ASSOCIATION GENETICS APPROACHES FOR THE IDENTIFICATION OF GENES ASSOCIATED TO BARLEY AGRONOMIC TRAITS IN A MEDITERRANEAN ENVIRONMENT

Barley (Hordeum vulgare) is one of the four most economically important cereal crops worldwide. It serves as a major animal feed crop, with smaller amounts used for malting and in health food. Among environmental factors influencing barley production, drought is recognized as the most common in the Mediterranean area and this problem is expected to worsen with on-going climate changes. Breeding for drought resistance is therefore an important objective to ensure stable crop yields. In this context, genetic and molecular dissection of drought tolerance is expected to lead to the identification of key genes/loci and favourable alleles through exploration of biodiversity. Association genetics focuses on the identification of links between phenotypic traits and genetic markers with the aim to identify QTLs and locate the underlying genes in the genome. In general, different barley genotypes are expected to carry different loci at genomic regions of interest. The probability of chromosomal recombination between two loci is proportional to the physical distance between those loci. General objective of this project was the identification of loci subtending major agronomic traits under different water regimes, using an association genetics approach. To this end, we considered a phenotypic panel consisting of 83 barley cultivars representing European diversity for drought tolerance. This germplasm collection included 2 and 6 rows winter and spring barleys, that were previously evaluated for plant height, flowering time and yield under irrigated and rainfed conditions over three successive years (Rizza et al., 2004). At the beginning of this project, an initial objective was to evaluate the potential association between allelic variants of candidate genes (CGs) selected for their known roles in drought responses and phenotypic variation for barley grain yield under different watering regimes. Thus, we re-sequenced an initial set of 3 CGs -HvCbf2, HvCbf4a and HvCbf7- and we identified 4 Single Nucleotide Polymorphisms (SNPs) in the coding region of HvCbf4a. In parallel, we tried to assess the genetic diversity and structure of our barley panel by utilizing the low cost molecular marker approach of Amplified Fragment Length Polymorphism (AFLP). Incorporation of structure information in association analysis is important to prevent recovery of false associations. However, when we run structure analyses using our AFLP data, we failed to recover the classical barley subdivisions reported in the literature indicating that these markers may not be reliable for our purposes. For this reason and considering the limited chances of detecting association with few CGs, we took advantage of the recently established iSELECT Infinium\uae Illumina 9k SNP platform (Comadran et al., 2012) to carry out a Genome Wide Association Study (GWAS) on our barley panel. Filtering out the monomorphic and failed-genotyped markers resulted in the identification of a total of 4,661 SNPs distributed over the 7 barley chromosomes. Population stratification was investigated with a subset of 260 SNPs selected as highly informative using admixture model implemented in Structure software. Accordingly, three main subgroups were identified corresponding to winter-2rows, winter-6rows and spring-2rows barleys, respectively. Association between barley genotypic data and flowering date (FD) was processed using general linear model and afterward compared with mixed linear model. Quantitative Trait Loci (QTLs) controlling grain yield (GY), flowering date (FD), and plant height (PH) were identified using the general linear model. Our results provide a starting point for the identification of potentially useful genes and markers for future applications in barley breeding schemes

International longitudinal registry of patients with atrial fibrillation and treated with rivaroxaban: RIVaroxaban Evaluation in Real life setting (RIVER)

Background Real-world data on non-vitamin K oral anticoagulants (NOACs) are essential in determining whether evidence from randomised controlled clinical trials translate into meaningful clinical benefits for patients in everyday practice. RIVER (RIVaroxaban Evaluation in Real life setting) is an ongoing international, prospective registry of patients with newly diagnosed non-valvular atrial fibrillation (NVAF) and at least one investigator-determined risk factor for stroke who received rivaroxaban as an initial treatment for the prevention of thromboembolic stroke. The aim of this paper is to describe the design of the RIVER registry and baseline characteristics of patients with newly diagnosed NVAF who received rivaroxaban as an initial treatment. Methods and results Between January 2014 and June 2017, RIVER investigators recruited 5072 patients at 309 centres in 17 countries. The aim was to enroll consecutive patients at sites where rivaroxaban was already routinely prescribed for stroke prevention. Each patient is being followed up prospectively for a minimum of 2-years. The registry will capture data on the rate and nature of all thromboembolic events (stroke / systemic embolism), bleeding complications, all-cause mortality and other major cardiovascular events as they occur. Data quality is assured through a combination of remote electronic monitoring and onsite monitoring (including source data verification in 10% of cases). Patients were mostly enrolled by cardiologists (n = 3776, 74.6%), by internal medicine specialists 14.2% (n = 718) and by primary care/general practice physicians 8.2% (n = 417). The mean (SD) age of the population was 69.5 (11.0) years, 44.3% were women. Mean (SD) CHADS2 score was 1.9 (1.2) and CHA2DS2-VASc scores was 3.2 (1.6). Almost all patients (98.5%) were prescribed with once daily dose of rivaroxaban, most commonly 20 mg (76.5%) and 15 mg (20.0%) as their initial treatment; 17.9% of patients received concomitant antiplatelet therapy. Most patients enrolled in RIVER met the recommended threshold for AC therapy (86.6% for 2012 ESC Guidelines, and 79.8% of patients according to 2016 ESC Guidelines). Conclusions The RIVER prospective registry will expand our knowledge of how rivaroxaban is prescribed in everyday practice and whether evidence from clinical trials can be translated to the broader cross-section of patients in the real world

Bacteria-inducing legume nodules involved in the improvement of plant growth, health and nutrition

Bacteria-inducing legume nodules are known as rhizobia and belong to the class Alphaproteobacteria and Betaproteobacteria. They promote the growth and nutrition of their respective legume hosts through atmospheric nitrogen fixation which takes place in the nodules induced in their roots or stems. In addition, rhizobia have other plant growth-promoting mechanisms, mainly solubilization of phosphate and production of indoleacetic acid, ACC deaminase and siderophores. Some of these mechanisms have been reported for strains of rhizobia which are also able to promote the growth of several nonlegumes, such as cereals, oilseeds and vegetables. Less studied are the mechanisms that have the rhizobia to promote the plant health; however, these bacteria are able to exert biocontrol of some phytopathogens and to induce the plant resistance. In this chapter, we revised the available data about the ability of the legume nodule-inducing bacteria for improving the plant growth, health and nutrition of both legumes and nonlegumes. These data showed that rhizobia meet all the requirements of sustainable agriculture to be used as bio-inoculants allowing the total or partial replacement of chemicals used for fertilization or protection of crops

Genetic dissection of heading date and yield under Mediterranean dry climate in barley (Hordeum vulgare L.)

Understanding the genetics of drought tolerance is a major goal of plant breeders, especially in the Mediterranean region due to its sensitivity to drought and climate changes. Here we investigated genomic regions associated with heading date and yield under rainfed (R) and irrigated (I) conditions, along with some drought tolerance indices derived from phenotypic data collected on a panel of 83 European barley cultivars evaluated in field trials in Foggia, southern Italy, for three growing seasons. A genome wide association analysis (GWAS) was carried out using gene-based SNPs incorporated in the Illumina iSELECT Chip with known map position. A total of 4169 polymorphic SNPs were considered after filtering out SNPs with >10 % missing data and <10 % minimum allele frequency. Heritability was high for heading date and moderate for yield. Principle components analysis partitioned the barley germplasm in three subgroups characterized by different row-type and growth habit. A significant association with heading date was detected on chromosome 2HS, where the different alleles of associated markers were able to significantly affect heading date in different cultivars. Moreover, GWAS uncovered a significant association with yield and relative yield under R conditions on chromosome 5HS. These results provide a clue for further analysis on genetic control of barley flowering time and production in response to water stress under Mediterranean dry climate

Persistence of chloroquine-resistant haplotypes of Plasmodium falciparum in children with uncomplicated Malaria in Lagos, Nigeria, four years after change of chloroquine as first-line antimalarial medicine.

BACKGROUND: In Nigeria, despite the change in National malaria drug policy to artemisinin combination therapy (ACT) in 2005 due to widespread chloroquine resistance, chloroquine (CQ) is still widely used in the treatment of malaria because it is cheap, affordable and accessible. The use of ACT for the management of uncomplicated malaria is currently being promoted. The employment of genetic markers to track circulating chloroquine-resistant parasites are useful in elucidating likely poor efficacy of chloroquine, especially in settings where it is not recommended for the treatment of uncomplicated falciparum malaria. This study determined the prevalence of pfcrt haplotypes and point mutations in pfmdr1 genes four years after the change in antimalarial treatment policy from CQ to the ACTs in Lagos, a commercial city in South-West, Nigeria. METHODS: This was a cross sectional study on uncomplicated malaria in children less than 12 years that presented with fever and other symptoms suggestive of malaria. Parasite DNA was extracted from 119 patients out of 251 children who were positive for Plasmodium falciparum by microscopy and amplified. The occurrence of haplotypes was investigated in pfcrt gene using probe-based qPCR and single nucleotide polymorphisms in pfmdr1 gene using nested PCR. RESULTS: One hundred and nine (109) of the 119 children with P falciparum infection (91.6%) harbourd parasites with the mutant pfcrt haplotype (CVIET). Out of this, 4.2% comprised a mixture of genotypes encoding CVMNK and CVIET, while 4.2% had the wild type (CVMNK). Furthermore, the frequency of point mutations in pfmdr1 was 62.2% and 69.0% for codons Y86 and F184 respectively. There were no mutations at codons 1034, 1042 and 1246 of the Pfmdr1 genes. CONCLUSION: The high frequency of the CQ-resistant haplotypes (CVIET) and mutations in Pfmdr1 associated with CQ resistance in P. falciparum among these children suggest that CQ-resistant parasites are still in circulation. Continuous use of chloroquine may continue to increase the level of mutations in pfcrt and pfmdr1genes. There is need to strengthen current case management efforts at promoting ACT use as well as urgently restricting access to chloroquine by the National drug regulatory agency, National Agency for Food Drug Administration and Control (NAFDAC). VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2069472010142303

Collection of SARS-CoV-2 Virus from the Air of a Clinic Within a University Student Health Care Center and Analyses of the Viral Genomic Sequence

The progression of COVID-19 worldwide can be tracked by identifying mutations within the genomic sequence of SARS-CoV-2 that occur as a function of time. Such efforts currently rely on sequencing the genome of SARS-CoV-2 in patient specimens (direct sequencing) or of virus isolated from patient specimens in cell cultures. A pilot SARS-CoV-2 air sampling study conducted at a clinic within a university student health care center detected the virus vRNA, with an estimated concentration of 0.87 virus genomes L(-1) air. To determine whether the virus detected was viable ('live'), attempts were made to isolate the virus in cell cultures. Virus-induced cytopathic effects (CPE) were observed within two days post-inoculation of Vero E6 cells with collection media from air samples; however, rtRT-PCR tests for SARS-CoV-2 vRNA from cell culture were negative. Instead, three other fast-growing human respiratory viruses were isolated and subsequently identified, illustrating the challenge in isolating SARS-CoV-2 when multiple viruses are present in a test sample. The complete SAR-CoV-2 genomic sequence was nevertheless determined by Sanger sequencing and most closely resembles SARS-CoV-2 genomes previously described in Georgia, USA. Results of this study illustrate the feasibility of tracking progression of the COVID-19 pandemic using environmental aerosol samples instead of human specimens. Collection of a positive sample from a distance more than 2 m away from the nearest patient traffic implies the virus was in an aerosol