites (SSR), have been developed for plant species or variety identification genotyping and for identification of their ingredients in the final food products. Here, we have applied High Resolution Melting (HRM) analysis coupled with four microsatellite markers to facilitate the identification of protected geographic indication (PGI) common bean variety �Plake Megalosperma Prespon� (�PMP�). The four microsatellite loci used were informative and were used to generate a unique melting curve profile of microsatellites for each variety tested. These microsatellite markers enabled the distinction and identification of the PGI (common bean variety �PMP�). Hence, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in common beans.

Abstract: The Leguminoseae family includes several economically important genera like legumes. Legumes are the second most important agricultural family after cereals in terms of harvested area and production. Legumes are key elements in the Mediterranean diet and as winter annuals with nitrogen fixing capacity, they are extremely important for sustainable agriculture worldwide. We report here the application of the Bar-HRM (Barcode DNA High Resolution Melting) analysis method with the universal plant DNA barcoding region trnL which allowed the identification, adulteration and quantification of major Greek and Mediterranean in general bean species. Bar-HRM detected Lupinus spp. adulterants in Glycine max flour as low as 1:100. Moreover, the method was coupled with the rapid Phire PCR kit that does not require prior DNA purification. This makes the method a very fast and effective tool for barcoding Legumes and particularly for the crops examined not only for their authenticity but for quantitative detection of purity of their seeds or their processed food and feed products.

Abstract: Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of �Fava Santorinis� are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO �Fava Santorinis�. Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-�Fava Santorinis� in �Fava Santorinis� commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO �Fava Santorinis� and to quantitatively detect adulterations in �Fava Santorinis� with other relative commercial �Fava� food products.

Abstract:

Abstract: Legume species are part of a very important agricultural family, second only to cereals. Their importance for sustainable agriculture worldwide comes from their nitrogen fixing ability. They include mainly annual grain crops but also very important perennial forage and pasture species. Given their small size seed admixture and adulteration is a common problem, lowering the forage value, creating weed components in the grassland and causing digestive problems to animals. Here we report the application of the Bar-HRM (Barcode-DNA High Resolution Melting) analysis method using the universal nuclear plant DNA barcoding region ITS2 for the identification, adulteration and quantification of the main pasture species. Bar-HRM detected Medicago lupulina adulterants in Trifolium pratense seeds as low as 1:100. In conclusion, Bar-HRM analysis could be a faster, with higher resolution and cost effective alternative method to authenticate forage and pasture species and quantitatively detect the purity of their seeds or their feed products.

Abstract:

Abstract: Wild cherry (Prunus avium L.) is a widespread, partially asexual, noble hardwood European species characterized by a scattered distribution, small population sizes, and human exploitation for its valuable wood. These characteristics, especially at the southern limits of the species natural distribution where additional varying stresses may occur, render P. avium populations prone to potential stochastic, genetic, and demographic events. In this study, we used dominant inter simple sequence repeat (ISSR) and codominant simple sequence repeat (SSR) markers to infer the genetic structure of P. avium. Five populations from northern Greece were evaluated based on 46 ISSR and 11 SSR loci. Populations presented a relatively high level of genetic variation, with a mean genetic diversity of H e�=�0.166 and H e�=�0.740 regarding ISSR and SSR analysis, respectively. We observed moderate population differentiation for ISSR (G ST�=�0.113) and SSR (F ST�=�0.097) markers. AMOVA also detected significant differentiation among populations for ISSRs (Φ ST�=�0.338) and SRRs (Φ ST�=�0.162). According to linkage disequilibrium analysis, estimates of effective population size were generally sufficient for maintaining extant genetic variability and evolutionary potential. A possible bottleneck was detected for only one population. In general, it appears that despite the particular characteristics of the P. avium populations studied, genetic stochasticity events were not apparent. The studied populations, located at the rear edge of the species European distribution, reveal a wealth of genetic variation that is very valuable for the genetic conservation of local adaptive gene complexes, especially under contemporary climatic change scenarios.

Abstract: The aim of this work was to setup a DNA based method coupled with High Resolution Melting (HRM) analysis for rice products traceability using five different microsatellite markers to genotyping Basmati and non-Basmati varieties. We also exploit the obtained information to detect the presence of Basmati varieties in commercial rice products. Additionally we used the 8 bp deletion in badh2 gene in combination with HRM to both DNA-typing of the Basmati and non-Basmati varieties and to quantitate accurately adulteration of Basmati rice products with non-Basmati rice products. HRM proved to be a very sensitive tool to genotype rice varieties and detect admixtures as well as able to detect a ratio of 1:100 of non-fragrance in fragrance rice. In conclusion HRM analysis can be a higher resolution, cost effective, alternative method compared to other techniques that could be extended to quantify adulterations in rice varieties and commercial rice food products.

Abstract: DNA based methods have been employed recently for plant species identification and their ingredients in the final food products. The aim of this work was to setup a microsatellite-based method not only to distinguish sweet cherry cultivars but also to compare different techniques for DNA isolation and DNA fragment analysis for verifying the presence of protected designation of origin (PDO) sweet cherry, at the cultivar level, in sweet cherry processed products. Thirteen microsatellites were tested and the combination of the amplification profiles of six of them, characterised by high polymorphism and simple electrophoretic patterns, enabled to distinguish and identify a PDO sweet cherry cultivar used for sweet cherry products. The obtained amplicons were all in the range of 68-258 bp, and were analyzed by capillary electrophoresis (CE) and High Resolution Melting (HRM) analysis. We compare the results obtained by both methods and analyze the differences observed. Dendrograms were constructed using data from HRM analysis and fragment analysis indicating a higher resolution capacity of the HRM method compared to fragment analysis. Moreover, costs, throughput and difficulties to implement HRM analysis and CE methods in the laboratory are discussed. In conclusion HRM analysis can be a cost effective alternative method, with higher resolution, not only to genotype sweet cherry cultivars but also to extend the use to process sweet cherry products using microsatellite markers. (C) 2010 Elsevier Ltd. All rights reserved.

Abstract: Quince is a deciduous tree known to the countries around the Mediterranean since antiquity. Nowadays, quince is used as an ornamental plant, and as a rootstock for pear trees, with its fruit being appreciated mainly for production of jam and sweets rather than for raw consumption. Quince leaves contain compounds with antioxidant, antimicrobial and anticancerous properties that have been the focus of recent research on pharmaceutical and medical uses as well as for food preservatives. An orchard has been established in Greece, composed of quince varieties (Cydonia oblonga, N = 49) collected from different sites of the country (mainly from home gardens), constituting a unique quince gene bank collection for southeast Europe. We made a phenotypic analysis using 26 morphological plus seven agronomical descriptors coupled with molecular techniques in order to examine the genetic diversity within the collection. Principal component analysis using the 33 descriptors identified 10 components explaining the existence of more than 70% of the total variation. Subsequent cluster analysis classified most of the previously identified productive varieties of the quince orchard in the same clade of a dendrogram. Molecular analysis generated by 13 inter-simple sequence repeat primers amplified 139 bands, including 109 polymorphic bands, indicating a level of polymorphism of 79%; mean gene diversity was calculated to be 0.309. Using stepwise multiple regression analysis, a number of markers significantly associated with fire blight susceptibility, yield, mean fruit weight, citric acid content, soluble solid content, and fruit drop were identified. Hence, data extracted by multiple regression analysis could be useful in marker-assisted breeding programs, especially when no previous genetic information is available.

Abstract: Extensive studies on the dry fruits of the model plant arabidopsis (Arabidopsis thaliana) have revealed various gene regulators of the development and dehiscence of the siliques. Peach pericarp is analogous to the valve tissues of the arabidopsis siliques. The stone (otherwise called pit) in drupes is formed through lignification of the fruit endocarp. The lignified endocarp in peach can be susceptible to split-pit formation under certain genetic as well as environmental factors. This phenomenon delays processing of the clingstone varieties of peach and causes economical losses for the peach fruit canning industry. The FRUITFULL (FUL) and SHATTERPROOF (SHP) genes are key MADS-box transcription protein coding factors that control fruit development and dehiscence in arabidopsis by promoting the expression of basic helix-loop-helix (bHLH) transcription factors like SPATULA (SPT) and ALCATRAZ (ALC). Results from our previous studies on peach suggested that temporal regulation of PPERFUL and PPERSHP gene expression may be involved in the regulation of endocarp margin development. In the present study a PPERSPATULA-like (PPERSPT) gene was cloned and characterized. Comparative analysis of temporal regulation of PPERSPT gene expression during pit hardening in a resistant and a susceptible to split-pit variety, suggests that this gene adds one more component to the genes network that controls endocarp margins development in peach. Taking into consideration that no ALC-like genes have been identified in any dicot plant species outside the Brassicaceae family, where arabidopsis belongs, PPERSPT may have additional role(s) in peach that are fulfilled in arabidopsis by ALC.

Abstract: It is important to couple phenotypic analysis with genetic diversity for germplasm conservation in gene bank collections. The use of molecular markers supports the study of genetic marker-trait associations of biological and agronomic interest on diverse genetic material. In this report, 19 Greek traditional sweet cherry cultivars and two international cultivars, which were used as controls, were grown in Greece and characterized for 17 morpho-physiological traits, 15 simple sequence repeat (SSR) loci and 10 inter simple sequence repeat (ISSR) markers. To our knowledge, this is the first report on molecular genetic diversity studies in sweet cherry in Greece. Principal component analysis (PCA) of nine qualitative and eight quantitative morphological parameters explain over 77.33% of total variability in the first five axes. The SSR markers yielded a combined matching probability ratio (MPR) of 9.569 � e�12. The 15 SSR loci produced a total of 92 alleles. Ten ISSR primers generated 91 bands, with an average of 9.1 bands per primer. Expected heterozygosity (gene diversity) values of 15 SSR loci and 10 ISSR markers averaged at 0.683 and 0.369, respectively. Based on stepwise multiple regression analysis (MRA), SSR alleles were found associated with harvest time and fruit polar diameter. Furthermore, three ISSR markers were correlated with fruit harvest and soluble solids and four ISSR markers were correlated with fruit skin color. Stepwise MRA identified six SSR alleles associated with harvest time with a high correlation (P < 0.001), with linear associations with high F values. Hence, data analyzed by the use of MRA could be useful in marker-assisted breeding programs when no other genetic information is available.

Abstract: Prunus avium cultivars widely used in northern Greece were investigated in terms of inter- and intra-cultivar genetic variation and DNA fingerprinting. Based on 11 simple sequence repeats loci, the average number of alleles per locus (N-a = 2.82), probability of identity (P-ID = 0.327), polymorphic information content (0.451) and expected heterozygosity (H-e = 0.494) were within the range reported in similar studies. The most informative markers were BPPCT039 and EMPa018. The cultivars were clearly separated in both an unweighted pair group method with arithmetic mean dendrogram and a multivariate space ordination. Any two cultivars differed on the average at 6.30 loci. The null hypothesis of zero intra-cultivar variability was tested and could not be rejected. Two cultivars (Tragana Edessis and Tragana Sarakinon) were genetically similar, but not identical. This study, the first of its kind for sweet cherry in Greece, presents a useful molecular tool for resolving issues of intra-cultivar variability and synonimity and provides a warranty of genetic identity in the handling and management of local traditional germplasm.

Abstract: As genetic improvement of forest trees still relies heavily on the selection of elite individuals from natural populations, 36 Prunus avium plus trees originating from four northern Greek populations were established in a gene conservation plantation. Their fingerprinting, genetic diversity and relationships were investigated based on 14 variable microsatellite loci. Notable amounts of genetic diversity were observed (P = 93.3, N-a = 3.270). The allelic variation detected was sufficient for unambiguous DNA fingerprinting. Mean polymorphic information content and diversity index were 0.473 and 0.536, respectively. The total value of the probability of identity was 3.13 x 10(-9). A principal coordinate analysis showed the formation of five groups in a two-dimensional multivariate space that explained 56.3 per cent of the total variation. Groups corresponded to populations, although the selected plus trees from one population corresponded to two distinct groups. The wild cherry germplasm collection was compared with a collection of five of the most widely known sweet cherry cultivars of northern Greece, by employing 11 commonly variable microsatellite loci. Results of a principal coordinate analysis indicated the diverse origin of this material as sweet cherry cultivars formed a short independent cluster, which was conspicuously differentiated from all wild cherry individuals in a graph of the first two principal coordinates.

Abstract: Sweet cherry is self-incompatible due to having a gametophytic self-incompatibility system. S alleles in the style and pollen determine possible crossing relationships. Knowledge of the S allele constitution of cultivars is important for sweet cherry growers and breeders. Recently, molecular methods have been developed to distinguish between S alleles in sweet cherry. The S allele genotypes of 21 sweet cherry cultivars widely grown in Greece, including 19 not previously genotyped, were determined based on their S-RNase gene sequences using PCR analysis. Eight different S alleles in ten combinations were distinguished and two new S-genotypes (S1S13 and S4S30) were documented. Four alleles, S-1, S-3, S-4, and S-9 were widespread and together were responsible for 85% of the S-haplotypes. Therefore many of the cultivar combinations were semi-compatible. In Greece, semi-compatibility was shown to correlate with low yields. However, the cultivar 'Hybrid Tragana Edessis x Unknown' (S3S13) and the cultivar 'Kapsiotika' (S2S5) carry rare S-haplotypes and are therefore fully cross-compatible with most of the cultivars analysed.