GWA mapping reveals loci associated with natural circadian
variation
We used the online GWA-portal to perform association mapping on the
three circadian traits found to be significantly affected by genetic
variation (Seren, 2018). We used an Accelerated Mixed Model (AMM) to
account for population structure, although the other association models
(linear and non-parametric) gave similar results (See Supplementary
Figure 6). Pseudo-heritability estimates for each trait were given as an
output of the GWA analysis: period=71%, RAE=37% and phase=13%.
GWA identified multiple genomic regions associated with period, phase
and RAE as shown in Figure 3. We used –log10(p ) 6.5 as an
arbitrary p-score cut-off to select SNPs for further investigation. This
threshold is conservative compared to several other previously published
studies in Arabidopsis (Kooke et al., 2016; Proietti et al.,
2018; van Rooijen et al., 2015). We investigated known core circadian
and flowering time genes to see whether these were significantly
associated with any of the traits measured. Other than ELF3 (see
below), none of these genes fell within the window of association for
significant SNPs. Genes in regions 30kb upstream or downstream of the
most significant SNPs were considered as potential gene candidates and
were selected for further analysis based on their previously attributed
functionality and designated GO term. Genes involved in circadian
rhythms, flowering time or chloroplast regulation were given prevalence
(details in Supplementary File 2). The most significant associations had
three SNPs with a –log10(p ) score of 10.4-11.5, found on
chromosome 4 associated with period variation. Within this interval we
identified a non-synonymous SNP in the gene COLD-REGULATED GENE
28, a gene that has previously been identified as a negative regulator
of several core clock genes (PRR7, TOC1, PRR5 and ELF4 )
and is also implicated in the trade-off between flowering-time and
freezing tolerance (X. Li et al., 2016; Wang et al., 2017). The
substitution resulted in a tryptophan (W) to serine (S) amino acid
change at position 58 within the second exon of COR28 (Figure
4A). This had a SIFT score of 0 indicating a highly probable deleterious
effect on protein function. 16 accessions in this study had the minor
allele, all found in the South of Sweden. The 58S accessions had a
period 1.29h longer than the 58W accessions (t(17.4)=-7.46,p< 0.001, Welch Two Sample t-test) (see Figure 4B) and
mostly belonged to the genomic sub-group PC.A. The long-period of
cluster PC.C therefore cannot be explained by variation in this SNP. We
used the online tool Polymorph 1001 to look for other variants with the
serine substitution and found only 5 other variants not assayed in this
study (making 21 in total), all of which were also from the South of
Sweden (Figure 4C).
We used flowering time data by Li et al. 2010 (Y. Li et al., 2010) and
Sasaki et al. 2016 (Sasaki et al., 2015) to look for flowering time
differences between the accessions carrying the COR28 -58W major
allele and the -58S minor allele (see Table 1). We found that the
accessions with COR28 -58S had significantly extended flowering
times under simulated seasons for Sweden and Spain (Li) and under a 10°C
and 16°C long-day temperature regime (Sasaki) (see Figure 4D). This
complements previous findings that COR28 is a flowering promoter
and that modifications to this gene increase flowering time as well as
lengthening period.
[Table 1]
Other significant SNPs associated with period (chromosome 3) and phase
(chromosome 1) identified gene candidates involved in chloroplast
function (PARC6, SCO2, DNF, AT1G71015 and FAB1C ). The DF
circadian output we used to assay these traits is based on oscillating
activation phases of PSII (Goltsev V, Zaharieva I, Chernev P, 2009;
Jursinic, 1986) and therefore natural variation in genes regulating
chloroplast functionally could also affect the DF output in these
candidates.
A RAE associated SNP on chromosome 2 was found 5726bp upstream from a
SNP in ELF3 previously characterized as the ELF3-Shaallele. Our associated SNP was found to be under strong linkage
disequilibrium with the ELF3-Sha allele
(R2=0.86). The alanine-to-valine transition in amino
acid position 362 has been associated with naturally occurring
alterations to periodicity and robustness in accessions from Central
Asia, specifically Tajikistan (Anwer et al., 2014). Here, 13 Swedish
accessions were shown to carry the ELF-Sha allele and had mean
RAE ratios 0.032 higher on average than for the other accessions. We
substantiate evidence that ELF3-Sha accessions have lower
rhythmicity and extend the global range of this allele into Northern
Sweden. A surprising kinship between Arabidopsis accessions from
Northern Sweden and Central Asia has been previously demonstrated
through analysis of global population structure and indicates that the
presence of this allele has not evolved convergently between the two
populations (Nordborg et al., 2005).