Cowan, S. | Search bioRxiv

bioRxiv2024

Genetic mapping of the powdery mildew resistance gene Pm13 on oat chromosome 1D

Powdery mildew, caused by the biotrophic fungus Blumeria graminis DC. f. sp. avenae, is a widespread disease of oats, especially in the temperate regions of Western and Central Europe, and the use of resistant varieties is the most sustainable way to ensure stable yields. Therefore, the identification of robust and effective resistance to powdery mildew is of great interest for oat breeding. In contrast to race-specific resistance genes, adult plant resistance (APR) is generally considered to be more durable. The oat variety Firth, as well as related varieties such as Husky or Flamingstip, contains an unknown APR gene, which was previously located on chromosome 1D using DArT markers. The aim of this study was to confirm and refine the chromosomal location of this resistance gene, tentatively named Pm13. To this end, two independent experiments were carried out using different genetic material under natural infection conditions in the field: genome-wide association mapping (GWAS) in a diverse set of 250 oat lines grown in ten environments and QTL mapping in a HuskyxAVE1284 bi-parental population grown in three environments. Both approaches identified a QTL for powdery mildew resistance on the distal end of chromosome 1D in the hexaploid Sang oat genome. The locus explained up to 15 % of the phenotypic variance in GWAS and 64 % of the phenotypic variance in QTL mapping. Comparison of field data with results from laboratory leaf segment tests confirmed that Pm13 does indeed confer APR. The sequence information of the identified linked markers may allow the development of molecular markers useful for early selection of oat lines with high levels of APR.

plant biology↗

bioRxiv2021

Clinicopathologic features of a feline SARS-CoV-2 infection model parallel acute COVID-19 in humans

The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. This study validates a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with severe COVID-19 in humans. Intra-tracheal inoculation of concentrated SARS-CoV-2 caused infected cats to develop clinical disease consistent with that observed in the early exudative phase of COVID-19. A novel clinical scoring system for feline respiratory disease was developed and utilized, documenting a significant degree of lethargy, fever, dyspnea, and dry cough in infected cats. In addition, histopathologic pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were observed due to SARS-CoV-2 infection, imitating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation exists between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were quantified in nasal turbinates, distal trachea, lung, and various other organs. Natural ACE2 expression, paired with clinicopathologic correlates between this feline model and human COVID-19, encourage use of this model for future translational studies. Author SummaryIdentifying an ideal animal model to study COVID-19 has been difficult, and current models come with challenges that restrict their potential in translational studies. Few lab animals naturally express the receptors necessary for viral infection (ACE2), and many fail to manifest clinical signs or pathology similar to that seen in humans. Other models (non-human primates, mink) are ideal for disease and transmission studies, but are restricted by cost, husbandry challenges, and scarce availability. Alternatively, cats naturally express ACE2 receptors, are naturally infected with SARS-CoV-2 and can transmit virus from cat-to-cat. Prior to this study, cats infected by oral/nasal routes have not displayed significant clinical disease or lung pathology. However, we demonstrate that direct inoculation of concentrated SARS-CoV-2 virus in the trachea of cats induces analogous clinical and pathologic features to hospitalized patients with acute COVID-19. Our results show that infected cats exhibit significant clinical signs during experimental infection (coughing, increased respiratory effort, lethargy, and fever) and exhibit extensive lung lesions that mimic severe COVID-19 pathology such as diffuse alveolar damage and hyaline membrane formation - highlighting the immeasurable potential for this feline model to address translational approaches for COVID-19 and to better understand the role of cats in transmission and disease.

microbiology↗

bioRxiv2018

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

We generated new genetic tools to efficiently tag genes in Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on CRE recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette.\n\nThis sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

genetics↗