Genetic architecture of heritable leaf microbes

Background

Host-associated microbiomes are shaped by both their environment and host genetics, and often impact host performance. The scale of host genetic variation important to microbes is largely unknown, yet fundamental to the community assembly of host-associated microbiomes, and with implications for the eco-evolutionary dynamics of microbes and hosts. Using Ipomoea hederacea, Ivy-leaved morning glory, we generated matrilines differing in quantitative genetic variation and leaf shape, which is controlled by a single Mendelian locus. We then investigated the relative roles of Mendelian and quantitative genetic variation in structuring the leaf microbiome, and how these two sources of genetic variation contributed to microbe heritability.

Results

We found that despite large effects of the environment, both Mendelian and quantitative genetic host variation were important in contributing to microbe heritability, and that the cumulative small effect genomic differences due to matriline explained as much or more microbial variation than a single large effect locus. Furthermore, our results are the first to suggest that leaf shape itself contributes to variation in the abundances of some microbes in the leaf microbiome.

Conclusions

The genetic architecture of plant-associated microbiomes depends on both quantitative genetic variation and Mendelian traits, with similar contributions to microbe heritability. Our results demonstrate the roles of different scales of host genetic variation in the assembly of a natural microbiome. The genetic basis and heritability of a host’s microbial phenotype is important for host evolution and ecology because microbes can affect host fitness, and because it can influence reciprocal selection between hosts and microbiomes. Additionally, when host-associated microbiomes have heritability, then this suggests they have the capacity to evolve as other host traits might, with potentially adaptive functions.

Methods
Overall goal: Using Ipomoea hederacea, Ivy-leaved morning glory, we generated matrilines differing in quantitative genetic variation and leaf shape, which is controlled by a single Mendelian locus. We then investigated the relative roles of Mendelian and quantitative genetic variation in structuring the leaf microbiome in a common garden, and how these two sources of genetic variation contributed to microbe heritability.

Breeding design: We used seeds derived from a cross by Campitelli and Stinchcombe (2013), where individuals from the two alternate homozygous leaf shape phenotypes (i.e., fully lobed or whole) were collected from North Carolina, USA, selfed for seven generations to generate homozygous parents (P1), and then crossed with each other (Figure 1). A single F1 was allowed to self-fertilize, producing F2 progeny. We scored F2 plants for leaf shape, and allowed them to self-fertilize; we refer to all the selfed progeny of an individual F2 plant as a “matriline.”  We used F3 seeds, set by F2 plants we had identified as heterozygous for leaf shape, as our experimental seeds.

Data collection: In 2021, we planted a total of 240 I. hederacea seeds from 82 matrilines (2-3 seeds/matriline) in a common garden at Koffler Scientific Reserve (www.ksr.utoronto.ca) in Ontario, Canada. We scarified seeds then planted them in a greenhouse in peat pots containing soil from the field. We planted the first cohort on June 4th and a second cohort on July 2nd, because of poor germination in the first cohort. After the majority of plants in a given cohort germinated (approximately one week), we transplanted the pots into the field. The 218 surviving plants consisted of 83 individuals from the first cohort, and 135 individuals from the second cohort. On September 1st, we collected similarly-sized leaves within one foot from the ground by cutting them with sterilized scissors into a sterile plastic bag, after which they were stored in a -80°C freezer until DNA extraction.

Sequencing and analysis: We performed extractions using the whole leaves with QIAGEN DNeasy® PowerSoil® Pro Kits; both epiphytic and endophytic microbes were extracted. We sent samples to Génome Québec (Montréal, Canada) for Illumina MiSeq PE 250bp 16S rRNA gene amplicon sequencing on the conserved hypervariable V4 region (primer pair 515F-806R). Raw microbiome sequence data is available at NCBI’s Sequence Read Archive under the BioProject accession number PRJNA1107536. We used Quantitative Insights Into Microbial Ecology 2 (QIIME2) v.2022.2 to trim the sequences for quality and we denoised the sequences with DADA2. Using QIIME2, we removed amplicon sequence variants (ASVs) that had fewer than 10 reads across all samples, and assigned taxonomy using the ‘sklearn’ feature classifier and Greengenes 16S rRNA gene V4 region reference, then filtered out reads assigned as cyanobacteria and mitochondria to remove plant DNA. Finally, we constructed a phylogeny using QIIME2’s MAFFT and FastTree 2 to obtain a rooted tree.