Discriminant Analyses of the Polyphenol Content of American

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Discriminant Analyses th Polyphenol Content of American Elderberry Juice from Multiple Environments Provide Genotype Fingerprint Mitch C. Johnson, Matheus Dela Libera Tres, Andrew L. Thomas, George E. Rottinghaus, and C Michael Greenlief J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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Journal of Agricultural and Food Chemistry

Discriminant Analyses of the Polyphenol Content of American Elderberry Juice from Multiple Environments Provide Genotype Fingerprint

Mitch C. Johnson†,‡, Matheus Dela Libera Tres⊥, Andrew L. Thomas‡,§, George E. Rottinghaus‡,ǁ, and C. Michael Greenlief *,†,‡ †

Department of Chemistry, University of Missouri, Columbia, MO, 65211.

‡

Center for Botanical Interaction Studies, University of Missouri, Columbia, MO, 65211. Department of Food Science and Engineering, University of Sao Paulo, Pirassununga SP,

⊥

05508, Brazil. §

Division of Plant Sciences, Southwest Research Center, University of Missouri, Mt. Vernon, MO, 65712.

ǁ

Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO, 65211.

*

Corresponding author: Email: [email protected] Phone: (573) 882-3288

1 ACS Paragon Plus Environment


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Abstract

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The cultivation of American elderberry (Sambucus nigra subsp. canadensis) continues to

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increase as the use of this botanical has expanded. It is well understood that elderberries contain

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a variety of polyphenols, including anthocyanins, which have purported health benefits.

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However, information is lacking regarding the impact of environmental, management, and

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genotypic factors on the quantity and type of polyphenols and anthocyanins produced.

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Quantification of sixteen polyphenols including eight anthocyanins present in juice from three

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genotypes of American elderberry grown at two Missouri sites from 2013−2014 was performed.

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Large variances in anthocyanin and other polyphenol content were observed between the

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different harvest seasons, locations and genotypes. Although specific phytochemical trends due

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to those factors were not apparent, a discriminant analysis was able to correctly identify 45 of 48

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juice samples by genotype, based on their polyphenol profiles. This type of characterization

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could be beneficial in elderberry authentication studies and to help develop and document high-

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quality dietary supplement products with specific phytochemical contents.

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KEYWORDS: elderberry, anthocyanin, polyphenol, mass spectrometry, UHPLC, discriminant

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analysis, solid phase extraction, Sambucus

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INTRODUCTION American elderberry [Sambucus nigra L. subsp. canadensis (L.) Bolli] has emerged as a

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popular crop for producing dietary supplements, natural food colorants, wines, and other

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commercial products from both its fruit and flowers.1 Although European elderberry (Sambucus

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nigra L. subsp. nigra) has been studied extensively for its bioactive components and potential

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human health benefits, less is known about the American elderberry; especially its bioactive

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components.2-4 Elderberries, including the American subspecies, are a rich source of

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polyphenols, which are responsible for some of the purported health benefits associated with

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their antioxidant activity.5 Antioxidants have the ability to scavenge free radicals, reducing the

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amount of oxidative stress that can cause cardiovascular diseases, cancer, and neurological

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disorders over a long period of time.6

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Multivariate techniques, such as discriminant analysis, are providing new information,

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such as chemical profiles, location identification, and sample authentication. Multivariate

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statistical methods, for example, have been used previously to develop a chemical profile of

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ginseng and to classify commercial ginseng products.7 Ultra-high performance liquid

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chromatography – quadrupole time of flight (UHPLC-QTOF) mass spectrometry analysis

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revealed ten compounds that were unique among three ginseng species. Yuk et al. also used this

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method to determine which species of ginseng were found in various commercial herbal

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supplements. In a different study, Italian saffron (Crocus sativus L.) samples from five growing

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locations were analyzed for various flavonoids and several other bioactive compounds using

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high-performance liquid-chromatography (HPLC).8 Classification to the actual saffron growing

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location using a discriminant analysis was performed with 88% accuracy.



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Several studies have investigated the phytochemical profile of elderberry fruit and its

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juice,5,9,10 elderberry extracts,11 and elderberry based dietary supplements.12 Previous studies on

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American elderberry have found significant differences in polyphenol content within vegetative

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tissues13 and within fruit and fruit juice10,14,15 based on environmental (e.g., location, climate, soil

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type, precipitation) and crop management factors. This confirms that, in addition to genetics,

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environmental variations can potentially influence the phytochemical profile of American

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elderberry. This work investigates the potential effect genotype, growing location and year-to-

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year factors have on the polyphenol content of American elderberry juice. A complete analysis

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of all polyphenols is shown for the 2013 and 2014 growing seasons. Partial polyphenol data for

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2012 and 2015 are also presented. Discriminant analysis is used to help show that different

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cultivars of elderberry can be determined based on their polyphenol content. Additional data is

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presented that shows wide variation in polyphenol content is observed with growing year and

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location.

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MATERIALS AND METHODS

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Plant Material. American elderberry fruit for this study was grown at the University of

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Missouri’s Southwest Research Center near Mt. Vernon (MV) and Missouri State University’s

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State Fruit Experiment Station at Mountain Grove (MG), both in southern Missouri. Three

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commercially available elderberry cultivars (Adams II, Bob Gordon, and Wyldewood) were

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used. Plants were propagated from our own mother plants in late winter, 2011, and transplanted

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to the sites in June, 2011. Experimental plots contained four plants of the same cultivar, planted

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1.2 m apart. At each site, 48 plots were established in four rows, with plots separated 2.4 m

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within and 3.1 m between rows. The three cultivars were each assigned to 16 of the 48 plots in a


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completely randomized manner. During the growing seasons, plants were irrigated via drip lines

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to provide 2.5 to 4.0 cm water per week when rainfall was lacking. Weeds and pests were

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managed using standard horticultural practices.16 During the establishment year (2011),

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inflorescences were removed to encourage development of healthy roots and structure.

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Ripe fruit for this study was collected during four consecutive growing seasons (2012–

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2015). The fruit was collected in late August when the fruit was in full ripeness. The elderberry

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fruit was transported to lab under refrigeration, where it was stored at -20°C. The fruit was later

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thawed and de-stemmed by hand. The juice was prepared using fruit that showed uniform

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ripeness (visually) and approximately the same sized berries. The fruit was hand-pressed using a

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French press into individual aliquots of juice. All other debris, pulp, and seeds were discarded.

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The juice was then filtered through a nylon filter (0.22 µm), and 1.5 mL of each replicate was

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individually aliquotted into 1.5 mL microcentrifuge tubes, and then re-frozen and stored at -20°C

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until analysis as previously described17.

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Chemicals. Water, acetonitrile, and formic acid were all HPLC grade and purchased from Fisher

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Scientific (Fair Lawn, NJ, USA). Trifluoroacetic acid (TFA; 99%) was obtained from Acros

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Organics (Morris Plains, NJ, USA). Cyanidin-3-O-glucoside analytical standard (≥ 95%),

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chlorogenic acid (reference standard), rutin (HPLC grade), neo-chlorogenic acid (analytical

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standard grade), crypto-chlorogenic acid (analytical standard grade), quercetin 3-rutinoside

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(analytical standard grade), quercetin 3-glucoside (analytical standard grade), kaempferol 3-

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rutinoside (analytical standard grade), isorhamnetin 3-rutinoside (analytical standard grade), and

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isorhamnetin 3-glucoside (analytical standard grade) were purchased from Sigma Aldrich (St.

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Louis, MO, USA).



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Solid Phase Extraction (SPE). Juice samples were thawed in an oven at 60°C for 5 min. This

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treatment yielded the same results as thawing the juice at room temperature for 30 min. A

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modification of the SPE protocol previously used by He and Giusti was used for polyphenol

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separation.18 200 µL of juice was diluted with 200 µL of water (0.01% TFA) and vortexed. An

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Oasis C18 SPE cartridge (Waters, Milford, MA, USA) was first washed with 5 mL of water

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(0.01% TFA). Then the diluted juice sample and an additional 5 mL of water (0.01% TFA) were

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added to the cartridge. The sample fraction was then collected after 10 mL of methanol (0.01%

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TFA) was run through the cartridge. The final methanolic fraction was dried under a constant

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flow of nitrogen gas. Samples were reconstituted in water (2% acetic acid) for HPLC analysis.

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Anthocyanins were separated from the juice using a previously developed method19. Briefly,

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mixed mode cation-exchange SPE was used (Oasis MCX 3cc, 60mg, Waters, Milford, MA,

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USA) and extractions performed using a Supelco Visiprep vacuum manifold.

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HPLC-UV Analysis of Polyphenols. The HPLC method was adapted from that that of Kim and

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Lee20 and is briefly summarized. The HPLC system consisted of a Hitachi L-7100 pump, a

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Hitachi L-7200 autosampler (20 µL injection), and a Hitachi L-7400 UV detector (detection

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wavelength 320 nm) with a Synergi™ 4 µm Hydro-RP 80Å (2.0 x 150 mm) column

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(Phenomenex, Torrance, CA, USA) fitted with a SecurityGuard C18 (ODS) 4.0 x 3.0 mm guard

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column (Phenomenex). The mobile phase used was: (A) water (2% acetic acid) and (B) 50:50

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water/acetonitrile (0.5% acetic acid). The gradient was 2% B initially until 2 min, then linearly

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ramped to 40% B from 2-52 min, 100% B from 53-58min, and 2% B from 59-63 min. The

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mobile phase flow rate was 0.6 mL/min and system was run at room temperature. Data were

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recorded and processed by a Hitachi D-7000 data acquisition package with ConcertChrom

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software on a microcomputer. Standards of chlorogenic acid and rutin were prepared at 0, 10, 25,


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50, 100 and 200 µg/mLand quantification was done by comparing the area under the

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chromatographic peaks of elderberry samples to the calibration curves. Polyphenols monitored

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were neo-chlorogenic acid, chlorogenic acid, crypto-chlorogenic acid, quercetin 3-rutinoside,

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quercetin 3-glucoside, kaempferol 3-rutinoside, isorhamnetin 3-rutinoside, and isorhamnetin 3-

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glucoside. Comparison of a polyphenol to the chlorogenic acid and rutin standard curves allowed

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for the determination of an average response factor for each compound. The compound are

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reports as rutin/chlorogenic acid equivalents.

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UHPLC-MS/MS Analysis of Anthocyanins. A previously described UHPLC-electrospray

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ionization mass spectrometry/mass spectrometry (UHPLC-ESI-MS/MS) method was used for

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anthocyanin analysis.19 A full analytical method validation was performed including the analysis

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of limit of quantitation, recovery, matrix- effect, linearity, and intra/inter-day reproducibility.

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Cyanidin-3-O-glucoside standards were prepared at 1, 10, 50, 100, 250, 500, and 1,000 ng/mL.

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Quantification was performed using the area under the mass spectral peak for individual multiple

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reaction monitoring ion channels for each anthocyanin and comparing it to a standard curve.

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Individual anthocyanins monitored were cyanidin 3-O-coumaroyl-sambubioside-5-glucoside,

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cyanidin based anthocyanin, cyanidin 3-O-sambubioside-5-glucoside, cyanidin 3-O-coumaroyl-

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sambubioside, cyanidin 3-O-sophoroside, cyanidin 3-O-rutinoside, cyanidin 3-O-sambubioside,

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and cyanidin 3-O-glucoside. Multiple reaction monitoring ion chromatograms of each

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anthocyanin are shown in Figure 1. The individual anthocyanin concentrations are reported as

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cyanidin 3-O-glucoside equivalents. The anthocyanins are identified by their MS/MS spectra and

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are consistent with previously published results3,14,21.

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Discriminant Analysis. Anthocyanin and polyphenol data were normalized to the same

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concentration units and represented as individual aliquots. Discriminant analysis (DA) was



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performed using the XLSTAT data analysis software in Microsoft Excel, version 14.5.3

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(Microsoft, Santa Rosa, CA, USA) at a tolerance of 0.0001, p-value 287.1

785.3 > 287.1

889.4 > 287.1

%

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Figure 1

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Discriminant Analysis Including 2014 Anthocyanin and Polyphenol Data at Mt. Vernon 10 8 6

F2 (43.68 %)

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Adams II

2

Bob Gordon

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Wyldewood

‐2 ‐4 ‐6 ‐8 ‐10 ‐10

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‐6

‐4

‐2

0

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F1 (56.32 %)

Figure 2



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Discriminant Analysis Including 2013 Anthocyanin and Polyphenol Data at Mt. Vernon 12 10 8 6

F2 (30.42 %)

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A

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Adams II

0 ‐2

Bob Gordon

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Wyldewood

‐6 ‐8 ‐10 ‐12 ‐18

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‐12

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F1 (69.58 %)

Discriminant Analysis Including 2014 Anthocyanin and Polyphenol Data at Mountain Grove 20 15

F2 (28.97 %)

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B

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Adams II

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Bob Gordon ‐5

Wyldewood

‐10 ‐15 ‐20 ‐15

‐10

‐5

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F1 (71.03 %)

Figure 3


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TOC graphic 81x27mm (300 x 300 DPI)


Discriminant Analyses of the Polyphenol Content of American

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