Gene annotation for the significantly associated SNPs
Of the 1458 SNPs associated with environmental gradients, functions
could be assigned for 788 (54%), while the rest had no matches in
available gene ontology databases. We found that 283 SNPs with
identifiable functions belonged to protein types that may be directly
related to drought tolerance or other environmental responses (Fig. 4).
We categorized these genes into five main functional groups: (a) the
ubiquitination pathway, (b) seed, pollen and ovule formation, (c) cell
wall formation, (d) stress responses, and (e) cell division and growth.
Other associated SNPs with known functions were in or near transcription
factors and genes with expression-regulating functions.
Many of the SNPs associated with TMAX, TMIN, CWD, and PCK4 were in or
near genes in the protein ubiquitination pathway or the jasmonic acid
synthesis response pathways (Fig. 4 and Table S2), both of which are
involved in responses to biotic or abiotic stress (Creelman & Mullet
1995; Lyzenga & Stone 2012; Stone 2014). CWD and PCK4 were also
associated with SNPs in or near genes involved in seed dormancy, cell
wall organization, and the abscisic acid (ABA) signaling pathway, which
have been previously linked to drought responses in trees (Moranet al. 2017b). Genes involved in reproduction, including pollen
and ovule formation, were associated with TMAX, TMIN, and PCK4. Genes
involved in vascular tissue formation, growth regulation, and stress
responses were associated with TMAX and PCK4. Genes involved in stomatal
regulation and pathogen responses were associated with TMIN and PCK4.
Further biotic and abiotic stress response genes were associated with
PCK4, as were genes involved in nutrient transport, photosynthesis,
respiration, sugar synthesis, and light responses (Table S2).
Of the 817 SNPs associated with seedling control (wet treatment) trait
values and 1,154 SNPs associated with trait drought responsiveness, 43%
and 51% could be assigned functions by gene ontology (Additional file
2: Table S3 and Table S4). Many of the same functional categories of
genes associated with the environment were also related to measured
phenotypes. This includes ubiquitination, seed development, cell wall
organization, stress response, cell division (Fig. 4, 5, 6), and
transcription factors. However, there was no overlap in specific SNPs
identified.
The control treatment levels of the two stomatal traits were associated
with genes involved in ubiquitination, cell wall organization or
modification, growth and development, and ABA response. Control
root-to-shoot ratio was associated with genes involved in biotic &
abiotic stress responses, cell wall organization or modification, cell
division or differentiation, lateral root formation, and ubiquitination.
Control height growth had no associated SNPs, and root length was only
associated with one SNP located in a gene involved in ubiquitination
(Fig. 5). However, drought responsiveness of height growth, shoot
weight, and root length were associated with all five functional
categories (Fig. 6). Drought responsiveness of the two stomatal traits
was associated with genes involved in stress responses, cell wall
formation/organization, cell division/differentiation, and root
formation.
Besides the five main functional groups of genes with SNPs associated
with climatic, phenotypic, and drought response variables, several other
functional groups were identified in the GEA and GPA annotation results
(Table S2, S3, and S4). For example, 111 (14%) of the environmentally
associated SNPs, 53 (6%) of SNPs associated with control traits, and
121 (12%) of the SNPs associated with trait drought responses were in
genes relating to ATP binding or protein kinases. It was also fairly
common for associated SNPs to be in genes associated with RNA/DNA
binding, metal ion binding, translation, and protein transport.