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Heritability of floral color in bouncing bet

Dana Dudle, Mary Ritke, and Sandra Davis
DePauw University, University of Indianapolis

Introduction
Bouncing bet produces flowers that change color from white to pink as they transition from male to female phase.  Our previous research demonstrated that the extent of floral color change depends on exposure to sunlight.  Plants in full sun produce female-phase flowers that are pinker than those on shaded plants.  The pink color of flowers is associated with production of anthocyanin, a reaction catalyzed by the enzyme anthocyanidin synthase (ANS).  Diruanl pollinators visit white or pale flowers more often than pink flowers.  This preference indicates a potential fitness disadvantage associated with increased pinkness when pollinator activity limits seed set.  Why does floral color change persist in the face of pollinator-medicated selection against pink flowers?  Two potential explanations are (1) a lack of heritable variation in floral color and (2) a low production cost of anthocyanin if flowers synthesize all necessary enzymes early in development.

Research Questions
Is flower color heritable in bouncing bet?
Does anthocyanadin synthase (ANS) expression vary with floral gender, genotype, and/or light environment? 

Methods
We grew 15 clones of 8 distinct genotypes of bouncing bet in sun and 60% shade in an experimental garden in 2013.  Four genotypes were "pink" and four were "pale."  Flower color was quantified by extracting anthocyanin from petals and measuring absorbance using a spectrophotometer.  Flower color was compared between sexual phases, among genotypes, and between light environments.  Heritability of flower color was estimated by comparing environmental variance with total variance among and within clones.  RNA was extracted from flower petals.  cDNA was synthesized using oligo dT primers and a recombinant reverse transcriptase.  RT-qPCR was conducted using cDNA samples and an ANS primer pair.  Expression of ANS was calculated with respect to actin (ACT) expression.  

Results
We found clear evidence for heritable variation in flower color.  We estimated the heritability of anthcyanin concentration to be 0.41 in sun plots.  We also found variation in plasticity among genotypes in response to differences in sun exposeure.  For example a "pale" and a "pink" genotype had similar phenotypes when grown in full sun but differed significantly when grown in 60% shade.  The increase in pigmentation in some genotypes was due in part to an increase in ANS expression.  "Pink" genotypes had significantly higher levels of ANS expression than "pale" genotypes in female-phase flowers.  When "pale" genotypes were exposed to the sun, they significantly increased expression of ANS in female-phase flowers.  But ANS expression did not differ between sun and shade environments in "pink" genotypes.  There was no difference in ANS expression between male-phase and female-phase flowers on the same plant.