Abstract
Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a.
Highlights
Flower color has played a pivotal role in our current understanding of biology since Mendel’s discovery of the inheritance of flower color in Pisum sativum (Mendel, 1866; Ellis et al, 2011)
After BLAST+ identification against all known genes of A. thaliana, multiple putative paralogs were identified for seven genes producing a total of 29 anthocyanin biosynthetic pathway (ABP)-related loci (Table 2)
The basic helix-loop-helix (bHLH) locus is closely related to the AN1 locus of Petunia and TT8 locus from Arabidopsis, both regulators of the ABP
Summary
Flower color has played a pivotal role in our current understanding of biology since Mendel’s discovery of the inheritance of flower color in Pisum sativum (Mendel, 1866; Ellis et al, 2011). Changes in the color of anthocyanins (e.g., shifts from blue to red flowers) and the loss of floral anthocyanins (producing white flowers) can be traced from ecological interactions in the field to the biochemical and molecular basis for these changes (Tanaka et al, 2008; Davies, 2009; Hopkins and Rausher, 2011; Zhao and Tao, 2015). These changes can result from mutations in core structural genes or regulatory loci (Sobel and Streisfeld, 2013). It is expected that null coding mutations will be more frequent within species; and cis-regulatory mutations between species (Stern and Orgogozo, 2008), which has been demonstrated in polymorphic populations of some species such as Mimulus lewisii (Wu et al, 2013)
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