GENETIC VARIABILITY IN Sechium spp. (Cucurbitaceae) EVALUATED WITH AFLP MARKERS

There are few studies in Mexico aimed at evaluating the genetic variability of Sechium spp. Despite certain biological variants are reported with very high potential to develop antineoplastic supplements to treat public health conditions. Using the Amplified Fragment Length Polymorphism (AFLP) technique, the genetic variability of a sample of 95 accessions of three species of Sechium (S. edule, S. chinantlense, S. compositum) was evaluated, with leaf DNA from the Banco Nacional de Germoplasma de Sechium edule en Mexico. Four combinations of AFPL were applied (EcoRI + ACC/MseI + CAC, EcoRI + ACC/MseI + CAT, EcoRI + ACC/MseI + CGC, and EcoRI + ACC/MseI + CGG). DNA samples were classified into three groups based on the flavour of the fruit (sweet, neutral, bitter). An average of 47.91% polymorphism, 0.16 heterozygosity, 32.83 number of polymorphic bands, and a zero Wright fixation index (Fst) was obtained. The evidence showed that the domesticated accessions (sweet, neutral) were separated from the bitter-taste genotypes. A monophyletic tree was generated with the genetic distance matrix and the neighbour-joining methodology. Analyses showed S. edule as the root taxon, deriving S. compositum and S. chinantlense as subgroups, and suggesting that there is not enough differentiation to treat them as separate species. The evaluated sample showed that there is no apparent reproductive barrier for genetic cross breeding. Genotypes behaved as a complex with evolutive dynamism; that genetic complexity would allow the design of new variants.

Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking and combatting their spread. Here, we use a genetic cross in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies novel alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor, cky-1, in ivermectin resistance. This gene is within a locus under selection in ivermectin resistant populations worldwide; functional validation using knockout and gene expression experiments supports a role for cky-1 overexpression in ivermectin resistance. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode, and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and recovery in a parasitic nematode

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and recovery in parasitic helminths.

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus

Aspergillus fumigatus is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant A. fumigatus strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences—comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP–SNP interactions impacting progeny growth at different AMB concentrations.

Divergent patterns of selection on metabolite levels and gene expression

Background Natural selection can act on multiple genes in the same pathway, leading to polygenic adaptation. For example, adaptive changes were found to down-regulate six genes involved in ergosterol biosynthesis—an essential pathway targeted by many antifungal drugs—in some strains of the yeast Saccharomyces cerevisiae. However, the impact of this polygenic adaptation on metabolite levels was unknown. Here, we performed targeted mass spectrometry to measure the levels of eight metabolites in this pathway in 74 yeast strains from a genetic cross. Results Through quantitative trait locus (QTL) mapping we identified 19 loci affecting ergosterol pathway metabolite levels, many of which overlap loci that also impact gene expression within the pathway. We then used the recently developed v-test, which identified selection acting upon three metabolite levels within the pathway, none of which were predictable from the gene expression adaptation. Conclusions These data showed that effects of selection on metabolite levels were complex and not predictable from gene expression data. This suggests that a deeper understanding of metabolism is necessary before we can understand the impacts of even relatively straightforward gene expression adaptations on metabolic pathways.

PENGGUNAAN KARTU PERSILANGAN GENETIKA TRIHIBRIDA SEBAGAI MEDIA PEMBELAJARAN BIOLOGI DAPAT MENINGKATKAN HASIL BELAJAR DAN AKTIVITAS SISWA KELAS XII MADRASAH ALIYAH NEGERI BINJAI

The purpose of this study was to increase student activity in Biology lessons. Based on research and data processing, it was found that the use of the Trihybrid Genetic Crosses card (three different traits) as a Biology learning medium, can improve the learning outcomes of class XII students of Madrasah Aliyah Negeri Binjai in the 2019/2020 school year in Biology lessons on Trihybrid Genetic Crosses (three different traits). ), this can be seen from the calculation of the correlation value in the first cycle between the pre-test to the first daily test of 0.827 with a t-test value of 8.333, and the second cycle between the post-test to the second daily test of 0.943 with a t-test value of 16.075. The use of the Trihybrid Genetic Cross card (three different traits) as a Biology learning medium, can increase the activity of class XII students of Madrasah Aliyah Negeri Binjai in the 2019/2020 school year in Biology lessons on Trihybrid Genetic Crosses (three different traits), this can be seen from the presence students during classroom action research (CAR), from 90% of the first cycle increased to 95% and 100% of the second cycle. Key words : Trihybrid Genetic Cross Card as Biology Learning Media, Learning Outcomes, Activities

Temperature-Inducible Precision Guided Sterile Insect Technique

Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains which requires maintenance and sexing of two strains in a factory. Therefore, to further advance pgSIT by removing this crossing step, here we describe a next-generation Temperature-Inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true-breeding strain for TI-pgSIT that eliminates the requirement for sex sorting, a feature that may help further automate production at scale.

Virulence shift in a sexual clade of Type X Toxoplasma infecting Southern Sea Otters

How virulent protozoal pathogens capable of causing overt disease are maintained in nature is an important paradigm of eukaryotic pathogenesis. Here we used population genetics and molecular methods to study the evolution and emergence of a marine invasion of new genetic variants of Toxoplasma gondii, referred collectively as Type X (HG12). 53 Toxoplasma isolates were obtained from mustelids that stranded between 1998-2004 with toxoplasmosis (ranging from chronic infection to fatal encephalitis). Over 74% of the sea otters collected throughout their geographic range were infected with Type X as determined by multi-locus PCR-DNA sequencing. Depending on the locus investigated, Type X strains possessed one of three allelic types that had independently assorted across the strains examined; either genetically distinct alleles, referred to as “γ” or “δ”, or a Type II allele. Phylogenetic incongruence among locus-specific trees, genome-wide CGH array and WGS analyses confirmed that Type X is a sexual clade of natural recombinants that resemble F1 progeny from a genetic cross between Type II and a mosaic of two distinct “γ” or “δ” ancestries. A single Type X genotype (19/53; 36%) had expanded in sea otters largely as subclinical chronic infections, but it was highly pathogenic to mice (LD100= 1 parasite). To determine whether murine virulence genes could be mapped within this naturally occurring population, we performed a genome scan and identified four QTLs with LOD scores greater than 4.0. Targeted disruption of ROP33, the strongest candidate from among 16 genes within the highest QTL on Chromosome VIIa established ROP33 as a murine virulence locus. The ability of this highly pathogenic clone to expand and cause the majority of sea otter infections supports a virulence shift model whereby generalist pathogens like Toxoplasma utilize their sexual cycles to produce new strains with an expanded biological potential. Such a trait enables pathogens to extend their host range or be naturally selected within their vast intermediate host range to maximize their transmission. Our work thus establishes a rationale for how virulent strains can be maintained cryptically in nature across a pathogen’s broad host range, and act as reservoirs for epidemic disease.ImportanceWaterborne outbreaks of protozoal parasites are increasingly causing fatal disease in a wide range of animals, including humans. Population expansion of felids near marine estuarine environments has led to increased exposure of marine wildlife to highly infectious Toxoplasma gondii oocysts shed in the feces of cats that are dispersed by storm events. In North America Toxoplasma is thought to possess a highly clonal population structure dominated by 4 clonal lineages (I, II, III, and X). Population genetic analysis of 53 Toxoplasma isolates collected longitudinally from mustelids infected with Toxoplasma that stranded between 1998-2004 identified a majority of otters (74%) to be infected with Type X Toxoplasma, and that Type X is not a clonal lineage, but rather a recombinant clade of strains consistent with a recent genetic cross that produced at least 12 distinct haplotypes. Importantly, one Type X haplotype expanded in 36% of otters across their geographic range and caused relatively benign infections, however it was highly pathogenic to mice. A genome scan was performed to identify a new virulence locus, a secreted serine threonine kinase (ROP33), that is pathogenic in mice, but not sea otters. Our data support a virulence shift model whereby generalist pathogens like Toxoplasma utilize their sexual cycles to produce virulent strains that are maintained cryptically in nature, according to their differential capacity to cause disease within the pathogen’s broad intermediate host range. This type of “zoonotic selection” has important public health implications. Strains capable of causing fatal infections can persist in nature by circulating as chronic infections in intermediate host species that act as reservoirs for epidemic disease.

FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type-specific profiling of protein–DNA interactions in Drosophila melanogaster

Targeted DamID (TaDa) is an increasingly popular method of generating cell-type-specific DNA-binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye color. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly reproducible DamID-binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin-modifying proteins within the Drosophila genome.