# Pooled enrichment sequencing identifies diversity and evolutionary pressures at NLR resistance genes within a wild tomato population

## TL;DR

This study uses sequencing to explore genetic diversity and evolutionary pressures at NLR resistance genes in a wild tomato population.

## Contribution

A cost-effective method for sequencing NLR genes in pooled samples is developed and validated in a wild tomato species.

## Key findings

- 13 out of 220 NLR genes show maintained polymorphism in the S. pennellii population.
- NLR genes display varying πN/πs ratios and site frequency spectra, indicating diverse evolutionary pressures.
- The method successfully identifies NLR genes under natural selection in a selfing and bottlenecked population.

## Abstract

Nod-like Receptors (NLRs) are Nucleotide-binding domain and Leucine rich Repeats (NB-LRR)-containing proteins that are important in plant resistance signaling. Many of the known pathogen Resistance (R)-genes in plants are NLRs and they can recognise directly or indirectly pathogen molecules. As such, divergence and copy number variants at these genes is found to be high between species. Within populations, positive and balancing selection are to be expected if plants coevolve with their pathogens. In order to understand the complexity of R-gene coevolution in wild non-model species, it is necessary to identify the full range of NLRs and infer their evolutionary history.

Here we investigate and reveal polymorphism occurring at 220 NLR genes within one population of the partially selfing wild tomato species S. pennellii. We use a combination of enrichment sequencing and pooling of ten individuals, to specifically sequence NLR genes in a resource and cost-effective manner. We focus on the effects which different of mapping and SNP calling software and settings have on calling polymorphisms in customized pooled samples. Our results are accurately verified using Sanger sequencing of polymorphic gene fragments. Our results indicate that some NLRs, namely 13 out of 220, have maintained polymorphism within our S. pennellii population. These genes show a wide range of πN/πs ratios and differing site frequency spectra. We compare our observed rate of heterozygosity to expectations for this selfing and bottlenecked population. We conclude that our method enables us to pinpoint NLR genes which have experienced natural selection in their habitat.