Co-segregation analysis
The co-segregation analysis was conducted using a Cleaved Amplified Polymorphic Sequences (CAPS) assay to genotype and DF to phenotype crossed and selfed individual plants.
Primers were designed to amplify a 252bp fragment within COR28exon 2 (COR28-F : 5’-ACAGTAAGTAACCCCGAACC-3’, COR28-R:5’-TTCTTGACAAAAGTCCCACTC-3’). These fragments were then digested using TaqI (Thermo Fisher Scientific, #ER0671, recognition site: T^CGA). TaqI was added to 10ul of PCR reaction mixture and incubated at 65C for 1.5h according to manufacturer’s instructions. Individuals homozygous for the COR28 SNP 58S (found in the parent St-0) are not recognised by TaqI and so presented as a single 252bp band on an agarose gel (3% Agarose, run for 1.5h at 50V). Individuals homozygous for theCOR28 SNP 58W (found in the parent T800) were fully cleaved resulting in two bands 92bp and 160bp in length. Heterozygous individuals therefore had three bands at 252bp, 160bp and 92bp clearly separated by electrophoresis. Restriction digest gels for identifying F2 plants can be viewed in Supplementary file 6. All plants in generations (F0-F1) were vernalised for 12 weeks at 4°C prior to growth at 22°C under long days (16:8h). Parent accessions St-0 and T800 were genotyped to check for homozygosity prior to crossing. All three heterozygous F1 individuals were (♀T800 x ♂St-0) and were also confirmed by the CAPS assay. These F1 plants were then selfed and the circadian rhythms of the F2 progeny were analysed using DF on detached leaves from 24 day old plants grown under 12:12h light:dark conditions at 22°C. Leaves were placed on 0.5% agar plates and were exposed to 24h constant L:L prior to imaging as described for the 96 well plates.
Results