Apple skin patterning is associated with differential expression of MYB10
© Telias et al; licensee BioMed Central Ltd. 2011
Received: 4 April 2011
Accepted: 20 May 2011
Published: 20 May 2011
Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') 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 MYB10, 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 MYB10 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 MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 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 (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 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 MYB10. Different levels of MYB10 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 MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.
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 [4, 5]. 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 [6, 7].
In apple, three research groups have independently identified an R2R3 MYB transcription factor responsible for anthocyanin accumulation in fruit. The loci have been named MYB1, MYB10 and MYBA [12, 15–17]. The coding region of MYBA is 100 and 98% identical to MYB1 and MYB10, respectively . In addition, MYB10 and MYBA have been mapped to the same region on linkage group 9 [15, 18]. Subsequent experiments have shown that MYB1, MYB10 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 MYB10 to exist as a single locus with MYBA and MYB1 representing alleles of the MYB10 locus.
Transcript levels of the MYB1 allele correlate with anthocyanin accumulation and are higher in red fruit peel sectors (more exposed to light) and in red peel cultivars than in green peel sectors or cultivars . Transcript levels of MYB1 increased in dark-grown apples once exposed to light, providing additional evidence of its role as an anthocyanin regulator. MYB1-1, a sequence variant of the MYB1 allele, co-segregates with red skin color [17, 21]. Transcription at the MYB10 locus strongly correlates with peel anthocyanin levels and this gene is able to induce anthocyanin accumulation in heterologous and homologous systems . In addition, MYB10 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 MYB10 and MYB1 [12, 17] (Figure 1). In apple, two candidate bHLH transcription cofactors (bHLH3 and bHLH33) are also needed for activating promoters of anthocyanin structural genes and MYB10 [12, 22].
Repressors of anthocyanin production were also identified within the MYB class of transcription factors, including MdMYB17 in apple , FaMYB1 in strawberry  and AtMYBL2 in Arabidopsis [25, 26]. FaMYB1 is up-regulated jointly with late anthocyanin pathway genes . Expression of AtMYBL2 is also coordinately up-regulated by the MYB-bHLH-WDR activation complex [26, 27]. In Arabidopsis a transcriptional regulatory loop has been postulated whereby AtPAP1 (MYB) is a positive regulator of AtTT8 (bHLH) , and AtTT8 is an activator of AtMYBL2 expression  which then negatively regulates the expression of AtTT8. It is suggested that the repressors' role is to balance anthocyanin levels produced at later stages of color response.
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 [34–41] 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 (e.g. through the introduction of an alternative transcription start site), 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 (unmethylated) and sectors of predominantly unpigmented cells (methylated). Sekhon and Chopra  identified a gene called Ufo1 that controls methylation levels in p1, 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, 5 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 ('Royal Gala' and 'Fireside'), a stably blushed cultivar ('Connell Red', a sport of 'Fireside') 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 MYB10. Sequence variation in the MYB10 region upstream of the translation start site (referred to as "promoter" for simplification) and coding region does not explain the observed phenotypes. The promoter and coding regions of MYB10 were examined in red and green stripes for DNA methylation levels and a 900 bp region, starting 1400 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 MYB10 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
Low sequence diversity in the MYB10coding 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 94 cDNA clones of the 'Honeycrisp' MYB10 coding region: 47 from a phenotypically uncharacterized 'Honeycrisp' fruit (harvested in late August when pigment pattern could not yet be conclusively determined), 24 from a mature striped and 23 from a mature blushed fruit. Ninety-two percent of the sequences obtained were 100% identical to MYB1-1, an allele of the MYB10 locus . We found three single nucleotide polymorphisms (SNP) 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 MYB10 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. MYB10 coding sequences from the striped cultivar Fireside (24 clones) and 'Connell Red' (23 clones), a stably blushed sport of 'Fireside', are identical to that of the most abundant version found in 'Honeycrisp' and the previously published MYB1-1 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 MYB10promoter region among apple cultivars
We amplified three fragments (-2029 to -1229, -1411 to -678, and -677 to 47; nucleotide positions on the Genbank accession EU518249 relative to translation start site) collectively spanning about 2 Kb of the MYB10 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 14 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 EU518249, the promoter of MYB10. Further upstream from this sequence, between positions -3038 and -2420 from the ATG translation start site of MYB10 (EU518249, 'Royal Gala') was a sequence with 85% identity to a Malus TRIM element (AY603367), a terminal-repeat retrotransposon in miniature . We checked for the presence of a TRIM element upstream of the MYB10 locus in 'Honeycrisp' (blushed and striped), 'Connell Red', 'Fireside', '1807' (green selection) and 'Geneva' (ultra red cultivar) via PCR, combining a primer designed from the TRIM element (TRIM forward primer) with one designed from the promoter region of MYB10 (primer -1873). Results confirmed the presence of the TRIM element in each of these cultivars in a position identical to that observed in 'Royal Gala' (Figure 5C). We subsequently cloned and sequenced three PCR products from 'Honeycrisp' (blushed and striped), 'Connell Red' and 'Fireside'. Half of the fragments yielded sequences showing 99% or more identity to the previously published (EU518249) upstream region of 'Royal Gala' MYB10. The other sequences were probably amplifications from insertions of similar TRIM elements located elsewhere in the genome, with percent identities to TRIM ranging from 40 to 56.5%.
We tested for TRIM transcript presence in blushed and striped 'Honeycrisp', 'Connell Red', 'Fireside', 'Geneva' (ultra red cultivar) and 'Honeygold' (green cultivar), and found it to be transcribed in all cases. However, a fragment spanning a portion of the TRIM element and extending 500 bp into the upstream region of MYB10 did not amplify from total RNA, indicating that transcription from the TRIM element did not extend into MYB10 in these cultivars. Overall, results indicated that neither the presence of the TRIM element in the MYB10 promoter region nor its transcription explained the differences in peel pigment accumulation among the cultivars studied.
Increased methylation levels in green stripes
In contrast to 'Honeycrisp' red and green stripes, we hypothesized that color differences between red (exposed to light) and green (unexposed to light) 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 (exposed) and green (unexposed) areas of blushed apples and red and green stripes. Results indicated no significant differences (p ≤ 0.05) between red and green regions of the peel of blushed apples. Interestingly, in two out of the three regions studied (-1411 to -1229 and -846 to -651), 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 (Figure 7).
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 (Figure 8B). Overall, CHH and CHG methylation was highest (20.2 and 16.9% respectively) and CG methylation was lowest (1.6%). A sequence alignment for the -1007 to -684 region is presented in Additional file 2.
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 MYB10, a transcription factor which regulates them. An additional gene evaluated in this study, MYB17, an apple transcription factor that represses anthocyanin synthesis  was transcribed in a similar manner to MYB10. As MYB17 is not elevated in green sectors relative to red, we considered MYB10, the main transcription factor regulating the pathway in apple [12, 15, 17, 21], to be the most likely candidate to be involved in peel variegation. We therefore sought to identify a mechanism responsible for MYB10 transcript level differences.
Variegation in apple peels is associated with MYB10methylation mosaicism
Our results indicate an inverse association between methylation levels in the MYB10 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 MYB10 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 MYB10 is associated with reduced transcript accumulation. Specifically, we propose that differential methylation of MYB10 promoter regions in red vs. green stripes of the 'Honeycrisp' peel results in differential accumulation of the MYB10 transcript, which in turn determines both transcription of anthocyanin structural genes and pigment accumulation. DNA methylation may affect MYB10 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 (i.e. fruit peel tissue does not derive from cells that have undergone recent meiosis), 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 MYB10 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 2.5 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 (<500 bp) a gene. The effect of a transposable element 2.5 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 MYB10.
Within the most methylated region of the MYB10 promoter in this study (-1411 to -555; Figure 5) are five putative E-box motifs  which are bHLH-related cis-acting elements (CACATG) [49, 50]. 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 MYB10 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 [51, 52]. 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 MYB10 promoter region in the green stripes, something that occurs only in some fruit and to varying degrees. In contrast, MYB10 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 MYB10, a transcription factor that regulates the anthocyanin pathway in apple. Different transcript levels of MYB10 in red versus green stripes are inversely associated with methylation levels in its promoter, especially along a 900 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 MYB10 promoter is more variable in the phenotypically plastic 'Honeycrisp' than in the more consistently striped 'Royal Gala'. Differential methylation of the 'Honeycrisp' MYB10 promoter alters accumulation of the MYB10 transcript, in turn altering both transcription of anthocyanin structural genes and pigment accumulation.
Materials and methods
Leaf samples of 'Honeycrisp', 'Connell Red', 'Fireside', '1807', 'Honeygold' and 'Geneva' apple were collected in early spring of 2005 and fruits of the same cultivars were collected at maturity during the 2005, 2006, 2007 and 2008 growing seasons from trees at the Horticultural Research Center in Chanhassen, Minnesota. In February 2008 ('Royal Gala') and 2010 ('Honeycrisp') fruits were harvested at Plant and Food Research orchards (Nelson, New Zealand). 'Royal Gala' apples grown in Chile were purchased in a Minnesota grocery store in April 2008 to be used for methylation experiments. For the MYB10 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 (redder) and -unexposed (greener) 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 1 ml methanol and 0.1% HCl. Samples were sonicated for 4 min, stored at room temperature in the dark for 3 h and then centrifuged at 3,000 × g. Aliquots of 1.0 ml were dried down to completion in a Labconco Centrivap Concentrator (Labconco, Kansas City, MO, USA). Samples were resuspended in 20% methanol (250 μl). Anthocyanins were identified by LC-MS analysis as described previously . Identification was based both on masses (M+) 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 303 Da (delphinidin), 287 Da (cyanidin) and 271 Da (pelargonidin). Neutral losses (i.e. mass differences between fragments) observed were 162 Da (hexoside sugar, e.g. galactose), 146 Da (deoxyhexoside sugar, e.g. xylose) and 132 Da (pentoside sugar, e.g. arabinose). MS cannot distinguish between sugars of the same mass (e.g. glucose/galactose). 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
Forward and reverse primers used in real-time PCR and RT-PCR analyses
Gene identifier (Genbank)
MYB10 -2029 to -1229
MYB10 -1411 to -678
MYB10 -677 to 47
MYB10 -1007 to -684
MYB10 -534 to -184
Mature 'Royal Gala' peel RNA was isolated by a method adapted from Chang et al. , quantified in a NanoDrop nd-100 spectrophotometer running software version 3.0.1 (NanoDrop Technologies, Wilmington, DE) and treated with DNA-free DNAse (Ambion, Austin, TX). For real time-PCR analysis, total RNA was reverse-transcribed into cDNA using the Super-Script III (Invitrogen Corp.) reverse transcriptase kit. Real-time PCR amplification and analysis were carried out using the Roche 480 LightCycler System (Roche Diagnostics, Mannheim, Germany). All reactions were performed using the LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics) following manufacturer instructions. Reactions were performed in triplicate using 5 μl 5X Master Mix, 1.0 μM each primer and 3 μl diluted cDNA. A negative nuclease-free water (Roche Diagnostics) control was included in each run. Primers used are the same as described above. PCRs used an initial denaturation step at 95°C for 5 min, followed by 50 cycles of denaturation for 10 s at 95°C, annealing for 10 s at 60°C and elongation for 20 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, (LightCycler 480, Software 1.5) 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 (ranging between 1.81 and 2.0) 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.
To study sequence diversity in the MYB10 coding region, fruit peel RNA and cDNA were obtained using the modified Lopez-Gomez and Gomez-Lim extraction method as described above. The MYB10 coding region was amplified using PfuUltra™ Hotstart DNA Polymerase (Stratagene, La Jolla, CA) using MYB10 cDNA primers (Table 1) and DNA template. Reactions were performed in a 50 μl total volume (15 ng template, 100 ng/μl each primer, 25 mM each dNTP, 2.5 units AmpliTaq™ (Applied Biosystems), 10X buffer provided by manufacturer and 25 mM MgCl2). PCRs used 35 cycles of 94°C 30 s, 50°C 30 s, 72°C 120 s (Gene Amp PCR system 9700, Applied Biosystems). Fragments were then A-tailed by incubating 3 μl PCR product with 1 μl AmpliTaq™ (Applied Biosystems), 1 μl buffer provided by manufacturer, 1 μl 2 mM dATP, and 1 μl sterile water for 24 minutes at 70°C. Fragments were then desalted through a MicroSpin™ S-200 HR column (Amersham Biosciences, Piscataway, NJ) according to manufacturer's recommendations. Desalted fragments were cloned into the pGEM®-T Easy Vector (Promega Corp.), also according to manufacturer's instructions. Plasmids were purified from 3 ml overnight cultures using the Wizard Plus SV Minipreps DNA Purification system (Promega Corp.). To verify insert size, 3 μl of plasmid DNA were digested in 1X manufacturer supplied buffer by 10 units EcoRI (Invitrogen) in a 10 μl total volume at 37°C for 1 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 3.2 pM of M13 forward or reverse primer. All nucleotide sequences were determined by Applied Biosystems BigDye Terminator version 3.1 cycle sequencing on an Applied Biosystems 3130xl or 3730xl automatic sequencer (Applied Biosystems) 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 (Windows 32 vs. 5.08; DNASTAR Inc, Madison, WI).
For characterization of the MYB10 region upstream of the translation start site (referred to as "promoter" for simplification), 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 (Qiagen). Three promoter regions were amplified using PfuUltra ™ Hotstart DNA Polymerase (Stratagene) using MYB10 primer pairs -2029/-1229, -1411/-678 and -677/47 (Table 1). Reactions were performed as described above, but without additional MgCl2. PCR fragments were then desalted through a MicroSpin™ S-300 HR column (Amersham Biosciences) according to manufacturer's recommendations and fragments from three independent replicate reactions per sample were sequenced directly using 3.2 pM of the corresponding forward and reverse primers, as detailed above.
Forward and reverse primers for apple genes used in McrBC/real-time PCR analyses
-2434 to -2281
-2254 to -2098
-2029 to -1873
-1874 to -1681
-1708 to -1426
-1411 to -1229
-1094 to -891
-991 to -776
-846 to -651
-704 to -555
-543 to -450
-465 to -316
-303 to -182
-168 to -45
-51 to 105
146 to 257
494 to 710
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 (2009 harvested apples) were subjected to bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corp., Irvine, CA) following manufacturer's recommendations. Two MYB10 promoter regions were amplified using MYB10 primer pairs -1007/-684 and -534/-184 (Table 1). PCRs were performed using Zymo Taq™ DNA Polymerase (Zymo Research Corp.) in a 50 μl total volume (1.0 μl bisulfite converted DNA as template, 0.6 μM each primer, 250 μM each dNTP, 2.0 units Zymo Taq, 1X buffer provided by manufacturer). PCRs used an initial denaturation step at 95°C for 10 min, followed by of 35 cycles of 95°C 30 s, 50°C 40 s (-1007 to - 684 fragment) or 55°C (-534 to - 184 fragment), 72°C 60 s, and a final elongation step at 72°C for 7 minutes. A secondary PCR was carried out using the same primers and conditions, and 1.0 μl of the primary PCR product. Fragments were then desalted through a MicroSpin™ S-300 HR column (Amersham Biosciences) according to manufacturer's recommendations. Desalted fragments were cloned into the pGEM®-T Easy Vector (Promega Corp.), also according to manufacturer's instructions. Bacterial colonies were frozen in 100 μl aliquots of Luria broth (Miller) solution with 10% glycerol in 96-well plates and shipped on dry ice to Beckman-Coulter Genomic Services (Beverly, MA) for Sanger sequencing. Percent methylation was calculated based on the methylation status of each cytosine within the two regions sequenced, using 12 to 24 clones per sample.
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.
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