Relationship between homoeologous regulatory and structural genes in allopolyploid genome - a case study in bread wheat

Abstract

Background: The patterns of expression of homoeologous genes in hexaploid bread wheat have been intensively studied in recent years, but the interaction between structural genes and their homoeologous regulatory genes remained unclear. The question was as to whether, in an allopolyploid, this interaction is genome-specific, or whether regulation cuts across genomes. The aim of the present study was cloning, sequence analysis, mapping and expression analysis of F3H (flavanone 3-hydroxylase - one of the key enzymes in the plant flavonoid biosynthesis pathway) homoeologues in bread wheat and study of the interaction between F3H and their regulatory genes homoeologues - Rc (red coleoptiles).

Results: PCR-based cloning of F3H sequences from hexaploid bread wheat (Triticum aestivum L.), a wild tetraploid wheat (T. timopheevii) and their putative diploid progenitors was employed to localize, physically map and analyse the expression of four distinct bread wheat F3H copies. Three of these form a homoeologous set, mapping to the chromosomes of homoeologous group 2; they are highly similar to one another at the structural and functional levels. However, the fourth copy is less homologous, and was not expressed in anthocyanin pigmented coleoptiles. The presence of dominant alleles at the Rc-1 homoeologous loci, which are responsible for anthocyanin pigmentation in the coleoptile, was correlated with F3H expression in pigmented coleoptiles. Each dominant Rc-1 allele affected the expression of the three F3H homoeologues equally, but the level of F3H expression was dependent on the identity of the dominant Rc-1 allele present. Thus, the homoeologous Rc-1 genes contribute more to functional divergence than do the structural F3H genes.

Conclusion: The lack of any genome-specific relationship between F3H-1 and Rc-1 implies an integrative evolutionary process among the three diploid genomes, following the formation of hexaploid wheat. Regulatory genes probably contribute more to the functional divergence between the wheat genomes than do the structural genes themselves. This is in line with the growing consensus which suggests that although heritable morphological traits are determined by the expression of structural genes, it is the regulatory genes which are the prime determinants of allelic identity.

Figure 1

The structure of wheat F3H. Different gene segments referred in the paper text and tables are indicated in the figure part (a), length of introns of the different T. aestivum F3H gene copies are indicated in part (b); partial sequences are extended with dotted lines, whereas solid lines correspond to sequences cloned and analysed in the present study.

Figure 2

F3H sequences comparison: (a) alignment of complete coding sequences of barley F3H [13] and wheat F3H1 and F3H2 and partial wheat F3H3 and F3H4 copies cloned in the present study (introns are not included into alignment); (b) similarity of part of F3H exon 2 (specified as segment 6 in Figure 1) from various plant species – the species from which F3H copies were cloned and analysed in the present study are underlined, others were obtained from GenBank; for species with more than one F3H gene, each copy is identified by a number in parentheses.

Figure 3

Gene divergence between the hexaploid wheat A and D genome F3H gene copies: (a) percentage of nucleotide substitutions in exons, (b) ratio of non-synonymous (Ka) to synonymous (Ks) nucleotide substitutions.

Figure 4

PCR profiles of the 'Chinese Spring' nulli-tetrasomic lines and the diploid donors of hexaploid wheat, amplified with F3H copy-specific primers. The length of the PCR products is given in base pairs to the right. Designations '1A', '1B' etc. correspond to 'nulli' chromosome in the certain nulli-tetrasomic line; 'Tu' – T. urartu, 'Aes' – Ae. speltoides, 'Aet' – Ae. tauschii.

Figure 5

Physical mapping of F3H loci in bread wheat performed using subset of T. aestivum cv. 'Chinese Spring' homoeologous group 2 chromosomes deletion lines. Microsatellite markers (Xgwm) designations are given to the right from each chromosome scheme, chromosome bin names are indicated to the left.

Figure 6

PCR profiles of 'Saratovskaya 29' (1), T. timopheevii (2) and 'Saratovskaya 29'x T. timopheevii introgression line 842 (3), amplified with gene copy-specific primers for T. aestivum F3H3 (a) and F3H4 (b) and T. timopheevii F3H2^t (c).

Figure 7

RT-PCR analysis of total F3H expression in four day old seedlings of (a) 'Chinese Spring' ('Hope' 7B) (1), 'TRI 2732' (2) and progeny of the cross 'Chinese Spring' ('Hope' 7B) × 'TRI 2732' (3–10); (b) substitution 'Chinese Spring' (Ae. tauschii 7D) (1), 'Chinese Spring' (2) and the 'Chinese Spring'/Ae. tauschii 7D introgression lines (3–8). Anthocyanin pigmentation in coleoptiles of the corresponding lines is shown above, whereas the status of chromosomes 7B (a) or 7D (b) of each line is indicated in the lower part of the panel.

Figure 8

F3H copy-specific RT-PCR analysis from four day old seedlings of 'Chinese Spring' ('Hope' 7B) (1), 'TRI 2732' (2) and progeny of the cross 'Chinese Spring' ('Hope' 7B) × 'TRI 2732' (3–10). Anthocyanin pigmentation in coleoptiles of the corresponding lines is shown below. The length of the RT-PCR products is given in base pairs to the right.

Figure 9

Quantitative RT-PCR analysis with respect to the various copies of F3H in 'Chinese Spring' (CS), 'Chinese Spring' ('Hope' 7A), 'Chinese Spring' ('Hope' 7B) and 'Mironovskaya 808' (M808).

Figure 10

Comparison of T. aestivum F3H copies vs homologous wheat ESTs. The highest identity value for each EST is indicated with black arrow.