DOI: 10.5061/dryad.n02v6wx9p
title: Genetic variation in host selectivity and adaptive strain enrichment in legume-rhizobia symbiosis

description: Mutualism breakdown can be prevented if partner species preferentially
 select and reward partners that provide greater benefit. We examined these
 two components using the legume Medicago truncatula and its
 nitrogen-fixing symbiont Sinorhizobium meliloti. The first dataset focuses
 on reanalyzing strain composition data from the nodules of 202 accessions
 to show significant genetic variation in the capacity of Medicago to
 restrict strain diversity when controlling for nodule number variation
 using a simulation. By performing an augmented image analysis of nodules
 pool images to produce scores of morphological parameters, we found that
 hosts with a suite of nodules traits, including shorter nodules, tended to
 be more selective. Using previously published SNP data for these ~200
 accessions, we performed a genome-wide association study on host
 selectivity to identify candidate genes. The second dataset uses two
 well-studied Medicago genotypes (A17 and R108) with contrasting nodule
 morphologies to assess the effectiveness of adaptive enrichment mechanisms
 by sampling the relative frequencies of rhizobial strains in pools of
 small nodules that we show have smaller rhizobia population sizescompared
 to large nodules. We pair these results with previous single-strain
 assessments of strain benefits to hosts to show that hosts enriched
 beneficial strains in large nodules, the host that formed larger and more
 variably sized nodules and thus had greater 'potential' to
 increase rhizobial populations was less effective. Together this package
 includes the data, code, and figures to reproduce our analysis.
description: # Genetic variation in host selectivity and adaptive strain enrichment in
 legume-rhizobia symbiosis This Dryad Repository supports and enables the
 reuse of Burghardt et al. 2026 data and associated analysis outputs,
 including raw phenotypic data, software, analysis code, and GWAS and
 statistical outputs. This repository included three major parts: 1) data
 and analysis of *Medicago* **host selectivity** and **nodule morphology**
 of 202 accessions of Medicago inoculated with a mixture of 
 86 *Sinorhizobium meliloti* strains. We provide code to assess how host
 selectivity is correlated with nodule morphology traits using LASSO
 regression and Boruta. 2) Results from a **genome-wide association
 analysis** of the host selectivity trait, and 3) data and analysis
 of *Medicago* **adaptive enrichment** of bacterial symbionts. The adaptive
 enrichment analysis includes measuring symbiotic traits (nodule length,
 size, branching, colony-forming units, nodule number, and strain relative
 frequencies in large and small nodule pools) on R108 and A17 hosts
 inoculated with a mixture of 68 rhizobial strains. We analyzed whether
 each host can enrich for beneficial rhizobial strains by comparing prior
 single-strain plant biomass measurements with strain frequencies in
 nodules. ## Host Selectivity Datasets and Code
 **NoduleMorphology_HapMap_ElasticNet.R** - Contains R code to conduct
 Medicago HapMap Panel rhizobial strain diversity analysis with host
 selectivity simulations, analysis of nodule traits, and relationships
 between nodule traits and host selectivity. This code generates Figure 2,
 S1, S3, and S4 as well as the information in Tables S1
 (**TableS1_CorrHS.csv** + **TableS1_CoV.csv**) and S3 from the manuscript.
 Takes as inputs **epstein_et_al_2022_S9.csv** and **df_summary.csv**
 **epstein_et_al_2022_S9.csv** - Contains previously published data
 describing the frequencies of 88 strains within initial inocula and nodule
 pools of the HapMap panel (HapMap Select & Resequence Experiment
 (Epstein *et al.*, 2022)). Columns include: * pot_or_sample- pot reference
 id from the greenhouse experiment in Epstein *et al* 2022- corresponds to
 ‘pot’ in the nodule morphology dataset (df_summary.csv)            *
 host_genotype- *Medicago truncatula* HapMap ID more information available
 on the MedicagoHapMap.org       * MAG746A… MAG97: relative frequency of
 each of the Sinorhizobium strains from Riley *et al* 2022 estimated in the
 nodules from each pot **S_RGWA_1.CSV** - Contains experimental design data
 from the 2020 HapMap Select & Resequence experiment described in
 Epstein et al. 2023. * pot - matches numbers
 in **epstein_et_al_2022_S9.csv** * geno - *Medicago truncatula* HapMap ID
 more information available on the MedicagoHapMap.org  * block -
 Experimental replicate block  **df_summary.csv** - The summary dataset
 compiled from nodule morphology ImageJ analysis output measures a subset
 of which are used in downstream analysis. * pot - matches numbers
 in **epstein_et_al_2022_S9.csv** - prop.harvest - proportion of nodules
 harvested and images from pot * Columns 3:137 - Summary statistics of
 nodule morphology traits measured in ImageJ including mean, 95.quant (95th
 percentile), 5.quant (5th percentile) median, var, min, max, range,
 scaled_range (range / mean). Also included are the number of
 nodule_raw_count, ttl.nodule.area, nodule_calc_count, and pred.prop.lobed
 (predicted proportion of lobed nodules). **TableS1_CoV.csv** * trait -
 Nodule morphological traits extracted from images of nodules pools *
 coeff.var - coefficient of variation among host genotypes for each trait
 **Table S1_CorHS.csv** * trait- nodule morphology trait inferred from
 nodule images for all host genotypes * correlation.coef- Pearson
 correlation coefficient with host selectivity * p_value – raw p-value 
             * sig_0.05 – y/n to indicate significant at 0.05
 threshold         * sig_correct – y/n to indicate significant after
 adjusting for multiple testing ## GWAS of Host Selectivity Trait Files for
 running the host selectivity GWAS pipeline (**host_selectivity_gwas.sh**)
 and record of settings for the GWAS run itself
 (**medtr_ulmm_host_selectivity.log.txt**), complete GWAS output files
 (**medtr_ulmm_host_selectivity.assoc.txt,
 medtr_ulmm_host_selectivity.csv**). **manhattan_plot.R,
 A17_R108_SNPsegregation.R**, and the summaries from of these analyses
 focused on significant SNPs, their associations, and linkage
 disequilibrium with other SNPs
 (**sig_LD_geneinfo_pval_beta.tsv, sig_mdtr.csv,** and **sig_mdtr.ld**).
 Significant SNP information is mostly summarized within
 **sig_LD_geneinfo_pval_beta.tsv.** The column names in the complete GWAS
 results in **medtr_ulmm_host_selectivity.assoc.txt** and
 **medtr_ulmm_host_selectivity.csv** overlap with the one detailed below.
 **sig_LD_geneinfo_pval_beta.tsv-** Includes additional annotation
 information and ref/alt SNPs calls gathered for significant SNPs in tab
 delimited format. Columns include: * snp- SNP location in the *Medicago
 truncatula* V5 genome assembly            * chr- Chromosome ID from the
 *Medicago truncatula* V5 genome assembly  * pos- SNP location        *
 pvalue- extracted from GEMMA likelihood ratio text * allele1- alternate
 allele           * allele0- reference accession allele      * af-
 frequency of the reference allele in the HapMap population      * beta-
 effect size of association with host selectivity  * se- standard
 error       * p_score – combination of p-value and effect size * start-
 closest annotated gene start     * end- closest annotated gene end *
 dist_from_start – distance of SNP from gene start * dist_from_end size –
 distance of SNP from gene end * score- NA        * strand- plus or minus
 DNA strand        - source- EuGene: integrative gene finder for eukaryotic
 and prokaryotic genomes from INRAE       - feature - All are CDS (Coding
 sequences) - frame- 0,1,2 - gene_id- from *Medicago truncatula* V5 genome
 assembly   - gene_name- If historically has one otherwise *Medicago
 truncatula* V5 genome assembly geneID - PRODUCT- based on
 annotation         - GO- GO terms from EuGene   - EC- KEGG Enzyme from
 EuGene         - ACTIVITY - prediction from EuGene         - PUBMED- -
 papers on the gene from EuGene                    - IPR-  InterPro
 Database Prediction                 - ALT- nucleotide found in
 non-reference accession     - REF- nucleotide found in the reference A17 
 - HM001… HM316 – binary descriptor of whether each of the accession from
 the Medicago HapMap panel has the reference allele (0) or the alternate
 allele (1). Two alleles are included because Medicago are diploid but
 highly selfing. **medtr_ulmm_host_selectivity.assoc.txt** - complete ulmm
 output of host selectivity means associated with SNPs containing
 information for all SNPs tested. Column names overlap with significant
 association files above. **medtr_ulmm_host_selectivity.csv** - Reduced
 ulmm output of host selectivity means associated with SNPs. Column names
 overlap with significant association files above. **manhattan_plot.R**- R
 code used generate the manhanttan plot. **A17_R108_SNPsegregation.R**. R
 Code to compile ref/alternate SNP calls across all HapMap accessions for
 the significant GWAS associations. Takes as
 inputs **sig_LD_geneinfo_pval_beta.tsv** and **HapMap_SNPs.csv**
 **HapMap_SNPs.csv** - CSV format of SNP markers included in the
 *M.* *truncatula* GWAS with genotypes as rows and SNPs as columns with
 names after the *Medicago truncatula* V5 genome assembly. ## Host Adaptive
 Enrichment Datasets **NodSize_A17_R108.R** - Contains R code for
 comparisons between A17 and R108 within Figure 3, 4, S5, S6, and S7 and
 associated analyses producing Tables S4, S5, S6, and S7. Takes as inputs
 **NodulePools_SizeData.txt**,
 **SingleNoduleColonies.txt, NoduleSizeClassInfo.txt, SingleStrain_phenotype_summary.tsv,** and **freq_C68.txt**. **NodulePools_SizeData.txt** - Overall characteristics of A17 and R108 nodule size pools (e.g., number of nodules and wet weight) * Plant_genotype- A17 or R108 * Community- *Sinorhizobium* community * Pot_replicate- number from 7-12 * Harvest_date- month/day/year * Person- Intials of harvestor * Size- catagory (small, medium, large) * Nodule_number - number of nodules in the pool * Nodule_weight_g - nodule wet weight in grams **NoduleSize_HarvestData.txt**- Overall summary of harvest data from A17 and R108 adaptive enrichment experiment. * Plant_genotype- A17 or R108 * Community- *Sinorhizobium* inoculum used   * Pot_replicate- numbers (7-12)           * Harvest_date- month/day/year         * Person- Intials of harvester    * Plant_num- number of plants in the pot          * Flowering_num- number of flowers at harvest          * Seeding_num- number of seeds at harvest   * Leaf_Color- qualitative measure of leaf yellowing- sign of N deficiency       * Nod_num- number of nodules harvested      * Plants_harvested- number of plants harvested         * Nod_Num_white- number of white nodules in pool * Nod_weight_g - total nodule wet weight in grams * Veg_dryweight_g        * Root_dryweight_g      * Small_Nod_Num - small pool nodule number           * Small_Nod_wieght- small nodule pool wet weight in grams             * Med_Nod_Num - medium pool nodule number                   * Med_Nod_wieght- medium nodule pool wet weight in grams                     * Large_Nod_Num- large pool nodule number             * Large_Nod_wieght- large nodule pool wet weight in grams **SingleNoduleColonies.txt** - Colony-forming units estimated from individual crushed A17 and R108 nodules of each size class. ~ 3 replicate dilution series were conducted. * Plant_genotype- A17 or R108 * Pot_replicate- numbers (7-12) * Size- nodule size category (small, medium, large)     * Plate- replicate            * Dilution- dilution level counted          * Count- number of colonies on the dilution plate       * Colonies- estimated number of colony-forming units from the nodule **NoduleSizeClassInfo.txt** - Measurements of A17 and R108 nodule length and number of lobes from 10 randomly chosen nodules of each size category from each replicate pot. * Plant_genotype- A17 or R108 * Community- *Sinorhizobium* inoculum used * Pot_replicate- numbers (7-12)           * Size- nodule size category (small, medium, large)     * Lobes_num- number of nodule lobes * Length_mm- length of nodule in millimeters **freq_C68.txt** - Frequencies of a community of 68 *Sinorhizobium* strains within nodules communities of A17 and R108 small, medium, and large pools and for the initial strain communities. These values are inferred from HARP (see Burghardt *et. al., *AEM 2023 for details, Kessner *et. al., *2013 for reference to method) * pool- includes all sample details separated by underscores community of 68 strains (C68), host genotype (A17,R108, or initial), and treatment (s=small nodule, m=medium nodules, b=big nodules, X= all nodules) and replicate pot (#) * columns USDA1157....USDA1719 refer to all the strains in the community and their inferred relative frequency in the pool **SingleStrain_phenotype_summary.tsv** - Single-strain plant growth benefit data for 68 strains from a previously published experiment (Burghardt *et al.* 2018) * plant_genotype- Medicago genotype (A17 or R108) * strain- *Sinorhizobium meliloti* strain name on NCBI * nodule- number of nodules centered and normalized based on dataset overall mean * nodule_raw- mean number of nodules for each host x strain combination  * nodabove- mean number of nodules in the top half of the root system centered and normalized based on dataset overall mean           * nodabove_raw mean number of nodules in the top half of the root system            * nodred- mean number of red nodules in the top half of the root system centered and normalized based on dataset overall mean. * nodred_raw- mean number of red nodules in the top half of the root system         * weight- mean aboveground plant dry weight in N-free conditions centered and normalized based on dataset overall mean       * weight_raw- aboveground plant weight in N-free conditions ## Code/Software R is required to run the **NoduleMorphology_HapMap_ElasticNet.R** and **NodSize_A17_R108.R**; the scripts was created using R version 4.5.1. Annotations are provided throughout the script through 1) library loading, 2) dataset loading and cleaning, 3) analyses, and 4) figure creation. ## Usage notes **Python** is required for .Py scripts, **R** is required to open .R scripts, .sh file runs on the command line, **Microsoft Excel** can be used to view .csv, .txt, .tsv files ## Works referencing this dataset Burghardt, LT; Sydow, P; Sutherland, J; Epstein, B; and Tiffin, P. (2026) Genetic variation in host selectivity and adaptive strain enrichment in legume-rhizobia symbiosis, Proceedings of the Royal Society B, [https://doi.org/10.1098/rspb.2025.2851](https://doi.org/10.7717/peerj.17048)