Article pubs.acs.org/JAFC
Genetic Diversity among Mandarins in Fruit-Quality Traits Livnat Goldenberg,†,§ Yossi Yaniv,‡ Tatiana Kaplunov,† Adi Doron-Faigenboim,‡ Ron Porat,*,† and Nir Carmi‡ †
Department of Postharvest Science of Fresh Produce, ARO, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel Department of Fruit Tree Crops, ARO, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel § Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel ‡
ABSTRACT: A detailed phenotypic analysis of fruit-quality traits was conducted among 46 mandarin varieties within the Israeli Citrus breeding collection, belonging to genetically different natural subgroups, including common mandarin (C. reticulata Blanco), clementine (C. clementina Hort. ex. Tan), satsuma (C. unshiu Marcovitch), Mediterranean mandarin (C. deliciosa Tenore), King mandarin (C. nobilis Loureiro), and mandarin hybrids, such as tangor (C. reticulata × C. sinensis) and tangelo (C. reticulata × C. paradisi). Evaluated qualities included physical attributes (size, shape, color, peel thickness, and seed number); physiological properties (ripening period, peelability, and segmentation); nutritional and biochemical composition (vitamin C, phenol, flavonoid, and carotenoid contents and total antioxidant activity); and sensory attributes (total soluble solids and acid levels, flavor preference, sweetness, sourness, and fruitiness). The results indicated wide genetic variability in fruit-quality traits among mandarin varieties and natural subgroups, and statistical and hierarchical clustering analysis revealed multiple correlations among attributes. Such phenomic analysis is an obligatory requirement for identification of molecular markers for distinct fruitquality traits and for selection of appropriate parents for future breeding programs. KEYWORDS: citrus, flavor, fruit quality, mandarin, nutritional quality, phenomics
■
of genomic and phenotypic data.14,15 Worth notice is that the term “phenomics” relates to a new, emerging research discipline, intended to measure the entire physical and biochemical traits of a particular organism.16 To obtain the necessary information regarding phenotypic analysis of fruit-quality traits of mandarins, we conducted a comprehensive analysis of fruit-quality traits among 46 distinct mandarin varieties within the Israeli citrus breeding collection. The studied fruit included 30 common mandarin varieties, four satsumas, two clementines, five tangors, two tangelos, two Mediterranean mandarins, and the King mandarin variety. The tested traits included physical (size, shape, color, peel thickness, and seed number), physiological (ripening period, peelability, and segmentation), nutritional and biochemical (vitamin C, phenol, flavonoid and carotenoid contents, and total antioxidant activity), and sensory (total soluble solids and acid levels, flavor preference, sweetness, sourness, and fruitiness) attributes. The presented data encompass the wide diversity in fruit-quality traits within the mandarin group.
INTRODUCTION During the past decade, there has been a continuous rise in consumption and global marketing of fresh, easy-to-peel mandarins, with forecast production of more than 24 million tons per year.1 Mandarins, together with pummelo and citron, are considered to be the original Citrus ancestors, from which all other citrus species have evolved.2−4 According to Hodgson’s classification,5 the mandarin group is divided into various natural subgroups, including common mandarin (C. reticulata Blanco), satsuma mandarin (C. unshiu Marcovitch), king mandarin (C. nobilis Loureiro), Mediterranean mandarin (C. deliciosa Tenore), smallfruited mandarin (C. indica, C. tachibana, and C. reshni), and mandarin hybrids including tangor (C. reticulata × C. sinensis) and tangelo (C. reticulata × C. paradisi). In addition to the above classification, clementine (C. clementina Hort. ex. Tan) is also considered to be a unique and distinct subgroup of mandarins.6,7 It is known that there is wide genetic diversity in fruit-quality traits among citrus species, such as orange (C. sinensis), pummelo (C. maxima), mandarin (C. reticulata), lemon (C. limon), and citron (C. medica). For example, it was reported that there are large genetic differences in fruit color and carotenoid pigments, bioactive compounds, and aroma volatile contents between different citrus groups and even among varieties.8−11 Nevertheless, as far as we are aware, there has not yet been any detailed systematic study that specifically evaluated the diversity in fruitquality traits among various mandarin subgroups and varieties. In light of recent advances in genomics and sequencing of citrus genomes, including the identification of thousands of single-nucleotide-polymorphism (SNP) DNA markers with high coverage of the citrus genome,6,12,13 it became crucially necessary to conduct a high-throughput phenotypic analysis (phenomics) of citrus fruit-quality traits, in order to enable reliable comparison © 2014 American Chemical Society
■
MATERIALS AND METHODS
Plant Material. Fruit of 46 different mandarin varieties were obtained from the Israeli citrus breeding collection at the Agricultural Research Organization, Volcani Center, Bet Dagan, Israel. The fruit of each variety were harvested at optimal maturity, as determined from a combination of maturity indices, including fruit size, color, and taste, i.e., loss of acidity, as well as the breeding team’s previous experience. The mandarin varieties included 30 common mandarins (C. reticulata Received: Revised: Accepted: Published: 4938
January 14, 2014 May 4, 2014 May 7, 2014 May 7, 2014 dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
Table 1. Diversity in Ripening Time, Fruit Weight and Shape, and Ease of Peelability and Segmentation among Mandarin Varieties and Subgroups variety
harvest date
Common mandarin (C. reticulata Blanco) Rishon 23.10.12 Admoni 24.10.12 Michal 13.11.12 Tami 13.11.12 Fallglo 14.11.12 Hinanit 02.12.12 Lee 02.12.12 Fairchild 09.12.12 Idit 11.12.12 Merav 17.12.12 Vered 18.12.12 Dancy 27.12.12 Nova 27.12.12 Ponkan 27.12.12 Yifat 13.01.13 Nectar 13.01.13 Cami 13.01.13 Wilking 13.01.13 Ora 16.01.13 Winola 16.01.13 Yafit 16.01.13 Afourer 24.01.13 Orit 03.02.13 Odem 03.02.13 Nurit 04.02.13 Sigal 11.02.13 Fortune 19.02.13 Shani 19.02.13 Novel 04.03.13 Hadas 17.03.13 Clementine (C. clementina Hort. ex. Tan.) GPP 23.10.12 Nour 09.12.12 Tangor (C. reticulata × C. sinensis) Niva 26.11.12 Tacle 01.01.13 Temple 01.01.13 Kiyomi 04.02.13 Murcott 11.02.13 Tangelo (C. reticulata × C. paradisi) Orlando 27.12.12 Minneola 01.01.13 Satsuma (C. unshiu Marcovitch) Okitsu 16.10.12 Owari 23.10.12 Nucellar 31.10.12 Imamura 26.12.12 Mediterrenean (C. deliciosa Tenore) Avana 01.01.13 Tardivo 24.01.13 King (C. nobilis Loureiro) King 27.02.13 a
weight (g)
shape (hight/diameter)
peelabilitya(1−5)
segmentationa (1−5)
29 29 32 32 32 35 35 36 36 37 37 38 38 38 41 41 41 41 41 41 41 42 44 44 44 45 46 46 48 50
138.1 ± 4.8 122.4 ± 2.9 70.8 ± 1.3 83.5 ± 2.2 148.9 ± 3.4 76.2 ± 1.8 112.3 ± 1.9 82.3 ± 2.2 125.0 ± 3.3 153.5 ± 4.3 165.3 ± 6.1 98.8 ± 3.2 123.6 ± 3.5 114.0 ± 2.4 155.9 ± 2.3 99.3 ± 4.1 121.9 ± 3.6 118.4 ± 3.3 153.0 ± 3.7 219.5 ± 5.5 127.9 ± 2.7 97.8 ± 3.5 146.1 ± 3.5 113.5 ± 3.6 185.1 ± 3.2 93.4 ± 1.9 150.8 ± 3.9 117.9 ± 3.3 167.5 ± 3.6 132.8 ± 4.9
0.85 ± 0.02 0.83 ± 0.01 0.84 ± 0.01 0.85 ± 0.01 0.76 ± 0.01 0.81 ± 0.01 0.84 ± 0.01 0.85 ± 0.01 0.68 ± 0.01 0.77 ± 0.01 0.71 ± 0.02 0.70 ± 0.01 0.84 ± 0.01 0.72 ± 0.01 0.75 ± 0.01 0.70 ± 0.01 0.87 ± 0.01 0.73 ± 0.01 0.74 ± 0.01 0.70 ± 0.02 0.86 ± 0.01 0.70 ± 0.01 0.75 ± 0.01 0.76 ± 0.01 0.69 ± 0.01 0.66 ± 0.01 0.81 ± 0.02 0.83 ± 0.01 0.81 ± 0.01 0.78 ± 0.02
3 3 3 3 3 1 2 1 3 3 2 4 2 5 3 3 3 4 4 4 3 4 3 3 1 5 3 3 3 3
3 3 4 3 3 3 2 2 4 3 3 4 2 5 3 4 2 4 4 3 3 4 3 3 3 4 3 3 3 3
29 36
84.1 ± 1.9 57.8 ± 1.5
0.79 ± 0.03 0.87 ± 0.01
3 3
3 4
34 39 39 44 45
109.9 ± 2.7 137.4 ± 7.1 117.6 ± 2.9 187.2 ± 5.6 127.6 ± 3.2
0.85 ± 0.01 0.76 ± 0.02 0.80 ± 0.01 0.86 ± 0.01 0.75 ± 0.01
1 2 2 3 3
2 2 1 1 3
38 39
155.3 ± 2.5 167.7 ± 6.9
0.84 ± 0.01 0.89 ± 0.02
1 3
2 3
28 29 30 38
134.6 ± 4.1 95.1 ± 2.4 93.4 ± 2.8 96.9 ± 3.1
0.78 ± 0.01 0.79 ± 0.01 0.76 ± 0.01 0.71 ± 0.01
5 5 5 5
5 5 5 5
39 42
81.0 ± 3.5 64.9 ± 3.0
0.69 ± 0.01 0.78 ± 0.01
5 3
4 3
47
120.7 ± 3.7
0.80 ± 0.01
4
3
time from blooming (weeks)
Data are means of three evaluators’ scores: 1 = very difficult, 5 = very easy.
Blanco), two clementines (C. clementina Hort. ex. Tan), five tangors (C. reticulata × C. sinensis), two tangelos (C. reticulata × C. paradisi), four satsumas (C. unshiu Marcovitch), two Mediterranean mandarins (C. deliciosa Tenore), and a King mandarin (C. nobilis Loureiro) (Table 1). Mandarin varieties not belonging purely to any specific subgroup, such
as hybrids between different mandarin subgroups, were classified as common mandarins. Fruit Weight, Shape, and Peel Thickness. Fruit weight was measured with a semianalytical scale (Citizen, Mumbai, India), and fruit height and diameter were measured with millimetric calipers. 4939
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
Figure 1. Diversity among mandarin varieties and subgroups in fruit seed number. (A) Photographs of ‘Havana’, ‘Michal’, and ‘Nectar’ mandarins containing high and moderate numbers and zero seeds, respectively. (B) Seed numbers of various mandarin varieties. Data are means ± SE of 10 fruit. Thicknesses of the albedo (the inner white part of the peel), flavedo (the outer colored part of the peel), and the total peel were measured with a Leica MZ FLIII stereomicroscope (Leitz, Wetzlar, Germany) with the NIS-Elements Imaging software. All data are means ± SE of 10 fruit. Seed Number and Peelability. The number of seeds per fruit was evaluated after cutting the fruit in half and extracting the juice with a hand extractor. Ease of peelability and segmentation were evaluated subjectively on a scale of 1 (very difficult) to 5 (very easy) by three laboratory personnel. The presented data are means ± SE of 10 fruit. Color Measurement. Peel color was measured with a CR-310 Chroma Meter (Minolta, Tokyo, Japan), and the results are expressed as hue angle (H°), in which 0° represents red color, 45° orange, 90° yellow, and 120° green. Data are means ± SE of 10 fruit. TSS, Acidity, and Vitamin C. Total soluble solids (TSS) content in the juice was determined with a PAL-1 digital refractometer (Atago, Tokyo, Japan), and acidity percentages were measured by titration to pH 8.3 against 0.1 M NaOH, with a CH-9101 automatic titrator (Metrohm, Herisau, Switzerland). Each measurement included four replications, each of juice collected from three different fruit (total of 12 fruit per measurement). Vitamin C (ascorbic acid) content in the juice was determined by titration against 2,6-dichlorophenolindophenol, as described previously.17 Ascorbic acid levels were determined by comparing the titration volumes of mandarin juices with those of 0.1% ascorbic acid (SigmaAldrich, St. Louis, MO, USA), and results are expressed as milligrams of ascorbic acid per 100 mL of juice. Each measurement included four replications, each of juice collected from three different fruit (total of 12 fruit per measurement). Total Phenols and Flavonoids. Extraction of phenols and flavonoids was performed by stirring 1 mL of juice samples with 9 mL of 80% methanol for 30 min at room temperature, followed by centrifugation at 10000g for 10 min. Total phenolics were determined by the Folin−Ciocalteu method.18 Briefly, the reaction mixtures included 0.2 mL of juice methanol extracts, 0.2 mL of Folin−Ciocalteu reagent, and 7 mL of 7% Na2CO3. The reaction mixtures were incubated for 90 min at room temperature, and the absorbance at 750 nm was measured by comparison with a prepared blank by means of a spectrophotometer. Total phenolic contents were expressed as gallic acid equivalents. Total flavonoids were determined as described by Shin et al.19 Briefly, the reaction mixtures included 1.0 mL of juice methanol extracts, 0.3 mL of 5% NaNO2, and 0.3 mL of AlCl3. The reaction mixtures were incubated for 10 min at room temperature, after which 2 mL of 1 N
NaOH was added to stop the reaction, and the total volume was adjusted to 10 mL by adding double-distilled water. Flavonoid contents were measured by comparing the absorbance at 510 nm against that of a prepared blank, and the results were expressed as catechin equivalents. Total phenol and flavonoid contents are presented as means ± SE of four replications, each of juice collected from three different fruit (total of 12 fruit per measurement). Total Carotenoid Content. Total carotenoids were extracted according to Lee and Castle.20 In general, 1 mL of juice was homogenized with 2 mL of hexane/acetone/ethanol extraction solution (2:1:1 by volume) for 30 s and centrifuged for 5 min at 6500 rpm at 5 °C. Afterward, 100 mL of the colored top layer was diluted with 900 mL of hexane, and absorbance was measured at 450 nm with a spectrophotometer. Total carotenoid levels were calculated according to an equation provided by Schiedt and Liaaen-Jensen,21 with a β-carotene extinction coefficient of E1% = 2400. Total carotenoid contents are presented as means ± SE of three replications, each of juice collected from three different fruit (total of 9 fruit per measurement). Antioxidant Activity. Total antioxidant activity was determined by using the ABTS*+ radical cation assay.22 The reaction mixture included 1 mL of 75 μM K2O8S2 and 150 μM 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) dissolved in acetate buffer, pH = 4.3, and 10 μL of juice samples. The reaction mixtures were incubated for 15 min at room temperature. Afterward, total antioxidant activities of juice samples were compared with a 1 mM Trolox solution by determining the degree of disappearance of the blue color by comparing the absorbance at 734 nm against that of a prepared blank. Trolox equivalent (TE) was calculated according to the following formula:
TE value (mM) = (Abs sample − Abs blank) /(Abs standard − Abs blank) The results were expressed as Trolox equivalent antioxidant capacity (TEAC), calculated as
TEAC (μM TE/g) = (TE × V )/(1000 × M ) where V = sample volume and M = sample weight. Antioxidant activity levels are presented as means ± SE of four replications, each of juice collected from three different fruit (total of 12 fruit per measurement). Sensory Evaluations. Fruit sensory quality was tested on the day of harvest. The fruit were peeled and segments were separated, cut 4940
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
Figure 2. Diversity among mandarin varieties and subgroups in peel thickness. (A) Photographs of ‘Minneola’ and ‘Lee’ mandarins exhibiting thick and thin peel, respectively. (B) Total peel, albedo, and flavedo thicknesses of various mandarin varieties. Data are means ± SE of 10 fruit. Pearson correlation values were calculated and matched against total peel thickness; double asterisks (**) indicate significance at P ≤ 0.01.
Figure 3. Diversity among mandarin varieties and subgroups in fruit color. (A) Photographs of ‘Owari’, ‘Ora’, and ‘Odem’ mandarins exhibiting green, orange, and reddish color, respectively. (B) Peel color of various mandarin varieties. Data are means ± SE of 10 fruit. was evaluated by a trained panel consisting of 10 members, five males and five females, aged 25 to 62, who routinely perform taste tests of
into halves, and placed into covered glass cups. Each sample comprised a mixture of cut segments prepared from five different fruit. Fruit taste 4941
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
Figure 4. Diversity among mandarin varieties and subgroups in antioxidative activity and total vitamin C, phenols, flavonoids, and carotenoids levels. Data are means ± SE of four replications, each of juice from three different fruit. Pearson correlation values were calculated and matched against total antioxidant activity; single (*) and double (**) asterisks indicate significance at P ≤ 0.05 and P ≤ 0.01, respectively.
■
citrus fruit.23,24 Each panelist assessed the taste attributes of the samples according to an unstructured 100 mm scale, on which the anchor points ‘very weak’ and ‘very strong’ for each attribute and/sensory data were recorded as distances (mm) from the origin. The samples were identified by means of randomly assigned threedigit codes. In addition, panelists were requested to rate overall fruit flavor preference on a scale of 1 to 9, where 1 = very bad and 9 = excellent. Statistical Analysis. One-way analysis of variance (ANOVA) was applied with the Microsoft Office Excel program. Hierarchical clustering and heat-map analysis were performed with the Expander 4.1 software,25 and Pearson correlations were calculated with the R software (http:// www.r-project.org).
RESULTS
Diversity in Time of Ripening, Fruit Weight, Shape, and Peelability. We evaluated fruit-quality traits of 46 mandarin varieties within the Israeli Citrus breeding collection; they belonged to genetically differing natural subgroups, comprising 30 common mandarins (C. reticulata Blanco), two clementines (C. clementina Hort. ex. Tan), five tangors (C. reticulata × C. sinensis), two tangelos (C. reticulata × C. paradisi), four satsumas (C. unshiu Marcovitch), two Mediterranean mandarins (C. deliciosa Tenore), and one King mandarin (C. nobilis Loureiro) (Table 1). We observed great genetic variability in ripening time, which was spread over 6 months from October through March. 4942
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
Figure 5. Diversity among mandarin varieties and subgroups in TSS and acidity contents and ripening ratios. Data are means ± SE of four replications, each of juice from three different fruit. Pearson correlation values were calculated and matched against acidity levels; double asterisks (**) indicate significance at P ≤ 0.01.
The earliest-ripening varieties, harvested in October, were the new Israeli breeding varieties ‘Rishon’ and ‘Admoni’, as well as the clementine ‘GPP’, and three satsumas, ‘Okitsu’, ‘Owari’, and ‘Nucellar Satsuma’ (Table 1). The latest-ripening varieties, harvested in March, were the common mandarins ‘Novel’ and ‘Hadas’ (Table 1). There was also great genetic diversity in fruit weight: the variety with the largest fruit (219 g) was ‘Winola’, whereas the one with the smallest (58 g) was ‘Nour’ clementine (Table 1). We further observed great variability in fruit shape, i.e., height/ diameter ratio. Fruit of some varieties, including ‘Minneola’ tangelo, ‘Kiyomi’ tangor, ‘Nour’ clementine, and ‘Cami’ and ‘Yafit’ common mandarins, were nearly round, with height/diameter ratios above 0.85, whereas, in contrast, other varieties such as ‘Sigal’ were rather flat, with a height/diameter ratio of just 0.66 (Table 1). Another important quality attribute of mandarins is ease of peelability and segment separation. We observed great variability in ease of peeling: some varieties, including all four satsumas, as well as ‘Ponkan’, ‘Havana’, and ‘Sigal’, were very easy to peel, whereas others, such as the ‘Niva’ tangor, ‘Orlando’ tangelo, and the common mandarins ‘Fairchild’, ‘Hinanit’, and ‘Nurit’, were very difficult to peel (Table 1). It was very easy to separate the segments of all four satsumas, as well as those of ‘Ponkan’, but very difficult to separate those of some others, especially the tangors ‘Temple’ and ‘Kiyomi’ (Table 1). Diversity in Seed Number. The number of seeds per fruit is a very important quality trait, as consumers prefer seedless fruit, and we observed great variability in fruit seed number. Five varieties had an average of more than 20 seeds per fruit, with ‘Avana’ Mediterranean mandarin containing an average of 25 seeds per fruit (Figure 1). In contrast, seven mandarin varieties averaged ≤1 seed per fruit (Figure 1). Worth noticing is that the ‘Nectar’ common mandarin variety was obligatorily seedless, even under the unfavorable conditions of a mixed orchard with many pollinators (Figure 1). Diversity in Peel Thickness. We observed substantial intervariety differences in peel thickness, which ranged from
Figure 6. Diversity among mandarin varieties and subgroups in flavor preference and sensations of ‘sweetness’, ‘sourness’, and ‘fruitiness’. Data are means ± SE of scores given by 10 trained testers. Pearson correlation values were calculated and matched against flavor preference scores; double asterisk (**) indicates significance at P ≤ 0.01.
2.0 to 5.9 mm (Figure 2). Furthermore, we observed significant correlations between overall peel thickness and thickness of the albedo (R = 0.92) and flavedo tissues (R = 0.84) (Figure 2). Most of the varieties that had a thick albedo also had a thick flavedo, and vice versa. However, some varieties differed in this respect. For example, the ‘Tardivo’ Mediterranean mandarin had a relatively thin albedo but a thick flavedo (Figure 2). Diversity in Peel Color. The vast majority of mandarin varieties exhibited the typical mandarin orange color, with measurable hue angles between 50° and 70° (Figure 3). In contrast, the early ripening varieties, including ‘Rishon’ common mandarin, ‘GPP’ clementine, and three satsuma varieties, ‘Okitsu’, ‘Owari’, and ‘Nucellar Satsuma’, were harvested when their peel color was mostly green, with a persisting hue angle of ≥100° (Figure 3). Worth notice is that the ‘Odem’ and ‘Shani’ common mandarins had a unique deep reddish-orange color, with a hue angle of 40−45° (Figure 3). Diversity in Fruit Nutritional Quality. The key nutritional components of citrus fruit that contribute to its overall antioxidative activity are vitamin C, phenols, flavonoids, and carotenoids. We observed significant variability among mandarin varieties and subgroups in their bioactive contents and total antioxidative activity. The total antioxidative activity ranged between 1.5 and 3.5 μM TE g−1 (Figure 4). Juice vitamin C levels were relatively high (i.e., between 45 and 60 mg per 100 mL) in most tangors as well as in a few common mandarin varieties, and the highest level (78 mg per 100 mL) was found in juice of ‘Tacle’ tangor (Figure 4). In contrast, juice from several common mandarin varieties, as well as ‘Minneola’ tangelo, ‘Okitsu’ 4943
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
Figure 7. Hierarchical clustering of mandarin varieties and subgroups and heat-map analysis of fruit-quality traits. Red and green colors represents high and low levels, respectively. The units in the color scale are standard deviations. The various mandarin natural subgroups are listed in different colors at the right.
Figure 8. Hierarchical clustering and Pearson correlations among all fruit-quality traits. Red and green colors represent positive and negative correlations, respectively. Pearson correlations were calculated with the R software (http://www.r-project.org).
to 804 mg L−1 in ‘Tacle’ tangor (Figure 4). Worth notice is that most tangors had relatively high phenol levels (Figure 4). We also observed wide variability among mandarin juices in their total flavonoid content, from just 10 mg L−1 in ‘Afourer’ common
satsuma, and ‘King’ mandarin, contained relatively low vitamin C levels of 22−24 mg per 100 mL (Figure 4). It was found that total phenol levels in mandarin juices ranged from just 375 mg L−1 in ‘Afourer’ common mandarin up 4944
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
mandarin up to 45 mg L−1 in ‘Orlando’ tangelo (Figure 4). However, we did not detect any clear differences among mandarin subgroups in their flavonoid contents. We further observed wide genetic diversity among mandarin juices in their total carotenoid contents: the juices of some mandarin varieties, such as ‘Fortune’, were pale yellow in color and contained only 3 mg L−1, whereas the juices of other varieties, such as ‘King’ mandarin and ‘Imamura’ satsuma, were dark orange in color and contained carotenoids at more than 30 mg L−1 (Figure 4). Overall, we found R values of 0.60, 0.47, 0.30, and 0.01, respectively, for the correlations between total antioxidative activity of mandarin juices and vitamin C, phenol, flavonoid, and carotenoid levels. Thus, it seems that vitamin C provides the highest contribution to total antioxidant activity of mandarin fruit (Figure 4). Diversity in Juice TSS, Acidity, and Flavor Perception. The taste of citrus fruit is principally governed by the levels of sugars and acids in the juice sacs, as well as the fruit ripening ratio. We observed remarkable differences among mandarins in their juice acidity levels, from as low as 0.5% in ‘Odem’ common mandarin to nearly 2.0% in ‘Vered’ common mandarin and ‘Temple’ tangor (Figure 5). The overall juice TSS levels ranged from as low as 9.6% in ‘Okitsu’ satsuma to 15.8% in ‘Fairchild’ common mandarin (Figure 5). These observed differences in juice TSS and acidity levels resulted in remarkable variability in fruit ripening ratios, from below 6.7 in the common mandarins ‘Vered’, ‘Hadas’, and ‘Winola’, the tangors ‘Temple’ and ‘Kiyomi’, and ‘King’ mandarins, up to 23 in ‘Odem’ common mandarin (Figure 5). Overall, sensory analysis revealed wide genetic variability among mandarin flavor preferences, ranging from a relatively high score of 7.5 (on a 1−9 scale) for several common mandarin varieties such as ‘Michal’, down to scores below 5.0 for ‘Owari’ satsuma and ‘Fallglo’ and ‘Rishon’ common mandarins (Figure 6). In addition, descriptive sensory analysis tests conducted with the aid of a trained panel indicated differences among mandarins in their elicited sensations of ‘sweetness’, ‘sourness’, and ‘fruitiness’ (Figure 6). Furthermore, we observed significant positive correlations between flavor preference and perceptions of sweetness (R = 0.75) and fruitiness (R = 0.88) (Figure 6). Hierarchical Clustering and Heat-Map Analysis. Hierarchical clustering of mandarin varieties and subgroups and heatmap analysis of fruit-quality traits demonstrated that the distinct mandarin varieties fell into two major subclusters: the first cluster included some common mandarins, satsumas, clementines, and Mediterranean mandarins; the second cluster included additional common mandarins, King mandarin, tangors, and tangelos (Figure 7). Furthermore, it can be seen that the different satsuma, clementine, Mediterranean mandarin, tangor, and tangelo varieties were clustered closely together, thus demonstrating the genetic diversity in fruit-quality traits among the various mandarin subgroups (Figure 7). Hierarchical clustering and Pearson correlations among all of the 28 examined fruit-quality parameters demonstrated that the various quality attributes can be grouped into several subclusters (Figure 8). For example, it can be seen that peel traits, such as albedo, flavedo, and total peel thickness, were clustered together with ease of peelability and segment separation, which suggests that these traits are mutually related (Figure 8). Furthermore, many of the biochemical characteristics, including levels of vitamin C, phenols, and flavonoids, as well as total antioxidant activity, were also clustered together (Figure 8). Finally, it was
Table 2. Outlines of the Diversity Observed among Mandarin Varieties and Subgroups in Fruit-Quality Traits range of variability quality trait
lowest score
Physical Traits weight 57.8 height 39.2 diameter 49.5 shape 0.66 peel color 42.4 peel thickness 2.02 albedo 0.98 flavedo 0.56 seed number 0 Physiological Traits ripening time 28 peelability 1 segmentation 1 splitting 0 Biochemical Composition antioxidative activity 1.5 vitamin C 22.2 phenols 375 flavonoids 10.4 carotenoids 2.9 Sensory Traits TSS 9.6 acid 0.5 ripening ratio 6.4 sweet 3 sour 1.3 bitter 0.1 gummy 0.9 juicy 4.9 fruity 2.9 flavor 4.4
highest score
units
219.5 68.3 87.6 0.89 112 4.9 3.15 2 25
g mm mm height/diameter ratio H° mm mm mm seeds/fruit
50 5 5 3 3.6 77.8 804 44.9 33.1 15.8 2 23.2 7.6 6.1 1.7 4.1 7.9 7.2 7.5
weeks after blooming scale of 1−5 scale of 1−5 scale of 0−3 μM TE g−1 mg/100 mL mg L−1 mg L−1 mg L−1 % % TSS/acid ratio scale of 0−10 scale of 0−10 scale of 0−10 scale of 0−10 scale of 0−10 scale of 0−10 scale of 1−9
found that flavor preference was clustered together with TSS levels and perceptions of ‘sweetness’ and ‘fruitiness’, whereas acidity and ‘sourness’ were clustered separately (Figure 8).
■
DISCUSSION A detailed phenotypic analysis study (phenomics) of fruit-quality traits is an obligatory requirement to enable designation of SNPs for marker-assisted breeding of new varieties with improved fruitquality traits, as well as for selection of appropriate parents for future breeding programs.12,13,26,27 In this respect, the present study provided the most comprehensive phenotypic analysis to date of fruit-quality traits among genetically distinct mandarin varieties and subgroups. The data yielded by this study highlight the inclusive genetic diversity among mandarins, in their fruit-quality traits (Table 2). For example, ripening time can range from 28 to 50 weeks after blooming; fruit weight from 58 to 219 g; peel thickness from 2.0 to 4.9 mm; fruit shape from nearly round to flat; peelability from very easy to very difficult; and seed number from zero to 25 (Table 2). Regarding fruit nutritional attributes, we found that vitamin C levels ranged from 22 to 78 mg per 100 mL; phenols from 375 to 804 mg per 100 mL; flavonoids from 10 to 45 mg per 100 mL; and carotenoids from 3 to 33 mg per 100 mL (Table 2). Finally, TSS levels may range from 9.6% to 15.8%; acidity from 4945
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
Journal of Agricultural and Food Chemistry
Article
0.5% to 2.0%; and flavor preference from 4.4 through 7.5 on a 1−9 scale (Table 2). These phenotypic analysis data are of great importance for citrus breeders worldwide, as they could assist in selection of appropriate varieties as putative parents in future breeding programs. For example, for breeding early-season varieties it would be preferable to choose ‘Rishon’, ‘Admoni’, or satsumas as putative parents, whereas for breeding late-season varieties it would be preferable to choose ‘Hadas’ (Table 1). In the case of breeding for high vitamin C content, it would be preferable to choose ‘Tacle’ tangor or other tangors as putative parents (Figure 4). Regarding the genetic variability in fruit-quality traits among different natural mandarin subgroups, hierarchical clustering analysis revealed clear evolutionary differences among mandarins (Figure 7). For example, satsuma mandarins are early ripening, are very easy to peel, and contain few seeds, and their juice is deep orange in color and contains high carotenoid pigment contents (Table 1; Figures 1, 4). Clementines also ripen early and have relatively small fruit (Table 1). Tangors (orange × mandarin hybrids) are relatively difficult to peel, but have high vitamin C and phenol contents and high antioxidative activity levels (Table 1; Figure 4). Tangelos (grapefruit × mandarin hybrids) have a relatively thick peel (Figure 2), Mediterranean mandarins have many seeds (Figure 1), and King mandarin has many seeds and high carotenoid and acidity contents (Figures 1, 4, 5).
■
(9) Cano, A.; Medina, A.; Bermejo, A. Bioactive compounds in different citrus varieties. Discrimination among cultivars. J. Food Compos. Anal. 2008, 21, 377−381. (10) Gonzalez-Mas, M. C.; Rambla, J. L.; Alamar, M. C.; Gutierrez, A.; Granell, A. Comparative analysis of the volatile fraction of fruit juice from different citrus species. PLoS One 2011, 6. (11) Goodner, K. L.; Rouseff, R. L.; Hofsommer, H. J. Orange, mandarin, and hybrid classification using multivariate statistics based on carotenoid profiles. J. Agric. Food Chem. 2001, 49, 1146−1150. (12) Gmitter, F. G.; Chen, C.; Machado, M. A.; de Souza, A. A.; Ollitrault, P.; Froehlicher, Y.; Shimizu, T. Citrus genomics. Tree Genet. Genome 2012, 8, 611−626. (13) Ollitrault, P.; Terol, J.; Garcia-Lor, A.; Berard, A.; Chauveau, A.; Froelicher, Y.; Belzile, C.; Morillon, R.; Navarro, L.; Brunel, D.; Talon, M. SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (Rutaceae), phylogenetic inferences and perspectives for genetic mapping. BMC Genomics 2012, 13. (14) Schauer, N.; Semel, Y.; Roessner, U.; Gur, A.; Balbo, I.; Carrari, F.; Pleban, T.; Perez-Melis, A.; Bruedigam, C.; Kopka, J.; Willmitzer, L.; Zamir, D.; Fernie, A. R. Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement. Nat. Biotechnol. 2006, 24, 447−454. (15) White, J. W.; Andrade-Sanchez, P.; Gore, M. A.; Bronson, K. F.; Coffelt, T. A.; Conley, M. M.; Feldmann, K. A.; French, A. N.; Heun, J. T.; Hunsaker, D. J.; Jenks, M. A.; Kimball, B. A.; Roth, R. L.; Strand, R. J.; Thorp, K. R.; Wall, G. W.; Wang, G. Y. Field-based phenomics for plant genetics research. Field Crop Res. 2012, 133, 101−112. (16) Furbank, R. T.; Tester, M. Phenomics−technologies to relieve the phenotyping bottleneck. Trends Plant Sci. 2011, 16, 635−644. (17) Hiromi, K.; Kuwamoto, C.; Ohnishi, M. A rapid sensitive method for the determination of ascorbic acid in the excess of 2,6dichlorophenolindophenol using a stopped-flow apparatus. Anal. Biochem. 1980, 101, 421−426. (18) Singleton, V. L.; Orthofer, R.; Lamuela-Raventós, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Meth. Enzymol. 1999, 299, 152−178. (19) Shin, Y.; Liu, R. H.; Nock, J. F.; Holliday, D.; Watkins, C. B. Temperature and relative humidity effects on quality, total ascorbic acid, phenolics and flavonoid concentrations, and antioxidant activity of strawberry. Postharv. Biol. Technol. 2007, 45, 349−357. (20) Lee, H. S.; Castle, W. S. Seasonal changes of carotenoid pigments and color in Hamlin, Earlygold, and Budd blood orange juices. J. Agric. Food Chem. 2001, 49, 877−882. (21) Schiedt, K.; Liaaen-Jensen, S. Carotenoids Vol. 1A: Isolation and Analysis; Birkhauser: Basel, Switzerland, 1995. (22) Miller, N. J.; Rice-Evans, C. A. Factors influencing the antioxidant activity determined by the ABTS(center dot+) radical cation assay. Free Radical Res. 1997, 26, 195−199. (23) Benjamin, G.; Tietel, Z.; Porat, R. Effects of rootstock/scion combinations on the flavor of citrus fruit. J. Agric. Food Chem. 2013, 61, 11286−11294. (24) Tietel, Z.; Lewinsohn, E.; Fallik, E.; Porat, R. Elucidating the roles of ethanol fermentation metabolism in causing off-flavors in mandarins. J. Agric. Food Chem. 2011, 59, 11779−11785. (25) Shamir, R.; Sharan, R. Algorithmic approaches to clustering gene expression data. in Current Topics in Computational Biology; Jiang, T., Smith, T., Xu, Y., Zhang, M. Q., Eds.; MIT Press: Cambridge, MA, 2002; pp 269−299. (26) DiStefano, G.; La Malfa, S.; Gentile, A.; Wu, S. B. EST-SNP genotyping of citrus species using high-resolution melting curve analysis. Tree Genet. Genomes 2013, 9, 1271−1281. (27) Fujii, H.; Shimada, T.; Nonaka, K.; Kita, M.; Kuniga, T.; Endo, T.; Ikoma, Y.; Omura, M. High-throughput genotyping in citrus accessions using an SNP genotyping array. Tree Genet. Genomes 2013, 9, 145−153.
AUTHOR INFORMATION
Corresponding Author
*(R. Porat) Tel: 972-3-9683617. Fax: 972-3-9683622. E-mail:
[email protected]. Notes
This article is contribution no. 680/13 from the Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel. The authors declare no competing financial interest.
■
REFERENCES
(1) United States Department of Agriculture. Citrus: World Markets and Trade; http://apps.fas.usda.gov/psdonline/circulars/citrus.pdf (accessed December 2013). (2) Barkley, N. A.; Roose, M. L.; Krueger, R. R.; Federici, C. T. Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theor. Appl. Genet. 2006, 112, 1519−1531. (3) Barret, H. C.; Rhodes, A. M. A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives. Syst. Bot. 1976, 1, 105−136. (4) Federici, C. T.; Fang, D. Q.; Scora, R. W.; Roose, M. L. Phylogenetic relationships within the genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis. Theor. Appl. Genet. 1998, 96, 812−822. (5) Hodgson, R. W. Horticultural varieties of citrus. In The Citrus Industry, Vol. 1; Reuther, W., Webber, H. J., Batchler, L. D., Eds.; University of California: Berkeley, CA, 1967; pp 431−588. (6) Aleza, P.; Juarez, J.; Hernandez, M.; Pina, J. A.; Ollitrault, P.; Navarro, L. Recovery and characterization of a Citrus clementina Hort. ex Tan. ‘Clemenules’ haploid plant selected to establish the reference whole Citrus genome sequence. BMC Plant Biol. 2009, 9, 110. (7) Tanaka, T. Fundamental discussion of citrus classification. Stud. Citrolog. 1977, 14, 1−6. (8) Bermejo, A.; Cano, A. Analysis of nutritional constituents in twenty citrus cultivars from the Mediterranean area at different stages of ripening. Food Nutr. Sci. 2012, 3, 639−650. 4946
dx.doi.org/10.1021/jf5002414 | J. Agric. Food Chem. 2014, 62, 4938−4946
