The overall goal of this project is to identify immune genes under balancing selection in a wild rodent (the bank vole), and to investigate why there is balancing selection on these genes. Immune genes are important targets of balancing selection, i.e. natural selection that acts to maintain genetic variation. However, the causes of balancing selection are not well understood. In this project we have re-sequenced a population sample of bank voles to identify immune genes under balancing selection. We now aim to continue this project along two lines: (i) Investigate what determines which immune genes are under balancing selection. Specifically, we are interested in whether the level and/or tissue specificity of gene expression affects the likelihood that a gene is under balancing selection. To test this, we have performed RNA-sequencing of 10 tissues in a set of bank voles, and will now use this to test for an association between gene expression and balancing selection. (ii) Investigate the functional effects of polymorphisms in immune genes under balancing selection. To this end, we have performed ex vivo assays of gene expression of bank vole splenocytes (white blood cells) stimulated by two different bacteria (Borrelia or Streptococcus). The primary aim of this study is to test if polymorphisms in genes under balancing selection (as identified in the first subproject) are associated with cis-regulatory variation, by testing for allele-specific expression in response to stimulation with Borrelia or Streptococcus. In particular, we are interested in finding polymorphisms where patterns of allele-specific expression are opposite in response to Borrelia and Streptococcus, something that would indicate antagonistic pleiotropy for the response to these two pathogens. Such antagonistic pleiotropy is thought to be an important factor leading to balancing selection on immune genes, but has rarely been demonstrated experimentally. Our study species, the bank vole (Myodes glareolus), is widely distributed in Europe and is a popular study species for various ecological and evolutionary questions. Råberg’s group has been using the bank vole as study species for research on the evolutionary ecology of host-pathogen interactions since 2006. In 2017 we performed whole-genome resequencing at ca 40× using Illumina HiSeq X of 30 individual bank voles from our study population in southern Sweden. This data has been used for identifying immune genes under balancing selection. In 2021, we performed in vitro assays of splenocyte responses to bacteria by RNA-sequencing. All in all 169 samples from 63 bank voles were sequenced. In 2023, we have performed RNA-sequencing of 10 tissues from 2-5 males and 2-5 females from both summer and winter, to measure level and breadth of gene expression (all in all 95 samples). During 2023 we also submitted a sample from a grey-sided vole (a closely related species to the bank vole) for genome sequencing; this will be used as outgroup to enhance power of selection analyses. The storage project will be used for analyses of these four data sets.