Trait mapping in diverse arthropods by bulked segregant analysis

Bulked segregant analysis (BSA) is a cross-based method for genetic mapping in sexually reproducing organisms. The method's use of bulked (pooled) samples markedly reduces the genotyping effort associated with traditional linkage mapping studies. Further, it can be applied to species with life histories or physical attributes (as for micro-insects) that render genetic mapping with other methods impractical. Recent studies in both insects and mites have revealed that advanced BSA experimental designs can resolve causal loci to narrow genomic intervals, facilitating follow-up investigations. As high-quality genomes become more widely available, BSA methods are poised to become an increasingly important tool for the rapid mapping of both monogenic and polygenic traits in diverse arthropod species

High-resolution QTL mapping in Tetranychus urticae reveals acaricide-specific responses and common target-site resistance after selection by different METI-I acaricides

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific

Convergent evolution of cytochrome P450s underlies independent origins of keto-carotenoid pigmentation in animals

Keto-carotenoids contribute to many important traits in animals, including vision and coloration. In a great number of animal species, keto-carotenoids are endogenously produced from carotenoids by carotenoid ketolases. Despite the ubiquity and functional importance of keto-carotenoids in animals, the underlying genetic architectures of their production have remained enigmatic. The body and eye colorations of spider mites (Arthropoda: Chelicerata) are determined by β-carotene and keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant of the spider mite Tetranychus kanzawai that, as shown by chromatography, lost the ability to produce keto-carotenoids. We employed bulked segregant analysis and linked the causal locus to a single narrow genomic interval. The causal mutation was fine-mapped to a minimal candidate region that held only one complete gene, the cytochrome P450 monooxygenase CYP384A1, of the CYP3 clan. Using a number of genomic approaches, we revealed that an inactivating deletion in the fourth exon of CYP384A1 caused the aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is orthologous across mite species of the ancient Trombidiformes order where carotenoids typify eye and body coloration, suggesting a deeply conserved function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a cytochrome P450 of the CYP2 clan, has been identified as a carotenoid ketolase in birds and turtles. Our study shows that selection for endogenous production of keto-carotenoids led to convergent evolution whereby cytochrome P450s were independently co-opted in vertebrate and invertebrate animal lineages

Long-term population studies uncover the genome structure and genetic basis of xenobiotic and host plant adaptation in the herbivore Tetranychus urticae

Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA carboxylase inhibitor, in populations from a cross between a spirodiclofen-resistant and a spirodiclofen-susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA carboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies

Effects of conjugative plasmids on the ecology and evolution of microbial communities

Thesis (Ph.D.)--University of Washington, 2022CHAPTER 1. To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need reliable estimates of their rate of transfer between bacterial cells. Current assays to measure transfer rate are based on deterministic modeling frameworks. However, some cell numbers in these assays can be very small, making estimates that rely on these numbers prone to noise. Here we take a different approach to estimate plasmid transfer rate, which explicitly embraces this noise. Inspired by the classic fluctuation analysis of Luria and Delbrück, our method is grounded in a stochastic modeling framework. In addition to capturing the random nature of plasmid conjugation, our new methodology, the Luria-Delbrück method (‘LDM’), can be used on a diverse set of bacterial systems, including cases for which current approaches are inaccurate. A notable example involves plasmid transfer between different strains or species where the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias or constrain current transfer estimates, thereby limiting our capabilities for estimating transfer in microbial communities. In contrast, the LDM overcomes obstacles of traditional methods by avoiding restrictive assumptions about growth and transfer rates for each population within the assay. Using stochastic simulations and experiments, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to the most widely used methods, which can produce estimates that differ from the LDM estimate by orders of magnitude. CHAPTER 2. Genes that undergo horizontal gene transfer (HGT) evolve in dramatically different genomic backgrounds as they move between hosts, which is in stark contrast to genes that evolve under strict vertical inheritance. Given the ubiquity of HGT in microbial communities, it is notable that the effects of host-switching on gene evolution have been largely understudied. Here, we present a novel framework to examine the consequences of host switching on gene evolution depending on the existence and form of host-dependent mutational effects. We started exploring the effects of HGT on gene evolution by focusing on a well-known antibiotic resistance gene (encoding a beta-lactamase) commonly encoded on conjugative plasmids found in Enterobacteriaceae pathogens. By reconstructing the resistance landscape for a small set of mutationally connected alleles in three species (Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae), we uncovered that the landscape topography was overwhelmingly aligned with very low levels of host-dependent mutational effects. By simulating gene evolution with and without HGT using the species-specific empirical landscapes, we found that evolutionary outcomes were similar despite HGT. These findings suggest that mobile genes adapting in one species can lead to adaptation in another species. In such a case, vehicles of cross-species HGT enable a distributed form of genetic evolution across a bacterial community, where species can ‘crowdsource’ adaptation from other community members

Evolutionary "crowdsourcing": alignment of fitness landscapes allows for cross-species adaptation of a horizontally transferred gene

<p>This repository accompanies the publication of <i><strong>Evolutionary "crowdsourcing": alignment of fitness landscapes allows for cross-species adaptation of a horizontally transferred gene</strong></i> by Kosterlitz et. al. This research project explores the cross-species adaptation of a horizontally transferred gene through evolutionary "crowdsourcing." The repository provides all relevant data, code, and figures associated with the publication, enabling users to replicate the results and explore the findings in-depth.</p&gt

De novo genome assemblies of lemon selected and wild-type selected mites from a segregating T. kanzawai population and its lemon and wild-type parent.

The segregating mite population was generated by crossing Jp-inbred-lemon to Jp2-WT and was used to genetically map the lemon phenotype in Tetranychus kanzawai. After approximately 10-12 generations, a total of three replicates were collected that consisted of 1100, 900 and 500 adult lemon females. In parallel, 1500 wild-type females were selected and pooled into a single sample. From these four populations and the two parental strains, genomic DNA was extracted and assembled using the CLC Genomics Workbench 9.0.1 (https://www.qiagenbioinformatics.com/)

Localized de novo transcriptome assemblies of CYP384A1 of a T. kanzawai population that segregates for lemon pigmentation.

RNA was extracted from 110 lemon and wild-type adult females in two replicates using an RNeasy Minikit (Qiagen). RNA reads that aligned to CYP384A1 were assembled into contigs using Trinity 2.5.1

β-carotene and astaxanthin levels detected by HPLC in wild-type and lemon T. kanzawai.

For each treatment 30 females were collected in three replicates and homogenized in 1 ml of acetone. The homogenate was filtrated using a glass syringe with a membrane possessing a pore size of 0.45 μm. The filtrate was dried under a nitrogen gas flow and dissolved in 300 μl of methanol. Five μl of the solution was used for HPLC analysis. Carotenoids were quantified by monitoring the absorbance at 450 nm. External calibration curves were constructed with authentic standards

Data from: Convergent evolution of cytochrome P450s underlies independent origins of keto-carotenoid pigmentation in animals

Keto-carotenoids contribute to many important traits in animals, including vision and coloration. In a great number of animal species, keto-carotenoids are endogenously produced from carotenoids by carotenoid ketolases. Despite the ubiquity and functional importance of keto-carotenoids in animals, the underlying genetic architectures of their production have remained enigmatic. The body and eye colorations of spider mites (Arthropoda: Chelicerata) are determined by β-carotene and keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant of the spider mite Tetranychus kanzawai that, as shown by chromatography, lost the ability to produce keto-carotenoids. We employed bulked segregant analysis and linked the causal locus to a single narrow genomic interval. The causal mutation was fine-mapped to a minimal candidate region that held only one complete gene, the cytochrome P450 monooxygenase CYP384A1, of the CYP3 clan. Using a number of genomic approaches, we revealed that an inactivating deletion in the fourth exon of CYP384A1 caused the aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is orthologous across mite species of the ancient Trombidiformes order where carotenoids typify eye and body coloration, suggesting a deeply conserved function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a cytochrome P450 of the CYP2 clan, has been identified as a carotenoid ketolase in birds and turtles. Our study shows that selection for endogenous production of keto-carotenoids led to convergent evolution whereby cytochrome P450s were independently co-opted in vertebrate and invertebrate animal lineages