Periodic Reporting for period 3 - POLYADAPT (Molecular-genetic mechanisms of extreme adaptation in a polyphagous agricultural pest) Reporting period: 2021-06-01 to 2022-11-30 Summary of the context and overall objectives of the project Generalist (polyphagous) herbivores can feed and reproduce on many different plant species and include some of the most pesticide resistant and notorious pests in agriculture. An evolutionary link between host plant range and the development of pesticide resistance has been suggested. Although crucial for devising efficient crop protection strategies, the mechanisms underlying rapid adaptation are not well understood, especially in generalists. The spider mite Tetranychus urticae is a global pest known to feed on 1,100 different hosts from 140 plant families, including most major crops. With experimental advances and new tools developed for T. urticae, we are now poised for fundamental advances in understanding the molecular genetic make-up of adaption in generalist pests. We will generate a large collection of fully inbred and resistant mite strains and describe the sampled genomic variation in the context of selection and adaptation. We will study gene regulation mechanisms and quantify cis versus trans regulation of gene expression on a genome wide scale. We will then create a unique population resource of 200 recombinant inbred lines (RILs) that will allow us to map master regulators of gene expression (an eQTL analysis) and construct a gene-regulatory network of adaptation responsive genes. In a highly replicated experimental evolution study, combined with Bulk Segregant Analysis (BSA), we will uncover, without a prior hypothesis, the genomic loci that underlie complex cases of resistance and plant adaptation. A core set of adaptation genes will be validated by functional expression and high-throughput interaction assays. Further validation will come from the development of genome editing tools. In summary, POLYADAPT will exploit the genomic tools now available for spider mites to elucidate regulatory and causal variants underlying the extreme adaptation potential of polyphagous pests. This will in the long term lead to innovative methods of pest management Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Inbred lines have been generated, phenotyped, sequenced and assembled.Allele specific expression in F1 progeny of crosses has allowed to look at cis versus trans regulation of gene expression. A manuscript has been published. Test crosses are performed in preparation of eQTL mapping.An eQTL experiment has been set up, RNA sequenced, and data has been analysed. Master regulators of gene expression have been identified. A manuscirpt is being written.A number of Bulk segregant mapping studies have been set up to unbiasedly map resistance loci in the genome. QTLs have been uncovered for a number of resistance cases. A number of QTL studies was published. A review outlining the method has been published recently.Pipelines for validation of resistance genes have been set up, mainly the E. coli and yeast expression systems. Crispr/Cas9 transformation of spider mites has been achieved, and the method has been greatly improved.A Xenopus oocyte pipeline for electrophysilogy has been set up, mutations have been validated.Yeast-1Hybrid protocols are being designed, but switching to gene editing is considered and explored. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We will unravel the molecular-genetic mechanisms that allow generalist herbivores to rapidly adapt to new hosts and pesticides. The development of acaricide resistance will be the main evolutionary process under study in POLYADAPT. Firstly, because it is of direct and crucial importance for agriculture, but also because it is a unique herbivore micro-evolutionary model and parallels between acaricide resistance and host plant adaptation will also be central to our study. With experimental advances and newly developed tools for T. urticae, many of which have come from my laboratory, we are now poised for fundamental advances. To make the critical jump to the next level, we will generate a large collection of fully inbred and resistant mite strains and describe the sampled genomic variation in the context of selection and adaptation. We will cross some of these genetically diverse inbred lines and quantify cis versus trans regulation of gene expression in an allele-specific design by sequencing RNA from F1 populations. We will then generate a collection of recombinant inbred lines (RILs) that will be used to map master regulators of gene expression (an eQTL analysis) and construct a regulatory network. Inbred lines will be used in an experimental evolution setup combined with Bulk Segregant Analysis (BSA) mapping to uncover the genomic loci that underlie both simple and complex cases of resistance and plant adaptation. This forward genetic approach will define loci regardless of prior hypotheses (functional coding variants, regulation variants, etc.). Finally and importantly, validation of the results in functional assays will be a prime focus and we will also devise protocols for genetic transformation and genome editing in the spider mite.Key questions: What is the level of intraspecific variation in resistant lines (copy number, allelic variation, etc.)? Which gene families are under positive or balancing selection, and can we detect selective sweeps for resistance? Are gene-expression changes caused by many cis-acting changes or rather a few trans-regulatory changes? Which loci underlie complex cases of acaricide resistance and differences in plant performance? Spider mite, foto J. van Arkel
