Use of genetic markers for the detection of off-types for DUS phenotypic traits in the inbreeding crop, barley

Detection of crop off-types is of interest for multiple uses, including the assessment of uniformity for new plant variety applications during distinctness, uniformity and stability (DUS) testing for the awarding of plant breeders’ rights (PBR). Here, we investigate whether genetic markers, in this case Kompetitive Allele-Specific PCR (KASP), can be used for the identification off-types for phenotypes assessed for DUS in the inbreeding cereal crop, barley (Hordeum vulgare). To demonstrate proof of principle, KASP markers diagnostic for phenotypic expression of nine DUS phenotypes, and DNA from two barley varieties (‘Pelican’ and ‘Felicie’) carrying contrasting alleles at each marker were used. We found that for the majority of markers, it was possible to robustly call alleles down to template DNA concentrations of 2 ng, but not ≤ 0.2 ng. When used in mixtures of DNA consisting of ‘Felicie’ DNA spiked with different concentrations of ‘Pelican’ DNA, robust allele calling was possible in DNA mixtures down to 18 ng:2 ng. Collectively, this demonstrates that where diagnostic markers are available, molecular identification of a single off-type for a given DUS trait within a bulk of ten individuals should be possible. We validated this assumption, with all of the diagnostic genetic markers investigated found to robustly detect DUS off-types at a frequency of 10% in DNA extracted from tissue collected from pools of 10 individuals. Ultimately, this work demonstrates that, where diagnostic polymorphisms are known for DUS traits, KASP markers should be able to robustly detect off-types or cross-contamination within DNA samples from a diploid inbred species down to 10%. While just two varieties that contrasted for the eight DUS targeted were investigated in this study, as the markers used are diagnostic for their relevant phenotype (or a proportion of the variation observed for that phenotype), in theory the approach should be valid for any variety studied— although the introduction of novel alleles via spontaneous mutation or more exotic germplasm pools may mean that marker sets would need to be periodically added to or updated. However, we nevertheless demonstrate the principle that, for a subset of DUS traits, molecular markers can now be robustly used as a tool towards determining all three components of the DUS testing process in barley. These results are relevant for the assessment of varietal uniformity by crop breeders, crop testing authorities and germplasm maintenance, as well as highlighting the potential use of bulk samples rather than individual plant samples for assessment of distinctness by molecular methods

A Novel Sphingomonas sp. Isolated from Argan Soil for the Polyhydroxybutyrate Production from Argan Seeds Waste

Polyhydroxybutyrate (PHB) is a biodegradable bio-based polymer synthesized by microorganisms under unfavorable conditions from agro-industrial residues as a source of carbon. These aspects make the bio-based polymer attractive for the mass production of biodegradable plastics, and a definitive replacement for petroleum-based plastics. The aim of this work was to characterize the putative PHB-producing bacterium 1B isolated from the argan soil, to identify the polymer produced, and quantify the PHB production using argan seeds waste. DNA extraction, PCR, and Sanger sequencing were conducted for the molecular identification of strain 1B; the residual biomass and the PHB quantification were measured and compared in the presence of simple sugars and pretreated argan seeds waste. The 1B growth and PHB synthesis were optimized by selecting physical and nutritional parameters: temperature, incubation time, pH, NaCl concentration, and nitrogen sources concentrations. A preliminary characterization of the bio-based polymer extracted was conducted by UV–Visible spectrophotometry and FTIR analysis. The strain 1B was identified as belonging to the genus Sphingomonas. The PHB final yield was higher in a growth culture enriched with argan waste (3.06%) than with simple sugars. The selected conditions for the bacterial optimal growth incremented the PHB final yield to 6.13%, while the increase in the argan residue concentration from 1 to 3% in a larger culture volume led to the PHB final yield of 8.16%. UV–Visible spectrophotometry of the extracted sample reported a remarkable peak at 248 nm, as well as FTIR spectra analysis, showed peaks at 1728 and 1282 wavenumber/cm. Both preliminary characterizations demonstrated that the extracted sample is the bio-based polymer polyhydroxybutyrate. The results reported in this work reveal how the costless available argan seeds can be used for polyhydroxybutyrate production using a novel Sphingomonas species

Current limitations and future prospects of detection and biomonitoring of NIS in the Mediterranean Sea through environmental DNA

The biodiversity of the Mediterranean Sea is currently threatened by the introduction of Non-Indigenous Species (NIS). Therefore, monitoring the distribution of NIS is of utmost importance to preserve the ecosystems. A promising approach for the identification of species and the assessment of biodiversity is the use of DNA barcoding, as well as DNA and eDNA metabarcoding. Currently, the main limitation in the use of genomic data for species identification is the incompleteness of the DNA barcode databases. In this research, we assessed the availability of DNA barcodes in the main reference libraries for the most updated inventory of 665 confirmed NIS in the Mediterranean Sea, with a special focus on the cytochrome oxidase I (COI) barcode and primers. The results of this study show that there are no barcodes for 33.18% of the species in question, and that 45.30% of the 382 species with COI barcode, have no primers publicly available. This highlights the importance of directing scientific efforts to fill the barcode gap of specific taxonomic groups in order to help in the effective application of the eDNA technique for investigating the occurrence and the distribution of NIS in the Mediterranean Se

Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.</p

Dr. Tibaldi, et al reply

We are pleased by the interest of Ozen and colleagues1 for our work2 and appreciate their choice to test our predictive model of response to anakinra in their cohort of children with systemic juvenile idiopathic arthritis (sJIA). The authors used the same definition of complete clinical response (CCR) as in our study and quantified the activity of systemic disease through the systemic manifestation score (SMS) that we developed2

Exploring the Biodiversity of a European NATURA 2000 Mediterranean Lagoon through eDNA Metabarcoding

Coastal lagoons are considered important habitats both for ecological functions and biodiversity worldwide. Thus, they provide relevant ecosystem services and valuable natural resources. However, coastal lagoons are highly susceptible to anthropogenic pressures that can cause biodiversity losses and require specific biomonitoring programs as well as management measures. In this research, we applied environmental DNA (eDNA) metabarcoding to investigate the biodiversity of a poorly known Mediterranean lagoon included in the European Natura 2000 Network. We used the cytochrome oxidase I (COI) gene marker to capture the entire biodiversity of this highly diversified aquatic coastal environment. With a low sampling effort and rapid laboratory practices, a large amount of valuable biodiversity data was generated and analyzed. Interestingly, this straightforward and broad molecular surveying of biodiversity unveiled a wide variety of taxonomic groups, such as benthic macroinvertebrates, zooplankton, phytoplankton, and macroalgae, which are frequently used as ecological indicators. We were able to detect species that were previously morphologically identified, as well as species never identified before. This research underlines the validity of eDNA metabarcoding in assessing the biodiversity in a poorly known and protected Mediterranean lagoon ecosystem, as well as in identifying the early warnings of environmental stressors. Finally, the research highlights the need to investigate multiple target genes and primers set for a larger analysis of specific species

Exploring the Biodiversity of a European NATURA 2000 Mediterranean Lagoon through eDNA Metabarcoding

Coastal lagoons are considered important habitats both for ecological functions and biodiversity worldwide. Thus, they provide relevant ecosystem services and valuable natural resources. However, coastal lagoons are highly susceptible to anthropogenic pressures that can cause biodiversity losses and require specific biomonitoring programs as well as management measures. In this research, we applied environmental DNA (eDNA) metabarcoding to investigate the biodiversity of a poorly known Mediterranean lagoon included in the European Natura 2000 Network. We used the cytochrome oxidase I (COI) gene marker to capture the entire biodiversity of this highly diversified aquatic coastal environment. With a low sampling effort and rapid laboratory practices, a large amount of valuable biodiversity data was generated and analyzed. Interestingly, this straightforward and broad molecular surveying of biodiversity unveiled a wide variety of taxonomic groups, such as benthic macroinvertebrates, zooplankton, phytoplankton, and macroalgae, which are frequently used as ecological indicators. We were able to detect species that were previously morphologically identified, as well as species never identified before. This research underlines the validity of eDNA metabarcoding in assessing the biodiversity in a poorly known and protected Mediterranean lagoon ecosystem, as well as in identifying the early warnings of environmental stressors. Finally, the research highlights the need to investigate multiple target genes and primers set for a larger analysis of specific species

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum. A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the beta-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families

Environmental DNA detects biodiversity and ecological features of phytoplankton communities in Mediterranean transitional waters

Abstract Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and evolvability of natural communities. Transitional water ecosystems are more sensitive to habitat reduction and degradation and, thus, are more exposed to biodiversity declines requiring biodiversity monitoring programs for their conservation. Environmental DNA (eDNA) metabarcoding represents a high-throughput tool for biodiversity assessment that is facilitating data collection for biodiversity monitoring. In this study, we applied, for the first time, eDNA metabarcoding in a Mediterranean coastal lagoon to assess the ecological features of eukaryotic phytoplankton communities. We sampled water in seven different lagoon sites and amplified the extracted DNA with primers targeting the variable region 4 (V4) of the 18S rRNA gene marker. The results demonstrated the validity of eDNA studies to provide insights into lagoon phytoplankton composition, establish the structure and spatial variation of phytoplankton communities, and evaluate its correlation to abiotic factors. Finally, the genetic distances analysis suggests that the different spatial distribution of OTUs, at least for the Tetraselmis genus, reflects the genetic background