Genetic Diversity Assessment in Plants from Reduced Representation Sequencing Data.

Genetic diversity refers to the variety of genetic traits within a population or a species. It is an essential aspect of both plant ecology and plant breeding because it contributes to the adaptability, survival, and resilience of populations in changing environments. This chapter outlines a pipeline for estimating genetic diversity statistics from reduced representation or whole genome sequencing data. The pipeline involves obtaining DNA sequence reads, mapping the corresponding reads to a reference genome, calling variants from the alignments, and generating an unbiased estimation of nucleotide diversity and divergence between populations. The pipeline is suitable for single-end Illumina reads and can be adjusted for paired-end reads. The resulting pipeline provides a comprehensive approach for aligning and analyzing sequencing data to estimate genetic diversity.

Identification of Novel Induced Mutations in Seed and Vegetatively Propagated Plants from Reduced Representation or Whole Genome Sequencing Data.

Treatment of plants with chemical mutagens results primarily in the production of novel single nucleotide variants. Mutagenesis is a mostly random process and as such plants derived from mutagenesis of different seeds or in vitro material are expected to accumulate different mutations. An important step in the creation of a mutant population for forward or reverse genetics is the choice of treatment conditions (e.g., dosage) such that sufficient mutations accumulate while not adversely affecting propagation of the plant. DNA sequencing provides a quick method to evaluate the effect of different treatment conditions and their effect on the density and spectrum of accumulated mutations. Whole genome sequencing or reduced representation sequencing is carried out followed by mapping to a reference genome and production of a Variant Call Format (VCF) file. We provide here a method for generating a multi-sample VCF from mutagenized plants and describe a new tool to streamline the process of recovering unique induced mutations and determining their possible effect on gene function.

A General Protocol for Gene Expression Analysis in Chemically Mutant Coffee Plants in Response to Rust (Hemileia vastatrix Berk & Broome) Infection.

Coffea spp. is the source of one of the most widely consumed beverages in the world. However, the cultivation of this crop is threatened by Hemileia vastatrix Berk & Broome, a fungal disease, which reduces the productivity and can cause significant economic losses. In this protocol, coffee leaf segment derived from a chemical mutagenesis process are inoculated with uredospores of the pathogen. Subsequently, the gene expression changes are analyzed over the time (0, 5, 24, 48, and 120 h) using quantitative real-time polymerase chain reaction (RT-qPCR). The procedures and example data are presented for expression analysis in the CaWRKY1 gene. This procedure can be applied for quantitative analysis of other genes of interest to coffee breeders and scientists for elucidating the molecular mechanisms involved in the interaction between the plant and pathogen, potentially leading to the development of more efficient approaches for managing this disease.

Transcriptomic Data Analysis Using the Galaxy Platform: Coffee (Coffea arabica L.) Flowers as Example.

Coffee, an important agricultural product for tropical producing countries, is facing challenges due to climate change, including periods of drought, irregular rain distribution, and high temperatures. These changes result in plant water stress, leading to significant losses in coffee productivity and quality. Understanding the processes that affect coffee flowering is crucial for improving productivity and quality. In this chapter, we describe a protocol for transcriptome analysis using available Internet software, mainly in the Galaxy Platform, using RNA-Seq data from flowers collected from different parts of the coffee tree. The methods presented in this chapter provide a comprehensive protocol for transcriptome analysis of differentially expressed genes from flowers of coffee plant. This knowledge can be utilized in coffee genetic improvement programs, particularly in the selection of cultivars that are tolerant to water deficit.

Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Hepatitis E and diaphragmatic dysfunction: Case series and review of the literature.

INTRODUCTION: The causes of diaphragmatic paresis are manifold. An association between neuralgic amyotrophy (NA) and hepatitis E virus (HEV) infection has been reported. We wondered about the prevalence of diaphragmatic disfunction and hepatitis E infection in our clinic. METHODS: From July 1st, 2020 to August 31st, 2023, patients presenting with diaphragmatic dysfunction and simultaneous clinical symptoms of an acute NA, or a history of NA, as well as patients with previously unexplained diaphragmatic dysfunction were examined for HEV infection. RESULTS: By August 31st, 2023, 13 patients with diaphragmatic dysfunction and HEV infection were diagnosed (4 women, 9 men). Mean age was 59 ± 10 years. Liver values were normal in all patients. The median latency to diagnosis was five months (range: 1-48 months); nine patients, 4 of them with typical symptoms of NA, presented with acute onset three patients showed bilateral diaphragmatic dysfunction. All patients had a positive IgG immunoblot. Seven patients, three with NA, had an elevated hepatitis E IgM titer and six of them also a positive IgM immunoblot. In all cases, O2C hepatitis genotype 3 was identified. In eight cases, all those with a high IgG titer >125, the O2 genotype 1 was also detected. CONCLUSION: NA that shows involvement of the phrenic nerve resulting in diaphragmatic dysfunction and dyspnoea, may be associated with HEV infection. The observation of 13 patients with diaphragmatic dysfunctions and HEV infection within a period of three years indicates a high number of undetected HEV-associated diaphragmatic dysfunction in the population, especially in the absence of NA symptoms. Therefore, even in diaphragmatic dysfunction without NA symptoms and causative damaging event, HEV infection should be considered, as it may represent a subform of NA with only phrenic nerve involvement. Therapy of HEV-associated diaphragmatic dysfunction in the acute phase is an open question. In view of the poor prognosis for recovery, antiviral therapy should be discussed. However, no relevant data are currently available.

Outcomes of accelerated corneal cross-linking for pediatric and adult keratoconus: a comparative study.

PURPOSE: To compare the visual, refractive, and topographic outcomes of a high irradiance accelerated corneal crosslinking (ACXL) protocol after a 12-month follow-up between pediatric and adult patients with progressive keratoconus (KC). METHODS: Retrospective, comparative, cohort study. Patients with KC were divided into two groups: pediatric (≤ 18 years) and adult (> 18 years). All of them were managed with epi-OFF ACXL (30 mW/cm2, 8 min, pulsed 1:1 on and off = 7.2 J/cm2). Visual, refractive, and topographic values were measured preoperatively and at 1, 3, 6, and 12 months postoperative. KC progression, defined as a Kmax increase of ≥ 1D during follow-up, was recorded. RESULTS: Eighty-nine eyes (53 patients) were included for analysis; 45 (50.6%) eyes were from pediatric patients and 44 (49.4%) from adults. At one-year follow-up, pediatric patients experienced significantly higher rates of progression (22.2% vs. 4.5%, p = .014). Contrariwise, female gender (Beta = - 3.62, p = .018), a baseline uncorrected visual acuity of Snellen ≥ 20/60 (Beta = - 5.96, p = .007), and being ≥ 15 years at ACXL treatment (Beta = - 0.31, p = .021) were associated with non-progressive disease. A significant improvement in best-corrected visual acuity, Kmin, Km, and Kmax was recorded in both groups. Overall, 86.5% of eyes from both groups showed Kmax stabilization or improvement. CONCLUSIONS: Despite the similarity in visual, refractive, and topographic outcomes in both groups, younger age was associated with KC progression after ACXL at one year of follow-up.

Can environmental factors increase oxidative responses in fish exposed to polycyclic aromatic hydrocarbons (PAHs)?

Relations among polycyclic aromatic hydrocarbons (PAHs), biomarkers of oxidative stress (lipid peroxidation, glutathione, and glutathione S-transferase activity), and the possible influence of environmental factors (temperature, pH, and salinity) were assessed in situ for specimens of Ramnogaster arcuata, a native estuarine fish. PAH levels found in the muscular tissue of R. arcuata ranged from 0.7 to 293.4 ng g-1 wet weight with petrogenic and pyrolytic inputs. Lipid peroxidation in the liver showed positive correlations (P < 0.05) with total PAHs (r = 0.66), 3-ring (r = 0.66) and 4-ring PAHs (r = 0.52) and glutathione in muscle (r = 0.58). Significant positive correlations (P < 0.05) were also evidenced between muscular glutathione with total (r = 0.62) and 3-ring PAHs (r = 0.75). Hepatic glutathione S-transferase negatively correlated with 4-ring PAHs (r = -0.58). These correlations suggest that lipid peroxidation and muscular glutathione could be good biomarkers for complex mixtures of PAHs, and hepatic glutathione S-transferase could be a suitable biomarker for 4-ring PAHs. Furthermore, significant correlations (P < 0.05) of environmental factors with PAH levels and biomarkers were observed, especially pH with 3-ring PAHs (r = -0.65), lipid peroxidation (r = -0.6), glutathione in the liver (r = -0.73) and muscle (r = -0.75); and temperature with 2-ring PAHs (r = -0.75) and glutathione in muscle (r = 0.51). The data suggest an influence of physicochemical parameters which could be driving a shift in PAH toxicity in R. arcuata. These results are essential for an integrated understanding of ecotoxicology and could help to predict environmental effects in present and future scenarios of ocean warming and acidification.

New dockside eDNA based protocol to detect the seaweed Asparagopsis armata evaluated by stakeholders.

Early detection of invasive species is crucial to deal effectively with biological invasions in ports, which are hotspots of species introductions. In this study, a simplified end-time PCR methodology conducted on eDNA from water samples was developed for rapid detection of the invasive seaweed Asparagopsis armata (four hours from water collection to result visualization). It was tested dockside in four international Spanish ports in presence of stakeholders, whose feedback was obtained to explore the real applicability of this biotechnology. Although biological invasions were not a main concern for them, results indicate a unanimous approval of the methodology by the stakeholders, having detected the presence of A. armata in three of the ports. Stakeholders suggested further developments for easier application of the tool and multiple species detection, to be adopted for the control of invasive species in ports.

Epidemiology, clinical features, and classification of 3,404 patients with uveitis: Colombian Uveitis Multicenter Study (COL-UVEA).

PURPOSE: To describe the epidemiology, clinical features, and classification of uveitis in a large cohort of Colombian patients. METHODS: Data were collected from seven ophthalmological referral centers in the four main cities in Colombia. The study included patients with a confirmed diagnosis of uveitis from January 2010 to December 2022. Information on demographics, ophthalmic examination findings, uveitis classification, and etiology was recorded. RESULTS: The study reviewed 3,404 clinical records of patients with uveitis. The mean age at diagnosis was 41.1 (SD 19.0) years, and 54.2% of the patients were female. Overall, 1,341(39.4%) were infectious, 626 (18.4%) non-infectious, and four masquerade syndromes (0.1%). The most common types of uveitis were unilateral (66.7%), acute (48.3%), and non-granulomatous (83%). Anterior uveitis was the most common anatomical localization (49.5%), followed by posterior uveitis (22.9%), panuveitis (22.3%), and intermediate uveitis (5.2%). A diagnosis was established in 3,252 (95.5%) cases; idiopathic was the most common cause (27.7%), followed by toxoplasmosis (25.3%) and virus-associated uveitis (6.4%). The age group between 30 and 50 exhibited the highest frequency of uveitis. CONCLUSION: This multicenter study comprehensively describes uveitis characteristics in Colombian patients, providing valuable insights into its demographic and clinical features. The study findings emphasize the need to continue updating the changing patterns of uveitis to improve diagnosis and treatment strategies for diseases associated with intraocular inflammation.

Autoantibodies neutralizing GM-CSF in HIV-negative Colombian patients infected with Cryptococcus gattii and C. neoformans.

Background: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Autoantibodies (auto-Abs) neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera from Colombian patients with non-HIV related cryptococcosis in a retrospective national cohort collected from 1997 to 2016. Methods: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs in 30 HIV (-) adults presenting cryptococcosis (13 by C. gattii, and 17 by C. neoformans). Results: We detected auto-Abs neutralizing GM-CSF in the plasma of 9 out of 13 (69%) patients infected with C. gattii and 1 out of 17 (6%) patients with C. neoformans. Conclusions: We report ten Colombian patients with cryptococcosis due to auto-Abs neutralizing GM-CSF. Nine of the ten patients were infected with C. gattii, and only one with C. neoformans.

Oligo-barcode illuminates holocentric karyotype evolution in Rhynchospora (Cyperaceae).

Holocentric karyotypes are assumed to rapidly evolve through chromosome fusions and fissions due to the diffuse nature of their centromeres. Here, we took advantage of the recent availability of a chromosome-scale reference genome for Rhynchospora breviuscula, a model species of this holocentric genus, and developed the first set of oligo-based barcode probes for a holocentric plant. These probes were applied to 13 additional species of the genus, aiming to investigate the evolutionary dynamics driving the karyotype evolution in Rhynchospora. The two sets of probes were composed of 27,392 (green) and 23,968 (magenta) oligonucleotides (45-nt long), and generated 15 distinct FISH signals as a unique barcode pattern for the identification of all five chromosome pairs of the R. breviuscula karyotype. Oligo-FISH comparative analyzes revealed different types of rearrangements, such as fusions, fissions, putative inversions and translocations, as well as genomic duplications among the analyzed species. Two rounds of whole genome duplication (WGD) were demonstrated in R. pubera, but both analyzed accessions differed in the complex chain of events that gave rise to its large, structurally diploidized karyotypes with 2n = 10 or 12. Considering the phylogenetic relationships and divergence time of the species, the specificity and synteny of the probes were maintained up to species with a divergence time of ~25 My. However, karyotype divergence in more distant species hindered chromosome mapping and the inference of specific events. This barcoding system is a powerful tool to study chromosomal variations and genomic evolution in holocentric chromosomes of Rhynchospora species.

Enhancing Ocular Surface in Dry Eye Disease Patients: A Clinical Evaluation of a Topical Formulation Containing Sesquiterpene Lactone Helenalin.

The aim of this work was to assess the tolerability, safety, and efficacy of an ophthalmic topical formulation containing helenalin from Arnica montana and hyaluronic acid 0.4% (HA) in patients with mild-to-moderate Dry Eye Disease (DED) exhibiting positive Matrix Metalloproteinase 9 (MMP-9) test results. Tolerability and safety were evaluated in 24 healthy subjects. Participants were instructed to apply one drop of the formulation three times a day in the study eye, for 2 weeks, followed by a clinical follow-up of 21 days. Efficacy was studied in 48 DED patients randomized into Study (Group 1/receiving the studied formulation) or Control (Group 2/Receiving HA 0.4% eye lubricant) groups for 1 month. Assessments included an MMP-9 positivity test, conjunctival impression cytology (CIC), Ocular Surface Disease Index (OSDI), non-invasive film tear breakup time (NIBUT), non-invasive average breakup time (NIAvg-BUT), ocular surface staining, Schirmer's test, and meibomiography. A crossover design with an additional 1-month follow-up was applied to both groups. Healthy subjects receiving the studied formulation exhibited good tolerability and no adverse events. Regarding the efficacy study, Group 1 exhibited a statistically significant reduction in the MMP-9 positivity rate compared to Group 2 (p < 0.001). Both Group 1 and Group 2 exhibited substantial improvements in OSDI and NIBUT scores (p < 0.001). However, Group 1 demonstrated a significant improvement in NI-Avg-BUT and Schirmer's test scores (p < 0.001), whereas Group 2 did not (p > 0.05). Finally, after the crossover, the proportion of MMP-9-positive subjects in Group 1 increased from 25% to 91.6%, while Group 2 showed a significant decrease from 87.5% to 20.8%. Overall, the topical formulation containing sesquiterpene helenalin from Arnica montana and hyaluronic acid was well tolerated and exhibited a favorable safety profile. Our formulation reduces DED symptomatology and modulates the ocular surface inflammatory process; this is evidenced by the enhancement of CIC, the improvement of DED-related tear film status, and the reduction of the MMP-9 positivity rate.

Correction: Arias et al. An Introduction to Diopatra, the Amazing Ecosystem Engineering Polychaete. Biology 2023, 12, 1027.

The authors wish to make an erratum to the published version of the paper [...].

PAH levels in sediments from a coastal area heavily subjected to anthropogenic pressure (Asturias, north of Spain).

Ninety-two sediment samples collected along the Asturias coastline (north of Spain), were studied based on their concentrations of 16PAHs. Concentrations of Σ16PAH showed an average of 12.650 mg kg-1 d.w., which is higher than most other studies conducted around the world. The origins of PAHs present in the sediments are mainly from fuel combustion in industrial processes. The main source of PAH to the coastal system seems to be the Nalón River, which played a significant past role related to different industrial activities, highlighting thermal power stations located in the basin. On the other hand, the Avilés Estuary, hotspot of the regional heavy metallurgical industry was the area with the highest concentrations of Σ16PAH, with an average of 5 to 6 times higher than the rest studied. The risk assessment of Σ16PAH concentrations in the study area showed a high potential risk of contamination transfer to other environmental compartments.

Spatial distribution and characteristics of plastic pollution in the salt marshes of Bahía Blanca Estuary, Argentina.

This study delves into the magnitude and attributes of plastic pollution in the salt marshes of the Bahía Blanca Estuary, Argentina, with a specific focus on its spatial distribution. The investigation included the evaluation of microplastics (1-5 mm), mesoplastics (5-25 mm) and macroplastics (25-100 mm), discovering elevated levels along the high salt marsh strandline compared to low salt marsh and mudflat areas. Notably, the abundance of plastic reached staggering levels, reaching up to 20,060 items/m2 in the vicinity of an illegal dumpsite. Microplastics, particularly in the 2-4 mm range, were dominant, and the main plastic components were high-density polyethylene (HDPE), low-density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS). Plastic films emerged as the predominant plastic type, while the presence of pellets hinted at potential sources such as illegal dumping and port-related activities. This contamination could be largely attributed to inappropriate waste management practices and urban runoff, which pose a substantial ecological threat to these ecosystems. Urgent remedial action is essential to protect these marshes, underscoring the critical need for comprehensive wetland management and educational initiatives to ensure their long-term sustainability.