Use of Wild Genotypes in Breeding Program Increases Strawberry

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Use of Wild Genotypes in Breeding Program Increases Strawberry Fruit Sensorial and Nutritional Quality Jacopo Diamanti,† Luca Mazzoni,‡ Francesca Balducci,† Roberto Cappelletti,† Franco Capocasa,† Maurizio Battino,‡ Gary Dobson,§ Derek Stewart,§ and Bruno Mezzetti*,† †

Department of Agricultural, Food and Environmental Sciences, Università Politecnica delle Marche, Via Brecce Bianche, 60100 Ancona, Italy ‡ Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche, Università Politecnica delle Marche, Via Ranieri 65, 60131 Ancona, Italy § James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland, United Kingdom S Supporting Information *

ABSTRACT: This study evaluated 20 advanced selections, derived from a strawberry interspecific backcross program, and their parents for fruit weight, commercial yield, acidity, sugar content, antioxidant capacity, and phenol and anthocyanin contents. Phytochemical profiling analysis was performed to determine the compositional characteristics of the improved selections in comparison with their parents and an important commercial variety (‘Elsanta’). Advanced selections showed substantial improvement for agronomic and nutritional quality parameters. From the profiling analysis there was evidence for specific improvements in fruit phytochemical contents; new advanced selections had substantially increased fruit flavonol, anthocyanin, and ellagitannin contents compared to their parent cultivar ‘Romina’ and, for flavonols and ellagitannins, compared to a standard cultivar ‘Elsanta’. Such results confirm that an appropriate breeding program that includes wild strawberry germplasm can produce new strawberry cultivars with a well-defined improvement in fruit nutritional and nutraceutical values. KEYWORDS: breeding, Fragaria virginiana subsp. glauca, anthocyanins, polyphenols, profiling

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quercetin derivatives, followed by kaempferol derivatives.17 These polyphenols have a protective role in reducing the bioavailability of carcinogen agents.18 Flavanols do not occur naturally as glycosides but are found in strawberry as monomers (catechins) or polymers (proanthocyanidins or condensed tannins). Recently, interest in flavanol content in fruits and in their metabolic fate after ingestion has arisen because of the antioxidant, antiallergic, antimicrobial, and antihypertensive properties that have been demonstrated.19 Together with anthocyanins, hydrolyzable tannins, in particular ellagitannins and gallotannins, are the most common phenolic compounds in strawberry.5 The oligomer sanguiin H-6 has been described as the most representative hydrolyzable tannin in strawberry and raspberry.20 Finally, other phenolic compounds, such as phenolic acids, occur in strawberries. These compounds occur in small quantities and are mainly derivatives of hydroxycinnamic acid (caffeic acid) and hydroxybenzoic acid (gallic acid).5,20−23 Ellagic acid in strawberry can occur both in the free form and esterified to glucose, the form in which they occur in hydrolyzable ellagitannins,11 and has anticarcinogenic and antimutagenic activities.24

INTRODUCTION Over the past few decades increasing attention has been paid to the consumption of fruits and vegetables as natural sources of bioactive molecules. In fact, several epidemiological studies have demonstrated the correlation between the dietary consumption of fruits and vegetables and a lower risk of chronic pathologies, such as cancer, cardiovascular and neurodegenerative diseases, obesity, and inflammation.1−4 Among fruits, berries have a high content of phytochemical compounds, and strawberries in particular are a natural source of minerals, vitamins, dietary fibers, and polyphenolic constituents such as phenolic compounds, a broad group of biologically active compounds that have many biological potentialities in vivo and in vitro.5−10 The main class of phenolic compounds in strawberries is represented by the flavonoids, followed by hydrolyzable tannins and finally phenolic acids.5 Anthocyanins, flavonols, and flavanols are the most abundant classes of compounds belonging to flavonoids.11 In strawberry, more than 25 different anthocyanin pigments have been detected, their distribution differing among varieties and selections,12 but the major anthocyanins in cultivated strawberry are pelargonidin-3-glucoside, pelargonidin-3-rutinoside, and cyanidin glucoside. These anthocyanin compounds have radical scavenging activity and have been associated with a decrease in the incidence of cancer, heart disease, and a number of other health benefits.13 The flavonol content and composition in strawberry have been the subject of several studies.14−16 The most abundant flavonols identified are © 2014 American Chemical Society

Special Issue: 2013 Berry Health Benefits Symposium Received: Revised: Accepted: Published: 3944

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Table 1. Families, Corresponding Parents, and Number of Offspring Evaluated for Fruit Sensorial and Nutritional Quality progenitors family

mother

AN07,003 AN07,216

AN94,414,52 AN00,239,55

× ×

father

no. of selections

Romina Romina

11 9

type of crossing F2 backcross F3 backcross

BC2 BC3

fruit with higher contents of phytochemicals combined with interesting sensorial characteristics, together with agronomic performances as required at a commercial level. The phytochemical composition of the genotypes with the most potential, and of their parents used in the intraspecific (cv. ‘Romina’) and interspecific backcrossing (BC) (AN94,414,52 and AN00,239,55), together with the important commercial variety ‘Elsanta’, were further characterized by HPLC-PDA-MS analyses. The prevalent type of phytochemicals relevant to the improvement of fruit nutritional value was determined.

Total antioxidant capacity (TAC) is a very common parameter for the evaluation of antioxidant activity strictly related to the amount of bioactive compounds in strawberry. TAC provides a measure of the radical scavenger activity of the fruit and is therefore a quality parameter related to the healthfulness of the fruit. The antioxidant capacity of strawberries has been shown to be due to the activity of high amounts of antioxidant compounds such as polyphenols and vitamin C5. As demonstrated for fruit sensorial traits, the composition of antioxidant compounds can vary considerably among strawberry cultivars,25−29 so breeding and/or biotechnology have potential for obtaining better tasting and more nutritious fruits.30−32 The breeding process can be successful if the variability and heritability of bioactive compounds is assured for the progenies derived from parental fruits. The biotechnological approach is a methodology that is able to provide a genetic improvement through modification of specific biosynthetic pathways.33 The application of such technology is still mostly limited to functional studies on specific pathways,34 whereas the commercial exploitation of new products is highly limited by public concerns and biosafety rules now standing for the commercial release of new genetically modified products. In any case, a deep knowledge of both the cultivated and wild genetic resources, which may be utilized for genetic and genomic studies, is an essential prerequisite for obtaining new cultivars of high interest for both the fresh market and the processing industry.35 Recently, the possibility to improve the fruit content of such phytochemicals by traditional breeding programs has been demonstrated36 and, to this end, the detailed characterization of the genetic resources to be used in the cross combination was an important component. The inclusion in breeding programs of wild species with a genetic background able to produce progeny that have increased health-related phytochemicals is also important.37 Furthermore, if the nutritional quality of strawberry is enhanced in association with favorable sensorial parameters (such as titratable acidity, sugar content, fruit firmness, and aroma), the consumption of fruit by consumers will be encouraged, leading to strawberries having a positive effect on health. Consequently, the biochemical characterization and the biomedical health validation of new products resulting from new genetic material, characterized by increased content of health-related phytochemicals, is an important issue. With this aim, high competencies in compositional analyses are needed to give a complete profile of the health-related phytochemicals present in the new type of fruit. Such an approach will complement a description of the potential health benefits of the fruit. The aim of this work was to evaluate the fruit sensorial and nutritional quality of 20 selections derived from a strawberry interspecific backcross breeding program. All selections were evaluated for their fruit sensorial (color, firmness, total soluble sugars, and acidity) and nutritional parameters (phenol and anthocyanin contents and total antioxidant capacity) by quantitative spectrometry analysis. Data on nutritional parameters allowed the identification of selections that produce

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MATERIALS AND METHODS

Plant Material. New selections originated from an interspecific backcross program having as common parents the cv. ‘Romina’, the F1 selection AN94,414,52 from the interspecific cross of Fragaria × ananassa (Don) × Fragaria virginiana glauca (FVG), and AN00,239,55, a BC2 selection produced by backcrossing the F1 selection AN94,414,52 with 91,143,5 (F. × ananassa advanced selection) (Table 1) were planted in 2011 in nonfumigated soil at the P. Rosati University experimental farm, Agugliano (Ancona, Italy), and grown in open-field conditions using the plastic hill culture production system. Selections were planted as frigo plants in single plots of six plants each, cultivated with the standard Integrated Pest Management system, then harvested and evaluated in May 2012. In the same field, under the same conditions, the corresponding parents of each cross combination were also grown. Fruit samples of fully red berries were harvested at the second, third, and fourth main pickings. In the evaluation, plant productivity (fruit weight and commercial plant production), fruit sensorial (fruit firmness, solid soluble content, titratable acidity, and color), and nutritional parameters (fruit TAC, phenol content, and anthocyanin content) were taken into account. Fruit sensorial and nutritional quality was determined on fresh (color and firmness) and on frozen (total soluble sugars and acidity) samples. HPLC-PDA-MS was performed on freeze-dried fruit and included the commercial variety ‘Elsanta’, shipped by express mail to James Hutton Institute in the United Kingdom. Chemicals. Methanol 99% ACS-ISO for analysis and bromothimol blue were purchased from Carlo Erba Reagenti (Milano, Italy). Acetonitrile, formic acid, double-distilled water, Folin−Ciocalteu reagent, sodium carbonate anhydrous, potassium chloride, sodium acetate, chloridric acid, glacial acetic acid, dihydrogen potassium phosphate, dipotassium hydrogen phosphate, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+), 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), potassium persulfate, 3,4,5-trihydroxybenzoic acid (gallic acid), and sodium hydroxide were purchased from Sigma-Aldrich (Sigma-Aldrich s.r.l., Milano, Italy). Pelargonidin-3-O-glucoside chloride and kaempferol-3glucoside were purchased from Extrasynthese (Genay, France). Strawberry Production Parameters. In 2012, strawberry plant production was evaluated in the single plot of all selections by measuring commercial production, total weight (g) of ripe fruit with commercial diameter (⌀ ≤ 0.22 mm) and undamaged, and fruit weight, average weight (g), of 20 commercial fruits from each harvest. Strawberry Sensorial Parameters. Fruit sensorial quality of selections and parents was analyzed on the harvesting day, at each of the second, third, and fourth harvests, on 10 commercial fruits by measuring the following parameters: (a) soluble solids (SS), determined using a hand-held refractometer (ATAGO), results expressed as °Brix; (b) titratable acidity (TA), determined from 10 mL of juice diluted with distilled water (1:2 v/v) and titrated with 0.1 N NaOH solution, to pH 8.2, and expressed as milliequivalents 3945

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Figure 1. Principal component analysis: (A, left) variable vector distributions; (B, right) case distributions. Biplot of qualitative (SS, TA, and firmness) and nutritional parameters (TAC, TPH, and ACY) of the selections and the corresponding parents of the interspecies cross populations. Factors 1 and 2 explain 62.77% of the data variation. (mEQ) of NaOH per 100 g of fresh weight (FW); (c) fruit firmness, measured by a penetrometer 327 (Effegi,̀ Ravenna, Italy), results expressed as grams; (d) fruit color, determined by a Minolta Chromameter CR 400, for two faces of 10 ripe, undamaged, and uniform fruits of each harvest. The instruments measured three parameters L* (luminescence), a* (red tone), and b* (yellow tone). Data were elaborated and expressed as chroma index and hue angle. A high chroma index indicated pale fruit and a low chroma index represented dark fruit, whereas a high hue angle indicated yellow fruit and a low hue angle denoted red fruit. Strawberry Nutritional Parameters. Fruit Extraction Method. As described by Diamanti,37 briefly, from a pool of fruit for each genotype, harvested in 2012 and stored at −20 °C, 10 ripe-commercial fruits were sampled and from each fruit two opposite slices were cut and milled into small pieces. Ten grams of this blend was weighed and placed in a tube for extraction with methanol (1:4, fruit/methanol, w/ w), including two subsequent steps. The first step consisted of homogenization, with an Ultraturrax T25 homogenizer, of fruit blend in 20 mL of methanol (Janke and Kunkel, IKA Labortechnik, Staufen, Denmark). The homogenized suspension was continuously agitated for 30 min in the dark. The suspension was centrifuged at 4500g for 10 min (Centrifuge Rotofix32, Hettich Zentrifugen, Tuttlingen, Denmark), and 3 × 1 mL of the supernatant was collected and stored in three amber vials at −20 °C. For a more exhaustive extraction, the pellet of the fruit was extracted a second time by adding another 20 mL of methanol and repeating the previously described procedure. The supernatant was collected and added to the previous ones, and 3 × 1 mL per sample was stored in amber vials at −20 °C. Total Antioxidant Capacity (TAC). Fruit extract of each genotype was used to analyze TAC by using the ABTS assay, according to a previously validated procedure.38,39 ABTS, a chromogen and colorless substance, is changed into its colored monocationic radical form (ABTS•+) by an oxidative agent. Addition of antioxidants reduces ABTS•+ into its colorless form. The extent of decolorization as a percentage of inhibition of ABTS•+ is determined as a function of concentration and calculated relative to the reactivity of Trolox, a water-soluble vitamin E analogue. The experiment was performed in triplicate, and antioxidant activity was expressed as millimoles of Trolox equivalent (TE) per kilogram of fresh pulp weight ± standard error (SE). The calibration was calculated as the linear regression from the dose response Trolox standard. Total Phenol Content (TPH). Fruit TPH was evaluated on fruit extracts by the Folin−Ciocalteu reagent method40 using gallic acid as standard for the calibration curve. Briefly, a test tube (glass) was filled with 7.0 mL of water, then 1 mL of the diluted sample (1:20) was added, followed by the addition of 500 μL of Folin−Ciocalteu reagent,

and the mixture was vortexed. After 3 min, 1.5 mL of sodium carbonate (0.53 mol/L) was added, and the tube was mixed again and then stored in the dark for 60 min. The absorbance of the sample was measured at 760 nm after exactly 60 min. Results were calculated and expressed as milligrams of gallic acid per kilogram of fresh fruit ± SE on three replications, by using a standard curve with a range of values of standards of 10−50 mg of gallic acid (GA)/L. Anthocyanin Content (ACY). Fruit total ACY was measured on fruit extracts by using the pH differential shift method.41 The assay is based on the anthocyanins’ property to change intensity of hue depending on pH shifting. The samples were diluted to a 1:10 ratio with potassium chloride (pH 1.00) and with sodium acetate (pH 4.50), and then the corresponding maximum absorbance for both solutions was measured, respectively, at λ = 500 nm and λ = 700 nm. Analyses were performed in triplicate, and results were expressed as milligrams of pelargonidin-3-glucoside (the most representative anthocyanin compound in strawberry) per kilogram of fresh weight (mg of Pel-3-Glu/kg FW) ± SE. Analysis of Polyphenolics by HPLC-PDA-MS. Sample Preparation. Strawberry samples were frozen at harvest and then freezedried by using a Freezone (Labconco) freeze-drying system. Samples were stored in a −20 °C freezer until analysis. Prior to use, doubledistilled water (2 mL) and 0.5% formic acid in acetonitrile (2 mL) were added to the sample, (500 mg) in a 15 mL polypropylene centrifuge tube. Samples were shaken on an orbital shaker for 30 min and centrifuged at 4000 rpm for 5 min, and the supernatant was transferred to another 15 mL centrifuge tube. The extraction was repeated with water (1 mL) and 0.5% formic acid in acetonitrile (1 mL) and the supernatant was added to the first one. After centrifugation at 13200 rpm for 5 min, 0.5 mL aliquots were transferred to microfuge tubes and stored in a −20 °C freezer. The solvent was removed from the sample at 45 °C using a Speed Vac, completely dried, using a freeze-drier, and stored in a −20 °C freezer until analysis. Prior to analysis, the sample was dissolved in 475 μL of 0.5% formic acid in water/acetonitrile (70:30, v/v), 25 μL of morin internal standard (1 mg mL−1 in methanol) was added, and, following vortexing and centrifugation at 13200 rpm for 3 min, 0.4 mL was transferred to a 0.45 μm filter vial. The sample was injected via an autosampler at 6 °C onto a HPLC column Synergi 4 μ Hydro-RP 80A (150 mm × 2.0 mm; 4 μm reversed-phase HPLC column, Phenomenex) at 30 °C, linked to a PDA system (Thermo Accela) and then an electrospray ionization ion trap mass/Fourier transform spectrometer (Thermo LTQ Orbitrap XL). The mobile phase was a gradient with (A) 0.1% (v/v) aqueous formic acid and (B) 0.1% (v/v) formic acid in acetonitrile/water (50:50, v/v), the flow rate was 300 μL min−1, and the gradient was as follows: 0−4 min, 95% A; 4−28 min, 3946

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3947

BC2 BC2 BC2 BC2 BC2 BC2 BC2 BC2 BC2 BC2 BC2 BC3 BC3 BC3 BC3 BC3 BC3 BC3 BC3 BC3 BC2 parent BC3 parent F. × a parent

AN07,003,51 AN07,003,52 AN07,003,53 AN07,003,54 AN07,003,55 AN07,003,56 AN07,003,57 AN07,003,58 AN07,003,59 AN07,003,60 AN07,003,61 AN07,216,52 AN07,216,53 AN07,216,54 AN07,216,55 AN07,216,56 AN07,216,57 AN07,216,58 AN07,216,60 AN07,216,61 AN94,414,52 AN00,239,55 Romina

10.2 9.2 9.7 10.4 11.1 10.9 9.6 10.3 11.3 11.7 9.9 8.2 7.1 10.6 9.9 7.3 8.6 9.1 8.2 8.2 10.1 11.1 7.5

abcde bcdef abcde abcd abc abc abcde abcde ab a abcde efg g abcd abcde fg defg cdefg efg efg abcde abc fg

soluble solidsb 12.7 9.3 8.5 10.0 8.4 10.6 10.4 8.7 10.8 8.1 11.3 9.6 10.3 13.9 14.8 10.1 10.6 10.2 16.2 9.5 15.6 20.8 10.2

cd de e de e de de e de e de de de bc bc de de de b de b a de

titratable acidityc 361 295 373 419 370 363 421 315 394 437 395 342 446 370 398 340 358 327 417 383 366 363 520

cdefgh h bcdefg bcde cdefg cdefgh bcd gh bcdef bc bcdef defgh b cdefgh bcdef efgh cdefgh fgh bcde bcdefg cdefgh cdefgh a

firmnessd 16.0 18.7 13.0 16.0 19.2 15.7 17.1 18.9 17.6 22.4 14.7 16.9 22.4 18.0 16.6 17.9 20.0 21.1 21.2 16.1 29.9 20.6 13.2

ghil defg il ghil bdefg ghi fgh defg efgh c hil fgh bc defgh fgh defgh bcdef bcd bcd ghil a bcde l

TACe 2352 2611 2082 2418 2732 2338 2498 2243 2297 2992 2180 1584 1583 1381 1549 1696 1757 1834 1991 1718 2015 1937 1396

TPHf cde bc efgh cd b cde bcd defg def a defg lm lm m lm il il hil fghi il fghi ghi m

307 389 258 234 383 238 533 350 377 499 291 470 468 442 567 398 539 380 819 419 513 600 274

ACYg m i op p i p d l i e mn f f g c hi d i a gh de b no

33.9 35.5 37.5 40.3 36.1 40.2 34.8 37.2 35.7 32.3 36.6 31.4 32.5 32.1 36.4 34.6 34.5 35.5 33.0 32.4 33.2 36.8 34.9

L* cdefg bcdef b a bcde a bcdefg bc bcdef fg bcd g fg fg bcde bcdefg bcdefg bcdef efg fg defg bc bcdef

47.7 49.5 52.5 50.2 47.2 51.7 40.4 50.5 47.4 45.5 47.7 39.3 38.9 40.0 42.7 39.7 44.0 44.5 37.9 39.5 44.7 43.7 45.1

abcd abc a ab abcd a efghi ab abcd bcde abcd ghi hi fghi defghi ghi cdefgh cdefg i ghi cdefg defgh bcdef

chroma 29.0 29.5 32.7 35.5 33.0 35.6 29.0 31.9 32.0 27.1 30.4 25.6 29.2 23.6 31.2 30.6 28.4 30.6 28.1 25.2 29.3 32.5 29.7

h° bcd bcd ab a ab a bcd ab ab cde bc de bcd e bc bc bcd bc bcd de bcd ab bcd

a Results are expressed as means. Data followed by different letters are significantly different. SNK test P < 0.05. b°Brix. cmequiv NaOH/100 g. dg. emmol TE/kg FW. fmg GA/kg FW. gmg pel-3-glu/kg FW.

backcross generation

genotype

Table 2. Qualitative, Quantitative, and Color Analysis of Selections and Respective Parentsa

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50% A; 28−32 min, 0% A; 32−34 min, 0% A; 34−36 min, 95% A; 36− 45 min, 95% A. The PDA detector range was 200−600 nm, and channels were (A) 280 nm (general polyphenols), (B) 365 nm (flavonols), and (C) 520 nm (anthocyanins). Mass spectrometer mass range was m/z 120−2000 with alternative full scan MS and MS/MS (data-dependent scan). The sample was run twice, in positive ion and negative ion modes. Identification of polyphenols was based on PDA characteristics and mass spectra including accurate MS and MS/MS data, and comparison to the PDA, MS, and elution times of authentic standards when available. Quantification of flavonols and anthocyanins was based on the area of [M − 1]− and [M + 1]+ ions, respectively, using calibration curves generated for standards of kaempferol-3-Oglucoside and pelargonidin-3-O-glucoside, respectively. Relative levels of ellagitannins and flavan-3-ols were based on areas of [M − 1]− ions, assuming that different compounds within each class had similar response factors. Experimental Trial and Statistical Analysis. Fruit sensorial (SS, TA, fruit firmness, fruit color) and nutritional (TAC, TPH, ACY) parameters were analyzed in triplicate for each selection and for their corresponding parents. All of these data were subjected to the two-way nested analysis, the MANOVA test. The analysis of variance was performed for genotype as random effects. The differences were calculated according to the SNK test and were considered significant at p ≤ 0.05. To evaluate also the association among the fruit qualitative and nutritional parameters of the samples and their respective parents, PCA was used. As described by Diamanti et al.,37 factor loading values ≥0.7 were used to identify the most important variables and observations in each dimension (PC). Variables and observations (Figure 1) that are closest to each other in the same geometric plane of the biplot are considered as interrelated, and therefore they are distant from those to which they are negatively related or not related. The greater the distance of a vector from the origin of the axis, the higher the correlation of the variable with the PC represented in that dimension. All analyses were performed using STATISTICA software (Statsoft Inc., Tulsa, OK, USA).

2). The BC3 selections AN07,216,60 and AN07,216,55 also had high values, intermediate between the two parental lines ‘Romina’ and AN00,239,55. Two other selections, AN07,003,59 and AN07,216,57, had lower values with respect to their respective “mothers” (AN94,414,52 and AN00,239,55 respectively), but similar values with respect to their “father” ‘Romina’. It is interesting to note that all of the selections showed lower values of titratable acidity compared with their female parents. The lowest values were registered for selections AN07,003,55, AN07,003,53, AN07,003,58, and AN07,003,60. ‘Romina’ fruit had the highest value of firmness; none of the new selections were able to reach it, AN07,003,52 having the lowest value (Table 2). The selection that reached the closest value to that of ‘Romina’ was AN07,216,53 followed by AN07,216,60, from the BC3 family. With regard to color, L*, chroma index, and hue angle were evaluated for all of the selections and parents (Table 2). Interestingly, the AN07,003 family (BC2) generally showed the highest values for L*, chroma index, and hue angle. In particular, fruit of selection AN07,003,53 possessed the highest values for chroma index, whereas AN07,003,56 and AN07,003,54 had the highest hue angle and L* value, respectively. In contrast, fruits of BC3 (AN07,216 family) generally had the lowest values for L* (AN07,216,52), chroma index (AN07,216,60), and hue angle (AN07,216,54). AN00,239,55 had the highest L* value among parents, whereas AN94,414,52 had the lowest. ‘Romina’ had average values for all of the color parameters analyzed. Interestingly, the AN07,003 family (BC2) showed a higher average value of all color parameters compared to both parents. In contrast, the AN07,216 family (BC3) had lower values of all color parameters compared to its parents. Fruit Nutritional Quantitative Analysis. The quantitative analysis performed to evaluate the nutritional quality of backcross genotypes involved in the study have highlighted main differences among them, specifically between parents and progenies, thus allowing the selection of those genotypes that had shown the highest improvements for each parameter. Phenol content of strawberry genotypes revealed an improvement in all of the progenies of second-generation backcrossing in comparison with their parent ‘Romina’, whereas some genotypes revealed also an improvement with respect to the other parent AN94,414,52. On the contrary, there was a decline in phenol content in most genotypes of third backcross generation in comparison to the BC3 parent AN00,239,55, but most had higher values than the parent ‘Romina’ (Table 2). The most elevated values were shown by the AN07,003 family and in particular by selections AN07,003,60 and AN07,003,55. The results of ACY content revealed that only one genotype (AN07,216,60) showed an improvement in ACY content compared to its wild parent (AN00,239,55), whereas there was a decrease in ACY in most of the progenies, but at least the majority of the genotypes showed higher values than the parent ‘Romina’, as shown in Table 2. Selections that revealed the highest ACY content were AN07,216,60, AN07,216,55, and AN07,216,57 from BC2 and AN07,003,57 and AN07,003,60 from BC3. TAC analysis revealed modest increased values of almost all progenies of both backcrosses in comparison with the parent ‘Romina’ (Table 2), whereas compared to the F1 parent AN94,414,52 there was a decrease in the BC2 progeny and compared to the BC3 parent AN00,239,55 there was no significant improvement in BC3 progenies. The selections

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RESULTS AND DISCUSSION Commercial Production and Fruit Weight. All 20 selections had a low commercial production and fruit weight due to their backcross origin (BC2 and BC3). Selections of both backcrosses (AN07,003 and AN07,216 families) showed intermediate commercial production values with respect to their parents; values were higher than those of their wild parents AN94,414,52-F1 and AN00,239,55-BC1 (selections of AN07,003 family produced 422 g/plants against 92 g/plants for their AN94,414,52 parent; selections of AN07,216 family produced 484 g/plants against 188 g/plant for their AN00,239,55 parent), but lower than those of ‘Romina’, the only commercial variety. Furthermore, fruit weight values were lower for all of the selections and the wild parents (15.2 g for BC3 selections AN07,216, 11.2 g for their wild parent AN00,239,55, 11.2 g for BC2 selections of AN07,003 family, and 11.5 g for their wild parent AN94,414,52), compared with ‘Romina’ (21 g). Strawberry Sensorial Parameters. Fruit of selections AN07,003,60 and AN07,003,59 had very high soluble solids values (Table 2); in particular, they were significantly higher than the parent ‘Romina’. The lowest values of soluble solids were detected in AN07,216,53, AN07,216,56 and ‘Romina’, and in general fruit of all selections of the AN07,216 family presented lower values than AN07,003 selections. The parent AN00,239,55 had the highest value of titratable acidity, and that of the parent AN94,414,52 was also high, both having higher values than ‘Romina’, which possessed a medium value (Table 3948

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Table 3. Characterization of Polyphenols in Selections, the Respective Parents (Except AN94,414,52) and the Commercial Cultivar ‘Elsanta’ by HPLC-PDA-MS Romina flavonols (mg 100 g−1 FDW) quercetin glucuronide 34.9 quercetin hexoside 2.5 methylquercetin glucuronide 1.1 kaempferol glucoside 3.0 kaempferol glucuronide 7.7 kaempferol acetylglucoside 0.7 kaempferol coumaroyl hexoside 1 7.1 kaempferol coumaroyl hexoside 2 1.2 kaempferol rutinoside 0.5 total 58.8 anthocyanins (mg 100 g−1 FDW) pelargonidin glucoside 156.5 pelargonidin rutinoside 8.8 pelargonidin malonylglucoside 54.6 pelargonidin acetylglucoside 3.7 cyanidin glucoside 15.4 cyanidin rutinoside 0.3 total 239.4 ellagic acid conjugates (mg 100 g−1 FDW) ellagic acid 7.0 ellagic hexoside 1.0 ellagic deoxyhexoside 1 8.7 ellagic deoxyhexoside 2 1.8 methylellagic deoxyhexoside 0.0 total 18.5 ellagitannins (relative levels) bis-HHDP-hexosidea 0.32 HHDP-galloyl-hexoside 0.17 galloyl-bis-HHDP-hexosideb 0.48 sanguiin H6 0.15 Ac 0.63 Bd 0.07 total 1.81 flavan-3-ols (relative levels) catechin 0.35 proanthocyanidin dimerse 0.92 proanthocyanidin trimersf 0.39 total 1.67

AN00,239,55

AN07,003,59

AN07,216,57

AN07,216,60

Elsanta

41.1 2.4 1.8 6.9 13.7 0.2 7.4 1.2 1.4 76.2

37.6 2.2 2.1 4.9 11.8 0.3 9.1 1.8 0.9 70.8

35.0 2.5 2.4 4.3 10.0 1.1 4.9 1.0 0.0 61.1

33.9 2.4 1.9 4.2 14.1 0.8 5.7 0.8 0.5 64.4

2.0 0.4 0.1 6.6 3.7 3.4 14.1 2.9 0.0 33.3

366.1 29.3 3.2 2.9 20.8 0.0 422.4

282.8 19.7 5.9 1.2 36.9 0.6 347.1

284.3 0.0 108.1 4.7 22.6 0.0 419.6

331.9 16.3 97.7 0.1 29.6 0.4 476.0

303.6 0.0 147.0 0.1 5.9 0.0 456.6

4.4 1.8 8.1 2.8 0.0 17.1

6.8 1.2 6.8 2.2 13.0 30.0

4.0 0.5 9.0 1.9 0.0 15.3

4.5 1.3 8.1 1.6 0.0 15.4

4.7 0.4 6.4 1.1 0.0 12.7

0.56 0.40 1.00 0.23 0.58 0.02 2.80

0.46 0.33 0.75 0.26 0.62 0.04 2.46

0.46 0.35 0.86 0.21 0.61 0.05 2.54

0.25 0.11 0.26 0.10 0.51 0.02 1.26

0.10 0.04 0.05 0.03 0.60 0.02 0.84

0.24 0.61 0.26 1.12

0.20 0.54 0.25 0.99

0.26 0.63 0.29 1.18

0.23 0.55 0.24 1.02

0.37 1.00 0.47 1.84

a Sum of four bis-HHDP-hexoside peaks. bSum of two galloyl-bis-HHDP-hexoside peaks. cEllagitannin with a mass spectrum similar to that of sanguiin H6. dEllagitannin with a mass spectrum similar to that of lambertianin C. eSum of five dimer peaks. fSum of five trimer peaks.

AN07,003,60, AN07,216,53, AN07,216,58, and AN07,216,60 had the highest TAC values. Fruits of selection AN07,003,60 also had the highest TPH value, whereas AN07,216,60 had the highest TPH value of all the BC3 selections and the highest ACY value of all genotypes. PCA. In the PCA biplot for fruit nutritional parameters of TAC, TPH, and ACY, combined with qualitative parameters of SS, TA, and firmness (Figure 1A), the selections and their parents are distributed in the four quadrants of the plot (Figure 1B). The female parent AN00,239,55 is located on the righthand side of the plot, near the ACY vector. The other female parent, AN94,414,52 was located slightly higher in the righthand side of the plot, near the ACY and TAC vectors. Finally, the common male parent ‘Romina’ is located on the lower left part of the plot, far from all of the vectors. The AN07,003 selections were mainly positioned in the central left-hand area of the plot, not far from the TPH vector

location. The only exception was AN07,003,60, which is located in the upper slightly right-hand side of the graph, where the firmness vector is located (Figure 1B). The selections of the AN07,216 family are mainly located in the lower right-hand quadrant (Figure 1B), where the SS vector is placed (Figure 1A). However, fruits of AN07,216,57 and AN07,216,60 were located in the upper right quadrant, slightly higher than the other AN07,216 selections, where the TAC and ACY vectors are also located. On the basis of the PCA, the three best selections were chosen for further characterization by HPLC-PDA-MS: AN07,003,59 BC2, which was characterized by medium TAC, TPH, and ACY values; AN07,216,57 BC3, which presented medium TAC value, medium/low TPH value, and high ACY; and AN07,216,60 BC3, which had the highest ACY and also one of the highest TAC and TPH values among the BC3 progenies. 3949

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Polyphenolic Composition by HPLC-PDA-MS. The phenolic compositional analyses of flavonols, anthocyanins, ellagic acid conjugates, ellagitannins, and flavan-3-ols (catechin and proanthocyanidins) were performed on the selected selections. Fruits of the two parents, AN00,239,55 and cv. ‘Romina’, and of ‘Elsanta’, a widely available commercial variety, were also profiled. The results (Table 3) showed that the BC2 parent AN00,239,55 and the BC2 selection AN07,003,59 contained the highest content of total flavonols, 2-fold higher than that of the commercial cultivar ‘Elsanta’. The levels in both BC3 selections were not enhanced compared to the parents. The flavonol profile was similar for all genotypes with the exception of ‘Elsanta’. Quercetin glucuronide was the main compound present in all genotypes except for ‘Elsanta’, in which the levels were up to about 20-fold less. Kaempferol glucuronide, the second main flavonol compound in strawberry, was higher in the selections and the parent AN00,239,55 than in the parent ‘Romina’ and much higher than in ‘Elsanta’. The highest content of kaempferol coumaroyl hexoside 1 was found in fruit of ‘Elsanta’ and then BC2 AN07,003,59. Another kaempferol coumaroyl hexoside was at lower levels and showed a distribution similar to that of the major component. Kaempferol glucoside content was highest in fruit of BC3 parent AN00,239,55 and ‘Elsanta’, but was not enhanced in the progeny and was particularly low in ‘Romina’. Quercetin hexoside and methylquercetin glucoronide were of low abundance, and there were only minor variations among genotypes, although they were at even lower levels in ‘Elsanta’. Kaempferol acetylglucoside was highest in ‘Elsanta’, but was lowest in fruit of AN00,239,55 and of BC2 AN07,003,59. Kaempferol rutinoside was most abundant in fruit of AN00,239,55, whereas it was not detected in AN07,216,57 and ‘Elsanta’. ACY profiling revealed marked differences in anthocyanin content, in both anthocyanins quantity and quality. The total ACY (Table 3) followed the same trend as the values obtained from the spectrophotometric assay (Table 2). The levels in the selections were higher than in ‘Romina’ but not the parent AN00,239,55 or ‘Elsanta’. Pelargonidin glucoside, the major anthocyanin, was about twice as abundant in the interspecific BC genotypes compared to ‘Romina’ and ‘Elsanta’. Interestingly, pelargonidin malonylglucoside was observed in high amounts in fruit of ‘Elsanta’ and both BC3 selections AN07,216,57 and AN07,216,60, followed by ‘Romina’, whereas very low levels were detected in AN00,239,55 (BC3 parent) and BC2 AN07,003,59; it was enhanced in both BC selections compared to both parents. Cyanidin glucoside was detected in highest amounts in BC2 and then in BC3 selections, whereas the lowest value was detected in fruit of ‘Elsanta’. Pelargonidin rutinoside was present in AN00,239,55, BC2 AN07,003,59, and BC3 AN07,216,60, and a small amount was found also in ‘Romina’, whereas it was not detected in the other BC3 selection or in ‘Elsanta’. Pelargonidin acetyl-glucoside was a minor anthocyanin in fruits of AN00,239,55, AN07,216,57, and ‘Romina’, followed by AN07,003,59, whereas only a very low concentration was detected in AN07,216,60 and ‘Elsanta’. Finally, cyanidin rutinoside was detected, at very low levels, only in fruits of AN07,003,59, AN07,216,60, and ‘Romina’. The pattern of ellagic acid conjugates was similar for all genotypes, with the exception of BC2 selection AN07,003,59, which was the only genotype to contain a methylellagic deoxyhexoside (Table 3). Ellagic deoxyhexoside 1 was the most prevalent

compound in the majority of genotypes, but there were no marked differences in the levels among genotypes. Ellagic acid was also a major component, and the highest values were evident for ‘Romina’ and BC2 AN07,003,59. Another ellagic deoxyhexoside and an ellagic hexoside were detected at lower levels in all genotypes. Ellagitannins were highest in AN00,239,55, AN07,003,59, and AN07,216,57 and lowest in ‘Elsanta’. The levels were enhanced in the BC2 selection (AN07,003,59) and one BC3 selection (AN07,216,57) compared to the parent ‘Romina’ but not the BC3 parent AN00,239,55. Galloyl-bis-HHDP-hexoside and unidentified ellagitannin A (with a mass spectrum similar to that of sanguiin H6) were the major components, followed by bis-HHDP-hexoside, in all genotypes except ‘Elsanta’, where ellagitannin A predominated. Among the flavan-3-ols, catechin and proanthocyanin dimers and trimers were identified, and the dimers predominated. Larger proanthocyanidin compounds were detected but they were not characterized in this study. The levels were highest in ‘Romina’ and ‘Elsanta’ and were at similar levels in the BC2 and BC3 selections compared to the BC3 parent. Most studies on strawberry genetic improvement have focused their attention on the evaluation and amelioration of agronomical and productive parameters. Only recently has attention shifted increasingly to the development of more healthful fruits, with emphasis on sensorial and nutritional characteristics. With this aim, a detailed study on the content of fruit bioactive compounds is a crucial aspect in evaluating new genetic material, because the profiling of at least the most important phytochemicals present in the fruit can provide a better estimation of the health-promoting potential of new future commercial material. The utilization of wild strawberries as a source of genetic variability for the improvement of nutritional value is being demonstrated as an important breeding strategy.37 A problem with such wild germplasm is the lack of other important commercial attributes with respect to fruit yield, size, and firmness. In fact, several studies have recently demonstrated that breeding programs based on the use of wild and cultivated strawberries led to the achievement of selections with better nutritional qualities compared to the existing cultivated strawberry,42 but then a long-term backcross program is needed to associate the improved nutritional traits with the commercially important agronomic and sensorial traits. Contemporary strawberry breeding programs, with the specific focus on improving strawberry nutritional quality, are using TPH, ACY, and TAC as preliminary screening methods. Then, more detailed compositional analyses are used to better characterize the phytochemicals that are responsible for the potential health benefits of the more interesting new genotypes. In the present study such an approach was taken to evaluate progenies from two backcross generations (BC2 and BC3) of an interspecific strawberry breeding program. The commercial production and the average fruit weight of all selections and their respective parents were the first parameters to be analyzed. It is interesting that for commercial production, the F. × ananassa parent ‘Romina’ showed a very high value compared to the other wild-derived parents and to all BC2 and BC3 selections. However, the breeding program has generated selections that, even if they possess lower values than ‘Romina’, have an average commercial production much higher than their wild parents. Also, for average fruit weight (see the Supporting Information), ‘Romina’ had the highest value, whereas all other genotypes had lower values. Indeed, the 3950

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values for TAC and ACY were shown by the wild-derived parents AN94,414,52 and AN00,239,55. As a consequence, average TAC and ACY values of selections AN07,003 and AN07,216 were statistically lower than those of their respective wild parents, but significantly higher compared to their “father” ‘Romina’. Furthermore, the TPH values of AN07,003 (BC2) selections and AN07,216 (BC3) selections were significantly higher than those of ‘Romina’, and that of AN07,003 was even higher than that of its “mother” AN94,414,52. The PCA biplot analysis for nutritional and qualitative parameters clearly showed the distributions of the BC2 and BC3 selections and of their respective parents. Selections of the AN07,003 family were mainly located in the left upper quadrant (Figure 1B), where the TPH vector is located (Figure 1A). In fact, all of the higher values of TPH were registered for selections belonging to the AN07,003 family (Table 2). On the contrary, selections of the AN07,216 family are mainly located in the right side of the blot (Figure 1B), where ACY and TAC vectors are located (Figure 1A). This is in accordance with the values in Table 2, where selections of the AN07,216 family possessed higher values for both of these parameters. On the basis of the results of the nutritional quantitative analysis, one BC2 and two BC3 selections, together with ‘Romina’, AN00,239,55, and the commercial cultivar ‘Elsanta’, were further analyzed by HPLC-PDA-MS for polyphenolic characterization and quantification. For flavonols content, all selected genotypes showed a modest increase in total flavonols compared to the “father” ‘Romina’ and much higher compared to ‘Elsanta’. This finding represented a crucial result in this study, because it demonstrated that the interspecific crossing of cultivated strawberry with a wild-derived selection such as AN00,239,55, which has a high concentration of flavonols, generated BC2 (AN07,003,59) and BC3 (AN07,216,60) progeny with flavonol levels higher than the cultivated parent ‘Romina’ while retaining the overall pattern of individual flavonols, which was also similar to that of the wild parent (but different from that of Elsanta). The enhancement of flavonols in AN07,003,59 was mainly due to increases in quercetin glucuronide, kaempferol glucuronide, and a kaempferol coumaroyl hexoside, whereas in AN07,216,60 kaempferol glucuronide alone was mainly responsible. For the anthocyanins, the effect of breeding is even more evident than what was observed for flavonols. The total anthocyanin content of selections was up to 2-fold higher (highest was in AN07,216,60) than in ‘Romina’. In BC2 selections (AN07,216,57 and AN07,216,60) the increase was mainly due to the two major anthocyanins, pelargonidin malonylglucoside and pelargonidin glucoside, whereas in the BC3 selection (AN07,003,59) only pelargonidin glucoside was the major contributor. Cyanidin glucoside was increased in all selections by up to 2-fold, and likewise pelargonidin rutinoside was enhanced in all selections, except AN07,216,57 where it was absent, compared to ‘Romina’, and to an even greater extent compared to ‘Elsanta’. It is interesting that the anthocyanin profile of ‘Romina’ was retained in the BC3 selections with discrepancies only in minor anthocyanins (pelargonidin rutinoside and pelargonidin acetylglucoside were either absent or at trace levels in AN07,216,57 and AN07,216,60, respectively), but the BC2 selection was notable in the low level of pelargonidin malonylglucoside. The level of ellagic acid conjugates in the BC3 selections was not increased compared to the parents, although the overall profile of individual compounds was retained. Increases for the BC2 selection

two backcross generations showed a breeding progress comparable with their corresponding parents. In fact, BC2 selections (AN07,003) had higher average production values than their parent AN94,414,52, and the BC3 selections (AN07,216) possessed average higher value compared to their parent AN00,239,55. Such results indicate an improvement in production parameters from the second to the third backcross generation, but to achieve the common commercial standards at least a further backcross generation is needed. On the contrary, fruit from the BC2 and BC3 generations had, in general, higher values for sensorial quality parameters compared with the commercial parent. In fact, for soluble solids, ‘Romina’ had the lowest value (7.5 °Brix) compared to the wild parents and the majority of selections. Interestingly, the backcross selection BC2 AN07,003, derived from ‘Romina’ and AN94,414,52, showed a higher value (10.4 °Brix) compared to both ‘Romina’ and AN94,414,52 (10.1 °Brix, statistically different only from ‘Romina’). Conversely, the BC3 AN07,216 selections had a much lower value (8.6 °Brix) than their parent AN00,239,55 (11.1 °Brix), but at least slightly higher than that of ‘Romina’. These data indicate that the backcrossing program can be associated with a loss of fruit sugar contents that had previously been improved in the parents. With regard to the titratable acidity, the wild parents had very high values compared to the majority of selections and ‘Romina’. Fruit from most BC2 and BC3 selections did not show significant differences compared to ‘Romina’, but in some cases, such as for fruit of AN07,216,54, AN07,216,55, and AN07,216,60, titratable acidity is significantly higher than that of ‘Romina’, indicating that the interspecific cross of this cultivar with the wild selections improved the titratable acidity of the BC3 selection families (AN07,216). Fruit firmness was improved in both BC2 and BC3 selections compared to ‘Romina’. Although ‘Romina’ had a very high value (520 g), the interspecific cross with wild genotypes produced BC2 (377 g for AN07,003) and BC3 (376 g for AN07,216) selections with relatively low values that were generally very similar and not statistically different from their respective wild parents (363 g for AN00,239,55 and 366 g for AN94,414,52); the exception was AN07,216,53, which had a statistically higher value than the wild parent. With regard to color, the BC2 AN07,003 family had the highest values for all the parameters evaluated; in particular, AN07,003,53 for chroma index (52.5), AN07,003,54 for L* (40.3), and AN07,003,56 for hue angle (35.6). In this case, the backcrossing of wild and cultivated germplasm generated progeny that showed much higher values of all the color parameters compared to both parents, closer to the red brilliant type of color requested by the market for fresh strawberries. The situation is completely different for the BC3 family AN07,216; in fact, AN07,216,60 showed the lowest values of chroma index (37.9), AN07,216,52 the lowest value of L* (31.4), and AN07,216,54 the lowest value of hue angle (23.6). For this family it is clear that the breeding program did not improve the color of the progeny; indeed, the average values of AN07,216 family were lower than in both parents (‘Romina’ and AN00,239,55) for all color parameters. The nutritional quantitative analysis performed on parents and selections demonstrated well the effect of genetic improvement, through the utilization of wild germplasm. In fact, for TAC, TPH, and ACY, the lowest values were always detected in fruit of ‘Romina’. On the contrary, the highest 3951

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Funding

AN07,003,59 were due to the unexpected high level of methylellagic deoxyhexoside, an ellagic acid conjugate totally absent in all other selections and parents, that probably originated from FVG but could be easily lost in backcrossing considering that it had already been lost in fruit of BC3 selections. Ellagitannins were enhanced in AN07,003,59 and one of the BC3 selections (AN07,216,57) compared to ‘Romina’, but the overall profile was maintained. Flavan-3-ol levels were not increased in the selections compared to ‘Romina’. The approach used for breeding strawberry nutritional quality is appropriate for (a) screening large populations by using spectrophotometric parameters (TPH, ACY, and TAC) and (b) characterizing the phytochemical content of the new selections chosen from those with the highest spectrophotometric values. With such an approach it is possible to concentrate effort on the characterization of fruit phytochemical content of only the more interesting selections. Wild FVG can make an important contribution to improving strawberry fruit content of important health-related phytochemicals combined with high values of sensorial traits. However, a well-defined backcross program is needed to conserve such improvements in fruit nutritional quality while recovering the other agronomic traits (in particular yield, size, and firmness) that are lost when the Fragaria × ananassa × FVG interspecific cross is performed. The HPLC-PDA-MS compositional study performed on the three BC selections enabled the characterization of bioactive compounds responsible for the improvement of the nutritional value of their fruit. They all had increased levels of flavonols and anthocyanins, two selections had enhanced ellagitannin contents, and the BC2 selection had increased content of ellagic acid conjugates (due to methylellagic deoxyhexoside, detected only in this selection), compared to the cultivated parent, ‘Romina’. Furthermore, it is interesting to note that at least the contents of total flavonols and ellagitannins in all selected selections and respective parents are higher compared to the commercial variety ‘Elsanta’. The knowledge now available for the three new selections, intensely characterized for their fruit compositional content, will be used to better define their use as breeding lines in future breeding programs or even for the release of the best ones as commercial varieties. In conclusion, it has been demonstrated that the progenies obtained from two halfsubcross combinations of cultivated strawberry ‘Romina’ and wild-derived selections AN94,414,52 and AN00,239,55 have productive, sensorial, and, in particular, nutritional qualities, and it is encouraging that breeding programs can be utilized to ameliorate the characteristics of such fruits. In particular, the breeding of cultivated with wild genotypes has generated progenies with a commercial production much higher than the wild parents, but with sensorial, nutritional characteristics, and polyphenolic content much higher than those of the cultivated cultivar.

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The research work was carried out thanks to the support of EU FP7 EUBerry Project 265942. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED TAC, total antioxidant capacity; BC, backcrossing; SS, soluble solids; TA, titratable acidity; FW, fresh weight; TPH, total phenol content; ACY, anthocyanin content; FVG, Fragaria virginiana glauca

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ASSOCIATED CONTENT

S Supporting Information *

Table S-1. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(B.M.) Phone: +39 071 220 4933. Fax: +39 071 220 4685. Email: [email protected]. 3952

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dx.doi.org/10.1021/jf500708x | J. Agric. Food Chem. 2014, 62, 3944−3953