Apostolos Kalivas

Heterotopic expression of B-class floral homeotic genes PISTILLATA/GLOBOSA supports a modified model for crocus (Crocus sativus L.) flower formation

For uncovering and understanding the molecular mechanisms controlling flower development in cultivated Crocus sativus and particularly the transformation of sepals in outer whorl (whorl 1) tepals, we have cloned and characterized the expression of a family of five PISTILLATA/GLOBOSA-like (PI/GLO-like) MADS-box genes expressed in the C. sativus flower. The deduced amino acid sequences of the coded proteins indicated high homology with members of the MADS-box family of transcription factors, and particularly with other members of the PI/GLO family of MADS-box proteins that control floral organ identity. PI/GLO expression studies in cultivated C. sativus uncover the presence of PI/GLO transcripts not only in the second and third whorls of flower organs as expected, but also in the outer whorl tepals that are the sepals in most typical flowers. This heterotopic expression of both B-class genes: PI/GLO and AP3/DEF, known to form heterodimers for stamens and petals (petaloid inner whor l–whorl 2-tepals in C. sativus), explains the homeotic transformation of sepals into outer whorl tepals in this species. Analysis of PI/GLO sequences from C. sativus for putative targets to known micro-RNAs (miRNAs) showed that the target site for ath-miRNA167 found in Arabidopsis thaliana PI is not present in C. sativus, however, the PI/GLO sequences may be regulated by an ath-miRNA163.

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.

Is the genetic diversity of small scattered forest tree populations at the southern limits of their range more prone to stochastic events? A wild cherry case study by microsatellite-based markers

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 He = 0.166 and He = 0.740 regarding ISSR and SSR analysis, respectively. We observed moderate population differentiation for ISSR (GST = 0.113) and SSR (FST = 0.097) markers. AMOVA also detected significant differentiation among populations for ISSRs (ΦST = 0.338) and SRRs (ΦST = 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.

Isolation, Characterization, and Expression Analysis of an NAP-Like cDNA from Crocus (Crocus sativus L.)

In Arabidopsis, it has been shown that the B-class MADS-box genes are expressed in the developing whorl 2 petals and whorl 3 stamens throughout the ontogeny of these organs. APETALA3/PISTILLATA (AP3/PI) heterodimers act as an inducer of a regulator-coding gene called NAP, a homologue to NAC-like transcription factor genes, required for meristem establishment, separation of floral organs, and leaf senescence. In monocots like crocus (Crocus sativus) cultivated for saffron production, the expression of B-class MADS-box CsatAP3/CsatPI genes extends to whorl 1 where petaloid tepals are formed. We report here for the first time the cloning and characterization of an NAC-like gene, named CsatNAP, from crocus. The sequence alignment indicated that CsatNAP protein contains the typical domain structure of plant NAC proteins consisted of the conserved five subdomains of the N-terminal NAC domain. Phylogenetic analysis revealed that CsatNAP protein falls in the subgroup NAP of the NAC proteins. Expression analysis of crocus NAP indicates that it is expressed in whorl 1, supporting the hypothesis that also in crocus, AP3/PI-like is associated with the expression of Crocus NAP gene. The expression patterns of CsatNAP cDNA were studied in flowers and different flower organs as well as in leaves and different part of leaves during senescence. The CsatNAP transcripts were detected in whole flowers and flower organs. Promoter analysis of CsatNAP revealed a number of putative common cis-regulatory elements and, among them, two CArG boxes indicative of its control by MADS box transcription factors known to bind on CArG sites. Micro-RNA (miRNA) target analysis showed that the NAP sequence of Crocus contain a possible target site for miRNA164. Furthermore, CsatNAP showed increased expression in senescence leaves compared to the green ones.

Isolation of a CENTRORADIALIS/TERMINAL FLOWER1 homolog in saffron (Crocus sativus L.): characterization and expression analysis

Genes in the phosphatidyl-ethanolamine-binding protein (PEBP) family are instrumental in regulating the fate of meristems and flowering time. To investigate the role of these genes in the monocotyledonous plant Crocus (Crocus sativus L), an industrially important crop cultivated for its nutritional and medicinal properties, we have cloned and characterized a CENTRORADIALIS/TERMINAL FLOWER1 (CEN/TFL1) like gene, named CsatCEN/TFL1-like, the first reported CEN/TFL1 gene characterized from such a perennial geophyte. Sequence analysis revealed that CsatCEN/TFL1 shows high similarity to its homologous PEBP family genes CEN/TFL1, FT and MFT from a variety of plant species and maintains the same exon/intron organization. Phylogenetic analysis of the CsatCEN/TFL1 amino acid sequence confirmed that the isolated sequences belong to the CEN/TFL1 clade of the PEBP family. CsatCEN/TFL1 transcripts could be detected in corms, flower and flower organs but not in leaves. An alternative spliced transcript was also detected in the flower. Comparison of expression levels of CsatCEN/TFL1 and its alternative spliced transcript in wild type flower and a double flower mutant showed no significant differences. Overexpression of CsatCEN/TFL1 transcript in Arabidopsis tfl1 plants reversed the phenotype of early flowering and terminal flowering of the tfl1 plants to a normal one. Computational analysis of the obtained promoter sequences revealed, next to common binding motifs in CEN/TFL1-like genes as well as other flowering gene promoters, the presence of two CArG binding sites indicative of control of CEN/TFL1 by MADS-box transcription factors involved in crocus flowering and flower organ formation.

famRCA-RACE: a rolling circle amplification race for isolating a family of homologous cDNAs in one reaction and its application to obtain NAC genes transcription factors from crocus (Crocus sativus) flower.

We describe an improvement of the RCA-RACE (rolling circle amplification—rapid amplification of cDNA ends) method, called family RCA-RACE (famRCA-RACE). The method is based on the generation of circular cDNA fragments, followed by rolling circle amplification of the circular cDNA using ϕ29 DNA polymerase and the application of PCR using degenerate outworking primers, designed for a conserved region of homologous genes, that allows the isolation of homologous cDNA sequences expressed in the mRNA preparation in a single polymerase chain reaction (PCR). As an example we present the isolation of seven NAC-like transcription factors cDNA sequences expressed in Crocus sativus flower, used for saffron production. Sequence alignment revealed that CsatNAC proteins contain the typical domain structure of plant NAC proteins, consisting of the conserved N-terminal NAC domain used to design the primers and the five subdomains. Phylogenetic analysis revealed that CsatNAC proteins fall in subgroup I of the NAC family of proteins.

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.

Comparative metagenomics reveals alterations in the soil bacterial community driven by N-fertilizer and Amino 16® application in lettuce

Nutrients in the form of fertilizers and/or other additives such as amino acids, dramatically influence plant development and growth, plant nutrient composition and the level of soil pollution. Moreover, the treatment of soil microbiota is emerging as a new strategy in plant breeding to achieve desirable traits. Thus, integrated study of fertilizer application and soil microbiota might lead to a better understanding of soil-plant interactions and inform the design of novel ways to fertilize plants. Herein we report metagenomics data for soil microbiota in lettuce (Lactuca sativa) treated with fertilizer, amino acids or their combinations as follows: N-fertilizer + Amino16®, Amino16®, N-fertilizer and no treatment control. Data have been deposited in the NCBI Sequence Read Archive (SRA) (accession number: PRJNA388765).