The circadian rhythm is an endogenous regulatory mechanism (autonomous oscillator) that enables the plants to synchronize their internal biological processes with the changes in daily temperature and light conditions of the external environment. It also participates in the regulation of photosynthesis, carbohydrate biosynthesis, biotic and abiotic stress responses. The regulatory mechanism of the plant circadian rhythm has already been significantly explored in Arabidopsis and many homologous genes have been described in wheat, but only a few of these have been studied more intensively.

Therefore, (1) we determined the daily expression patterns of a major circadian clock gene (CCA1). Three winter wheat cultivars with different plant development and genetic diversities were examined (‘Mv Toborzó’ /AT1, early heading/, from Hungary, ‘Tommi’ /AT3, late heading/, from Germany and ‘Charger’ /AT20, late heading/, from Great Britain) under four controlled environmental conditions (18 °C vernalized/unvernalized, 25 °C vernalized in the phytotron and 14-18 C vernalized in the greenhouse) applying long photoperiod (16h). The leaf sampling started at 6:00 a.m. (1 h after the start of the light period - ASLP), and it was carried out every three hours, for two consecutive days (48h) using two-week old plants.

The relative gene expression was normalized with three housekeeping genes. In parallel to the gene transcription studies (2) we also carried out in silico researches using an online database (https://plants.ensembl.org). The sequenced region of CCA1 was analyzed and compared with CLC Genomics Workbench 3.6.5. program to determine the possible mutations. In addition, the phylogenetic tree of CCA1 was also constructed with MEGA 11 software to examine the possible evolutionary diversities between the homologue genes of Arabidopsis and two cereal species (Hordeum vulgare and Triticum aestivum).

The expression of CCA1 showed a definite diurnal activity irrespective of genotypes. This finding was in a good agreement with the expression of homologue gene in Arabidopsis. In the case of the early cultivar (‘Mv Toborzó’) the peak expression of this gene nearly doubled at 3h ASLP under 18 C (vernalized). According to the phylogenetic analysis of CCA1, the genotypes were positioned in five distinctly separated subgroups.

A stronger association between the subgroups and the genomes of different species (7A, 7B, 7D of hexaploidy wheat, 7H of barley and 2G of Arabidopsis) was observed, that was due to the length variation in intron 3. A larger insert section of intron 3 region was identified in barley (1913 bp), which was even longer in hexaploid wheat (3300 – 3600 bp) compared to the relevant region of the homologue gene in Arabidopsis (479 bp). In addition, a few single nucleotide polymorphisms (SNPs) were also identified in the exon 5 of hexaploid wheat.

One non-functional mutation was found both in chromosome 7A in ′Landmarkʼ (4591 bp) and in chromosome 7B in ′Lancerʼ varieties (5240 bp). A synonymous mutation was found both in ′Chinese Springʼ (GGG (Gly)-GAG (Glu)) and in ′Lancerʼ (GTG (Val)-GCG (Ala)) in chromosome 7B. Moreover, both ′Kariegaʼ and ′Lancerʼ have a same functional mutation (GAC (Asp)-AAC (Asn) in chromosome 7D.

This research was supported by the National Research, Development and Innovation Office (NKFIH-FK-134234, NKFIH-K-129221 and NKFIH-K-142934) and János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00396/21/4).