BACKGROUND
apple peel color is one of the most important factors determining apple market acceptance.
in general, red cultivars are the most preferred, and within a cultivar more highly colored fruits are favored  <cit> .
consumer preferences vary from country to country and region to region: new zealand consumers prefer striped apples, consumers in nova scotia, canada prefer blushed apples, while consumers in british columbia, canada are more accepting of a range of apple types  <cit> .
peel pigments not only affect visual appeal, they also contribute to the fruit's nutritional value.
apples have been associated with lowered risks of cancer and cardiovascular diseases, which are thought to be caused by oxidative processes.
polyphenolics, including anthocyanins which are the red pigments in apple peels, have been found to be the major source of antioxidants in apple  <cit> .
antioxidants are mainly localized in the apple peel, but cultivars exhibit a wide variation in the distribution pattern  <cit> .
anthocyanin accumulation in apple peels can be affected by genetic, environmental, nutritional and cultural factors, the stage of maturity of the fruit, and by the microenvironment within the canopy  <cit> .
the main anthocyanin identified in apple skin is cyanidin 3-galactoside, while cyanidin 3-glucoside levels are very low  <cit> .
two categories of genes affect the biosynthesis of anthocyanin.
the first category encodes enzymes required for pigment biosynthesis , which have been widely studied in apple  <cit>  .
the second category is comprised of transcription factors, which are regulatory genes that influence the intensity and pattern of anthocyanin accumulation and control transcription of different biosynthetic genes.
at least three families, myb, bhlh and wdr, have been found to be involved in the regulation of anthocyanin synthesis, but the specific classes and genes involved vary depending on the species  <cit> .
in apple, three research groups have independently identified an r2r <dig> myb transcription factor responsible for anthocyanin accumulation in fruit.
the loci have been named myb <dig>  myb <dig> and myba  <cit> .
the coding region of myba is  <dig> and 98% identical to myb <dig> and myb <dig>  respectively  <cit> .
in addition, myb <dig> and myba have been mapped to the same region on linkage group  <dig>  <cit> .
subsequent experiments have shown that myb <dig>  myb <dig> and myba are likely to be allelic  <cit>  and more-over, at this locus in the current apple genome assembly, there is only one myb present  <cit> .
based on this evidence, in this research article, we consider myb <dig> to exist as a single locus with myba and myb <dig> representing alleles of the myb <dig> locus.
transcript levels of the myb <dig> allele correlate with anthocyanin accumulation and are higher in red fruit peel sectors  and in red peel cultivars than in green peel sectors or cultivars  <cit> .
transcript levels of myb <dig> increased in dark-grown apples once exposed to light, providing additional evidence of its role as an anthocyanin regulator.
myb1- <dig>  a sequence variant of the myb <dig> allele, co-segregates with red skin color  <cit> .
transcription at the myb <dig> locus strongly correlates with peel anthocyanin levels and this gene is able to induce anthocyanin accumulation in heterologous and homologous systems  <cit> .
in addition, myb <dig> co-segregates with the rni locus, a major genetic determinant of red foliage and red color in the core of apple fruit  <cit> .
consistently, the expression of several anthocyanin pathway genes was found to be regulated by myb <dig> and myb <dig>  <cit>  .
in apple, two candidate bhlh transcription cofactors  are also needed for activating promoters of anthocyanin structural genes and myb <dig>  <cit> .
repressors of anthocyanin production were also identified within the myb class of transcription factors, including mdmyb <dig> in apple  <cit> , famyb <dig> in strawberry  <cit>  and atmybl <dig> in arabidopsis  <cit> .
famyb <dig> is up-regulated jointly with late anthocyanin pathway genes  <cit> .
expression of atmybl <dig> is also coordinately up-regulated by the myb-bhlh-wdr activation complex  <cit> .
in arabidopsis a transcriptional regulatory loop has been postulated whereby atpap <dig>  is a positive regulator of attt <dig>   <cit> , and attt <dig> is an activator of atmybl <dig> expression  <cit>  which then negatively regulates the expression of attt <dig>  it is suggested that the repressors' role is to balance anthocyanin levels produced at later stages of color response.
'honeycrisp', an increasingly important apple cultivar developed at the university of minnesota, produces fruits that can adopt two basic peel color patterns: blushed or striped .
for the purposes of this study, fruits are defined as striped when the color alternates between vertically elongated regions in some or all portions of the peel.
fruits are termed blushed when the surface is partly covered with a red tinge that is not broken.
these two phenotypic categories are mutually exclusive.
in 'honeycrisp' both kinds of fruit may be present on the same tree, a characteristic that has not been described in other cultivars.
the molecular basis of this phenomenon is unknown.
different mechanisms can cause variegation in plants, including chimeras  <cit> , transposable element activity  <cit>  and cytosine methylation  <cit> .
previous results do not provide evidence for a chimeral source of variegation in the case of 'honeycrisp', since the phenotype is not stable after propagation  <cit>  as would be expected if changes were caused by a periclinal chimera.
microscopic observations indicated that the difference between stripes is due to a reduction in pigment accumulation in the paler stripes, both in the epidermis and in the first hypodermal layers  <cit> .
activation and suppression of transposable elements may be responsible for some of the genetic variation that occurs in peel color in pome fruits  <cit> .
transposable elements have been identified in apple  <cit>  but to date there is no evidence associating transposable elements with fruit peel variegation.
the presence of transposable elements can affect gene expression both at the transcriptional , and at the post-transcriptional level  <cit> .
cocciolone and cone  <cit>  reported that striped patterns of anthocyanin accumulation in maize were due to differential dna methylation in the 3' untranslated region of pl-bh, a myb transcription factor regulating anthocyanin accumulation.
methylation was found to be inversely correlated with pl-bh mrna levels in variegated plant tissues.
the authors hypothesized that early during development, the pl-bh gene would be differentially methylated and this methylation would be more or less maintained through subsequent cell divisions, producing clonal sectors in plant tissues of predominantly pigmented cells  and sectors of predominantly unpigmented cells .
sekhon and chopra  <cit>  identified a gene called ufo <dig> that controls methylation levels in p <dig>  a gene that regulates phlobaphene biosynthesis in maize, and whose activity may also produce variegation in the maize pericarp.
ectopic expression of p1-wr correlated with hypomethylation of an enhancer region,  <dig> kb upstream of the transcription start site.
it is not known whether methylation is responsible for color differences in apple.
we therefore sought to understand the molecular mechanism responsible for 'honeycrisp' color pattern regulation and instability.
we also included in this study two stably striped cultivars , a stably blushed cultivar  and other cultivars differing in the degree of peel pigmentation.
our results showed that variation in pigment accumulation between red and green stripes correlates with anthocyanin levels, and the steady state mrna levels of both the anthocyanin biosynthetic genes and the transcription factor myb <dig>  sequence variation in the myb <dig> region upstream of the translation start site  and coding region does not explain the observed phenotypes.
the promoter and coding regions of myb <dig> were examined in red and green stripes for dna methylation levels and a  <dig> bp region, starting  <dig> bp upstream of the predicted translation start site, was found to be highly methylated in both 'honeycrisp' and 'royal gala'.
red stripes were associated with lower methylation across the promoter of myb <dig> in 'honeycrisp' and to a lesser degree in 'royal gala', but no differences were found between blushed 'honeycrisp' green and red peel regions.
RESULTS
red stripes have higher anthocyanin accumulation and transcript levels of biosynthetic genes
red stripes of 'royal gala' and 'honeycrisp' contained approximately eight and four times as much anthocyanin as green stripes .
in all cases, the major anthocyanin detected was cyanidin-3-galactoside .
we subsequently compared the transcript levels of regulatory genes myb <dig>  myb <dig>  bhlh <dig> and bhlh <dig> and biosynthetic genes chs, chi, f3h, dfr <dig>  ldox, ufgt, in rna isolated from red and green stripes of 'royal gala' and 'honeycrisp' .
myb <dig> and myb <dig> transcript levels correlated with anthocyanin concentration in both 'honeycrisp' and 'royal gala', with higher mrna levels in red stripes as compared to green stripes .
transcript levels of structural genes followed the same pattern as those of myb <dig> and myb <dig>  levels of the two bhlh transcription factors did not differ in green and red stripes , and therefore correlated poorly with anthocyanin concentration.
these results reveal differential transcript accumulation of myb <dig> and myb <dig> in differentially pigmented stripes, which in turn results in a corresponding modulation of transcript levels of structural genes.
myb <dig> is a known activator of the apple anthocyanin pathway  <cit>  and myb <dig> has been shown to inhibit steps in the anthocyanin pathway  <cit>  and has high sequence similarity to atmyb <dig>  a repressor of the phenylpropanoid pathway  <cit> .
we decided to further characterize myb <dig> coding and upstream regions in order to determine whether sequence polymorphisms can explain different pigmentation patterns.
low sequence diversity in the myb <dig> coding region in 'honeycrisp', 'connell red' and 'fireside'
to study the possibility that sequence differences are the cause of differential color patterns in the peel, we sequenced a total of  <dig> cdna clones of the 'honeycrisp' myb <dig> coding region:  <dig> from a phenotypically uncharacterized 'honeycrisp' fruit ,  <dig> from a mature striped and  <dig> from a mature blushed fruit.
ninety-two percent of the sequences obtained were 100% identical to myb1- <dig>  an allele of the myb <dig> locus  <cit> .
we found three single nucleotide polymorphisms  that produce changes in protein sequence, but since each one appeared only once in our dataset, and in phenotypically different apples, they most likely represent amplification or sequencing errors.
these results indicate low levels of sequence diversity in the myb <dig> coding region in 'honeycrisp', with no evidence suggesting that the blushed/striped phenomenon is associated with modifications at the primary dna sequence level within the coding region.
myb <dig> coding sequences from the striped cultivar fireside  and 'connell red' , a stably blushed sport of 'fireside', are identical to that of the most abundant version found in 'honeycrisp' and the previously published myb1- <dig> sequence--supporting our conclusion that differences in primary dna sequence are not the source of differential patterns of apple peel pigment accumulation.
no size variation in myb <dig> promoter region among apple cultivars
we amplified three fragments  collectively spanning about  <dig> kb of the myb <dig> promoter.
pcr results did not indicate any fragment size differences among blushed and striped 'honeycrisp', 'connell red' and 'fireside' dna, suggesting no large insertion or deletions were present.
we sequenced the pcr products of each of these fragments from three independent reactions and found no sequence differences between blushed and striped 'honeycrisp', or between 'connell red' and 'fireside', although there were  <dig> snps between 'honeycrisp' and the other two cultivars.
neither presence nor transcription of a trim element explains apple peel phenotypic variation
within the plant & food malus gene database  <cit>  was a dna sequence identical to genbank accession eu <dig>  the promoter of myb <dig>  further upstream from this sequence, between positions - <dig> and - <dig> from the atg translation start site of myb <dig>  was a sequence with 85% identity to a malus trim element , a terminal-repeat retrotransposon in miniature  <cit> .
we checked for the presence of a trim element upstream of the myb <dig> locus in 'honeycrisp' , 'connell red', 'fireside', '1807'  and 'geneva'  via pcr, combining a primer designed from the trim element  with one designed from the promoter region of myb <dig> .
results confirmed the presence of the trim element in each of these cultivars in a position identical to that observed in 'royal gala' .
we subsequently cloned and sequenced three pcr products from 'honeycrisp' , 'connell red' and 'fireside'.
half of the fragments yielded sequences showing 99% or more identity to the previously published  upstream region of 'royal gala' myb <dig>  the other sequences were probably amplifications from insertions of similar trim elements located elsewhere in the genome, with percent identities to trim ranging from  <dig> to  <dig> %.
we tested for trim transcript presence in blushed and striped 'honeycrisp', 'connell red', 'fireside', 'geneva'  and 'honeygold' , and found it to be transcribed in all cases.
however, a fragment spanning a portion of the trim element and extending  <dig> bp into the upstream region of myb <dig> did not amplify from total rna, indicating that transcription from the trim element did not extend into myb <dig> in these cultivars.
overall, results indicated that neither the presence of the trim element in the myb <dig> promoter region nor its transcription explained the differences in peel pigment accumulation among the cultivars studied.
increased methylation levels in green stripes
dna samples from green and red stripes of 'honeycrisp'  and 'royal gala' were treated with the methylation-sensitive endonuclease mcrbc to ascertain whether the observed differences in transcript accumulation were associated with methylation differences in the promoter or coding region of myb <dig> .
mcrbc preferentially cuts dna containing methylcytosine on one or both strands, between two recognition sites .
mcrbc treated and mock-digested templates were compared using real-time pcr, and percent methylation was calculated.
in total,  <dig> fragments starting at the transposon insertion and spanning  <dig>  kb of the promoter region and three exons of myb <dig>  were evaluated.
results indicated that a region of the myb <dig> promoter, encompassing the fragments between nucleotide positions - <dig> and - <dig> is highly methylated  in both cultivars.
'connell red' and 'fireside' had low methylation  in the - <dig> to - <dig> fragment and high methylation  in the - <dig> to - <dig> fragment, indicating a similar pattern of myb <dig> methylation in these cultivars relative to those observed in 'royal gala' and 'honeycrisp' .
green stripes of 'honeycrisp'  showed higher overall methylation levels than red stripes throughout the promoter region .
the - <dig> to - <dig> fragment was omitted from this comparison since quantification in the mcrbc digested samples was highly variable due to extremely low template levels, indicating that this region was so highly methylated that treatment with mcrbc resulted in nearly complete digestion of the template dna.
sequence analysis indicated that differences in predicted methylation levels between regions were not due to difference in the number of potential mcrbc recognition sites .
similar results were obtained for  <dig> fruits, but overall methylation levels were higher than in  <dig> and differences between red and green stripes were even greater .
these results indicate that while methylation levels are variable between years, green stripes are consistently associated with higher methylation of myb <dig> promoter regions.
similar trends were observed in 'royal gala' for some of the fragments, except that the differences between red and green stripes were smaller.
in total, higher methylation levels were observed for 'royal gala' than 'honeycrisp' .
in contrast to 'honeycrisp' red and green stripes, we hypothesized that color differences between red  and green  regions of the peel of blushed apples are only due to light effects and not to differences in methylation levels.
we therefore compared methylation percentages in red  and green  areas of blushed apples and red and green stripes.
results indicated no significant differences  between red and green regions of the peel of blushed apples.
interestingly, in two out of the three regions studied , red stripes have methylation levels comparable to those in the exposed peel portions of blushed apples, while green stripes have methylation levels higher than those of red stripes or red and green regions of blushed apples .
bisulfite sequencing offers greater resolution than mcrbc-based methods for the detection of methylated cytosines.
building on mcrbc results, we next pursued bisulfite sequencing of myb <dig> promoter regions from 'honeycrisp' and 'royal gala'.
preliminary bisulfite sequencing experiments indicated that cytosine methylation in the promoter region of myb <dig> is found in all three methylation contexts .
to avoid amplification bias, we therefore designed degenerate pcr primers to target two different promoter regions.
this severely constrained areas that could be targeted, and amplification upstream of - <dig> was ultimately unsuccessful using unbiased primers.
a comparison of methylation levels between red and green stripes in the - <dig> to - <dig> and - <dig> to - <dig> regions confirmed that green stripes are more highly methylated than red stripes , with 80% and 65% of cytosines showing higher methylation levels in green than in red stripes in the - <dig> to - <dig> and - <dig> to - <dig> regions respectively .
further analysis of the - <dig> to - <dig> region indicated that clones obtained from green stripes have higher overall methylation levels than those obtained from red stripes .
observed methylation differences between red and green stripes are modest, but actual differences may be greater.
although great care was taken, manual isolation of red and green stripes from 'honeycrisp' peels was imprecise, resulting in tissue samples that were substantially enriched for red or green stripes but not entirely devoid of contaminating tissues.
thus, dna samples used for mcrbc- and bisulfite-based analyses, while predominantly derived from the target tissue  likely represent a mixture of dna, partially obscuring methylation differences between sources.
consistent with our preliminary results, different cytosine contexts did not exhibit distinct methylation patterns; all cytosine contexts showed high methylation levels in highly methylated regions and vice versa .
overall, chh and chg methylation was highest  and cg methylation was lowest .
a sequence alignment for the - <dig> to - <dig> region is presented in additional file  <dig>
discussion
anthocyanin genes transcript levels correlate with striped patterns
anthocyanin and transcript quantifications performed in this study suggested a possible mechanism controlling pigment patterns in apple fruit peels.
we have found that green stripes are associated with lower anthocyanin accumulation, which is explained by reduced transcript levels of all the anthocyanin pathway genes evaluated, including the structural genes in the pathway, and myb <dig>  a transcription factor which regulates them.
an additional gene evaluated in this study, myb <dig>  an apple transcription factor that represses anthocyanin synthesis  <cit>  was transcribed in a similar manner to myb <dig>  as myb <dig> is not elevated in green sectors relative to red, we considered myb <dig>  the main transcription factor regulating the pathway in apple  <cit> , to be the most likely candidate to be involved in peel variegation.
we therefore sought to identify a mechanism responsible for myb <dig> transcript level differences.
variegation in apple peels is associated with myb <dig> methylation mosaicism
our results indicate an inverse association between methylation levels in the myb <dig> promoter and anthocyanin accumulation in striped apple peels.
as previously suggested by cocciolone and cone  <cit>  for maize, we hypothesized that early in apple fruit development, differences in myb <dig> methylation are present among individual cells.
throughout fruit growth, these differentially methylated cells give rise to sectors of peel varying in their ability to accumulate anthocyanins.
our results indicate that dna methylation in the promoter of myb <dig> is associated with reduced transcript accumulation.
specifically, we propose that differential methylation of myb <dig> promoter regions in red vs. green stripes of the 'honeycrisp' peel results in differential accumulation of the myb <dig> transcript, which in turn determines both transcription of anthocyanin structural genes and pigment accumulation.
dna methylation may affect myb <dig> transcription through interference with the rna-polymerase transcription complex or by preventing binding of additional factors required for transcription.
in arabidopsis, genome wide studies of dna methylation have found that methylation within regulatory regions is rare and probably selected against, as it may interfere with transcription initiation  <cit> .
our results suggest that high levels of methylation in certain promoter regions of a key transcription factor in the flavonoid biosynthetic pathway in apple may play a regulatory role, but it is not inhibitory for gene activity.
it is possible that since apple trees are clonally propagated and stay in production for many years , mechanisms of epigenetic regulation might not be identical to what has been described in more widely studied species such as arabidopsis and rice.
different methylation levels early in apple fruit development could be mitotically maintained from those in the meristematic cells that gave origin to the fruit, or could result from demethylation or de novo methylation.
previous results in 'honeycrisp' suggest that there is at least some mitotic maintenance of methylation states, given that trees clonally propagated from buds on branches with exclusively blushed fruits, tend to produce a higher percentage of blushed fruit  <cit> .
nonetheless, results from the same study indicated that additional factors can influence the pattern in the peel, namely position of the fruit on the tree and crop load.
the presence of a trim transposable element in an upstream region of the myb <dig> promoter might influence the changes in methylation observed between different regions of the peel but neither its presence per se nor its transcription appears to be responsible for determining peel pigmentation.
the trim element identified in this study is located  <dig>  kb upstream of the predicted translation start site, and is present in 'honeycrisp', 'royal gala', and five other cultivars with peel pigmentation ranging from green to ultra red.
lippman et al.
<cit>  indicated that in arabidopsis transposable elements can determine epigenetic gene silencing when inserted within or very near  a gene.
the effect of a transposable element  <dig>  kb upstream of the coding region is unknown.
we did not find any evidence of transposable element sequences within the highly methylated promoter region of myb <dig>
within the most methylated region of the myb <dig> promoter in this study  are five putative e-box motifs  <cit>  which are bhlh-related cis-acting elements   <cit> .
methylation was absent at the three e-box motifs evaluated using bisulfite sequencing, but highly methylated areas occurred a few nucleotides upstream or downstream of these motifs.
this may suggest a potential regulatory role for one or more of these motifs.
an assessment of the presence of other types of epigenetic marks such as histone methylation can shed additional light on the mechanism involved in myb <dig> regulation.
our results show that methylation followed the same pattern in all three cytosine methylation contexts, with alternation of high and low methylation regions.
the high methylation levels observed for chh and chg sites, and low methylation levels for the cg context, indicate a pattern not representative of what is generally observed in flowering plants  <cit> .
broader survey of methylation patterns throughout the apple genome is warranted.
the unstable nature of pigment patterning in 'honeycrisp' could be a result of a more variable cell to cell methylation pattern than is present in other cultivars producing fruit with consistent pigment patterns, such as 'royal gala', 'fireside' and 'connell red'.
we speculate that the occurrence of stripes in 'honeycrisp' is a consequence of higher than normal methylation levels in the myb <dig> promoter region in the green stripes, something that occurs only in some fruit and to varying degrees.
in contrast, myb <dig> methylation levels and thus peel pigmentation remains more constant in 'royal gala'.
CONCLUSIONS
differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of myb <dig>  a transcription factor that regulates the anthocyanin pathway in apple.
different transcript levels of myb <dig> in red versus green stripes are inversely associated with methylation levels in its promoter, especially along a  <dig> bp region upstream of the translation start site.
although observed methylation differences are modest, trends are consistent across years and differences are statistically significant.
methylation might be associated with the presence of a trim element within the promoter region, but the presence of the trim element alone cannot explain the phenotypic variability observed in 'honeycrisp'.
we suggest that methylation in the myb <dig> promoter is more variable in the phenotypically plastic 'honeycrisp' than in the more consistently striped 'royal gala'.
differential methylation of the 'honeycrisp' myb <dig> promoter alters accumulation of the myb <dig> transcript, in turn altering both transcription of anthocyanin structural genes and pigment accumulation.
materials and methods
plant material
leaf samples of 'honeycrisp', 'connell red', 'fireside', '1807', 'honeygold' and 'geneva' apple were collected in early spring of  <dig> and fruits of the same cultivars were collected at maturity during the  <dig>   <dig>   <dig> and  <dig> growing seasons from trees at the horticultural research center in chanhassen, minnesota.
in february  <dig>  and  <dig>  fruits were harvested at plant and food research orchards .
'royal gala' apples grown in chile were purchased in a minnesota grocery store in april  <dig> to be used for methylation experiments.
for the myb <dig> characterization experiments, whole fruit peels were used.
for anthocyanin quantification, transcript analyses and methylation studies, red and green stripes were carefully separated using a razor blade.
since stripes cannot be absolutely classified as green or red, samples are more accurately described as "red stripe enriched" or "green stripe enriched".
both green and red stripes were obtained from the same region of the peel at each time, preventing the possibility that the "red stripe enriched" sample would also be enriched for fruit peel regions more exposed to light and vice versa.
for comparisons between different blushed fruit regions, light-exposed  and -unexposed  peel regions were separated.
for both blushed and striped fruit regions, peel tissue from at least two apples was pooled.
in all cases, leaves and peels were immediately frozen in liquid nitrogen and placed at -80°c before anthocyanins, dna or rna was extracted.
identification and quantification of anthocyanins
apple peel samples were finely ground in liquid nitrogen and then resuspended in  <dig> ml methanol and  <dig> % hcl.
samples were sonicated for  <dig> min, stored at room temperature in the dark for  <dig> h and then centrifuged at  <dig>  × g. aliquots of  <dig>  ml were dried down to completion in a labconco centrivap concentrator .
samples were resuspended in 20% methanol .
anthocyanins were identified by lc-ms analysis as described previously  <cit> .
identification was based both on masses  of molecular ions and characteristic fragments/neutral losses and comparison of retention times and fragmentation with authentic standards of cyanidin-3-o-glucoside and cyanidin-3-o-galactoside.
m+ fragments observed were  <dig> da ,  <dig> da  and  <dig> da .
neutral losses  observed were  <dig> da ,  <dig> da  and  <dig> da .
ms cannot distinguish between sugars of the same mass .
anthocyanins and other phenolic compounds were quantified by hplc as described previously  <cit> .
quantification was achieved by reference to standards of anthocyanins and other phenolic compounds, using lc-ms data to confirm identification of peaks.
real-time transcript analysis
mature 'honeycrisp' fruit peels were very finely ground in liquid nitrogen and rna was extracted using the lopez-gomez and gomez-lim extraction method  <cit>  as modified by mann et al.
<cit> .
briefly, after precipitation with  <dig> m licl, rna was collected by centrifugation at  <dig>  × g for  <dig> min, and second day licl washes were eliminated.
rna pellets were resuspended in  <dig> μl rnase free sterile water, potassium acetate was added to a final concentration of  <dig>  m, and the rna was precipitated with two volumes of absolute ethanol.
after incubation for at least  <dig> hour at 20°c, rna was pelleted by centrifugation  and resuspended in rnase free sterile water.
rna was treated with rq <dig> rnase-free dnase  and then purified using the rneasy rna clean-up procedure .
rna quantification was performed using a qubit™ fluorometer .
total rna was reverse-transcribed into cdna using the super-script iii  reverse transcriptase kit.
real-time pcr amplification and analysis were carried out using an applied biosystems  <dig> real-time pcr system .
reactions were performed in triplicate using  <dig> μl 2x itaq sybr green supermix with rox  master mix,  <dig> μl  <dig> mm of each primer,  <dig> μl template and nuclease-free water  to a final volume of  <dig> μl.
primers were designed to amplify actin chs, chi, f3h, dfr <dig>  ldox, ufgt, myb <dig>  myb <dig>  bhlh <dig> and bhlh <dig> .
a negative nuclease-free water control was included in each run.
pcrs used an initial denaturation step at 95°c for  <dig> min, followed by  <dig> cycles of denaturation for  <dig> s at 95°c and annealing and elongation for  <dig> s at 60°c.
fluorescence was measured at the end of each annealing step at 60°c.
amplification was followed by a melting curve evaluation.
the data were analyzed with the applied biosystems sequence detection software, version  <dig> .
<dig>  , and transcript levels were normalized to malus × domestica actin  to minimize variation in cdna template levels.
actin was selected for normalization due to its consistent transcript levels throughout leaf and fruit tissues, with crossing threshold  values changing by less than  <dig>  for each gene, a standard curve was generated using a cdna serial dilution, and the resultant pcr efficiency calculations  were used for relative transcript level analysis.
error bars shown in real-time pcr data are biological and technical replicates, representing the means ± se of three biological samples and three technical replicates.
analysis of variance  on real-time pcr data was performed using jmp®  <dig>  statistical software .
student's t-test was used to establish significant differences in transcript accumulation between biological replicates.
primers used to amplify anthocyanin biosynthetic enzymes, candidate transcription factors and trim transposable elements.
mature 'royal gala' peel rna was isolated by a method adapted from chang et al.
<cit> , quantified in a nanodrop nd- <dig> spectrophotometer running software version  <dig> .
<dig>  and treated with dna-free dnase .
for real time-pcr analysis, total rna was reverse-transcribed into cdna using the super-script iii  reverse transcriptase kit.
real-time pcr amplification and analysis were carried out using the roche  <dig> lightcycler system .
all reactions were performed using the lightcycler  <dig> sybr green i master mix  following manufacturer instructions.
reactions were performed in triplicate using  <dig> μl 5x master mix,  <dig>  μm each primer and  <dig> μl diluted cdna.
a negative nuclease-free water  control was included in each run.
primers used are the same as described above.
pcrs used an initial denaturation step at 95°c for  <dig> min, followed by  <dig> cycles of denaturation for  <dig> s at 95°c, annealing for  <dig> s at 60°c and elongation for  <dig> s at 72°c.
fluorescence was measured at the end of each annealing step at 72°c.
amplification was followed by a melting curve analysis with continual fluorescence data acquisition during the 65-95°c melt curve.
the raw data were analyzed with the lightcycler software,  and transcript levels were normalized to actin to minimize variation in cdna template levels.
for each gene, a standard curve was generated using a cdna serial dilution, and the resultant pcr efficiency calculations  were imported into relative transcript level analysis.
error bars shown in real-time pcr data are technical replicates, representing the means ± se of three replicate real-time pcr reactions.
anova on real-time pcr data was performed as described above.
myb <dig> characterization
to study sequence diversity in the myb <dig> coding region, fruit peel rna and cdna were obtained using the modified lopez-gomez and gomez-lim extraction method as described above.
the myb <dig> coding region was amplified using pfuultra™ hotstart dna polymerase  using myb <dig> cdna primers  and dna template.
reactions were performed in a  <dig> μl total volume , 10x buffer provided by manufacturer and  <dig> mm mgcl2).
pcrs used  <dig> cycles of 94°c  <dig> s, 50°c  <dig> s, 72°c  <dig> s .
fragments were then a-tailed by incubating  <dig> μl pcr product with  <dig> μl amplitaq™ ,  <dig> μl buffer provided by manufacturer,  <dig> μl  <dig> mm datp, and  <dig> μl sterile water for  <dig> minutes at 70°c.
fragments were then desalted through a microspin™ s- <dig> hr column  according to manufacturer's recommendations.
desalted fragments were cloned into the pgem®-t easy vector , also according to manufacturer's instructions.
plasmids were purified from  <dig> ml overnight cultures using the wizard plus sv minipreps dna purification system .
to verify insert size,  <dig> μl of plasmid dna were digested in 1x manufacturer supplied buffer by  <dig> units ecori  in a  <dig> μl total volume at 37°c for  <dig> h. the entire reaction was loaded and separated on 1% agarose gels in tbe buffer, stained with ethidium bromide, and photographed under uv light.
inserts were compared to dna standards of known size.
subsequently, undigested plasmids were sequenced using  <dig>  pm of m <dig> forward or reverse primer.
all nucleotide sequences were determined by applied biosystems bigdye terminator version  <dig>  cycle sequencing on an applied biosystems 3130xl or 3730xl automatic sequencer  at the university of minnesota dna biomedical genomics center's sequencing and analysis facility.
sequences were analyzed, assembled into contigs, and compared to known sequences using seqman™ ii .
for characterization of the myb <dig> region upstream of the translation start site , leaf tissues or fruit peels were very finely ground in liquid nitrogen, and dna was isolated using the haymes' method  <cit>  or using the dneasy plant mini kit .
three promoter regions were amplified using pfuultra ™ hotstart dna polymerase  using myb <dig> primer pairs -2029/- <dig>  -1411/- <dig> and -677/ <dig> .
reactions were performed as described above, but without additional mgcl <dig>  pcr fragments were then desalted through a microspin™ s- <dig> hr column  according to manufacturer's recommendations and fragments from three independent replicate reactions per sample were sequenced directly using  <dig>  pm of the corresponding forward and reverse primers, as detailed above.
to amplify the trim element in the cultivars studied, standard pcrs were performed using amplitaq™  in a  <dig> μl total volume ,  <dig> μm each dntp,  <dig>  units taq, 10x buffer provided by manufacturer).
pcrs used  <dig> cycles of 94°c  <dig> s, 55°c  <dig> s, 72°c  <dig> s. these same thermocycling conditions were used to study whether the trim element is transcribed in the cultivars studied.
the template in transcription studies consisted of cdna obtained as described above, and trim forward and reverse primers  or trim forward combined with myb <dig> - <dig> were used .
primers used to amplify different regions of the myb <dig> locus of apple
primer position is indicated relative to the atg translation start site.
methylation studies
peel genomic dna  from red or green stripes, or from red and green areas of blushed apples harvested in  <dig> and  <dig>  was digested with mcrbc  in  <dig> μl total volume including 1x neb <dig> buffer,  <dig>  mg/ml bovine serum albumin,  <dig> mm gtp and  <dig> u mcrbc or 50% glycerol .
digestions were incubated overnight at 37°c to ensure complete digestion and then incubated at 65°c for  <dig> min to halt enzyme activity.
real-time experiments were performed on mcrbc and mock digested template as described above for 'honeycrisp' and primers used are presented in table  <dig>  for each experiment, real-time pcr runs, including a control  and a mcrbc digested sample, were performed in triplicate, and two or three independent digestions were used.
percent methylation for individual samples was calculated as a function of the delta ct between control and mcrbc treated dna, using the formula:
student's t-test was used to establish significant differences in template amounts between biological replicates and subsequently calculate sample size.
the estimated sample size was used when determining whether significant differences occurred between red and green peel regions.
peel genomic dna from red or green stripes  were subjected to bisulfite conversion using the ez dna methylation-gold™ kit  following manufacturer's recommendations.
two myb <dig> promoter regions were amplified using myb <dig> primer pairs -1007/- <dig> and -534/- <dig> .
pcrs were performed using zymo taq™ dna polymerase  in a  <dig> μl total volume .
pcrs used an initial denaturation step at 95°c for  <dig> min, followed by of  <dig> cycles of 95°c  <dig> s, 50°c  <dig> s  or 55°c , 72°c  <dig> s, and a final elongation step at 72°c for  <dig> minutes.
a secondary pcr was carried out using the same primers and conditions, and  <dig>  μl of the primary pcr product.
fragments were then desalted through a microspin™ s- <dig> hr column  according to manufacturer's recommendations.
desalted fragments were cloned into the pgem®-t easy vector , also according to manufacturer's instructions.
bacterial colonies were frozen in  <dig> μl aliquots of luria broth  solution with 10% glycerol in 96-well plates and shipped on dry ice to beckman-coulter genomic services  for sanger sequencing.
percent methylation was calculated based on the methylation status of each cytosine within the two regions sequenced, using  <dig> to  <dig> clones per sample.
authors' contributions
at conceived of the study, participated in its design, carried out the molecular biology experiments and drafted the manuscript, jmb conceived of the study, participated in its design and coordination and helped draft the manuscript, aca participated in the design and coordination of the study and helped draft the manuscript, klw carried out dna and rna extractions and real-time experiments, des carried out anthocyanin quantification analysis and edited the manuscript, jmc carried out identification of anthocyanin compounds using lc-ms, rph participated in the design of the study and helped draft the manuscript and eeh participated in the design of the study and provided the majority of funding to complete the research.
all authors read and approved the final manuscript.
supplementary material
additional file 1
overall methylation of individual clones in the myb <dig> - <dig> to - <dig> promoter region.
percent methylation in the  <dig> clones obtained from red and green stripes is presented.
clones are sorted in ascending order according to methylation percentages.
regression lines for methylation levels in green and red stripes as a function of clone number are indicated as green and red dotted lines respectively, and highlight the higher values observed in green stripes as compared to red stripes.
click here for file
additional file 2
sequence alignment of myb <dig> and  <dig> individual clones in the - <dig> to - <dig> promoter region.
increased myb <dig> dna methylation in green stripes is evident when comparing the number of methylated cytosines in each nucleotide position .
yellow bands indicate the location of cytosines in myb <dig>  bars indicate the net difference in methylation  at a particular site.
green bars indicate that a larger number of green stripe-derived clones carry methylated cytosines in that particular nucleotide position; red bars indicate higher methylation in red stripe-derived clones.
panel b depicts a dna sequence alignment of myb <dig> clones obtained from bisulfite-treated dna from green stripes  and from red stripes .
methylated cytosines are highlighted in yellow in all the sequences.
methylation, which is mostly present at the 5' and 3' ends of this region, was observed in all cytosine contexts.
click here for file
acknowledgements
the authors are grateful to nathan springer, alan smith and james luby for their insightful suggestions, to dwayne jensen of p&fr, ruakura for assistance with lcms of phenylpropanoids, and to harpartap mann and richard espley for assistance with rna extraction and real-time protocols.
at gratefully acknowledges the guidance and professional support of christopher walsh, university of maryland.
support from the university of minnesota supercomputing institute is gratefully acknowledged.
this work was funded in part by the minnesota agricultural experiment station.

 apple peel color is one of the most important factors determining apple market acceptance. in general, red cultivars are the most preferred, and within a cultivar more highly colored fruits are favored. consumer preferences vary from country to country and region to region: new zealand consumers prefer striped apples, consumers in nova scotia, canada prefer blushed apples, while consumers in british columbia, canada are more accepting of a range of apple types. peel pigments not only affect visual appeal, they also contribute to the fruit's nutritional value. apples have been associated with lowered risks of cancer and cardiovascular diseases, which are thought to be caused by oxidative processes. polyphenolics, including anthocyanins which are the red pigments in apple peels, have been found to be the major source of antioxidants in apple. antioxidants are mainly localized in the apple peel, but cultivars exhibit a wide variation in the distribution pattern. anthocyanin accumulation in apple peels can be affected by genetic, environmental, nutritional and cultural factors, the stage of maturity of the fruit, and by the microenvironment within the canopy. the main anthocyanin identified in apple skin is cyanidin 3-galactoside, while cyanidin 3-glucoside levels are very low. two categories of genes affect the biosynthesis of anthocyanin. the first category encodes enzymes required for pigment biosynthesis, which have been widely studied in apple. the second category is comprised of transcription factors, which are regulatory genes that influence the intensity and pattern of anthocyanin accumulation and control transcription of different biosynthetic genes. at least three families, myb, bhlh and wdr, have been found to be involved in the regulation of anthocyanin synthesis, but the specific classes and genes involved vary depending on the species. in apple, three research groups have independently identified an r2r myb transcription factor responsible for anthocyanin accumulation in fruit. the loci have been named myb myb and myba. the coding region of myba is and 98% identical to myb and myb respectively. in addition, myb and myba have been mapped to the same region on linkage group. subsequent experiments have shown that myb myb and myba are likely to be allelic and more-over, at this locus in the current apple genome assembly, there is only one myb present. based on this evidence, in this research article, we consider myb to exist as a single locus with myba and myb representing alleles of the myb locus. transcript levels of the myb allele correlate with anthocyanin accumulation and are higher in red fruit peel sectors and in red peel cultivars than in green peel sectors or cultivars. transcript levels of myb increased in dark-grown apples once exposed to light, providing additional evidence of its role as an anthocyanin regulator. myb1- a sequence variant of the myb allele, co-segregates with red skin color. transcription at the myb locus strongly correlates with peel anthocyanin levels and this gene is able to induce anthocyanin accumulation in heterologous and homologous systems. in addition, myb co-segregates with the rni locus, a major genetic determinant of red foliage and red color in the core of apple fruit. consistently, the expression of several anthocyanin pathway genes was found to be regulated by myb and myb. in apple, two candidate bhlh transcription cofactors are also needed for activating promoters of anthocyanin structural genes and myb. repressors of anthocyanin production were also identified within the myb class of transcription factors, including mdmyb in apple, famyb in strawberry and atmybl in arabidopsis. famyb is up-regulated jointly with late anthocyanin pathway genes. expression of atmybl is also coordinately up-regulated by the myb-bhlh-wdr activation complex. in arabidopsis a transcriptional regulatory loop has been postulated whereby atpap is a positive regulator of attt, and attt is an activator of atmybl expression which then negatively regulates the expression of attt it is suggested that the repressors' role is to balance anthocyanin levels produced at later stages of color response.'honeycrisp', an increasingly important apple cultivar developed at the university of minnesota, produces fruits that can adopt two basic peel color patterns: blushed or striped. for the purposes of this study, fruits are defined as striped when the color alternates between vertically elongated regions in some or all portions of the peel. fruits are termed blushed when the surface is partly covered with a red tinge that is not broken. these two phenotypic categories are mutually exclusive. in'honeycrisp' both kinds of fruit may be present on the same tree, a characteristic that has not been described in other cultivars. the molecular basis of this phenomenon is unknown. different mechanisms can cause variegation in plants, including chimeras, transposable element activity and cytosine methylation. previous results do not provide evidence for a chimeral source of variegation in the case of'honeycrisp', since the phenotype is not stable after propagation as would be expected if changes were caused by a periclinal chimera. microscopic observations indicated that the difference between stripes is due to a reduction in pigment accumulation in the paler stripes, both in the epidermis and in the first hypodermal layers. activation and suppression of transposable elements may be responsible for some of the genetic variation that occurs in peel color in pome fruits. transposable elements have been identified in apple but to date there is no evidence associating transposable elements with fruit peel variegation. the presence of transposable elements can affect gene expression both at the transcriptional, and at the post-transcriptional level. cocciolone and cone reported that striped patterns of anthocyanin accumulation in maize were due to differential dna methylation in the 3' untranslated region of pl-bh, a myb transcription factor regulating anthocyanin accumulation. methylation was found to be inversely correlated with pl-bh mrna levels in variegated plant tissues. the authors hypothesized that early during development, the pl-bh gene would be differentially methylated and this methylation would be more or less maintained through subsequent cell divisions, producing clonal sectors in plant tissues of predominantly pigmented cells and sectors of predominantly unpigmented cells. sekhon and chopra identified a gene called ufo that controls methylation levels in p a gene that regulates phlobaphene biosynthesis in maize, and whose activity may also produce variegation in the maize pericarp. ectopic expression of p1-wr correlated with hypomethylation of an enhancer region, kb upstream of the transcription start site. it is not known whether methylation is responsible for color differences in apple. we therefore sought to understand the molecular mechanism responsible for'honeycrisp' color pattern regulation and instability. we also included in this study two stably striped cultivars, a stably blushed cultivar and other cultivars differing in the degree of peel pigmentation. our results showed that variation in pigment accumulation between red and green stripes correlates with anthocyanin levels, and the steady state mrna levels of both the anthocyanin biosynthetic genes and the transcription factor myb sequence variation in the myb region upstream of the translation start site and coding region does not explain the observed phenotypes. the promoter and coding regions of myb were examined in red and green stripes for dna methylation levels and a bp region, starting bp upstream of the predicted translation start site, was found to be highly methylated in both'honeycrisp' and'royal gala'. red stripes were associated with lower methylation across the promoter of myb in'honeycrisp' and to a lesser degree in'royal gala', but no differences were found between blushed'honeycrisp' green and red peel regions.  red stripes have higher anthocyanin accumulation and transcript levels of biosynthetic genes red stripes of'royal gala' and'honeycrisp' contained approximately eight and four times as much anthocyanin as green stripes. in all cases, the major anthocyanin detected was cyanidin-3-galactoside. we subsequently compared the transcript levels of regulatory genes myb myb bhlh and bhlh and biosynthetic genes chs, chi, f3h, dfr ldox, ufgt, in rna isolated from red and green stripes of'royal gala' and'honeycrisp'. myb and myb transcript levels correlated with anthocyanin concentration in both'honeycrisp' and'royal gala', with higher mrna levels in red stripes as compared to green stripes. transcript levels of structural genes followed the same pattern as those of myb and myb levels of the two bhlh transcription factors did not differ in green and red stripes, and therefore correlated poorly with anthocyanin concentration. these results reveal differential transcript accumulation of myb and myb in differentially pigmented stripes, which in turn results in a corresponding modulation of transcript levels of structural genes. myb is a known activator of the apple anthocyanin pathway and myb has been shown to inhibit steps in the anthocyanin pathway and has high sequence similarity to atmyb a repressor of the phenylpropanoid pathway. we decided to further characterize myb coding and upstream regions in order to determine whether sequence polymorphisms can explain different pigmentation patterns. low sequence diversity in the myb coding region in'honeycrisp','connell red' and'fireside' to study the possibility that sequence differences are the cause of differential color patterns in the peel, we sequenced a total of cdna clones of the'honeycrisp' myb coding region: from a phenotypically uncharacterized'honeycrisp' fruit, from a mature striped and from a mature blushed fruit. ninety-two percent of the sequences obtained were 100% identical to myb1- an allele of the myb locus. we found three single nucleotide polymorphisms that produce changes in protein sequence, but since each one appeared only once in our dataset, and in phenotypically different apples, they most likely represent amplification or sequencing errors. these results indicate low levels of sequence diversity in the myb coding region in'honeycrisp', with no evidence suggesting that the blushed/striped phenomenon is associated with modifications at the primary dna sequence level within the coding region. myb coding sequences from the striped cultivar fireside and'connell red', a stably blushed sport of'fireside', are identical to that of the most abundant version found in'honeycrisp' and the previously published myb1- sequence--supporting our conclusion that differences in primary dna sequence are not the source of differential patterns of apple peel pigment accumulation. no size variation in myb promoter region among apple cultivars we amplified three fragments collectively spanning about kb of the myb promoter. pcr results did not indicate any fragment size differences among blushed and striped'honeycrisp','connell red' and'fireside' dna, suggesting no large insertion or deletions were present. we sequenced the pcr products of each of these fragments from three independent reactions and found no sequence differences between blushed and striped'honeycrisp', or between'connell red' and'fireside', although there were snps between'honeycrisp' and the other two cultivars. neither presence nor transcription of a trim element explains apple peel phenotypic variation within the plant & food malus gene database was a dna sequence identical to genbank accession eu the promoter of myb further upstream from this sequence, between positions - and - from the atg translation start site of myb was a sequence with 85% identity to a malus trim element, a terminal-repeat retrotransposon in miniature. we checked for the presence of a trim element upstream of the myb locus in'honeycrisp','connell red','fireside','1807' and'geneva' via pcr, combining a primer designed from the trim element with one designed from the promoter region of myb. results confirmed the presence of the trim element in each of these cultivars in a position identical to that observed in'royal gala'. we subsequently cloned and sequenced three pcr products from'honeycrisp','connell red' and'fireside'. half of the fragments yielded sequences showing 99% or more identity to the previously published upstream region of'royal gala' myb the other sequences were probably amplifications from insertions of similar trim elements located elsewhere in the genome, with percent identities to trim ranging from to %. we tested for trim transcript presence in blushed and striped'honeycrisp','connell red','fireside','geneva' and'honeygold', and found it to be transcribed in all cases. however, a fragment spanning a portion of the trim element and extending bp into the upstream region of myb did not amplify from total rna, indicating that transcription from the trim element did not extend into myb in these cultivars. overall, results indicated that neither the presence of the trim element in the myb promoter region nor its transcription explained the differences in peel pigment accumulation among the cultivars studied. increased methylation levels in green stripes dna samples from green and red stripes of'honeycrisp' and'royal gala' were treated with the methylation-sensitive endonuclease mcrbc to ascertain whether the observed differences in transcript accumulation were associated with methylation differences in the promoter or coding region of myb. mcrbc preferentially cuts dna containing methylcytosine on one or both strands, between two recognition sites. mcrbc treated and mock-digested templates were compared using real-time pcr, and percent methylation was calculated. in total, fragments starting at the transposon insertion and spanning kb of the promoter region and three exons of myb were evaluated. results indicated that a region of the myb promoter, encompassing the fragments between nucleotide positions - and - is highly methylated in both cultivars.'connell red' and'fireside' had low methylation in the - to - fragment and high methylation in the - to - fragment, indicating a similar pattern of myb methylation in these cultivars relative to those observed in'royal gala' and'honeycrisp'. green stripes of'honeycrisp' showed higher overall methylation levels than red stripes throughout the promoter region. the - to - fragment was omitted from this comparison since quantification in the mcrbc digested samples was highly variable due to extremely low template levels, indicating that this region was so highly methylated that treatment with mcrbc resulted in nearly complete digestion of the template dna. sequence analysis indicated that differences in predicted methylation levels between regions were not due to difference in the number of potential mcrbc recognition sites. similar results were obtained for fruits, but overall methylation levels were higher than in and differences between red and green stripes were even greater. these results indicate that while methylation levels are variable between years, green stripes are consistently associated with higher methylation of myb promoter regions. similar trends were observed in'royal gala' for some of the fragments, except that the differences between red and green stripes were smaller. in total, higher methylation levels were observed for'royal gala' than'honeycrisp'. in contrast to'honeycrisp' red and green stripes, we hypothesized that color differences between red and green regions of the peel of blushed apples are only due to light effects and not to differences in methylation levels. we therefore compared methylation percentages in red and green areas of blushed apples and red and green stripes. results indicated no significant differences between red and green regions of the peel of blushed apples. interestingly, in two out of the three regions studied, red stripes have methylation levels comparable to those in the exposed peel portions of blushed apples, while green stripes have methylation levels higher than those of red stripes or red and green regions of blushed apples. bisulfite sequencing offers greater resolution than mcrbc-based methods for the detection of methylated cytosines. building on mcrbc results, we next pursued bisulfite sequencing of myb promoter regions from'honeycrisp' and'royal gala'. preliminary bisulfite sequencing experiments indicated that cytosine methylation in the promoter region of myb is found in all three methylation contexts. to avoid amplification bias, we therefore designed degenerate pcr primers to target two different promoter regions. this severely constrained areas that could be targeted, and amplification upstream of - was ultimately unsuccessful using unbiased primers. a comparison of methylation levels between red and green stripes in the - to - and - to - regions confirmed that green stripes are more highly methylated than red stripes, with 80% and 65% of cytosines showing higher methylation levels in green than in red stripes in the - to - and - to - regions respectively. further analysis of the - to - region indicated that clones obtained from green stripes have higher overall methylation levels than those obtained from red stripes. observed methylation differences between red and green stripes are modest, but actual differences may be greater. although great care was taken, manual isolation of red and green stripes from'honeycrisp' peels was imprecise, resulting in tissue samples that were substantially enriched for red or green stripes but not entirely devoid of contaminating tissues. thus, dna samples used for mcrbc- and bisulfite-based analyses, while predominantly derived from the target tissue likely represent a mixture of dna, partially obscuring methylation differences between sources. consistent with our preliminary results, different cytosine contexts did not exhibit distinct methylation patterns; all cytosine contexts showed high methylation levels in highly methylated regions and vice versa. overall, chh and chg methylation was highest and cg methylation was lowest. a sequence alignment for the - to - region is presented in additional file discussion anthocyanin genes transcript levels correlate with striped patterns anthocyanin and transcript quantifications performed in this study suggested a possible mechanism controlling pigment patterns in apple fruit peels. we have found that green stripes are associated with lower anthocyanin accumulation, which is explained by reduced transcript levels of all the anthocyanin pathway genes evaluated, including the structural genes in the pathway, and myb a transcription factor which regulates them. an additional gene evaluated in this study, myb an apple transcription factor that represses anthocyanin synthesis was transcribed in a similar manner to myb as myb is not elevated in green sectors relative to red, we considered myb the main transcription factor regulating the pathway in apple, to be the most likely candidate to be involved in peel variegation. we therefore sought to identify a mechanism responsible for myb transcript level differences. variegation in apple peels is associated with myb methylation mosaicism our results indicate an inverse association between methylation levels in the myb promoter and anthocyanin accumulation in striped apple peels. as previously suggested by cocciolone and cone for maize, we hypothesized that early in apple fruit development, differences in myb methylation are present among individual cells. throughout fruit growth, these differentially methylated cells give rise to sectors of peel varying in their ability to accumulate anthocyanins. our results indicate that dna methylation in the promoter of myb is associated with reduced transcript accumulation. specifically, we propose that differential methylation of myb promoter regions in red vs. green stripes of the'honeycrisp' peel results in differential accumulation of the myb transcript, which in turn determines both transcription of anthocyanin structural genes and pigment accumulation. dna methylation may affect myb transcription through interference with the rna-polymerase transcription complex or by preventing binding of additional factors required for transcription. in arabidopsis, genome wide studies of dna methylation have found that methylation within regulatory regions is rare and probably selected against, as it may interfere with transcription initiation. our results suggest that high levels of methylation in certain promoter regions of a key transcription factor in the flavonoid biosynthetic pathway in apple may play a regulatory role, but it is not inhibitory for gene activity. it is possible that since apple trees are clonally propagated and stay in production for many years, mechanisms of epigenetic regulation might not be identical to what has been described in more widely studied species such as arabidopsis and rice. different methylation levels early in apple fruit development could be mitotically maintained from those in the meristematic cells that gave origin to the fruit, or could result from demethylation or de novo methylation. previous results in'honeycrisp' suggest that there is at least some mitotic maintenance of methylation states, given that trees clonally propagated from buds on branches with exclusively blushed fruits, tend to produce a higher percentage of blushed fruit. nonetheless, results from the same study indicated that additional factors can influence the pattern in the peel, namely position of the fruit on the tree and crop load. the presence of a trim transposable element in an upstream region of the myb promoter might influence the changes in methylation observed between different regions of the peel but neither its presence per se nor its transcription appears to be responsible for determining peel pigmentation. the trim element identified in this study is located kb upstream of the predicted translation start site, and is present in'honeycrisp','royal gala', and five other cultivars with peel pigmentation ranging from green to ultra red. lippman et al. indicated that in arabidopsis transposable elements can determine epigenetic gene silencing when inserted within or very near a gene. the effect of a transposable element kb upstream of the coding region is unknown. we did not find any evidence of transposable element sequences within the highly methylated promoter region of myb within the most methylated region of the myb promoter in this study are five putative e-box motifs which are bhlh-related cis-acting elements. methylation was absent at the three e-box motifs evaluated using bisulfite sequencing, but highly methylated areas occurred a few nucleotides upstream or downstream of these motifs. this may suggest a potential regulatory role for one or more of these motifs. an assessment of the presence of other types of epigenetic marks such as histone methylation can shed additional light on the mechanism involved in myb regulation. our results show that methylation followed the same pattern in all three cytosine methylation contexts, with alternation of high and low methylation regions. the high methylation levels observed for chh and chg sites, and low methylation levels for the cg context, indicate a pattern not representative of what is generally observed in flowering plants. broader survey of methylation patterns throughout the apple genome is warranted. the unstable nature of pigment patterning in'honeycrisp' could be a result of a more variable cell to cell methylation pattern than is present in other cultivars producing fruit with consistent pigment patterns, such as'royal gala','fireside' and'connell red'. we speculate that the occurrence of stripes in'honeycrisp' is a consequence of higher than normal methylation levels in the myb promoter region in the green stripes, something that occurs only in some fruit and to varying degrees. in contrast, myb methylation levels and thus peel pigmentation remains more constant in'royal gala'.  differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of myb a transcription factor that regulates the anthocyanin pathway in apple. different transcript levels of myb in red versus green stripes are inversely associated with methylation levels in its promoter, especially along a bp region upstream of the translation start site. although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. methylation might be associated with the presence of a trim element within the promoter region, but the presence of the trim element alone cannot explain the phenotypic variability observed in'honeycrisp'. we suggest that methylation in the myb promoter is more variable in the phenotypically plastic'honeycrisp' than in the more consistently striped'royal gala'. differential methylation of the'honeycrisp' myb promoter alters accumulation of the myb transcript, in turn altering both transcription of anthocyanin structural genes and pigment accumulation. materials and methods plant material leaf samples of'honeycrisp','connell red','fireside','1807','honeygold' and'geneva' apple were collected in early spring of and fruits of the same cultivars were collected at maturity during the and growing seasons from trees at the horticultural research center in chanhassen, minnesota. in february and fruits were harvested at plant and food research orchards.'royal gala' apples grown in chile were purchased in a minnesota grocery store in april to be used for methylation experiments. for the myb characterization experiments, whole fruit peels were used. for anthocyanin quantification, transcript analyses and methylation studies, red and green stripes were carefully separated using a razor blade. since stripes cannot be absolutely classified as green or red, samples are more accurately described as "red stripe enriched" or "green stripe enriched". both green and red stripes were obtained from the same region of the peel at each time, preventing the possibility that the "red stripe enriched" sample would also be enriched for fruit peel regions more exposed to light and vice versa. for comparisons between different blushed fruit regions, light-exposed and -unexposed peel regions were separated. for both blushed and striped fruit regions, peel tissue from at least two apples was pooled. in all cases, leaves and peels were immediately frozen in liquid nitrogen and placed at -80°c before anthocyanins, dna or rna was extracted. identification and quantification of anthocyanins apple peel samples were finely ground in liquid nitrogen and then resuspended in ml methanol and % hcl. samples were sonicated for min, stored at room temperature in the dark for h and then centrifuged at × g. aliquots of ml were dried down to completion in a labconco centrivap concentrator. samples were resuspended in 20% methanol. anthocyanins were identified by lc-ms analysis as described previously. identification was based both on masses of molecular ions and characteristic fragments/neutral losses and comparison of retention times and fragmentation with authentic standards of cyanidin-3-o-glucoside and cyanidin-3-o-galactoside. m+ fragments observed were da, da and da. neutral losses observed were da, da and da. ms cannot distinguish between sugars of the same mass. anthocyanins and other phenolic compounds were quantified by hplc as described previously. quantification was achieved by reference to standards of anthocyanins and other phenolic compounds, using lc-ms data to confirm identification of peaks. real-time transcript analysis mature'honeycrisp' fruit peels were very finely ground in liquid nitrogen and rna was extracted using the lopez-gomez and gomez-lim extraction method as modified by mann et al.. briefly, after precipitation with m licl, rna was collected by centrifugation at × g for min, and second day licl washes were eliminated. rna pellets were resuspended in μl rnase free sterile water, potassium acetate was added to a final concentration of m, and the rna was precipitated with two volumes of absolute ethanol. after incubation for at least hour at 20°c, rna was pelleted by centrifugation and resuspended in rnase free sterile water. rna was treated with rq rnase-free dnase and then purified using the rneasy rna clean-up procedure. rna quantification was performed using a qubit™ fluorometer. total rna was reverse-transcribed into cdna using the super-script iii reverse transcriptase kit. real-time pcr amplification and analysis were carried out using an applied biosystems real-time pcr system. reactions were performed in triplicate using μl 2x itaq sybr green supermix with rox master mix, μl mm of each primer, μl template and nuclease-free water to a final volume of μl. primers were designed to amplify actin chs, chi, f3h, dfr ldox, ufgt, myb myb bhlh and bhlh. a negative nuclease-free water control was included in each run. pcrs used an initial denaturation step at 95°c for min, followed by cycles of denaturation for s at 95°c and annealing and elongation for s at 60°c. fluorescence was measured at the end of each annealing step at 60°c. amplification was followed by a melting curve evaluation. the data were analyzed with the applied biosystems sequence detection software, version., and transcript levels were normalized to malus × domestica actin to minimize variation in cdna template levels. actin was selected for normalization due to its consistent transcript levels throughout leaf and fruit tissues, with crossing threshold values changing by less than for each gene, a standard curve was generated using a cdna serial dilution, and the resultant pcr efficiency calculations were used for relative transcript level analysis. error bars shown in real-time pcr data are biological and technical replicates, representing the means ± se of three biological samples and three technical replicates. analysis of variance on real-time pcr data was performed using jmp® statistical software. student's t-test was used to establish significant differences in transcript accumulation between biological replicates. primers used to amplify anthocyanin biosynthetic enzymes, candidate transcription factors and trim transposable elements. mature'royal gala' peel rna was isolated by a method adapted from chang et al., quantified in a nanodrop nd- spectrophotometer running software version. and treated with dna-free dnase. for real time-pcr analysis, total rna was reverse-transcribed into cdna using the super-script iii reverse transcriptase kit. real-time pcr amplification and analysis were carried out using the roche lightcycler system. all reactions were performed using the lightcycler sybr green i master mix following manufacturer instructions. reactions were performed in triplicate using μl 5x master mix, μm each primer and μl diluted cdna. a negative nuclease-free water control was included in each run. primers used are the same as described above. pcrs used an initial denaturation step at 95°c for min, followed by cycles of denaturation for s at 95°c, annealing for s at 60°c and elongation for s at 72°c. fluorescence was measured at the end of each annealing step at 72°c. amplification was followed by a melting curve analysis with continual fluorescence data acquisition during the 65-95°c melt curve. the raw data were analyzed with the lightcycler software, and transcript levels were normalized to actin to minimize variation in cdna template levels. for each gene, a standard curve was generated using a cdna serial dilution, and the resultant pcr efficiency calculations were imported into relative transcript level analysis. error bars shown in real-time pcr data are technical replicates, representing the means ± se of three replicate real-time pcr reactions. anova on real-time pcr data was performed as described above. myb characterization to study sequence diversity in the myb coding region, fruit peel rna and cdna were obtained using the modified lopez-gomez and gomez-lim extraction method as described above. the myb coding region was amplified using pfuultra™ hotstart dna polymerase using myb cdna primers and dna template. reactions were performed in a μl total volume, 10x buffer provided by manufacturer and mm mgcl2). pcrs used cycles of 94°c s, 50°c s, 72°c s. fragments were then a-tailed by incubating μl pcr product with μl amplitaq™, μl buffer provided by manufacturer, μl mm datp, and μl sterile water for minutes at 70°c. fragments were then desalted through a microspin™ s- hr column according to manufacturer's recommendations. desalted fragments were cloned into the pgem®-t easy vector, also according to manufacturer's instructions. plasmids were purified from ml overnight cultures using the wizard plus sv minipreps dna purification system. to verify insert size, μl of plasmid dna were digested in 1x manufacturer supplied buffer by units ecori in a μl total volume at 37°c for h. the entire reaction was loaded and separated on 1% agarose gels in tbe buffer, stained with ethidium bromide, and photographed under uv light. inserts were compared to dna standards of known size. subsequently, undigested plasmids were sequenced using pm of m forward or reverse primer. all nucleotide sequences were determined by applied biosystems bigdye terminator version cycle sequencing on an applied biosystems 3130xl or 3730xl automatic sequencer at the university of minnesota dna biomedical genomics center's sequencing and analysis facility. sequences were analyzed, assembled into contigs, and compared to known sequences using seqman™ ii. for characterization of the myb region upstream of the translation start site, leaf tissues or fruit peels were very finely ground in liquid nitrogen, and dna was isolated using the haymes' method or using the dneasy plant mini kit. three promoter regions were amplified using pfuultra ™ hotstart dna polymerase using myb primer pairs -2029/- -1411/- and -677/. reactions were performed as described above, but without additional mgcl pcr fragments were then desalted through a microspin™ s- hr column according to manufacturer's recommendations and fragments from three independent replicate reactions per sample were sequenced directly using pm of the corresponding forward and reverse primers, as detailed above. to amplify the trim element in the cultivars studied, standard pcrs were performed using amplitaq™ in a μl total volume, μm each dntp, units taq, 10x buffer provided by manufacturer). pcrs used cycles of 94°c s, 55°c s, 72°c s. these same thermocycling conditions were used to study whether the trim element is transcribed in the cultivars studied. the template in transcription studies consisted of cdna obtained as described above, and trim forward and reverse primers or trim forward combined with myb - were used. primers used to amplify different regions of the myb locus of apple primer position is indicated relative to the atg translation start site. methylation studies peel genomic dna from red or green stripes, or from red and green areas of blushed apples harvested in and was digested with mcrbc in μl total volume including 1x neb buffer, mg/ml bovine serum albumin, mm gtp and u mcrbc or 50% glycerol. digestions were incubated overnight at 37°c to ensure complete digestion and then incubated at 65°c for min to halt enzyme activity. real-time experiments were performed on mcrbc and mock digested template as described above for'honeycrisp' and primers used are presented in table for each experiment, real-time pcr runs, including a control and a mcrbc digested sample, were performed in triplicate, and two or three independent digestions were used. percent methylation for individual samples was calculated as a function of the delta ct between control and mcrbc treated dna, using the formula: student's t-test was used to establish significant differences in template amounts between biological replicates and subsequently calculate sample size. the estimated sample size was used when determining whether significant differences occurred between red and green peel regions. peel genomic dna from red or green stripes were subjected to bisulfite conversion using the ez dna methylation-gold™ kit following manufacturer's recommendations. two myb promoter regions were amplified using myb primer pairs -1007/- and -534/-. pcrs were performed using zymo taq™ dna polymerase in a μl total volume. pcrs used an initial denaturation step at 95°c for min, followed by of cycles of 95°c s, 50°c s or 55°c, 72°c s, and a final elongation step at 72°c for minutes. a secondary pcr was carried out using the same primers and conditions, and μl of the primary pcr product. fragments were then desalted through a microspin™ s- hr column according to manufacturer's recommendations. desalted fragments were cloned into the pgem®-t easy vector, also according to manufacturer's instructions. bacterial colonies were frozen in μl aliquots of luria broth solution with 10% glycerol in 96-well plates and shipped on dry ice to beckman-coulter genomic services for sanger sequencing. percent methylation was calculated based on the methylation status of each cytosine within the two regions sequenced, using to clones per sample. authors' contributions at conceived of the study, participated in its design, carried out the molecular biology experiments and drafted the manuscript, jmb conceived of the study, participated in its design and coordination and helped draft the manuscript, aca participated in the design and coordination of the study and helped draft the manuscript, klw carried out dna and rna extractions and real-time experiments, des carried out anthocyanin quantification analysis and edited the manuscript, jmc carried out identification of anthocyanin compounds using lc-ms, rph participated in the design of the study and helped draft the manuscript and eeh participated in the design of the study and provided the majority of funding to complete the research. all authors read and approved the final manuscript. supplementary material additional file 1 overall methylation of individual clones in the myb - to - promoter region. percent methylation in the clones obtained from red and green stripes is presented. clones are sorted in ascending order according to methylation percentages. regression lines for methylation levels in green and red stripes as a function of clone number are indicated as green and red dotted lines respectively, and highlight the higher values observed in green stripes as compared to red stripes. click here for file additional file 2 sequence alignment of myb and individual clones in the - to - promoter region. increased myb dna methylation in green stripes is evident when comparing the number of methylated cytosines in each nucleotide position. yellow bands indicate the location of cytosines in myb bars indicate the net difference in methylation at a particular site. green bars indicate that a larger number of green stripe-derived clones carry methylated cytosines in that particular nucleotide position; red bars indicate higher methylation in red stripe-derived clones. panel b depicts a dna sequence alignment of myb clones obtained from bisulfite-treated dna from green stripes and from red stripes. methylated cytosines are highlighted in yellow in all the sequences. methylation, which is mostly present at the 5' and 3' ends of this region, was observed in all cytosine contexts. click here for file acknowledgements the authors are grateful to nathan springer, alan smith and james luby for their insightful suggestions, to dwayne jensen of p&fr, ruakura for assistance with lcms of phenylpropanoids, and to harpartap mann and richard espley for assistance with rna extraction and real-time protocols. at gratefully acknowledges the guidance and professional support of christopher walsh, university of maryland. support from the university of minnesota supercomputing institute is gratefully acknowledged. this work was funded in part by the minnesota agricultural experiment station.
most apple cultivars produce fruit with a defined fruit pigment pattern, but in the case of'honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. methylation may be associated with the presence of a trim retrotransposon within the promoter region, but the presence of the trim element alone cannot explain the phenotypic variability observed in'honeycrisp'. transcript levels of the major biosynthetic genes and myb a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. a survey of methylation levels throughout the coding region of myb and a kb region 5' of the atg translation start site indicated that an area bp long, starting bp upstream of the translation start site, is highly methylated. cytosine methylation was present in all three contexts, with higher methylation levels observed for chh and chg than for cg. comparisons of methylation levels of the myb promoter in'honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of myb different levels of myb transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. here we show that striped areas of'honeycrisp' and'royal gala' are due to sectorial increases in anthocyanin concentration. we suggest that methylation in the myb promoter is more variable in'honeycrisp' than in'royal gala', leading to more variable color patterns in the peel of this cultivar.
 some apple varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. most apple cultivars produce fruit with a defined fruit pigment pattern, but in the case of'honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. the causes of this phenomenon are unknown.  here we show that striped areas of'honeycrisp' and'royal gala' are due to sectorial increases in anthocyanin concentration. transcript levels of the major biosynthetic genes and myb a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. however, nucleotide changes in the promoter and coding sequence of myb do not correlate with skin pattern in'honeycrisp' and other cultivars differing in peel pigmentation patterns. a survey of methylation levels throughout the coding region of myb and a kb region 5' of the atg translation start site indicated that an area bp long, starting bp upstream of the translation start site, is highly methylated. cytosine methylation was present in all three contexts, with higher methylation levels observed for chh and chg than for cg. comparisons of methylation levels of the myb promoter in'honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes.  differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of myb different levels of myb transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. methylation may be associated with the presence of a trim retrotransposon within the promoter region, but the presence of the trim element alone cannot explain the phenotypic variability observed in'honeycrisp'. we suggest that methylation in the myb promoter is more variable in'honeycrisp' than in'royal gala', leading to more variable color patterns in the peel of this cultivar. 