Aliki Xanthopoulou

DNA barcode ITS2 coupled with high resolution melting (HRM) analysis for taxonomic identification of Sideritis species growing in Greece

Identification of genotypes in Sideritis is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Sideritis species. Species- and genotype-specific DNA markers are very useful for plant identification, breeding and preservation programs. Herein, a real-time polymerase chain reaction (PCR) of ITS2 barcode region coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on seven Sideritis species growing in Greece. The HRM assay developed in this study is a rapid and straightforward method for the identification and discrimination of the investigated Sideritis species. This assay is simple compared to other genotyping methods as it does not require DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of Sideritis species.

Microsatellite high-resolution melting (SSR-HRM) analysis for genotyping and molecular characterization of an Olea europaea germplasm collection

Olea europaea L. has been cultivated in the Mediterranean region for thousands of years and is of major economic importance. The origin of olive cultivars remains as complex to trace as their identification. Thus, their molecular characterization and discrimination will enable olive germplasm management. In addition, it would be a useful tool for authentication of olive products. High-resolution melting (HRM) analysis, coupled with five microsatellite markers, was integrated to facilitate molecular identification and characterization of main O. europaea cultivars collected from the National Olive Tree Germplasm Collection established in Chania, Greece. The five microsatellite loci used were highly informative and generated a unique melting curve profile for each of the 47 cultivars and for each microsatellite tested. In particular, three microsatellite markers (DCA03, DCA09 and DCA17), which generated 29 HRM profiles, were sufficient to genotype all the olive cultivars studied, highlighting their potential use for cultivar identification. Furthermore, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method well suited for molecular characterization of olive cultivars.

Summer Squash Identification by High-Resolution-Melting (HRM) Analysis Using Gene-Based EST–SSR Molecular Markers

Cucurbita pepo (squash, pumpkin, gourd), a worldwide cultivated vegetable of American origin, is extremely variable in fruit characteristics. Most of its widely grown commercial types are known as summer squashes and belong to the elongated forms of C. pepo ssp. pepo (Cocozelle, Vegetable marrow and Zucchini groups). Here, we have integrated the high-resolution-melting (HRM) analysis method with expressed sequence tags–simple sequence repeat (EST–SSR) marker genotyping, in order to facilitate the identification of 36 summer squash landraces originated from Greece. The six EST–SSR loci used were informative and generated a unique melting curve profile of EST-derived microsatellites for each accession allowing their comparison and classification. Moreover, HRM was highly informative, as by using only four microsatellite markers we were able to discriminate 36 summer squash landraces and by using six EST–SSRs. We were able to construct a highly informative and discriminative dendrogram where the 36 genotypes were classified in six distinct clusters. Furthermore, we acquired information about the genes containing the EST–SSRs using bioinformatics tools. We found that the EST–SSRs used in this study were hybridizing to genes involved in stress response to heavy metals and biotic stresses or the production of flavonoids or symporters of important nitrogen sources, like xanthine and uric acid amongst others. The results presented here suggest that the panel of EST–SSR markers used in combination with HRM analysis could be useful in a variety of applications, like squash biodiversity assessment but most importantly in managing squash germplasm to improve breeding programs.

Sweet Cherry Cultivar Identification by High-Resolution-Melting (HRM) Analysis Using Gene-Based SNP Markers

Single nucleotide polymorphisms (SNPs) provide an important tool for cultivar identification in studies of genetic diversity, but until now, the time-consuming and costly nature of DNA sequencing has limited the identification of new markers. Herein, we describe the application of high-resolution melting (HRM), a recent enhancement to traditional DNA melting analysis, for the characterization of polymerase chain reaction products and the identification of nine gene-based SNPs for distinguishing the main Greek sweet cherry cultivars. The expected heterozygosity value of nine SNPs averaged at 0.518. The combined power of discrimination for the SNP markers was 0.999969. The ability of HRM to accurately discern nucleotide changes in a DNA sequence makes it a cost- and time-effective alternative to traditional sequencing for the detection of gene-based SNPs.

Exploring priming responses involved in peach fruit acclimation to cold stress

Cold storage of fruit may induce the physiological disorder chilling injury (CI); however, the molecular basis of CI development remains largely unexplored. Simulated conditions of CI priming and suppression provided an interesting experimental system to study cold response in fruit. Peaches (cv. June Gold) at the commercial harvest (CH) or tree-ripe (TR) stages were immediately exposed to cold treatment (40 d, 0 °C) and an additional group of CH fruits were pre-conditioned 48 h at 20 °C prior to low-temperature exposure (pre-conditioning, PC). Following cold treatment, the ripening behaviour of the three groups of fruits was analysed (3 d, 20 °C). Parallel proteomic, metabolomic and targeted transcription comparisons were employed to characterize the response of fruit to CI expression. Physiological data indicated that PC suppressed CI symptoms and induced more ethylene biosynthesis than the other treatments. Differences in the protein and metabolic profiles were identified, both among treatments and before and after cold exposure. Transcriptional expression patterns of several genes were consistent with their protein abundance models. Interestingly, metabolomic and gene expression results revealed a possible role for valine and/or isoleucine in CI tolerance. Overall, this study provides new insights into molecular changes during fruit acclimation to cold environment.

De novo transcriptome assembly of two contrasting pumpkin cultivars

Cucurbita pepo (squash, pumpkin, gourd), a worldwide-cultivated vegetable of American origin, is extremely variable in fruit characteristics. However, the information associated with genes and genetic markers for pumpkin is very limited. In order to identify new genes and to develop genetic markers, we performed a transcriptome analysis (RNA-Seq) of two contrasting pumpkin cultivars. Leaves and female flowers of cultivars, ‘Big Moose’ with large round fruits and ‘Munchkin’ with small round fruits, were harvested for total RNA extraction. We obtained a total of 6 GB (Big Moose; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056882) and 5 GB (Munchkin; http://www.ncbi.nlm.nih.gov/Traces/sra/?run=SRR3056883) sequence data (NCBI SRA database SRX1502732 and SRX1502735, respectively), which correspond to 18,055,786 and 14,824,292 150-base reads. After quality assessment, the clean sequences where 17,995,932 and 14,774,486 respectively. The numbers of total transcripts for ‘Big Moose’ and ‘Munchkin’ were 84,727 and 68,051, respectively. TransDecoder identified possible coding regions in assembled transcripts. This study provides transcriptome data for two contrasting pumpkin cultivars, which might be useful for genetic marker development and comparative transcriptome analyses.

Genetic diversity and metabolic profile of Salvia officinalis populations: implications for advanced breeding strategies

AbstractMain conclusionAs a result of this work, we were able to characterize seven indigenous to GreeceSalvia officinalispopulations using genetic and metabolomic tools. These tools can be used to select the most promising genotypes, capable to design future breeding programs for high valuable varieties. An initial investigation was carried out to compare the genetic and metabolic diversity in S. officinalis grown in Greece and to discern the relationship between the two sets of data. Analysis of inter-simple sequence repeats (ISSR) revealed significant genetic differences among seven sage populations, which were grouped into three main clusters according to an UPGMA ISSR data-based dendrogram and Principle Coordinate Analysis. 80 loci were scored of which up to 90% were polymorphic at species level. According to the composition of their essential oil, the populations were classified into two chemotypes: 1.8 cineole/α-thujone and α-thujone/1.8 cineole. Additionally, a targeted ultra performance liquid chromatography (UPLC–MS/MS) method was used to qualify and quantify phenolic compounds in methanolic extracts of the seven sage genotypes according to which they were districted in six clusters among the sage populations. The main compounds characterizing the seven genotypes were rosmarinic acid and carnosol, followed by apigenin-7-O-glucoside (Ap7glc), and luteolin-7-O-glucoside (Lu7glc). The correlation between matrices obtained from ISSR data and metabolic profiles was non-significant. However, based on the differences in metabolic fingerprint, we aimed to define populations using as main selection criteria the high polyphenol content and desired essential oil composition, using state to the art analytical tools for the identification of parent lines for breeding programs.

Identification of Phytophthora species by a high resolution melting analysis: an innovative tool for rapid differentiation

A new molecular method via the high resolution melting (HRM) analysis of the Ypt1 gene non-coding regions was validated for ten Phytophthora species with a broad host range from forest trees to crop species. The melting curve analysis of the amplicons specifically grouped all species into 10 respective unique and distinct HRM curve profiles. The analysis of the normalised HRM melting curves, assigning P. nicotianae as a normalised reference genotype, revealed that the genotype similarities among all the species were adequately low, indicating that Ypt1 marker was sufficient to identify and differentiate the tested species. This HRM method is rapid and reproducible allowing the identification of Phytophthora species and the screening of eventual variants eliminating the separate steps and reducing the risk of contamination.

Fast and Accurate Screening of Solanum melongena with High-Resolution Melting Analysis for Resistance to Fusarium Wilt

ABSTRACT The causal agent of Fusarium wilt, Fusarium oxysporum Schlecht. f. sp. melongenae, produces a vascular disease of eggplant (Solanum melongena L.). Resistant germplasm with wide variation is essential for controlling this disease via breeding, and understanding the genetic background helps design breeding strategies. This study screened 36 S. melongena germplasm landraces and cultivars for Fusarium wilt resistance. Fusarium oxysporum is a particularly complex species composed of several pathogenic strains arranged into groups called formae speciales, which pose a challenge to developing resistant plant varieties. Screening and selecting resistant plant sources could play an important role in developing resistant or tolerant plant lines. High-resolution melting analysis coupled with a sequenced characterized amplified region marker for fast screening and accurate identification of Fusarium wilt–resistant eggplant has been developed. The method could be useful in breeding where great numbers of plants need to be tested during germplasm evaluation and for single plant selection. Breeders could use these findings to choose Fusarium wilt–resistant cultivars/landraces for their improvement.

Rapid and accurate identification of black aspergilli from grapes using high-resolution melting (HRM) analysis: Identification of black aspergilli from grapes using HRM analysis

BACKGROUND Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required. RESULTS The aim of this study was to develop a high-resolution melting polymerase chain reaction (HRM-PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species-specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi. CONCLUSION To our knowledge this is the first report on the development of an HRM-PCR assay for the identification of black Aspergillus species in grape samples.