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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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

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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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Journal of Agricultural and Food Chemistry

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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611.1 > 287.1

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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

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Bob Gordon

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Wyldewood

‐2 ‐4 ‐6 ‐8 ‐10 ‐10

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‐6

‐4

‐2

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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

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Bob Gordon

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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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Wyldewood

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F1 (71.03 %)

 

  Figure 3  

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TOC graphic 81x27mm (300 x 300 DPI)

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