Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Comparative analysis of cattle breeds as satellite cells donors for cultured beef

Background: Cultured meat is a promising new field with the potential for considerable environmental and animal welfare benefits. One technological approach to cultured meat production utilises the proliferative and differentiative capacity of muscle-derived satellite cells (SCs) to produce large volumes of cultured muscle tissue from small biopsies of donor animals. Differing genotypes between cattle breeds lead to predictable phenotypic traits, resulting in breeds being favoured for their respective meat or milk production characteristics in the livestock industry. However, whilst these breeds show significant differences in muscle growth, it is unclear whether the physiological differences observed between them in vivo are reflected in differences in SC behaviour in vitro, particularly with respect to proliferation, differentiation and cellular longevity, and hence whether particular breeds might represent preferred SC donors for a cultured beef bioprocess. Results: Comparing SCs isolated from five breeds (Belgian Blue, Holstein Friesian, Galloway, Limousin and Simmental), we found that the proliferation rates were largely unaffected by the donor breed. In contrast, potentially meaningful differences were observed in the kinetics and extent of myogenic differentiation. Furthermore, whilst differentiation dropped for all breeds with increasing population doublings (PDs), SCs from Belgian Blue and Limousin cattle showed significantly longer retention of differentiation capacity over long-term passaging. Conclusion: SCs from all breeds were able to proliferate and differentiate, although Limousin and (particularly) Belgian Blue cattle, both breeds commonly used for traditional meat production, may represent preferred donors for cultured beef production.

Adaptation to an amoeba host leads to Pseudomonas aeruginosa isolates with attenuated virulence

The opportunistic pathogen Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when co-incubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and non-adapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoeba as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoeba resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. Importance Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by bacterial predators, e.g. protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examine the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa . We show that long term co-incubation with protozoa resulted in mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures amongst host cell types as well as similar adaptation strategies.

Development and function explain the modular evolution of phalanges in gecko lizards

Selective regimes favouring the evolution of functional specialization probably affect covariation among phenotypic traits. Phalanges of most tetrapods develop from a conserved module that constrains their relative proportions. In geckos, however, biomechanical specializations associated with adhesive toepads involve morphological variation in the autopodium and might reorganize such modular structures. We tested two hypotheses to explain the modular architecture of hand bones in geckos, one based on developmental interactions and another incorporating functional associations related to locomotion, and compared the empirical support for each hypothetical module between padded and padless lineages. We found strong evidence for developmental modules in most species, which probably reflects embryological constraints during phalangeal formation. Although padded geckos exhibit a functional specialization involving the hyperextension of the distal phalanges that is absent in padless species, the padless species are the ones that show a distal functional module with high integration. Some ancestrally padless geckos apparently deviate from developmental predictions and present a relatively weak developmental module of phalanges and a strongly integrated distal module, which may reflect selective regimes involving incipient frictional adhesion in digit morphology. Modularity of digit elements seems dynamic along the evolutionary history of geckos, being associated with the presence/absence of adhesive toepads.

Optimized Method for Pseudomonas aeruginosa Integrative Filamentous Bacteriophage Propagation

Filamentous bacteriophages frequently infect Pseudomonas aeruginosa and alter its phenotypic traits, including virulence factors. The first step in examination of these phages is to obtain suspensions with high virus titer, but as there are no methods for integrative filamentous phage multiplication, the aim was to design, describe, and compare two methods for this purpose. As models, three strains of Pseudomonas aeruginosa, containing (pro)phages Pf4, Pf5, and PfLES were used (PAO1, UCBPP-PA14, and LESB58, respectively). Method 1 comprised propagation of phages in 6 L of bacterial culture for 48 h, and method 2 applied 600 mL culture and incubation for 6 days with centrifugation and addition of new medium and inoculum at 2-day intervals. In method 1, phages were propagated by culture agitation, followed by centrifugation and filtration (0.45 and 0.22 μm), and in method 2, cultures were agitated and centrifuged several times to remove bacteria without filtration. Regardless of the propagation method, supernatants were subjected to concentration by PEG8000 and CsCl equilibrium density gradient centrifugation, and phage bands were removed after ultracentrifugation and dialyzed. In the obtained suspensions, phage titer was determined, and concentration of isolated ssDNA from virions was measured. When propagation method 2 was compared with method 1, the phage bands in CsCl were much thicker, phage number was 3.5–7.4 logs greater, and concentration of ssDNA was 7.6–22.4 times higher. When phage count was monitored from days 2 to 6, virion numbers increased for 1.8–5.6 logs, depending on phage. We also observed that filamentous phage plaques faded after 8 h of incubation when the double layer agar spot method was applied, whereas the plaques were visible for 24 h on single-layer agar. Finally, for the first time, we confirmed existence of replicative form and virions of PfLES (pro)phage as well as its ability to produce plaques. Similarly, for the first time, we confirmed plaque production of Pf5 (pro)phage present in P. aeruginosa strain UCBPP-PA14. The described method 2 has many advantages and can be further improved and adopted for filamentous phages of other hosts.

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with training of multiple supervised classifiers, to predict severity on the basis of screened features. Feature importance analysis from tree-based models allowed to identify a handful of 16 variants with highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with good accuracy (ACC=81.88%; ROC_AUC=96%; MCC=61.55%). Principal Component Analysis (PCA) and clustering of patients on important variants orthogonally identified two groups of individuals with a higher fraction of severe cases. Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response, such as JAK-STAT, Cytokine, Interleukin, and C-type lectin receptor signaling. It also identified additional processes cross-talking with immune pathways, such as GPCR signalling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as “Respiratory or thoracic disease”, confirming their link with COVID-19 severity outcome. Taken together, our analysis suggests that curated genetic information can be effectively integrated along with other patient clinical covariates to forecast COVID-19 disease severity and dissect the underlying host genetic mechanisms for personalized medicine treatments.

Evaluation on the phenotypic diversity of Calamansi (Citrus microcarpa) germplasm in Hainan island

Calamansi or Philippine lime (Citrofortunella macrocarpa) is an important crop for local economic in Hainan Island. There is no study about Calamansi germplasm evaluation and cultivar development. In this study, Calamansi data were collected from 151 of Calamansi seedling trees, and 37 phenotypic traits were analyzed to investigate their genetic diversities. The cluster analysis and principal component analysis were conducted aiming to provide a theoretical basis for the Calamansi genetic improvement. The results of the diversity analysis revealed: (1) the diversity indexes for qualitative traits were ranged from 0.46–1.39, and the traits with the highest genetic diversity level were fruit shaped and pulp colored (H′ > 1.20); and the diversity indexes for quantitative traits ranged from 0.67–2.10, with the exception of a lower in fruit juice rate (1.08) and lower in number of petals (0.67). (2) The clustering analysis of phenotypic traits have arranged the samples into 4 categories: the first group characterized by fewer flesh Segment number per fruit (SNF) and more Oil cell number (OCN); the second group had 7 samples, all characterized with larger Crown breadth (CB), higher Yield per tree (YPT), the lager leaf, the higher Ascorbic acid (AA), and less Seed number per fruit (SNPF); the third group had 25 samples characterized by smaller Tree foot diameter (TFD),smaller Fruit shape index (FSI) and higher Total soluble solids (TSS) contain; the fourth group had 87 samples, they were characterized by shorter Petiole length (PEL), larger fruit, higher Juice ratio (JR), multiple Stamen number (SN) and longer Pistil length (PIL). (3) The principal component analysis showed the values of the first 9 major components characteristic vectors were all greater than 3, the cumulative contribution rate reach 72.20%, including the traits of single fruit weight, fruit diameter, tree height, tree canopy width etc. Finally, based on the comprehensive main component value of all samples, the Calamansi individuals with higher testing scores were selected for further observation. This study concludes that Calamansi seedling populations in the Hainan Island holds great genetic diversity in varies traits, and can be useful for the Calamansi variety improvements.

High Level of Phenotypic Differentiation of Common Yew (Taxus baccata L.) Populations in the North-Western Part of the Balkan Peninsula

Common or English yew (Taxus baccata L., Taxaceae) is a conifer species, native to Europe, northern Africa, Asia Minor and Caucasus. It is a dioecious, wind-pollinated and animal-dispersed tree, known for its high-quality wood and medicinal properties, albeit poisonous. The species is rare and has been legally protected at the European and national levels. In addition, its low population density and disjunct character of distribution have reinforced the need for its protection as regeneration is mostly lacking. The aim of this study was to phenotypically characterise the north-western Balkan yew populations. Phenotypic diversity was examined for seven populations, using morphometric analysis of nine phenotypic traits of needles. Descriptive and multivariate statistical analyses were conducted to evaluate the inter- and intrapopulation variability. In addition, to test correlations between geographic, climatic and phenotypic data, Mantel test was used. We identified a geographic structure across studied populations that exhibited high levels of variability on intra- and interpopulation levels. Two groups of populations have been defined and are consistent with previously described genetic divergent lineages from separate refugia. In addition, a significant correlation between phenotypic and geographic data were revealed, i.e., isolation by distance (IBD). However, the Mantel test revealed no significant correlation between morphometric and environmental data. In conclusion, our data reveal that the historical events and persistent IBD acted in combination to produce the morphological patterns observed in common yew populations in the north-western part of the Balkan Peninsula. Finally, we suggested conservation measures to be implemented on a stand level, with habitat preservation as the main goal. In addition, ex situ conservation should be considered, both in the form of collections and urban planting, as both provide additional gene pool reserves.

Identification of QTLs and Putative Candidate Gene Involved in Rhizome Enlargement of Asian lotus (Nelumbo Nucifera)

Lotus (Nelumbo) is perennial aquatic plant with nutritional, pharmacological, and ornamental significance. Rhizome is an underground lotus stem that act as a storage organ and as a reproductive tissue for asexual production. The enlargement of lotus rhizome is an important adaptive strategy for surviving the cold winter. The aims of this study were to identify quantitative trait loci (QTLs) for rhizome enlargement traits including rhizome enlargement index (REI) and number of enlarged rhizome (NER), and to uncover candidate genes associated with these phenotypic traits. A high-density genetic linkage map was constructed, consisting of 2,935 markers binned from 236,840 SNPs. A total of 14 significant QTLs were detected for REI and NER, which explained 6.67–22.28% of trait variance. Three QTL regions were repeatedly identified in at least two years, and a major QTL, designated cqREI-LG2, with a rhizome-enlargement effect and about 20% of the phenotypic contribution was identified across the three climatic years. A candidate NnBEL6 gene located within the confidence interval of cqREI-LG2 was considered to be putatively involved in lotus rhizome enlargement. The expression of NnBEL6 was exclusively induced by rhizome swelling. Sequence comparison of NnBEL6 among lotus varieties revealed a functional Indel site in its promoter that likely initiates the rhizome enlargement process. Transgenic potato assay was used to confirm the role of NnBEL6 in inducing tuberization. The successful identification QTLs and functional validation of NnBEL6 gene reported in this study will enrich our knowledge on the genetic basis of rhizome enlargement in lotus.