Additional file 1 of Molecular genetic examination of Circadian Clock Associated 1 gene (CCA1) in hexaploid wheat

Supplementary Material 1. Table S1. The generic primer sequences in the gene expression studies (qRT-PCR). Abbreviations: CCA1 – Circadian Clock-Associated 1, PGD – Phosphogluconate dehydrogenase, F=Forward, R=Reverse. Table S2. The gene codes of the DNA sequences used for the in silico approaches of the CCA1 gene in the studied plant species are listed below. Table S3. The list of primer sequences of the CCA1 gene for KASP genotyping. The genome specific primers were designed by PolyMarker. Abbreviations: CCA1 – Circadian Clock-Associated 1, F=FAM and V=VIC fluorescens dyes, C=Common primer, Tm=Melting temperature, red and green highlights: FAM and VIC fluorescens labeled sequences, the studied allele variants are indicated by red at the end of the primer sequences. Table S4. The list of A genome specific primer pairs used in the sequencing of the intron 3 of TaCCA1 gene. The primers were designed by Primer3. Abbreviations: CCA1 – Circadian Clock-Associated 1, F=Forward, R=Reverse, Tm=Melting temperature, bp=base pair. Table S5. The effects of the different factors on the variance components of the CCA1 gene (σ2 (%)) under two controlled environmental temperature values on 14-days-old winter wheat. The factor of Environment refers to the two ambient temperature levels 18 °C vs 25 °C. Abbreviations: The not-significant factors are indicated by ns (not significant). The asterisks denote the significance level. *P≤0.05; **P≤0.01; ***P≤0.001. Table S6. The allele distribution of the CCA1 gene in a diverse wheat population. The description column indicates the genotypes in the gene expression study. Table S7. Sequence-based comparision of the alternative isoform of the CCA1 gene among wheat its progenitors, and barley. Table S8. Sequence-based comparison of exon and intron regions of CCA1 gene between wheat and barley. Table S9. Categorization of the different cis-regulating motifs and their proportion in the intron 3 sequence before the alternative exon in barley HvCCA1-7H and hexaploid wheat TaCCA1-7A, TaCCA1-7B. and TaCCA1-7D genes. Table S10. Comparison between the sequenced intron 3 region of the CCA1 gene with cys-motifs and alternative isoform among Chinese Spring and the three examined hexaploid wheat genotypes. Abbreviations: CS=Chinese Spring, Yellow highlights=Set1F, Set2F primers, Red highlights=Set2R, Set3F primers, Green highlights=Set1R, Set3R, TATA-box is indicated by red letters, Low Temperature Response motif is indicated by blue letters, Bold letters=Sequence of the alternative exon, asterisk=conservation among the nucleotide positions. Fig. S1. The position of generic primer sequences in the structure of the CCA1 gene. Abbreviations: ATG=Start codon, TAG=Stop codon, yellow rectangles=exon sequences, lines=intron sequences, thick line=scale (500 bp), position of the (F) Forward and (R) Reverse primers indicated by red rectangles. Fig. S2. The daily gene expression dynamics of CCA1 in three winter wheat cultivars (AT1, a; AT3, b; and AT20, c) under two controlled environmental conditions: optimal 18 °C and supra-optimal 25 °C. Data points represent individual measurements from three independent biological replicates; solid lines indicate mean expression values; shaded areas denote ± SE. Grey rectangles indicate the dark periods. AT1 = Mv Toborzó, AT3 = Tommi, and AT20 = Charger. Cosinor-fitted curves were generated from the average expression values of each cultivar, and the mean amplitude (amp) and peak time were calculated from these fits. Fig. S3. (A) The positions and sequence-based comparison of the identified repeated motifs in the intron 3 sequence after the alternative exon among barley HvCCA1-7H and hexaploid wheat TaCCA1-7A, TaCCA1-7B and TaCCA1-7D genes. The sequences of TaCCA1-7A are shown beside the colored rectangles. (B) Determination of the evolutionary diversities among the three repeated motifs in corresponding intron 3 sequences of HvCCA1-7H, TaCCA1-7A genes. Abbreviations: CCA1 – Circadian Clock Associated, colored rectangles=repeated motifs, lines=intron sequences, SNP=Single-Nucleotid Polymorphism, bp=base pair, red bold letters indicate the polymorphisms between wheat and barley, A=Adenine, G=Guanine, T=Thymine, C=Cytosine, W=wheat, B=barley.