3.4 Genetic basis of temperature-dependent plasticity
Next, we used the SensyPAM platform to investigate the genetic basis for
the period and amplitude of circadian photosynthetic rhythms and their
thermal plasticity. To this end, we selected 285 wild barley of the B1K
collection (Hubner et al. , 2009) from different geographic
origins. We performed a GWAS analysis using the traits values per se;
comparing genetic signals between phenotypic plasticity of the circadian
parameters under OT and HT environments (see Methods; Table S3).
Moreover, since the analysis of RHL (this study; Figure 2 ) and
the ASHER populations (Bdolach et al., 2019) indicated a
significant effect of the plasmotype diversity on the photosynthetic
rhythm plasticity, we included sequencing information from isolated
chloroplast DNA for a portion of this panel (see Methods; TableS4 ).
We wanted to identify nuclear loci for which allelic diversity is
associated with plasticity of photosynthetic rhythms. First, we
increased the current coverage of Barley 1K genomic analysis by
harvesting genic and intergenic DNA variation from several sources (see
Supporting information and Methods). Prior to GWAS analysis, missing
values were imputed with the ”missForest” algorithm and filtered for
markers with missing data >0.2, minor allele frequency
< 0.03, monomorphic and multiallelic markers. The selection
process led to a final set of 13,786 informative markers for GWAS
analysis (Table S5 ). We wished to detect QTLs with persistent
effects across the two environments (OT and HT) but also with Q × E
effects, i.e., loci with specific effects to a certain environment
(Yamamoto & Matsunaga, 2021).
Briefly, the output of the analysis for each SNP included “Additive
Main Effect” (p.ame ), “All SNP effects” (p.all ),
“Interaction terms” (p.int ) and a Wald score for each
environment indicated the environment with the most significant effect
of the locus on the trait (Table S6 ). By setting a threshold of
LOD > 4, we found 102 signals for amplitude and only eight
for the period. Seven of the eight signals we identified for the period
are found in pall, p.int and with the highest Wald score under
HT. We could not detect signals above LOD 4 under OT in none of the
traits. There were 17 significant signals for interactions inP.all , and all except one were affecting the trait under HT.
There are five signals for the amplitude and seven for the period.
In a previous study, we identified several DOCs loci that
modulated the circadian clock output in the HEB interspecific mapping
population (Prusty et al., 2021). Interestingly, some of theseDOC loci are overlapping with signals in this current genome scan
of the B1K, including some that contain genes reported to be involved in
the circadian clock. Of note is the gene GIGANTEA (GI )
that resides in the long arm of chromosome 3. Previously, we identified
a large interval on chromosome 3, DOC3.2 , associated with
significant pleiotropic effects on the clock period and growth in the
field. Nevertheless, DOC3.2 , although harboring the barley
ortholog of the GI gene (Fowler et al., 1999), stretched
in the previous HEB population analysis to a distance of 45.98 Mbp
(Chr3H_35066186 – Chr3H_81047480). Here, in the GWAS in the B1K
collection, we identified SNPs around the GI gene that were
significantly associated with the amplitude of the clock and its
plasticity. In fact, the current SNP arrangement point to a causal
variation in 150 kb downstream to the GI gene. Phenotypic
analysis showed difference in the amplitude between two allele under HT
condition while no effect was assigned to the allele in OT condition
(Figure S5 ).