Nucleotide diversity across the full-length alignment of TTG1 was relatively high (π=0.01348) in comparison with the genome-wide estimate of nucleotide diversity for A. thaliana (π=0.0047 (Bakker, et al., 2006)). While also high in comparison with the gene-wide value for the trichome-specific R2R3 MYB transcription factor GL1 (π=0.0049; (Bloomer, et al., 2012)), a similarly high value of 0.011 was previously reported for AtMYC1 (Symonds, et al., 2011), a bHLH transcription factor acting in concert with TTG1 and GL1 in trichome initiation. Nucleotide diversity is variable across the TTG1 locus, with peaks in π as high as 0.07657 found within the intron (Figure 4.1). However, π is considerably lower in the 5’ UTR, exon and 3’ UTR; the gene-wide value of π across the coding region (exon) is similar to the genome- wide estimate at 0.0058. Peaks of nucleotide diversity in the sliding window analysis reflect a pattern of high frequency polymorphism which is particularly pronounced in the intron, separating TTG1 alleles into two major haplogroups referred to here as A and B. Such allelic dimorphism is commonly observed in A. thaliana, including in other epidermal development genes such as the R3 repressor ETC2 (Hilscher, et al., 2009), AtMYC1 (Symonds, et al., 2011),and GL1 (Bloomer, et al., 2012), and in a number of others including the phenylpropanoid pathway (Aguade, 2001), chitinase (Kawabe, et al., 1997) and R genes (Tian, et al., 2002; Mauricio, et al., 2003; Rose, et al., 2004), and chloroplast sequences (Yin, et al., 2010). Arabidopsis thaliana is thought to have been isolated in two glacial refugia before subsequently spreading back into central Europe (Sharbel et al. 2000; Beck et al. 2008), and divergence, secondary contact and recombination between diverged genomes has been
122 suggested as an explanation for the observed patterns of allelic dimorphism (Aguade, 2001; Symonds, et al., 2011).
With the exception of the high frequency pattern described above, the TTG1 coding region was highly conserved both in terms of nucleotide and amino acid sequence. None of the 19 coding region SNPs reflecting the pattern of high frequency polymorphism on which the A and B haplogroups are based are nonsynonymous. Of the four nonsynonymous SNPs elsewhere in the coding region, all are at positions conserved across A. thaliana, A. lyrata and three Brassica species based on cDNA sequences available from Genbank (data not shown). Notably, the replacement A311T is at a position completely conserved across both AtTTG1- type and PhAN11-type WD40 proteins from a range of both dicot and monocot taxa (Lu, et al., 2009; Pang, et al., 2009). Truncation of the C-terminal 25 amino acids of TTG1 is sufficient to abolish bHLH interaction (Payne, et al., 2000) and is the mutation underlying the strongest ttg1 mutant allele; two of the replacements identified here, A333S and A311T, fall near or within this region. Interestingly, the accession Lp2-2, which carries the A311T mutation, has one of the lowest trichome densities amongst the accessions phenotyped (data presented in Supplementary Material to Chapter 3, Figure 3.5 (Bloomer, et al., 2012)). This mutation is a candidate for a role in reducing trichome density, perhaps reducing TTG1 binding affinity with its bHLH partners.
In agreement with its highly pleiotropic role and with the level of conservation of amino acid sequence observed, a sliding window of Ka/Ks across the single exon of TTG1 suggests that evolution of the protein is highly constrained. Ka/Ks is much less than one across the entire coding region, indicating purifying selection acting on TTG1. Conversely, when calculated using all 96 accessions’ sequences, significant departure from the neutral model of evolution was not detected at TTG1 using either Tajima’s D or Fu and Li’s D* and F* statistics. TTG1 instead shows non-significant positive values for D, D* and F*, which could be interpreted as indicative of balancing selection. However, D, D* and F* are influenced by both selection and by demographic processes. An alternative interpretation, particularly given the hypothesised history of the species (Sharbel et al. 2000; Beck et al. 2008), is that the positive values obtained here reflect the admixture of two diverged populations, masking any signature of selection. Supporting this interpretation, when calculated separately for haplogroups A and B the values of D, D* and F* indicate purifying selection at TTG1. D* and F* are significantly
123 negative for haplogroup A across the coding region and cannot be calculated for haplogroup B due to absolute conservation of sequence among the accessions sampled. Consistent with the Ka/Ks analysis, these results suggest purifying selection acting on TTG1.
WDR proteins often act as protein scaffolds, interacting with and mediating interactions of a range of other proteins. Homologues of A. thaliana TTG1, which have generally conserved functions in regulating anthocyanin production (and a broader set of epidermal traits in some species) via interaction with bHLH and MYB transcription factors, show strong conservation of amino acid sequence across a diverse range of monocot and dicot plant species (Lu, et al., 2009; Pang, et al., 2009). Cross-species comparison of amino acid sequences has shown that that homologous WDR proteins are more similar to one another than homologous bHLHs (Carey, et al., 2004); further, the functional homologue of TTG1 in Ipomoea species, WDR1, exhibits a higher level of evolutionary constraint than its less pleiotropic bHLH and MYB partners (Streisfeld, et al., 2011). Similarly, the Ka/Ks analysis undertaken here indicates strong purifying selection at TTG1 while less pleiotropic genes within the pathway, the bHLH AtMYC1 and the MYB GL1, show evidence of positive or balancing selection with spikes of Ka/Ks>>1 (Symonds, et al., 2011; Bloomer, et al., 2012). This supports the proposal that, within regulatory pathways, those genes higher in regulatory hierarchies will be subject to stronger constraint (Martin, et al., 2010).