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Genetic variability in yellow pigment components in cultivated and wild tetraploid wheats
Journal of Cereal Science 50 (2009) 210–218
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Genetic variability in yellow pigment components in cultivated and wild tetraploid wheats A.M. Digesu` a, C. Platani a, L. Cattivelli a, G. Mangini b, A. Blanco b, * a b
CRA – Cereal Research Centre, S.S. 16 km 6757, 1100 Foggia, Italy Department of Environmental and Agro-Forestry Biology and Chemistry, sect. Genetic and Plant Breeding, University of Bari, Via Amendola 165/A, 70126 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2009 Received in revised form 1 May 2009 Accepted 15 May 2009
Yellow pigment concentration (YPC) in durum wheat is an important criterion in the assessment of semolina quality, particularly in determining the commercial and nutritional quality of end-products. Genetic variability of YPC and carotenoid components was analysed in 102 wild and cultivated tetraploid wheat accessions in two trials. Overall, modern cultivars showed significantly higher values of YPC compared to old cultivars and wild ssp. dicoccum and ssp. dicoccoides accessions. Total carotenoid concentration varied between 1.178 and 4.416 mg/g with an average of 2.460 mg/g. The portion of carotenoids amounted to 33.2% of the YPC in 80 wheat accessions examined in the 2006 trial. Lutein was the main component of carotenoids, followed by zeaxanthin and b-carotene. a-carotene and b-cryptoxanthin were minor components. Pigment concentration was negatively correlated with kernel weight and grain protein concentration. Significant positive correlations were found between b* index and YPC. Knowledge of the carotenoid composition and concentration is useful for wheat breeders in the development of cultivars with high yellow colour and enhanced phytochemical concentrations, and provides valuable information for evaluating contributions to health benefits from the consumption of durum wheat end-products. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids Yellow pigments HPLC T. turgidum
1. Introduction Yellow pigment concentration in durum wheat is a criterion in the assessment of semolina quality and is of particular importance in determining the commercial and nutritional quality of endproducts such as pasta. Yellow to amber colour is generally preferred by consumers rather than a brown or cream. Semolina colour is the result of carotenoid pigment concentration of the grain and any losses during storage of the grain or semolina due to carotenoid oxidative degradation by lipoxygenase during processing, as well as processing conditions (for a review see Pagnotta et al., 2005; Troccoli et al., 2000). Carotenoids are among the most important natural pigments, having wide distribution, different structure and numerous biological functions. In plants, they are part of the light harvesting
Abbreviations: HPLC, high-performance liquid chromatography; YPC, yellow pigment concentration. * Corresponding author. Tel.: þ39 0039 080 5442992; fax: þ39 0039 080 5442200. E-mail address: [email protected] (A. Blanco). 0733-5210/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.05.002
complexes, involved in photo-oxidative protection, serve as precursors of the hormone abscisic acid and provide the yellow, orange and red colours in many flowers and fruits (Della Penna and Pogson, 2006; Hirschberg, 2001). Two distinct classes of carotenoids are recognised: carotenes, which are tetraterpenoid hydrocarbons, and xanthophylls, which contain one or more oxygen groups (Van den Berg et al., 2000). In humans, the nutritional importance of carotenoids comes mainly from the pro-vitamin A activity of b-carotene, a-carotene, b-cryptoxanthin and others, with at least one intact non-oxygenated b-ionone ring (Zile, 1998). In addition to their role as precursors of vitamin A, carotenoids have been associated with a reduced risk of cancer, decrease in cardiovascular diseases, protection of the macula region of the retina and prevention of cataracts, and increased levels of iron absorption (Garcia-Casal, 2006; Granado et al., 2003; Le Marchand et al., 1993; Mares-Perlman et al., 2002). Several analytical procedures have been developed to evaluate yellow pigment concentration in durum semolina and pasta (see review by Fratianni et al., 2005). The most popular colour measurement instruments in the food industry are based on the colour-space system L*, a*, b* (CIE, Commission Internationale de l’Eclairage, 1986) due to the safety and rapidity of the technique
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
and good correlation between chemical determinations and reflectance measurements (Fratianni et al., 2005; Johnston et al., 1980). Yellow pigments are usually analysed by using ICC method 152 (ICC, 1990) or AACC method 14-50 (AACC, 2000), both based on the extraction of pigments from semolina or pasta with watersaturated n-butyl alcohol and subsequent spectrophotometrical determination. High-performance liquid chromatography (HPLC) is widely accepted as an accurate and sensitive technique to allow the separation and identification of carotenoid components (Panfili et al., 2004). Specific HPLC procedures to detect carotenoids in durum grain and end-products have been developed by Burkhardt and Bo¨hm (2007), Fratianni et al. (2005), Hentschel et al. (2002). There is little literature related to the HPLC analysis of carotenoids in durum wheat and data have been reported on carotenoid composition of small numbers of samples (from one to ten cultivars). The concentration of carotenoids is higher in durum (from 1.50 to 5.00 mg/g) than in bread wheat (from 0.50 to 2.00 mg/g) (Fratianni et al., 2005; Hentschel et al., 2002; Hidalgo et al., 2006; Leenhardt et al., 2006) as a result of genetic selection for high yellow pigment concentration in durum wheat breeding programs. Inheritance of grain or semolina pigment concentration is complex, but overall heritability of pigment concentration was found to be reasonably high (from 0.34 to 0.95) (Clarke et al., 2006). The objective of this study was to explore the genetic variability of yellow pigment concentration and carotenoid components of a tetraploid wheat collection including 102 accessions of wild and cultivated wheats belonging to different sub-species of Triticum turgidum L. This will provide information on the existing variability and genetic resources available to breeders to improve colour and nutritional value of pasta and other durum wheat endproducts.
2. Experimental 2.1. Plant materials A collection of 102 tetraploid wheat accessions (T. turgidum L.), including 52 old and modern cultivars of durum wheat (ssp. turgidum var. durum), 16 accessions of ssp. turgidum var. turanicum, 27 accessions of spp. dicoccum and 7 wild accessions of ssp. dicoccoides, were evaluated for yellow pigment concentration, yellow and brown indexes, some grain yield traits (yield per spike, kernel weight, grain protein concentration) over two years (2006 and 2007). Eighty accessions grown in 2006 were analysed by HPLC for chemical determination of carotenoid components. The wheat collection was grown in the field at the University of Bari at Valenzano (Bari, Italy), in a randomized complete block design with three replications. In both experiments, each accession was planted at a rate of 30 seeds per row 1 m long with 30 cm between rows. During the growing season, standard cultivation practices were used. Plots were hand harvested at maturity and grain yield per spike was calculated as grain yield per row/number of spikes per row (about 50–60 spikes). A 15 g seed sample per plot was used to determine 1000-kernel weight. Grain protein concentration, expressed as percentage of protein on a dry weight basis, was directly obtained on a 2 g sample of whole-meal flour using nearinfrared reflectance spectroscopy. Yellow pigments were analysed by AACC method 14-50, the colorimeter system and high-performance liquid chromatography (HPLC). To minimize the degradation of pigments by oxidative enzymes, the samples were ground in a laboratory mill (Cyclotec Sample Mill, Tecator) with a 1 mm sieve and the resulting whole flour stored at 4 C for a maximum of 24 h before analysis.
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2.2. Yellow pigment concentration The evaluation of yellow pigment concentration (YPC) was made according to AACC Approved Method 14-50 (AACC, 2000) with slight modifications, as described by Fares et al. (1991): 1 g of each sample was extracted with 5 mL of water-saturated n-butyl alcohol on an orbital incubator (Gallenkamp INR-200) for 3 h at 260 oscillations per minute. Samples were centrifuged for 7 min at 2417 xg (Beckman JA21) and absorbance of n-butyl alcohol extracts was measured by a Beckman DU-65 UV-spectrophotometer at 435.8 nm (the wavelength of maximum absorption of lutein, taking into account that this xanthophyll is the dominant carotenoid in durum wheat). To improve reliability of the analysis, calibration of the instrument was carried out by reading a blank solvent extraction. Pigment concentration was calculated using the extinction coefficient of lutein and the Lambert–Beer Law (Rodriguez-Amaya and Kimura, 2004). The reported data are the mean of three replications. 2.3. Brown and yellow indexes Brown and yellow indexes were determined using the reflectance colorimeter Chroma Meter CR-300 (Minolta) equipped with a pulsed xenon arc lamp. Absolute measurement of L*, a*, b* (CIE, 1986) coordinates in the Munsell colour system were taken using D65 lightning. Samples to be analysed were placed in a granular material support. Brown index was calculated as 100-L* (Borrelli et al., 2003). Results were the average of three replications. 2.4. Carotenoid components Lutein, zeaxanthin, b-cryptoxanthin, a-carotene and b-carotene standards were obtained from LGC Promochem (LGC Promochem, Milano, Italy). All other reagents were of analytical or HPLC grade and were purchased form Levanchimica (Bari, Italy). Stock solutions of carotenoid standards were dissolved in ethanol, degassed to remove oxygen, and stored at 20 C. Concentrations were determined spectrophotometrically using the Lambert–Beer Law (Rodriguez-Amaya and Kimura, 2004). Solutions were diluted in methanol: di-chloromethane (MeOH: DCM 45:55 v/v) to make calibration curves. Carotenoid pigment extraction was performed according to Konopka et al. (2005) with some modifications. Briefly, 2 g of each sample was extracted under nitrogen in a screwcapped tube by adding 4 mL of extraction buffer (hexane/acetone, 80:20 v/v) and 600 mL of butyled hydroxytoluene (BHT) (0.1% w/v) as an anti-oxidant. Samples were stirred for a few minutes and kept in the dark for 16 h. Samples were then centrifuged for 10 min at 3000 rpm (Heraeus Sepatech), supernatants placed in glass tubes and residues extracted once again. The organic layer was collected and filtered with a gyroscope filter for syringe PTFE (porosity of 0.45 mm). Then, 5 mL of extract was evaporated to dryness under a nitrogen stream. Finally, the dry residues were re-dissolved in 200 mL of MeOH: DCM (45:55 v/v). A sample volume of 20 mL was injected into an Agilent Technologies 1100 HPLC system equipped with an automatic sampler and a diode array detector (DAD). Separation was done on a YMC C30 column (250 4.6 mm i.d., particle size ¼ 5 mm). The mobile phase was methanol and methyltert-butyl ether (89:11 v/v) at a constant flow rate of 1 mL/min. The mobile phase was previously degassed by sonication for 10 min. Spectrophotometric detection was achieved by means of a diode array detector in the range 400–600 nm. Peaks were detected at 450 nm. Carotenoids were identified through their characteristic spectra and comparison of retention times with known standard solutions. Calibration curves were constructed using five different concentrations of lutein (between 0.125 and 22.05 mg/mL),
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zeaxanthin (between 0.6 and 6.45 mg/mL), b- cryptoxanthin (between 0.1 and 1 mg/mL), a-carotene (between 0.08 and 0.85 mg/ mL) and b-carotene (between 0.05 and 0.6 mg/mL). Carotenoid concentrations were calculated using a linear regression (concentration versus area) of the five-point standard curve. Results were the average of three replications. 2.5. Statistical analysis Standard ANOVA (mixed model analysis of variance with genotype as fixed factor and blocks as random factor) was performed using the software MSTAT-C. When significant differences were detected, Fisher’s least significant difference (LSD) was computed. Genetic variance (s2G) and environmental variance (s2E) were obtained by using the standard ANOVA. Heritability (h2) was estimated by the ratio s2G/s2P, where s2P is phenotypic variance (s2P ¼ s2G þ s2E). Pearson phenotypic correlation coefficients were calculated between yellow pigment concentration, yellow and brown indexes, carotenoid components and grain yield components in each environment. 3. Results 3.1. Yellow pigment concentration and colorimetric indexes Yellow pigment concentration (YPC) and yellow (b*) and brown (100-L*) indexes of the tetraploid wheat collection evaluated over two years (2006 and 2007) are reported in Table 1. Wheat accessions are grouped by sub-species and cultivated durums grouped by year of release. YPC varied between 3.8 and 10.6 mg/g in 2006 and between 3.9 and 12.3 mg/g in 2007, with average values of 6.7 and 7.1 mg/g, respectively. Durum wheat cultivars (ssp. turgidum var. durum) showed the highest mean values (7.2 mg/g in 2006 and 7.8 mg/g in 2007). The cvs. Primadur, Svevo, Grecale, Normanno, Duetto, Exeldur and Brindur had significantly higher YPC (>10 mg/g) than the others. Notably, all of the most popular cultivars in Italy (Simeto, Iride, Duilio, Ciccio, Claudio and Creso) showed a yellow pigment concentration higher than 6.5 mg/g, a value sufficient to meet the durum market needs. When the durum cultivars are grouped by year of release, the results indicated that most recent varieties had significantly higher YPC than old cultivars: mean values of YPC were 6.5 mg/g (2006) and 6.8 mg/g (2007) for cultivars released before the 1970s, and 8.1 mg/g (2006) and 8.5 mg/g (2007) for cultivars released in the last decade. This observation suggests that grain and semolina colour has become a sign of quality worthy of note in durum, and that, in the last two decades, breeders have focused particular attention on high YPC during selection of new cultivars. YPC of var. turanicum accessions was comparable to the value detected in the oldest durum wheat cultivars, with mean values of 5.8 mg/g (range 4.7– 8.5 mg/g) and 6.8 mg/g (range 4.9–10.9 mg/g) in 2006 and 2007, respectively. A relatively high value was found in the accession PI 306665 (8.5 mg/g) in 2006 and in PI 278350 (10.9 mg/g) in 2007. The ssp. dicoccum and ssp. dicoccoides accessions showed mean values significantly lower than the durum wheat cultivars (5.6 and 6.1 mg/g in 2006 and 2007, respectively, for ssp. dicoccum, and 5.7 and 5.9 mg/g in 2006 and 2007, respectively, for ssp. dicoccoides), with small ranges of variation. Yellow index (b*) showed the same trend as yellow pigment concentration, with a range of variation of 13.3–19.1 in 2006 and 11.5–17.9 in 2007. Durum wheat cultivars showed the highest average value (16.4 in 2006 and 14.9 in 2007). High b* values were observed for the cvs. Cosmodur, Exeldur, Svevo, Grecale and Ambral in 2006, and for the cvs. Exeldur, Svevo, Normanno and Primadur in 2007. Accessions of var. turanicum exhibited b* values similar to
those of old durum wheat cultivars (range 14.1–18.7 in 2006, and 12.9-17.0 in 2007), while accessions of ssp. dicoccum (range 11.5– 15.0 in 2007) and ssp. dicoccoides (range 11.7–15.2 in 2007) had the lowest b* values. Brown index (100 – L*) is a qualitative parameter that provides an indication about the polyphenol oxidase activity in wheat grain and semolina. The brown index had a generally opposite trend compared to yellow index, with range of variations between 14.4 and 19.6 in 2006 and 14.0 and 18.5 in 2007. Accessions of ssp. dicoccum had the highest mean values for both trials, while the lowest values were detected in the durum cultivars. 3.2. Carotenoid components Eighty wheat accessions grown in 2006 were analysed by HPLC to determine the total amount of carotenoids and the relative quantities of the main carotenoid components: lutein, zeaxanthin, b-cryptoxanthin, a-carotene and b-carotene (Table 2). Fig. 1 shows a typical HPLC chromatogram for carotenoids of a durum wheat sample (cv. Primadur). Total carotenoid concentration varied from 1.178 mg/g (Timilia) to 4.416 mg/g (Svevo) with an average of 2.460 mg/g. The highest mean value was found in durum cultivars (2.728 mg/g), while the lowest mean value was found in the sp. dicoccum accessions (1.515 mg/g). Durum cultivars released after 1991 had a significantly higher mean value (3.109 mg/g) than cultivars released in the period 1971–1991 (2.560 mg/g) or before 1971 (2.327 mg/g). HPLC identified lutein as the main carotenoid component in the examined tetraploid wheat accessions (average 1.605 mg/g), followed by zeaxanthin (0.133 mg/g) and b-carotene (0.014 mg/g), while a-carotene (0.011 mg/g) and b-cryptoxanthin (0.008 mg/g) were minor components, in several cases present only in traces. HPLC analysis showed the presence of significant amounts of other non-identified carotenoid compounds (0.688 mg/g) that were grouped into a single class named as ‘‘other components’’. Most of these compounds could be geometric isomers of lutein and zeaxanthin (cis–trans) or traces of violaxanthin and neoxanthin resulting from epoxidation of zeaxanthin. Wide ranges of variation for all identified carotenoid components were observed in the wheat accessions. Lutein ranged from 0.699 mg/g (Farvento) to 3.078 mg/g (Svevo). Durum cultivars had the highest mean value (1.852 mg/g), while var. turanicum, ssp. dicoccum and ssp. dicoccoides accessions showed average values much lower than durum (1.296, 0.876 and 1.068 mg/g, respectively). Zeaxanthin had a range of variation from 0.850 to 0.230 mg/g. Among the tetraploid wheat sub-species, ssp. dicoccum and ssp. dicoccoides had the highest mean values (0.164 and 0.148 mg/g, respectively). The accession MG3533/52 of ssp. dicoccoides showed the highest value (0.230 mg/g) of the whole collection. The range of variation of b-carotene concentration was 0.006– 0.033 mg/g. Durum cultivars showed the highest mean value (0.015 mg/g), the commercial cultivars Meridiano, Normanno, Grecale and Dylan showing interesting values higher than 0.027 mg/g. Var. turanicum, ssp. dicoccum and ssp. dicoccoides accessions had lower values than durum, with a mean value of 0.013 mg/g and range from 0.006 to 0.022 mg/g. However, taking into account the total carotenoid concentration, wild accessions of ssp. dicoccum and ssp. dicoccoides showed higher percentages of b-carotene (mean value 0.84%) than durum (mean value 0.54%). Interestingly, the accessions MG 5473 and MG 5444/235 showed 1.0 and 1.2% of b-carotene, respectively. a-carotene and b-criptoxanthin were found in small amounts, below the detection limit in several wheat accessions. Mean values of 0.011 mg/g (range 0.002–0.030 mg/g) and 0.008 mg/g (range 0.002–0.014 mg/g) were observed for a-carotene and b-criptoxanthin, respectively, in the accessions where the two carotenoid components were detected.
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Table 1 Average values, least significant difference (LSD) and heritability (h2B) of yellow pigment content (AACC method 14-50) and colorimetric indexes (brown index and yellow index) in a T. turgidum collection evaluated at Valenzano (Bari, Italy) over two years (2006 and 2007). Sub-species
Cultivar or accession
Year of release
ssp. turgidum var. durum
Brindur Cannizzo Ceedur Ciccio Claudio Cosmodur Exeldur Iride Agridur Meridiano Parsifal Svevo Vesuvio Cirillo Duetto Dylan Fiore Grecale Normanno Tiziana Virgilio
1991–2008
Mean Range Appulo Creso Valnova Valgerardo Karel Produra Sansone Tito Ambral Primadur Arcangelo Duilio Grazia Latino Neodur Ofanto Simeto Tresor Messapia
1971–1990
Mean Range Timilia Russello Aziziah Grifoni Capeiti Cappelli Hymera Trinakria Kiperounda Polesine Taganrog Shram 5 Mean Range
Before 1971
Yellow pigment content (mg/g)
Brown index (100-L*)
Yellow index (b*)
2006
2007
2006
2007
2006
2007
8.7 6.1 8.3 7.4 7.4 9.7 9.3 7.5 7.3 9.3 6.8 10.5 7.0 7.1 9.2 8.3 5.7 10.6 9.3 6.0 8.0
10.1 6.9 6.9 8.0 6.5 9.3 11.2 7.4 7.8 9.4 6.6 10.5 8.3 8.0 11.1 9.4 5.9 10.1 10.4 6.1 8.1
16.0 16.5 15.6 17.7 15.0 16.3 17.1 15.4 15.9 15.8 16.0 16.1 16.4 16.7 14.5 16.0 15.8 16.1 16.5 15.5 16.2
14.9 15.7 17.1 15.1 14.0 15.2 16.5 15.1 15.2 15.0 14.7 15.3 15.6 15.1 15.5 14.8 14.7 14.8 15.8 15.1 15.2
17.2 15.7 16.6 17.2 16.2 18.5 18.4 16.2 16.8 17.8 15.8 19.1 15.6 17.2 16.8 17.4 15.4 18.7 17.5 15.6 16.9
16.1 14.2 13.8 14.9 13.8 16.1 16.9 14.6 15.5 15.9 13.6 16.9 14.9 15.2 16.4 15.6 13.5 16.1 16.6 14.2 14.9
8.1 (5.7–10.6)
8.5 (5.9–11.2)
16.1 (14.5–17.7)
15.3 (14.0–17.1)
17.0 (15.4–19.1)
15.2 (13.5–16.9)
6.7 6.5 7.9 5.2 5.8 5.4 4.8 6.4 8.5 9.9 6.5 6.4 6.5 5.4 7.9 7.1 7.4 8.4 4.0
6.4 8.2 8.6 6.9 8.1 7.8 5.9 8.3 9.5 12.3 7.8 6.0 7.8 5.3 9.6 8.5 7.8 8.5 4.4
16.6 16.4 16.8 17.1 15.8 15.9 16.4 16.0 16.5 15.4 16.2 14.4 16.9 15.3 16.4 16.1 17.2 15.4 15.0
15.3 15.1 15.3 14.9 15.6 15.4 16.5 15.8 15.1 15.6 16.0 14.4 15.3 14.4 15.5 15.2 15.5 15.8 14.3
16.4 16.1 17.3 16.2 14.9 15.2 13.7 15.9 18.5 17.4 15.7 15.2 17.9 15.1 17.8 16.9 16.6 16.7 13.3
13.9 14.5 14.9 14.9 15.5 15.5 13.3 15.5 16.1 17.9 15.3 13.8 15.8 13.2 16.4 15.4 14.8 15.8 12.3
6.7 (4.0–9.9)
7.8 (4.4–12.3)
16.1 (14.4–17.2)
15.3 (14.3–16.5)
16.1 (13.3–18.5)
15.0 (12.3–17.9)
3.8 7.2 6.9 7.4 8.2 5.6 7.9 6.5 7.0 4.3 5.4 7.8
4.1 8.1 6.6 8.1 8.3 6.5 8.8 6.5 6.7 4.1 6.8 7.5
17.4 17.0 16.0 15.9 16.8 15.9 15.6 16.4 15.9 17.2 15.1 15.5
16.8 15.9 15.4 15.7 14.8 15.8 15.3 15.4 14.9 14.8 15.2 15.4
13.0 16.2 17.0 16.7 17.8 14.5 16.5 16.0 15.5 14.2 14.5 16.6
12.0 15.4 14.2 15.6 15.0 14.6 15.6 14.7 13.7 12.0 14.4 15.4
6.5 (3.8–8.2)
6.8 (4.1–8.8)
16.2 (15.1–17.4)
15.5 (14.8–16.8)
15.7 (13.0–17.8)
14.4 (12.0–15.6)
(continued on next page)
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
214 Table 1 (continued )
Brown index (100-L*)
Yellow index (b*)
Sub-species
Cultivar or accession
2006
2007
2006
2007
2006
2007
ssp. turgidum var. turanicum
Kamut Cltr 11390 PI 68287 PI 113392 PI 113393 PI 167481 PI 254206 PI 278350 PI 306665 PI 576854 PI 623629 PI 623656 PI 624209 PI 254204 PI 624429 PI 290530
6.5 7.1 5.9 5.0 5.5 6.8 5.4 6.7 8.5 6.2 5.4 4.9 4.7 4.8 4.9 4.9
6.9 7.8 8.1 5.6 5.1 6.8 5.6 10.9 9.1 7.9 4.9 5.6 6.1 6.9 6.3 5.1
15.4 15.6 15.9 16.0 16.5 14.9 16.3 16.9 15.8 16.8 15.9 16.6 16.6 15.8 16.5 16.8
14.9 14.9 14.8 14.6 16.3 14.5 15.3 15.0 15.4 15.8 15.0 14.9 16.3 16.5 16.1 14.1
15.7 16.7 16.1 15.5 15.5 15.5 15.3 14.1 18.7 16.0 15.0 15.0 14.4 14.5 14.2 15.4
15.5 14.9 15.2 13.6 13.8 14.3 14.0 17.0 16.2 15.0 13.0 13.6 14.2 15.8 14.6 12.9
Mean Range
5.8 (4.7–8.5)
6.8 (4.9–10.9)
16.1 (14.9–16.9)
15.3 (14.1–16.5)
15.5 (14.1–18.7)
14.6 (12.9–17.0)
Lucanica Farvento MG 4375 MG 5473 MG 5350 MG 3521 MG 4387 MG 5323 MG 5416/1 MG 5471/1 MG 5471/2 MG 5471/3 MG 15516/1 MG 15516/3 MG 15516/4 MG 15516/5 MG 15516/6 MG 15516/7 MG 15516/8 MG 15516/9 MG 15516/10 MG 15516/11 MG 5344/1 MG 5344/2 MG 5300/1 MG 5300/3 MG 5293/1
– – 5.5 – – – – 5.6 – – – – – – – – – – – – – – – – – – –
6.8 4.9 7.7 5.3 4.4 5.9 6.6 5.5 8.3 5.5 4.5 5.1 5.9 6.1 6.2 6.7 6.4 7.3 5.6 7.5 5.9 6.1 5.7 6.4 5.2 5.7 7.4
– – 19.6 – – – – 18.5 – – – – – – – – – – – – – – – – – – –
18.5 16.5 18.0 17.4 16.1 16.5 18.5 16.8 17.2 16.1 16.6 16.7 18.0 17.5 17.2 17.2 17.2 18.4 17.9 16.7 17.7 17.0 16.5 17.3 16.5 17.0 18.0
– – 14.0 – – – 14.3 – – – – – – – – – – – – – – – – – – –
12.8 11.5 13.3 12.4 11.9 13.5 13.3 13.1 14.5 12.5 12.1 12.2 13.8 14.3 14.1 13.9 13.9 15.0 13.9 14.8 14.2 14.1 12.0 12.9 12.9 13.5 12.7
Mean Range
5.6 (5.5–5.6)
6.1 (4.4–8.3)
19.1 (18.6–19.6)
17.2 (16.1–18.5)
14.2 (14.0–14.3)
13.3 (11.5–15.0)
MG MG MG MG MG MG MG
5.3 5.3 6.6 5.4 – – –
3.9 7.6 8.3 6.3 5.0 5.3 5.1
18.0 16.7 16.2 18.9 – – –
17.4 14.6 15.6 17.2 14.2 17.6 16.1
18.0 16.7 16.2 18.9 – – –
11.7 15.2 14.9 12.1 12.7 12.7 13.4
5.7 (5.3–6.6)
5.9 (3.9–8.3)
17.5 (16.2–18.9)
16.1 (14.2–17.6)
17.5 (16.2–18.9)
13.2 (11.7–15.2)
6.7 (3.8–10.6) 0.6 0.94
7.1 (3.9–12.3) 0.9 0.91
16.3 (14.4–19.6) 1.1 0.59
15.9 (14.0–18.5) 0.9 0.79
16.0 (13.3–19.1) 1.1 0.81
14.3 (11.5–17.9) 0.6 0.94
ssp. dicoccum
ssp. dicoccoides
4328/61 29896 5510/7 5444/235 4337/198 5445 4330/66
Mean Range Mean Range LSD (0.05) h2B – ¼ not evaluated.
Year of release
Yellow pigment content (mg/g)
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Table 2 Average values (mg/g), least significant difference (LSD), and heritability (h2B) of carotenoid components in a T. turgidum collection evaluated at Valenzano (Bari, Italy) in 2006. Sub-species
Cultivar or accession Year of release Lutein
Zeaxanthin
b-crypto-xanthin a-carotene
b-carotene
Other componentsa Total carotenoids
ssp.turgidum var. durum
Brindur Cannizzo Ceedur Ciccio Claudio Cosmodur Exeldur Iride Agridur Meridiano Parsifal Svevo Vesuvio Cirillo Duetto Dylan Fiore Grecale Normanno Tiziana Virgilio
0.112 0.085 0.138 0.108 0.113 0.126 0.160 0.118 0.116 0.110 0.100 0.125 0.093 0.096 0.156 0.122 0.092 0.129 0.129 0.176 0.114
0.013 0.007 0.006 0.012 0.009 0.011 0.011 0.009 0.013 0.013 0.013 0.009 0.006 0.008 0.006 0.010 tr 0.013 0.008 tr 0.008
0.024 0.014 0.012 0.024 0.012 0.023 0.019 0.013 0.013 0.033 0.013 0.023 0.015 0.014 0.018 0.029 0.008 0.027 0.029 0.014 0.010
0.799 0.596 0.653 0.654 0.729 0.904 0.800 0.660 0.872 1.060 0.623 1.159 0.827 0.523 0.850 0.849 0.198 1.060 1.153 0.749 0.631
1991–2008
Mean Range Appulo Creso Valnova Valgerardo Karel Produra Sansone Tito Ambral Primadur Arcangelo Duilio Grazia Latino Neodur Ofanto Simeto Tresor Messapia
1971–1990
Mean Range Timilia Russello Aziziah Grifoni Capeiti Cappelli Hymera Trinakria Kiperounda Polesine Taganrog Shram 5
ssp. turgidum var. turanicum
Before 1971
2.487 1.332 1.939 1.843 2.164 2.843 2.606 2.037 1.980 2.367 1.611 3.078 1.625 1.869 2.421 2.510 1.559 2.947 2.822 1.352 2.105
0.018 0.010 0.012 0.012 0.013 0.022 0.020 0.007 0.020 0.026 0.013 0.023 0.019 0.012 0.011 0.017 tr 0.029 0.016 0.008 0.009
3.453 2.044 2.760 2.653 3.039 3.929 3.617 2.844 3.014 3.608 2.373 4.416 2.585 2.523 3.462 3.536 1.857 4.205 4.157 2.299 2.877
2.167 0.120 0.010 (1.332–3.078) (0.805–0.176) (0.006–0.013)
0.016 0.018 0.779 (0.007–0.029) (0.008–0.033) (0.198–1.159)
3.109 (1.857–4.416)
1.655 1.385 2.331 1.394 1.365 1.270 1.301 1.588 2.094 2.394 1.843 1.412 1.985 1.299 2.087 2.038 1.920 2.405 0.843
0.007 0.011 0.011 0.008 0.003 0.011 0.007 0.007 0.010 0.011 0.007 0.018 0.013 0.010 0.012 0.013 0.010 0.015 tr
2.353 2.160 3.366 2.032 1.924 2.236 1.819 2.236 3.179 3.532 2.727 2.170 2.777 2.134 3.281 2.770 3.087 3.472 1.352
0.091 0.128 0.101 0.154 0.140 0.096 0.123 0.141 0.122 0.159 0.097 0.098 0.122 0.104 0.123 0.088 0.100 0.178 0.122
0.006 0.002 0.004 0.007 tr tr 0.002 0.004 0.007 0.008 0.008 0.014 0.004 0.008 0.009 0.008 0.006 0.011 tr
0.008 0.016 0.013 0.014 0.007 0.012 0.007 0.017 0.017 0.021 0.009 0.010 0.011 0.013 0.018 0.016 0.018 0.020 0.008
0.586 0.617 0.907 0.455 0.409 0.847 0.380 0.479 0.929 0.939 0.763 0.619 0.642 0.700 1.032 0.606 1.033 0.844 0.379
1.716 0.120 0.007 (0.843–2.405) (0.088–0.178) (0.002–0.014)
0.010 0.013 0.693 (0.003–0.018) (0.007–0.021) (0.379–1.032)
2.560 (1.352–3.532)
0.721 1.421 1.358 2.012 2.395 1.176 1.861 1.561 1.563 0.861 1.381 1.898
tr 0.008 0.007 0.014 0.010 0.008 0.015 0.012 0.018 tr 0.006 0.011
1.178 2.420 2.166 2.954 3.345 1.769 2.808 2.362 2.471 1.458 2.201 2.737
0.134 0.184 0.126 0.170 0.126 0.150 0.131 0.124 0.131 0.109 0.163 0.126
tr 0.006 0.007 0.006 0.009 tr 0.010 0.011 tr tr 0.006 tr
0.010 0.012 0.009 0.013 0.013 0.013 0.015 0.010 0.020 0.007 0.013 0.010
0.313 0.789 0.660 0.740 0.793 0.422 0.776 0.644 0.739 0.481 0.633 0.692
Mean Range
1.517 0.140 0.008 (0.721–2.395) (0.109–0.184) (0.006–0.011)
0.011 0.012 0.640 (0.006–0.018) (0.007–0.020) (0.313–0.793)
2.327 (1.178–3.345)
Kamut Cltr 11390 PI 68287 PI 113392 PI 113393 PI 167481 PI 254206 PI 278350 PI 306665 PI 576854
1.586 1.977 1.521 0.955 1.097 1.367 1.006 1.837 2.135 1.290
0.018 0.030 0.007 0.002 tr 0.007 tr 0.008 0.010 0.007
2.509 2.836 2.381 1.705 1.729 2.351 1.829 2.826 4.329 2.432
0.152 0.141 0.154 0.134 0.132 0.157 0.113 0.113 0.197 0.130
0.006 0.010 0.005 0.005 0.008 tr tr 0.014 0.008 0.004
0.020 0.020 0.015 0.009 0.006 0.016 0.013 0.022 0.015 0.014
0.727 0.658 0.679 0.600 0.487 0.804 0.697 0.832 1.964 0.988
(continued on next page)
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
216 Table 2 (continued ) Sub-species
ssp. dicoccum
Cultivar or accession Year of release Lutein
Zeaxanthin
b-crypto-xanthin a-carotene
b-carotene
Other componentsa Total carotenoids
PI PI PI PI PI PI
0.135 0.141 0.130 0.160 0.148 0.137
tr 0.005 tr tr tr 0.007
0.009 0.014 0.009 0.007 0.009 0.009
0.581 0.595 0.547 0.567 0.563 0.387
623629 623656 624209 254204 624429 290530
tr 0.007 0.002 tr tr tr
1.735 1.609 1.628 1.768 1.817 1.576
Mean Range
1.296 0.142 0.007 (0.848–2.315) (0.113–0.197) (0.004–0.014)
0.010 0.013 0.730 (0.002–0.030) (0.006–0.022) (0.387–1.964)
2.198 (1.576–4.329)
Farvento MG 5473 MG 5350 MG 3521 MG 5323
0.699 0.891 0.935 0.960 0.894
tr 0.006 tr 0.002 0.004
1.265 1.552 1.508 1.583 1.669
Mean Range
0.876 0.164 (0.699–0.960) (0.130–0.176)
ssp. dicoccoides MG MG MG MG MG MG MG MG
4328/61 29896 5510/7 5444/235 5445 4330/66 3533/52 4343
Mean Range Mean Range LSD (0.05) h2B
1.010 0.848 0.940 1.034 1.097 1.036
0.710 1.391 1.157 0.773 1.111 0.892 1.570 0.943
0.173 0.176 0.130 0.166 0.173
0.143 0.098 0.129 0.166 0.113 0.118 0.230 0.189
0.004 tr tr tr 0.004
tr 0.010 tr tr tr tr tr tr
1.068 0.148 (0.710–1.570) (0.098–0.230) 1.605 (0.699–3.078) 0.265 0.93
0.133 (0.850–0.230) 0.041 0.48
0.008 (0.002–0.014) 0.004 0.72
0.010 0.016 0.014 0.009 0.014
0.379 0.463 0.429 0.446 0.580
0.004 0.013 0.459 (0.002–0.006) (0.009–0.016) (0.379–0.580)
1.515 (1.265–1.669)
0.006 0.008 0.011 0.007 0.011 0.010 tr tr
1.276 2.083 2.114 1.424 1.856 1.748 2.491 1.510
0.009 0.014 0.016 0.017 0.015 0.016 0.017 0.009
0.408 0.561 0.801 0.461 0.606 0.712 0.674 0.368
0.009 0.014 0.574 (0.006–0.011) (0.009–0.017) (0.368–0.801)
1.823 (1.276–2.491)
0.011 (0.002–0.030) 0.006 0.79
2.460 (1.178–4.416) 0.300 0.94
0.014 (0.006–0.033) 0.007 0.57
0.688 (0.198–1.964) 0.279 0.63
tr ¼ traces. a Obtained as difference between total carotenoid and the sum of the identified components.
3.3. Heritability and correlation between yellow pigments and grain yield components Broad-sense heritability, as estimated on an accession mean basis, was found to be high for yellow pigment concentration (0.94 and 0.91 in 2006 and 2007, respectively) and yellow index (0.81 and 0.94 in 2006 and 2007, respectively), and relatively high for brown index (0.59 and 0.79 in 2006 and 2007, respectively) (Table 1). High heritability was also found for total carotenoid concentration (0.94) and lutein (0.93), intermediate values for b-cryptoxanthin (0.72) and a-carotene (0.79), and relatively low values for b-carotene (0.57) and zeaxanthin (0.48) (Table 2). Correlation analysis revealed highly significant and positive relationships (P 0.001) among single carotenoid components and
total carotenoid concentration determined by HPLC (data not shown). Total carotenoid concentration was strongly correlated with yellow pigment concentration (0.94; P 0.001) and with yellow index (0.88; P 0.001) (Table 3). A significant correlation was also observed between the last two parameters (0.88 and 0.90 in 2006 and 2007 trials, respectively; P 0.001). Significant negative correlations were observed for YPC and yellow index with grain protein concentration (P 0.01–0.001). These correlations were significant for each of the two trials, suggesting consistent relationships. Total carotenoid concentration, yellow pigment concentration and yellow index were found to be positively correlated with grain yield per spike (P 0.01–0.001). The three colour parameters showed negative correlation values with 1000kernel weight, but only the correlations between yellow pigment concentration and 1000-kernel weight in 2006, and between yellow index and 1000-kernel weight in 2007 were significant at P 0.05. Brown index was positively correlated with grain protein concentration (P 0.01 and P 0.001 in 2006 and 2007, respectively) and negatively correlated with grain yield per spike (P 0.01 and P 0.001 in 2006 and 2007, respectively), total carotenoid concentration (P 0.05) in 2006, yellow pigment concentration (P 0.05) and yellow index (P 0.01) in 2007. 4. Discussion and conclusions
Fig. 1. High-performance liquid chromatogram of carotenoids extracted from durum wheat cultivar Primadur.
Wheat breeding has generally focused on improving yield and yield stability, disease resistances and technological properties, mostly gluten quality and quantity, and to a lesser extent, colour. Yellow pigment of durum wheat semolina has recently received considerable attention to improve the yellow colour in end-products such as pasta, and the nutritional value of carotenoid
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
217
Table 3 Correlations between yellow pigment content (AACC method 14-50), total carotenoid content (HPLC), colorimetric indexes (brown and yellow indexes), grain protein content, kernel weight and grain yield per spike in a T. turgidum collection grown at Valenzano (Bari, Italy) in 2006 and 2007. Trait
Trial
Total carotenoid pigments
Yellow pigment content
2006
0.94***
Yellow index
2006 2007
0.88***
Brown index
2006 2007
0.24*
0.22 0.23*
0.16 0.33**
Grain protein content
2006 2007
0.40**
0.34** 0.32**
0.41*** 0.31**
Kernel weight
2006 2007
0.08
0.29* 0.17
0.21 0.24*
Grain yield per spike
2006 2007
0.49***
Yellow pigment content
Yellow index
Brown index
0.88*** 0.90***
0.34** 0.24**
0.38** 0.17
0.39** 0.61*** 0.01 0.13 0.31** 0.37***
*, **, ***: Significant differences at 0.05P, 0.01P and 0.001P, respectively.
components. Thus, increasing yellow colour is a valuable goal for improving the commercial and nutritional values of durum wheat products. Yellow pigment analysis of 102 accessions showed highly significant differences (P < 0.001) in the grain pigment concentration among the wheat genotypes in two trials. The results showed wide variation of YPC, ranging from the highest value of the elite cultivar Svevo (10.5 mg/g) of durum wheat to the lowest of the old cultivar Timilia (3.8 mg/g). Overall, modern commercial cultivars showed significantly higher values compared to traditional cultivars and wild ssp. dicoccum and ssp. dicoccoides accessions. This can be explained by the fact that modern durum breeding programs have selected genotypes with high pigment concentrations because yellow colour has been considered in the last two decades to be an important quality parameter with regard to pasta production. Although environmental factors can influence yellow pigment concentration, the genetic component is predominant because heritability values detected in 2006 and 2007 trials were more than 0.90, confirming previous studies (Clarke et al., 2006; Elouafi et al., 2001; Veronico et al., 2001) that underlined the polygenic nature but also the strong genotypic component of yellow pigment concentration in durum wheat. Total carotenoid concentration determined by HPLC varied between 1.178 and 4.416 mg/g (average 2.460 mg/g), in agreement with that found by others authors (Fortmann and Joiner, 1978; Fratianni et al., 2005; Hentschel et al., 2002; Hidalgo et al., 2006; Leenhardt et al., 2006) in durum and bread wheats. Higher average value (8.41 mg/g) was found in a collection of 54 accessions of einkorn wheat (T. monococcum L.) by Hidalgo et al. (2006), in part due to the lower kernel weight of einkorn in comparison to durum and to the negative relationship between carotenoid concentration and kernel weight (see later) found in cereals. Lutein (65.2%) was the main component of carotenoids, followed by zeaxanthin (5.4%) and b-carotene (0.6%); a-carotene (0.5%) and b-cryptoxanthin (0.3%) were minor components, in several cases present only in traces. HPLC analysis also showed the presence of significant quantities of other not identified carotenoid compounds (28.0%). Lutein was always found to be the major carotenoid in diploid, tetraploid and hexaploid wheats, even if the proportion varied greatly in different assessments (Adom et al., 2003; Fortmann and Joiner, 1978; Fratianni et al., 2005; Hentschel et al., 2002; Hidalgo et al., 2006; Leenhardt et al., 2006; Serpen et al., 2008). The relative proportion of zeaxanthin, b-cryptoxanthin, a-carotene and bcarotene reported by these authors varied from traces or not detectable quantities to significantly high proportion of a-carotene (6.3%) in einkorn wheat (Hidalgo et al., 2006), and of b-carotene (3.7%) (Fratianni et al., 2005) and zeaxanthin (8.5%) (Panfili et al.,
2004) in durum. The components with higher pro-vitamin A activity (b-carotene, a-carotene, b-cryptoxanthin) showed wide variation (from 0.015 to 0.072 mg/g) indicating the need for screening cultivated and wild germplasm to identify accessions with higher concentration of specific carotenoids, and the possibility of improving the pro-vitamin A activity of commercial cultivars via traditional breeding and selection. Noteworthy, the durum cultivar Meridiano and the var. turanicum accession Cltr11390 had the highest values of pro-vitamin A components expressed as absolute amount (0.072 and 0.060 mg/g, respectively) and as percentage of the total carotenoid concentration (2.0 and 2.1%, respectively). The apparently high differences in the relative proportion of carotenoid components reported in the literature may be attributed to genetic effects, environmental conditions and agronomic practices, but also to different extraction methods, internal standards, milling fractions, particle size of semolina and presence of peripheral parts, HPLC procedures, etc. Total carotenoid concentration, as determined by HPLC, compared with yellow pigment concentration, measured by AACC method 14-50, showed that the portion of carotenoids amounted to 33.2% of the yellow pigments in the 80 tetraploid wheat accessions examined in the 2006 trial. This implies that there are unknown colour-producing compounds in the durum extracts absorbing light at 435.6 nm. This observation is in accordance with results provided by Hentschel et al. (2002) and by Leenhardt et al. (2006). Hentschel et al. (2002) found that the carotenoid fraction of yellow pigment concentration amounted to only 30–50% in eight durum cultivars, and concluded that there are other compounds not yet identified that contribute considerably to the yellow colour of the grain extracts. In contrast, Burkhardt and Bo¨hm (2007) and Fratianni et al. (2005) showed close correspondence between reflectance measurements and total carotenoid concentration determined on durum cultivars by both the standard AACC 14-50 or ICC 152 methods and the HPLC procedure. Pigment concentration demonstrated a slightly negative correlation with kernel weight, as did yellow index in one trial. This negative correlation was also found by Alvarez et al. (1999), Clarke et al. (2006), Marais (1992), Veronico et al. (2001) and Zhang et al. (2008), and could be partly attributed to dilution effects, with increases in grain starch in large kernels reducing the proportion of pigments. The highly significant negative correlations observed between yellow pigment parameters with grain protein concentration are particularly important for durum breeding programs. The genetic basis of trait correlations may include single genes with pleiotropic effects or the tight linkage of several genes controlling different traits. Combining quantitative trait loci (QTLs) for high pigment concentration with those for high grain protein
218
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
concentration will require detailed information on the chromosomal position and effects of each QTL controlling all of these traits in the genetic material under study. In agreement with results published by Fratianni et al. (2005), highly significant correlations were found between reflectance measurement (b* index) and yellow pigment concentration determined by both standard AACC method and HPLC procedure, thus confirming the usefulness of yellow index as a fast and safe method for screening yellow pigments in large-scale wheat breeding programs. Indeed, the development of a rapid screen for kernel and semolina colour has facilitated the increased grain yellow colour of new durum cultivars developed in the last two decades, while marker-assisted selection may allow additional improvement in the future. Yellow index, although strongly related to pigment concentration, provides only relative and not absolute values of grain or semolina colour and does not provide information on the proportional composition of carotenoids. The HPLC technique, although expensive, resulted in a sensitive, selective and reliable method for determining the qualitative and quantitative distribution of carotenoid compounds in cereals (Hentschel et al., 2002; Panfili et al., 2004). In view of the increasing importance of nutritional aspects in the definition of food quality, complete information on the estimation of colour and nutritional value of grain, semolina and end-products of durum and bread wheats may be obtained by combining the b* index and HPLC techniques. The present survey of 102 accessions of wild and cultivated tetraploid wheats demonstrated marked variations in the carotenoid composition and concentration. Such information is useful for plant breeders in screening and selecting cultivars with high yellow colour and enhanced phytochemical concentrations, and provides valuable information for evaluating potential contributions to health from the consumption of durum wheat end-products. Acknowledgements This research was supported by grants from the Ministero dell’Universita` e della Ricerca, Italy, projects ‘FISR’ and ‘AGROGEN’, and by the EU grant DEVELONUTRI. References AACC (American Association of Cereal Chemists), 2000. AACC Official Method 14-50. In: Approved Methods of the American Association of Cereal Chemists, tenth ed. St. Paul, MN, USA. Adom, K.K., Sorrells, M.E., Liu, R.H., 2003. Phytochemical profiles and antioxidant activity of wheat varieties. Journal of Agricultural and Food Chemistry 51, 7825–7834. Alvarez, J.B., Martin, L.M., Martin, A., 1999. Genetic variation for carotenoid pigment content in the amphiploid Hordeum chilense Triticum turgidum conv. durum. Plant Breeding 118, 187–189. Borrelli, G.M., De Leonardis, A.M., Fares, C., Platani, C., Di Fonzo, N., 2003. Effects of modified processing conditions on oxidative properties of semolina dough and pasta. Cereal Chemistry 80, 225–231. Burkhardt, S., Bo¨hm, V., 2007. Development of a new method for the complete extraction of carotenoids from cereals with special reference to durum wheat (Triticum durum Desf.). Journal of Agricultural and Food Chemistry 55, 8295– 8301. CIE (Commission Internationale de l’Eclairage), 1986. Publication 15.2. Colorimetry, second ed. CIE Central Bureau Kegelgasse, Wien, Austria. 27-A-1030. Clarke, F.R., Clarke, J.M., McCaig, T.N., Knox, R.E., DePauw, R.M., 2006. Inheritance of yellow pigment concentration in four durum wheat crosses. Canadian Journal of Plant Science 86, 133–141. Della Penna, D., Pogson, B.J., 2006. Vitamin synthesis in plants: tocopherols and carotenoids. Annual Review of Plant Biology 57, 711–738.
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Genetic variability in yellow pigment components in cultivated and wild tetraploid wheats A.M. Digesu` a, C. Platani a, L. Cattivelli a, G. Mangini b, A. Blanco b, * a b
CRA – Cereal Research Centre, S.S. 16 km 6757, 1100 Foggia, Italy Department of Environmental and Agro-Forestry Biology and Chemistry, sect. Genetic and Plant Breeding, University of Bari, Via Amendola 165/A, 70126 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2009 Received in revised form 1 May 2009 Accepted 15 May 2009
Yellow pigment concentration (YPC) in durum wheat is an important criterion in the assessment of semolina quality, particularly in determining the commercial and nutritional quality of end-products. Genetic variability of YPC and carotenoid components was analysed in 102 wild and cultivated tetraploid wheat accessions in two trials. Overall, modern cultivars showed significantly higher values of YPC compared to old cultivars and wild ssp. dicoccum and ssp. dicoccoides accessions. Total carotenoid concentration varied between 1.178 and 4.416 mg/g with an average of 2.460 mg/g. The portion of carotenoids amounted to 33.2% of the YPC in 80 wheat accessions examined in the 2006 trial. Lutein was the main component of carotenoids, followed by zeaxanthin and b-carotene. a-carotene and b-cryptoxanthin were minor components. Pigment concentration was negatively correlated with kernel weight and grain protein concentration. Significant positive correlations were found between b* index and YPC. Knowledge of the carotenoid composition and concentration is useful for wheat breeders in the development of cultivars with high yellow colour and enhanced phytochemical concentrations, and provides valuable information for evaluating contributions to health benefits from the consumption of durum wheat end-products. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids Yellow pigments HPLC T. turgidum
1. Introduction Yellow pigment concentration in durum wheat is a criterion in the assessment of semolina quality and is of particular importance in determining the commercial and nutritional quality of endproducts such as pasta. Yellow to amber colour is generally preferred by consumers rather than a brown or cream. Semolina colour is the result of carotenoid pigment concentration of the grain and any losses during storage of the grain or semolina due to carotenoid oxidative degradation by lipoxygenase during processing, as well as processing conditions (for a review see Pagnotta et al., 2005; Troccoli et al., 2000). Carotenoids are among the most important natural pigments, having wide distribution, different structure and numerous biological functions. In plants, they are part of the light harvesting
Abbreviations: HPLC, high-performance liquid chromatography; YPC, yellow pigment concentration. * Corresponding author. Tel.: þ39 0039 080 5442992; fax: þ39 0039 080 5442200. E-mail address: [email protected] (A. Blanco). 0733-5210/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.05.002
complexes, involved in photo-oxidative protection, serve as precursors of the hormone abscisic acid and provide the yellow, orange and red colours in many flowers and fruits (Della Penna and Pogson, 2006; Hirschberg, 2001). Two distinct classes of carotenoids are recognised: carotenes, which are tetraterpenoid hydrocarbons, and xanthophylls, which contain one or more oxygen groups (Van den Berg et al., 2000). In humans, the nutritional importance of carotenoids comes mainly from the pro-vitamin A activity of b-carotene, a-carotene, b-cryptoxanthin and others, with at least one intact non-oxygenated b-ionone ring (Zile, 1998). In addition to their role as precursors of vitamin A, carotenoids have been associated with a reduced risk of cancer, decrease in cardiovascular diseases, protection of the macula region of the retina and prevention of cataracts, and increased levels of iron absorption (Garcia-Casal, 2006; Granado et al., 2003; Le Marchand et al., 1993; Mares-Perlman et al., 2002). Several analytical procedures have been developed to evaluate yellow pigment concentration in durum semolina and pasta (see review by Fratianni et al., 2005). The most popular colour measurement instruments in the food industry are based on the colour-space system L*, a*, b* (CIE, Commission Internationale de l’Eclairage, 1986) due to the safety and rapidity of the technique
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and good correlation between chemical determinations and reflectance measurements (Fratianni et al., 2005; Johnston et al., 1980). Yellow pigments are usually analysed by using ICC method 152 (ICC, 1990) or AACC method 14-50 (AACC, 2000), both based on the extraction of pigments from semolina or pasta with watersaturated n-butyl alcohol and subsequent spectrophotometrical determination. High-performance liquid chromatography (HPLC) is widely accepted as an accurate and sensitive technique to allow the separation and identification of carotenoid components (Panfili et al., 2004). Specific HPLC procedures to detect carotenoids in durum grain and end-products have been developed by Burkhardt and Bo¨hm (2007), Fratianni et al. (2005), Hentschel et al. (2002). There is little literature related to the HPLC analysis of carotenoids in durum wheat and data have been reported on carotenoid composition of small numbers of samples (from one to ten cultivars). The concentration of carotenoids is higher in durum (from 1.50 to 5.00 mg/g) than in bread wheat (from 0.50 to 2.00 mg/g) (Fratianni et al., 2005; Hentschel et al., 2002; Hidalgo et al., 2006; Leenhardt et al., 2006) as a result of genetic selection for high yellow pigment concentration in durum wheat breeding programs. Inheritance of grain or semolina pigment concentration is complex, but overall heritability of pigment concentration was found to be reasonably high (from 0.34 to 0.95) (Clarke et al., 2006). The objective of this study was to explore the genetic variability of yellow pigment concentration and carotenoid components of a tetraploid wheat collection including 102 accessions of wild and cultivated wheats belonging to different sub-species of Triticum turgidum L. This will provide information on the existing variability and genetic resources available to breeders to improve colour and nutritional value of pasta and other durum wheat endproducts.
2. Experimental 2.1. Plant materials A collection of 102 tetraploid wheat accessions (T. turgidum L.), including 52 old and modern cultivars of durum wheat (ssp. turgidum var. durum), 16 accessions of ssp. turgidum var. turanicum, 27 accessions of spp. dicoccum and 7 wild accessions of ssp. dicoccoides, were evaluated for yellow pigment concentration, yellow and brown indexes, some grain yield traits (yield per spike, kernel weight, grain protein concentration) over two years (2006 and 2007). Eighty accessions grown in 2006 were analysed by HPLC for chemical determination of carotenoid components. The wheat collection was grown in the field at the University of Bari at Valenzano (Bari, Italy), in a randomized complete block design with three replications. In both experiments, each accession was planted at a rate of 30 seeds per row 1 m long with 30 cm between rows. During the growing season, standard cultivation practices were used. Plots were hand harvested at maturity and grain yield per spike was calculated as grain yield per row/number of spikes per row (about 50–60 spikes). A 15 g seed sample per plot was used to determine 1000-kernel weight. Grain protein concentration, expressed as percentage of protein on a dry weight basis, was directly obtained on a 2 g sample of whole-meal flour using nearinfrared reflectance spectroscopy. Yellow pigments were analysed by AACC method 14-50, the colorimeter system and high-performance liquid chromatography (HPLC). To minimize the degradation of pigments by oxidative enzymes, the samples were ground in a laboratory mill (Cyclotec Sample Mill, Tecator) with a 1 mm sieve and the resulting whole flour stored at 4 C for a maximum of 24 h before analysis.
211
2.2. Yellow pigment concentration The evaluation of yellow pigment concentration (YPC) was made according to AACC Approved Method 14-50 (AACC, 2000) with slight modifications, as described by Fares et al. (1991): 1 g of each sample was extracted with 5 mL of water-saturated n-butyl alcohol on an orbital incubator (Gallenkamp INR-200) for 3 h at 260 oscillations per minute. Samples were centrifuged for 7 min at 2417 xg (Beckman JA21) and absorbance of n-butyl alcohol extracts was measured by a Beckman DU-65 UV-spectrophotometer at 435.8 nm (the wavelength of maximum absorption of lutein, taking into account that this xanthophyll is the dominant carotenoid in durum wheat). To improve reliability of the analysis, calibration of the instrument was carried out by reading a blank solvent extraction. Pigment concentration was calculated using the extinction coefficient of lutein and the Lambert–Beer Law (Rodriguez-Amaya and Kimura, 2004). The reported data are the mean of three replications. 2.3. Brown and yellow indexes Brown and yellow indexes were determined using the reflectance colorimeter Chroma Meter CR-300 (Minolta) equipped with a pulsed xenon arc lamp. Absolute measurement of L*, a*, b* (CIE, 1986) coordinates in the Munsell colour system were taken using D65 lightning. Samples to be analysed were placed in a granular material support. Brown index was calculated as 100-L* (Borrelli et al., 2003). Results were the average of three replications. 2.4. Carotenoid components Lutein, zeaxanthin, b-cryptoxanthin, a-carotene and b-carotene standards were obtained from LGC Promochem (LGC Promochem, Milano, Italy). All other reagents were of analytical or HPLC grade and were purchased form Levanchimica (Bari, Italy). Stock solutions of carotenoid standards were dissolved in ethanol, degassed to remove oxygen, and stored at 20 C. Concentrations were determined spectrophotometrically using the Lambert–Beer Law (Rodriguez-Amaya and Kimura, 2004). Solutions were diluted in methanol: di-chloromethane (MeOH: DCM 45:55 v/v) to make calibration curves. Carotenoid pigment extraction was performed according to Konopka et al. (2005) with some modifications. Briefly, 2 g of each sample was extracted under nitrogen in a screwcapped tube by adding 4 mL of extraction buffer (hexane/acetone, 80:20 v/v) and 600 mL of butyled hydroxytoluene (BHT) (0.1% w/v) as an anti-oxidant. Samples were stirred for a few minutes and kept in the dark for 16 h. Samples were then centrifuged for 10 min at 3000 rpm (Heraeus Sepatech), supernatants placed in glass tubes and residues extracted once again. The organic layer was collected and filtered with a gyroscope filter for syringe PTFE (porosity of 0.45 mm). Then, 5 mL of extract was evaporated to dryness under a nitrogen stream. Finally, the dry residues were re-dissolved in 200 mL of MeOH: DCM (45:55 v/v). A sample volume of 20 mL was injected into an Agilent Technologies 1100 HPLC system equipped with an automatic sampler and a diode array detector (DAD). Separation was done on a YMC C30 column (250 4.6 mm i.d., particle size ¼ 5 mm). The mobile phase was methanol and methyltert-butyl ether (89:11 v/v) at a constant flow rate of 1 mL/min. The mobile phase was previously degassed by sonication for 10 min. Spectrophotometric detection was achieved by means of a diode array detector in the range 400–600 nm. Peaks were detected at 450 nm. Carotenoids were identified through their characteristic spectra and comparison of retention times with known standard solutions. Calibration curves were constructed using five different concentrations of lutein (between 0.125 and 22.05 mg/mL),
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zeaxanthin (between 0.6 and 6.45 mg/mL), b- cryptoxanthin (between 0.1 and 1 mg/mL), a-carotene (between 0.08 and 0.85 mg/ mL) and b-carotene (between 0.05 and 0.6 mg/mL). Carotenoid concentrations were calculated using a linear regression (concentration versus area) of the five-point standard curve. Results were the average of three replications. 2.5. Statistical analysis Standard ANOVA (mixed model analysis of variance with genotype as fixed factor and blocks as random factor) was performed using the software MSTAT-C. When significant differences were detected, Fisher’s least significant difference (LSD) was computed. Genetic variance (s2G) and environmental variance (s2E) were obtained by using the standard ANOVA. Heritability (h2) was estimated by the ratio s2G/s2P, where s2P is phenotypic variance (s2P ¼ s2G þ s2E). Pearson phenotypic correlation coefficients were calculated between yellow pigment concentration, yellow and brown indexes, carotenoid components and grain yield components in each environment. 3. Results 3.1. Yellow pigment concentration and colorimetric indexes Yellow pigment concentration (YPC) and yellow (b*) and brown (100-L*) indexes of the tetraploid wheat collection evaluated over two years (2006 and 2007) are reported in Table 1. Wheat accessions are grouped by sub-species and cultivated durums grouped by year of release. YPC varied between 3.8 and 10.6 mg/g in 2006 and between 3.9 and 12.3 mg/g in 2007, with average values of 6.7 and 7.1 mg/g, respectively. Durum wheat cultivars (ssp. turgidum var. durum) showed the highest mean values (7.2 mg/g in 2006 and 7.8 mg/g in 2007). The cvs. Primadur, Svevo, Grecale, Normanno, Duetto, Exeldur and Brindur had significantly higher YPC (>10 mg/g) than the others. Notably, all of the most popular cultivars in Italy (Simeto, Iride, Duilio, Ciccio, Claudio and Creso) showed a yellow pigment concentration higher than 6.5 mg/g, a value sufficient to meet the durum market needs. When the durum cultivars are grouped by year of release, the results indicated that most recent varieties had significantly higher YPC than old cultivars: mean values of YPC were 6.5 mg/g (2006) and 6.8 mg/g (2007) for cultivars released before the 1970s, and 8.1 mg/g (2006) and 8.5 mg/g (2007) for cultivars released in the last decade. This observation suggests that grain and semolina colour has become a sign of quality worthy of note in durum, and that, in the last two decades, breeders have focused particular attention on high YPC during selection of new cultivars. YPC of var. turanicum accessions was comparable to the value detected in the oldest durum wheat cultivars, with mean values of 5.8 mg/g (range 4.7– 8.5 mg/g) and 6.8 mg/g (range 4.9–10.9 mg/g) in 2006 and 2007, respectively. A relatively high value was found in the accession PI 306665 (8.5 mg/g) in 2006 and in PI 278350 (10.9 mg/g) in 2007. The ssp. dicoccum and ssp. dicoccoides accessions showed mean values significantly lower than the durum wheat cultivars (5.6 and 6.1 mg/g in 2006 and 2007, respectively, for ssp. dicoccum, and 5.7 and 5.9 mg/g in 2006 and 2007, respectively, for ssp. dicoccoides), with small ranges of variation. Yellow index (b*) showed the same trend as yellow pigment concentration, with a range of variation of 13.3–19.1 in 2006 and 11.5–17.9 in 2007. Durum wheat cultivars showed the highest average value (16.4 in 2006 and 14.9 in 2007). High b* values were observed for the cvs. Cosmodur, Exeldur, Svevo, Grecale and Ambral in 2006, and for the cvs. Exeldur, Svevo, Normanno and Primadur in 2007. Accessions of var. turanicum exhibited b* values similar to
those of old durum wheat cultivars (range 14.1–18.7 in 2006, and 12.9-17.0 in 2007), while accessions of ssp. dicoccum (range 11.5– 15.0 in 2007) and ssp. dicoccoides (range 11.7–15.2 in 2007) had the lowest b* values. Brown index (100 – L*) is a qualitative parameter that provides an indication about the polyphenol oxidase activity in wheat grain and semolina. The brown index had a generally opposite trend compared to yellow index, with range of variations between 14.4 and 19.6 in 2006 and 14.0 and 18.5 in 2007. Accessions of ssp. dicoccum had the highest mean values for both trials, while the lowest values were detected in the durum cultivars. 3.2. Carotenoid components Eighty wheat accessions grown in 2006 were analysed by HPLC to determine the total amount of carotenoids and the relative quantities of the main carotenoid components: lutein, zeaxanthin, b-cryptoxanthin, a-carotene and b-carotene (Table 2). Fig. 1 shows a typical HPLC chromatogram for carotenoids of a durum wheat sample (cv. Primadur). Total carotenoid concentration varied from 1.178 mg/g (Timilia) to 4.416 mg/g (Svevo) with an average of 2.460 mg/g. The highest mean value was found in durum cultivars (2.728 mg/g), while the lowest mean value was found in the sp. dicoccum accessions (1.515 mg/g). Durum cultivars released after 1991 had a significantly higher mean value (3.109 mg/g) than cultivars released in the period 1971–1991 (2.560 mg/g) or before 1971 (2.327 mg/g). HPLC identified lutein as the main carotenoid component in the examined tetraploid wheat accessions (average 1.605 mg/g), followed by zeaxanthin (0.133 mg/g) and b-carotene (0.014 mg/g), while a-carotene (0.011 mg/g) and b-cryptoxanthin (0.008 mg/g) were minor components, in several cases present only in traces. HPLC analysis showed the presence of significant amounts of other non-identified carotenoid compounds (0.688 mg/g) that were grouped into a single class named as ‘‘other components’’. Most of these compounds could be geometric isomers of lutein and zeaxanthin (cis–trans) or traces of violaxanthin and neoxanthin resulting from epoxidation of zeaxanthin. Wide ranges of variation for all identified carotenoid components were observed in the wheat accessions. Lutein ranged from 0.699 mg/g (Farvento) to 3.078 mg/g (Svevo). Durum cultivars had the highest mean value (1.852 mg/g), while var. turanicum, ssp. dicoccum and ssp. dicoccoides accessions showed average values much lower than durum (1.296, 0.876 and 1.068 mg/g, respectively). Zeaxanthin had a range of variation from 0.850 to 0.230 mg/g. Among the tetraploid wheat sub-species, ssp. dicoccum and ssp. dicoccoides had the highest mean values (0.164 and 0.148 mg/g, respectively). The accession MG3533/52 of ssp. dicoccoides showed the highest value (0.230 mg/g) of the whole collection. The range of variation of b-carotene concentration was 0.006– 0.033 mg/g. Durum cultivars showed the highest mean value (0.015 mg/g), the commercial cultivars Meridiano, Normanno, Grecale and Dylan showing interesting values higher than 0.027 mg/g. Var. turanicum, ssp. dicoccum and ssp. dicoccoides accessions had lower values than durum, with a mean value of 0.013 mg/g and range from 0.006 to 0.022 mg/g. However, taking into account the total carotenoid concentration, wild accessions of ssp. dicoccum and ssp. dicoccoides showed higher percentages of b-carotene (mean value 0.84%) than durum (mean value 0.54%). Interestingly, the accessions MG 5473 and MG 5444/235 showed 1.0 and 1.2% of b-carotene, respectively. a-carotene and b-criptoxanthin were found in small amounts, below the detection limit in several wheat accessions. Mean values of 0.011 mg/g (range 0.002–0.030 mg/g) and 0.008 mg/g (range 0.002–0.014 mg/g) were observed for a-carotene and b-criptoxanthin, respectively, in the accessions where the two carotenoid components were detected.
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Table 1 Average values, least significant difference (LSD) and heritability (h2B) of yellow pigment content (AACC method 14-50) and colorimetric indexes (brown index and yellow index) in a T. turgidum collection evaluated at Valenzano (Bari, Italy) over two years (2006 and 2007). Sub-species
Cultivar or accession
Year of release
ssp. turgidum var. durum
Brindur Cannizzo Ceedur Ciccio Claudio Cosmodur Exeldur Iride Agridur Meridiano Parsifal Svevo Vesuvio Cirillo Duetto Dylan Fiore Grecale Normanno Tiziana Virgilio
1991–2008
Mean Range Appulo Creso Valnova Valgerardo Karel Produra Sansone Tito Ambral Primadur Arcangelo Duilio Grazia Latino Neodur Ofanto Simeto Tresor Messapia
1971–1990
Mean Range Timilia Russello Aziziah Grifoni Capeiti Cappelli Hymera Trinakria Kiperounda Polesine Taganrog Shram 5 Mean Range
Before 1971
Yellow pigment content (mg/g)
Brown index (100-L*)
Yellow index (b*)
2006
2007
2006
2007
2006
2007
8.7 6.1 8.3 7.4 7.4 9.7 9.3 7.5 7.3 9.3 6.8 10.5 7.0 7.1 9.2 8.3 5.7 10.6 9.3 6.0 8.0
10.1 6.9 6.9 8.0 6.5 9.3 11.2 7.4 7.8 9.4 6.6 10.5 8.3 8.0 11.1 9.4 5.9 10.1 10.4 6.1 8.1
16.0 16.5 15.6 17.7 15.0 16.3 17.1 15.4 15.9 15.8 16.0 16.1 16.4 16.7 14.5 16.0 15.8 16.1 16.5 15.5 16.2
14.9 15.7 17.1 15.1 14.0 15.2 16.5 15.1 15.2 15.0 14.7 15.3 15.6 15.1 15.5 14.8 14.7 14.8 15.8 15.1 15.2
17.2 15.7 16.6 17.2 16.2 18.5 18.4 16.2 16.8 17.8 15.8 19.1 15.6 17.2 16.8 17.4 15.4 18.7 17.5 15.6 16.9
16.1 14.2 13.8 14.9 13.8 16.1 16.9 14.6 15.5 15.9 13.6 16.9 14.9 15.2 16.4 15.6 13.5 16.1 16.6 14.2 14.9
8.1 (5.7–10.6)
8.5 (5.9–11.2)
16.1 (14.5–17.7)
15.3 (14.0–17.1)
17.0 (15.4–19.1)
15.2 (13.5–16.9)
6.7 6.5 7.9 5.2 5.8 5.4 4.8 6.4 8.5 9.9 6.5 6.4 6.5 5.4 7.9 7.1 7.4 8.4 4.0
6.4 8.2 8.6 6.9 8.1 7.8 5.9 8.3 9.5 12.3 7.8 6.0 7.8 5.3 9.6 8.5 7.8 8.5 4.4
16.6 16.4 16.8 17.1 15.8 15.9 16.4 16.0 16.5 15.4 16.2 14.4 16.9 15.3 16.4 16.1 17.2 15.4 15.0
15.3 15.1 15.3 14.9 15.6 15.4 16.5 15.8 15.1 15.6 16.0 14.4 15.3 14.4 15.5 15.2 15.5 15.8 14.3
16.4 16.1 17.3 16.2 14.9 15.2 13.7 15.9 18.5 17.4 15.7 15.2 17.9 15.1 17.8 16.9 16.6 16.7 13.3
13.9 14.5 14.9 14.9 15.5 15.5 13.3 15.5 16.1 17.9 15.3 13.8 15.8 13.2 16.4 15.4 14.8 15.8 12.3
6.7 (4.0–9.9)
7.8 (4.4–12.3)
16.1 (14.4–17.2)
15.3 (14.3–16.5)
16.1 (13.3–18.5)
15.0 (12.3–17.9)
3.8 7.2 6.9 7.4 8.2 5.6 7.9 6.5 7.0 4.3 5.4 7.8
4.1 8.1 6.6 8.1 8.3 6.5 8.8 6.5 6.7 4.1 6.8 7.5
17.4 17.0 16.0 15.9 16.8 15.9 15.6 16.4 15.9 17.2 15.1 15.5
16.8 15.9 15.4 15.7 14.8 15.8 15.3 15.4 14.9 14.8 15.2 15.4
13.0 16.2 17.0 16.7 17.8 14.5 16.5 16.0 15.5 14.2 14.5 16.6
12.0 15.4 14.2 15.6 15.0 14.6 15.6 14.7 13.7 12.0 14.4 15.4
6.5 (3.8–8.2)
6.8 (4.1–8.8)
16.2 (15.1–17.4)
15.5 (14.8–16.8)
15.7 (13.0–17.8)
14.4 (12.0–15.6)
(continued on next page)
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
214 Table 1 (continued )
Brown index (100-L*)
Yellow index (b*)
Sub-species
Cultivar or accession
2006
2007
2006
2007
2006
2007
ssp. turgidum var. turanicum
Kamut Cltr 11390 PI 68287 PI 113392 PI 113393 PI 167481 PI 254206 PI 278350 PI 306665 PI 576854 PI 623629 PI 623656 PI 624209 PI 254204 PI 624429 PI 290530
6.5 7.1 5.9 5.0 5.5 6.8 5.4 6.7 8.5 6.2 5.4 4.9 4.7 4.8 4.9 4.9
6.9 7.8 8.1 5.6 5.1 6.8 5.6 10.9 9.1 7.9 4.9 5.6 6.1 6.9 6.3 5.1
15.4 15.6 15.9 16.0 16.5 14.9 16.3 16.9 15.8 16.8 15.9 16.6 16.6 15.8 16.5 16.8
14.9 14.9 14.8 14.6 16.3 14.5 15.3 15.0 15.4 15.8 15.0 14.9 16.3 16.5 16.1 14.1
15.7 16.7 16.1 15.5 15.5 15.5 15.3 14.1 18.7 16.0 15.0 15.0 14.4 14.5 14.2 15.4
15.5 14.9 15.2 13.6 13.8 14.3 14.0 17.0 16.2 15.0 13.0 13.6 14.2 15.8 14.6 12.9
Mean Range
5.8 (4.7–8.5)
6.8 (4.9–10.9)
16.1 (14.9–16.9)
15.3 (14.1–16.5)
15.5 (14.1–18.7)
14.6 (12.9–17.0)
Lucanica Farvento MG 4375 MG 5473 MG 5350 MG 3521 MG 4387 MG 5323 MG 5416/1 MG 5471/1 MG 5471/2 MG 5471/3 MG 15516/1 MG 15516/3 MG 15516/4 MG 15516/5 MG 15516/6 MG 15516/7 MG 15516/8 MG 15516/9 MG 15516/10 MG 15516/11 MG 5344/1 MG 5344/2 MG 5300/1 MG 5300/3 MG 5293/1
– – 5.5 – – – – 5.6 – – – – – – – – – – – – – – – – – – –
6.8 4.9 7.7 5.3 4.4 5.9 6.6 5.5 8.3 5.5 4.5 5.1 5.9 6.1 6.2 6.7 6.4 7.3 5.6 7.5 5.9 6.1 5.7 6.4 5.2 5.7 7.4
– – 19.6 – – – – 18.5 – – – – – – – – – – – – – – – – – – –
18.5 16.5 18.0 17.4 16.1 16.5 18.5 16.8 17.2 16.1 16.6 16.7 18.0 17.5 17.2 17.2 17.2 18.4 17.9 16.7 17.7 17.0 16.5 17.3 16.5 17.0 18.0
– – 14.0 – – – 14.3 – – – – – – – – – – – – – – – – – – –
12.8 11.5 13.3 12.4 11.9 13.5 13.3 13.1 14.5 12.5 12.1 12.2 13.8 14.3 14.1 13.9 13.9 15.0 13.9 14.8 14.2 14.1 12.0 12.9 12.9 13.5 12.7
Mean Range
5.6 (5.5–5.6)
6.1 (4.4–8.3)
19.1 (18.6–19.6)
17.2 (16.1–18.5)
14.2 (14.0–14.3)
13.3 (11.5–15.0)
MG MG MG MG MG MG MG
5.3 5.3 6.6 5.4 – – –
3.9 7.6 8.3 6.3 5.0 5.3 5.1
18.0 16.7 16.2 18.9 – – –
17.4 14.6 15.6 17.2 14.2 17.6 16.1
18.0 16.7 16.2 18.9 – – –
11.7 15.2 14.9 12.1 12.7 12.7 13.4
5.7 (5.3–6.6)
5.9 (3.9–8.3)
17.5 (16.2–18.9)
16.1 (14.2–17.6)
17.5 (16.2–18.9)
13.2 (11.7–15.2)
6.7 (3.8–10.6) 0.6 0.94
7.1 (3.9–12.3) 0.9 0.91
16.3 (14.4–19.6) 1.1 0.59
15.9 (14.0–18.5) 0.9 0.79
16.0 (13.3–19.1) 1.1 0.81
14.3 (11.5–17.9) 0.6 0.94
ssp. dicoccum
ssp. dicoccoides
4328/61 29896 5510/7 5444/235 4337/198 5445 4330/66
Mean Range Mean Range LSD (0.05) h2B – ¼ not evaluated.
Year of release
Yellow pigment content (mg/g)
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Table 2 Average values (mg/g), least significant difference (LSD), and heritability (h2B) of carotenoid components in a T. turgidum collection evaluated at Valenzano (Bari, Italy) in 2006. Sub-species
Cultivar or accession Year of release Lutein
Zeaxanthin
b-crypto-xanthin a-carotene
b-carotene
Other componentsa Total carotenoids
ssp.turgidum var. durum
Brindur Cannizzo Ceedur Ciccio Claudio Cosmodur Exeldur Iride Agridur Meridiano Parsifal Svevo Vesuvio Cirillo Duetto Dylan Fiore Grecale Normanno Tiziana Virgilio
0.112 0.085 0.138 0.108 0.113 0.126 0.160 0.118 0.116 0.110 0.100 0.125 0.093 0.096 0.156 0.122 0.092 0.129 0.129 0.176 0.114
0.013 0.007 0.006 0.012 0.009 0.011 0.011 0.009 0.013 0.013 0.013 0.009 0.006 0.008 0.006 0.010 tr 0.013 0.008 tr 0.008
0.024 0.014 0.012 0.024 0.012 0.023 0.019 0.013 0.013 0.033 0.013 0.023 0.015 0.014 0.018 0.029 0.008 0.027 0.029 0.014 0.010
0.799 0.596 0.653 0.654 0.729 0.904 0.800 0.660 0.872 1.060 0.623 1.159 0.827 0.523 0.850 0.849 0.198 1.060 1.153 0.749 0.631
1991–2008
Mean Range Appulo Creso Valnova Valgerardo Karel Produra Sansone Tito Ambral Primadur Arcangelo Duilio Grazia Latino Neodur Ofanto Simeto Tresor Messapia
1971–1990
Mean Range Timilia Russello Aziziah Grifoni Capeiti Cappelli Hymera Trinakria Kiperounda Polesine Taganrog Shram 5
ssp. turgidum var. turanicum
Before 1971
2.487 1.332 1.939 1.843 2.164 2.843 2.606 2.037 1.980 2.367 1.611 3.078 1.625 1.869 2.421 2.510 1.559 2.947 2.822 1.352 2.105
0.018 0.010 0.012 0.012 0.013 0.022 0.020 0.007 0.020 0.026 0.013 0.023 0.019 0.012 0.011 0.017 tr 0.029 0.016 0.008 0.009
3.453 2.044 2.760 2.653 3.039 3.929 3.617 2.844 3.014 3.608 2.373 4.416 2.585 2.523 3.462 3.536 1.857 4.205 4.157 2.299 2.877
2.167 0.120 0.010 (1.332–3.078) (0.805–0.176) (0.006–0.013)
0.016 0.018 0.779 (0.007–0.029) (0.008–0.033) (0.198–1.159)
3.109 (1.857–4.416)
1.655 1.385 2.331 1.394 1.365 1.270 1.301 1.588 2.094 2.394 1.843 1.412 1.985 1.299 2.087 2.038 1.920 2.405 0.843
0.007 0.011 0.011 0.008 0.003 0.011 0.007 0.007 0.010 0.011 0.007 0.018 0.013 0.010 0.012 0.013 0.010 0.015 tr
2.353 2.160 3.366 2.032 1.924 2.236 1.819 2.236 3.179 3.532 2.727 2.170 2.777 2.134 3.281 2.770 3.087 3.472 1.352
0.091 0.128 0.101 0.154 0.140 0.096 0.123 0.141 0.122 0.159 0.097 0.098 0.122 0.104 0.123 0.088 0.100 0.178 0.122
0.006 0.002 0.004 0.007 tr tr 0.002 0.004 0.007 0.008 0.008 0.014 0.004 0.008 0.009 0.008 0.006 0.011 tr
0.008 0.016 0.013 0.014 0.007 0.012 0.007 0.017 0.017 0.021 0.009 0.010 0.011 0.013 0.018 0.016 0.018 0.020 0.008
0.586 0.617 0.907 0.455 0.409 0.847 0.380 0.479 0.929 0.939 0.763 0.619 0.642 0.700 1.032 0.606 1.033 0.844 0.379
1.716 0.120 0.007 (0.843–2.405) (0.088–0.178) (0.002–0.014)
0.010 0.013 0.693 (0.003–0.018) (0.007–0.021) (0.379–1.032)
2.560 (1.352–3.532)
0.721 1.421 1.358 2.012 2.395 1.176 1.861 1.561 1.563 0.861 1.381 1.898
tr 0.008 0.007 0.014 0.010 0.008 0.015 0.012 0.018 tr 0.006 0.011
1.178 2.420 2.166 2.954 3.345 1.769 2.808 2.362 2.471 1.458 2.201 2.737
0.134 0.184 0.126 0.170 0.126 0.150 0.131 0.124 0.131 0.109 0.163 0.126
tr 0.006 0.007 0.006 0.009 tr 0.010 0.011 tr tr 0.006 tr
0.010 0.012 0.009 0.013 0.013 0.013 0.015 0.010 0.020 0.007 0.013 0.010
0.313 0.789 0.660 0.740 0.793 0.422 0.776 0.644 0.739 0.481 0.633 0.692
Mean Range
1.517 0.140 0.008 (0.721–2.395) (0.109–0.184) (0.006–0.011)
0.011 0.012 0.640 (0.006–0.018) (0.007–0.020) (0.313–0.793)
2.327 (1.178–3.345)
Kamut Cltr 11390 PI 68287 PI 113392 PI 113393 PI 167481 PI 254206 PI 278350 PI 306665 PI 576854
1.586 1.977 1.521 0.955 1.097 1.367 1.006 1.837 2.135 1.290
0.018 0.030 0.007 0.002 tr 0.007 tr 0.008 0.010 0.007
2.509 2.836 2.381 1.705 1.729 2.351 1.829 2.826 4.329 2.432
0.152 0.141 0.154 0.134 0.132 0.157 0.113 0.113 0.197 0.130
0.006 0.010 0.005 0.005 0.008 tr tr 0.014 0.008 0.004
0.020 0.020 0.015 0.009 0.006 0.016 0.013 0.022 0.015 0.014
0.727 0.658 0.679 0.600 0.487 0.804 0.697 0.832 1.964 0.988
(continued on next page)
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
216 Table 2 (continued ) Sub-species
ssp. dicoccum
Cultivar or accession Year of release Lutein
Zeaxanthin
b-crypto-xanthin a-carotene
b-carotene
Other componentsa Total carotenoids
PI PI PI PI PI PI
0.135 0.141 0.130 0.160 0.148 0.137
tr 0.005 tr tr tr 0.007
0.009 0.014 0.009 0.007 0.009 0.009
0.581 0.595 0.547 0.567 0.563 0.387
623629 623656 624209 254204 624429 290530
tr 0.007 0.002 tr tr tr
1.735 1.609 1.628 1.768 1.817 1.576
Mean Range
1.296 0.142 0.007 (0.848–2.315) (0.113–0.197) (0.004–0.014)
0.010 0.013 0.730 (0.002–0.030) (0.006–0.022) (0.387–1.964)
2.198 (1.576–4.329)
Farvento MG 5473 MG 5350 MG 3521 MG 5323
0.699 0.891 0.935 0.960 0.894
tr 0.006 tr 0.002 0.004
1.265 1.552 1.508 1.583 1.669
Mean Range
0.876 0.164 (0.699–0.960) (0.130–0.176)
ssp. dicoccoides MG MG MG MG MG MG MG MG
4328/61 29896 5510/7 5444/235 5445 4330/66 3533/52 4343
Mean Range Mean Range LSD (0.05) h2B
1.010 0.848 0.940 1.034 1.097 1.036
0.710 1.391 1.157 0.773 1.111 0.892 1.570 0.943
0.173 0.176 0.130 0.166 0.173
0.143 0.098 0.129 0.166 0.113 0.118 0.230 0.189
0.004 tr tr tr 0.004
tr 0.010 tr tr tr tr tr tr
1.068 0.148 (0.710–1.570) (0.098–0.230) 1.605 (0.699–3.078) 0.265 0.93
0.133 (0.850–0.230) 0.041 0.48
0.008 (0.002–0.014) 0.004 0.72
0.010 0.016 0.014 0.009 0.014
0.379 0.463 0.429 0.446 0.580
0.004 0.013 0.459 (0.002–0.006) (0.009–0.016) (0.379–0.580)
1.515 (1.265–1.669)
0.006 0.008 0.011 0.007 0.011 0.010 tr tr
1.276 2.083 2.114 1.424 1.856 1.748 2.491 1.510
0.009 0.014 0.016 0.017 0.015 0.016 0.017 0.009
0.408 0.561 0.801 0.461 0.606 0.712 0.674 0.368
0.009 0.014 0.574 (0.006–0.011) (0.009–0.017) (0.368–0.801)
1.823 (1.276–2.491)
0.011 (0.002–0.030) 0.006 0.79
2.460 (1.178–4.416) 0.300 0.94
0.014 (0.006–0.033) 0.007 0.57
0.688 (0.198–1.964) 0.279 0.63
tr ¼ traces. a Obtained as difference between total carotenoid and the sum of the identified components.
3.3. Heritability and correlation between yellow pigments and grain yield components Broad-sense heritability, as estimated on an accession mean basis, was found to be high for yellow pigment concentration (0.94 and 0.91 in 2006 and 2007, respectively) and yellow index (0.81 and 0.94 in 2006 and 2007, respectively), and relatively high for brown index (0.59 and 0.79 in 2006 and 2007, respectively) (Table 1). High heritability was also found for total carotenoid concentration (0.94) and lutein (0.93), intermediate values for b-cryptoxanthin (0.72) and a-carotene (0.79), and relatively low values for b-carotene (0.57) and zeaxanthin (0.48) (Table 2). Correlation analysis revealed highly significant and positive relationships (P 0.001) among single carotenoid components and
total carotenoid concentration determined by HPLC (data not shown). Total carotenoid concentration was strongly correlated with yellow pigment concentration (0.94; P 0.001) and with yellow index (0.88; P 0.001) (Table 3). A significant correlation was also observed between the last two parameters (0.88 and 0.90 in 2006 and 2007 trials, respectively; P 0.001). Significant negative correlations were observed for YPC and yellow index with grain protein concentration (P 0.01–0.001). These correlations were significant for each of the two trials, suggesting consistent relationships. Total carotenoid concentration, yellow pigment concentration and yellow index were found to be positively correlated with grain yield per spike (P 0.01–0.001). The three colour parameters showed negative correlation values with 1000kernel weight, but only the correlations between yellow pigment concentration and 1000-kernel weight in 2006, and between yellow index and 1000-kernel weight in 2007 were significant at P 0.05. Brown index was positively correlated with grain protein concentration (P 0.01 and P 0.001 in 2006 and 2007, respectively) and negatively correlated with grain yield per spike (P 0.01 and P 0.001 in 2006 and 2007, respectively), total carotenoid concentration (P 0.05) in 2006, yellow pigment concentration (P 0.05) and yellow index (P 0.01) in 2007. 4. Discussion and conclusions
Fig. 1. High-performance liquid chromatogram of carotenoids extracted from durum wheat cultivar Primadur.
Wheat breeding has generally focused on improving yield and yield stability, disease resistances and technological properties, mostly gluten quality and quantity, and to a lesser extent, colour. Yellow pigment of durum wheat semolina has recently received considerable attention to improve the yellow colour in end-products such as pasta, and the nutritional value of carotenoid
` et al. / Journal of Cereal Science 50 (2009) 210–218 A.M. Digesu
217
Table 3 Correlations between yellow pigment content (AACC method 14-50), total carotenoid content (HPLC), colorimetric indexes (brown and yellow indexes), grain protein content, kernel weight and grain yield per spike in a T. turgidum collection grown at Valenzano (Bari, Italy) in 2006 and 2007. Trait
Trial
Total carotenoid pigments
Yellow pigment content
2006
0.94***
Yellow index
2006 2007
0.88***
Brown index
2006 2007
0.24*
0.22 0.23*
0.16 0.33**
Grain protein content
2006 2007
0.40**
0.34** 0.32**
0.41*** 0.31**
Kernel weight
2006 2007
0.08
0.29* 0.17
0.21 0.24*
Grain yield per spike
2006 2007
0.49***
Yellow pigment content
Yellow index
Brown index
0.88*** 0.90***
0.34** 0.24**
0.38** 0.17
0.39** 0.61*** 0.01 0.13 0.31** 0.37***
*, **, ***: Significant differences at 0.05P, 0.01P and 0.001P, respectively.
components. Thus, increasing yellow colour is a valuable goal for improving the commercial and nutritional values of durum wheat products. Yellow pigment analysis of 102 accessions showed highly significant differences (P < 0.001) in the grain pigment concentration among the wheat genotypes in two trials. The results showed wide variation of YPC, ranging from the highest value of the elite cultivar Svevo (10.5 mg/g) of durum wheat to the lowest of the old cultivar Timilia (3.8 mg/g). Overall, modern commercial cultivars showed significantly higher values compared to traditional cultivars and wild ssp. dicoccum and ssp. dicoccoides accessions. This can be explained by the fact that modern durum breeding programs have selected genotypes with high pigment concentrations because yellow colour has been considered in the last two decades to be an important quality parameter with regard to pasta production. Although environmental factors can influence yellow pigment concentration, the genetic component is predominant because heritability values detected in 2006 and 2007 trials were more than 0.90, confirming previous studies (Clarke et al., 2006; Elouafi et al., 2001; Veronico et al., 2001) that underlined the polygenic nature but also the strong genotypic component of yellow pigment concentration in durum wheat. Total carotenoid concentration determined by HPLC varied between 1.178 and 4.416 mg/g (average 2.460 mg/g), in agreement with that found by others authors (Fortmann and Joiner, 1978; Fratianni et al., 2005; Hentschel et al., 2002; Hidalgo et al., 2006; Leenhardt et al., 2006) in durum and bread wheats. Higher average value (8.41 mg/g) was found in a collection of 54 accessions of einkorn wheat (T. monococcum L.) by Hidalgo et al. (2006), in part due to the lower kernel weight of einkorn in comparison to durum and to the negative relationship between carotenoid concentration and kernel weight (see later) found in cereals. Lutein (65.2%) was the main component of carotenoids, followed by zeaxanthin (5.4%) and b-carotene (0.6%); a-carotene (0.5%) and b-cryptoxanthin (0.3%) were minor components, in several cases present only in traces. HPLC analysis also showed the presence of significant quantities of other not identified carotenoid compounds (28.0%). Lutein was always found to be the major carotenoid in diploid, tetraploid and hexaploid wheats, even if the proportion varied greatly in different assessments (Adom et al., 2003; Fortmann and Joiner, 1978; Fratianni et al., 2005; Hentschel et al., 2002; Hidalgo et al., 2006; Leenhardt et al., 2006; Serpen et al., 2008). The relative proportion of zeaxanthin, b-cryptoxanthin, a-carotene and bcarotene reported by these authors varied from traces or not detectable quantities to significantly high proportion of a-carotene (6.3%) in einkorn wheat (Hidalgo et al., 2006), and of b-carotene (3.7%) (Fratianni et al., 2005) and zeaxanthin (8.5%) (Panfili et al.,
2004) in durum. The components with higher pro-vitamin A activity (b-carotene, a-carotene, b-cryptoxanthin) showed wide variation (from 0.015 to 0.072 mg/g) indicating the need for screening cultivated and wild germplasm to identify accessions with higher concentration of specific carotenoids, and the possibility of improving the pro-vitamin A activity of commercial cultivars via traditional breeding and selection. Noteworthy, the durum cultivar Meridiano and the var. turanicum accession Cltr11390 had the highest values of pro-vitamin A components expressed as absolute amount (0.072 and 0.060 mg/g, respectively) and as percentage of the total carotenoid concentration (2.0 and 2.1%, respectively). The apparently high differences in the relative proportion of carotenoid components reported in the literature may be attributed to genetic effects, environmental conditions and agronomic practices, but also to different extraction methods, internal standards, milling fractions, particle size of semolina and presence of peripheral parts, HPLC procedures, etc. Total carotenoid concentration, as determined by HPLC, compared with yellow pigment concentration, measured by AACC method 14-50, showed that the portion of carotenoids amounted to 33.2% of the yellow pigments in the 80 tetraploid wheat accessions examined in the 2006 trial. This implies that there are unknown colour-producing compounds in the durum extracts absorbing light at 435.6 nm. This observation is in accordance with results provided by Hentschel et al. (2002) and by Leenhardt et al. (2006). Hentschel et al. (2002) found that the carotenoid fraction of yellow pigment concentration amounted to only 30–50% in eight durum cultivars, and concluded that there are other compounds not yet identified that contribute considerably to the yellow colour of the grain extracts. In contrast, Burkhardt and Bo¨hm (2007) and Fratianni et al. (2005) showed close correspondence between reflectance measurements and total carotenoid concentration determined on durum cultivars by both the standard AACC 14-50 or ICC 152 methods and the HPLC procedure. Pigment concentration demonstrated a slightly negative correlation with kernel weight, as did yellow index in one trial. This negative correlation was also found by Alvarez et al. (1999), Clarke et al. (2006), Marais (1992), Veronico et al. (2001) and Zhang et al. (2008), and could be partly attributed to dilution effects, with increases in grain starch in large kernels reducing the proportion of pigments. The highly significant negative correlations observed between yellow pigment parameters with grain protein concentration are particularly important for durum breeding programs. The genetic basis of trait correlations may include single genes with pleiotropic effects or the tight linkage of several genes controlling different traits. Combining quantitative trait loci (QTLs) for high pigment concentration with those for high grain protein
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concentration will require detailed information on the chromosomal position and effects of each QTL controlling all of these traits in the genetic material under study. In agreement with results published by Fratianni et al. (2005), highly significant correlations were found between reflectance measurement (b* index) and yellow pigment concentration determined by both standard AACC method and HPLC procedure, thus confirming the usefulness of yellow index as a fast and safe method for screening yellow pigments in large-scale wheat breeding programs. Indeed, the development of a rapid screen for kernel and semolina colour has facilitated the increased grain yellow colour of new durum cultivars developed in the last two decades, while marker-assisted selection may allow additional improvement in the future. Yellow index, although strongly related to pigment concentration, provides only relative and not absolute values of grain or semolina colour and does not provide information on the proportional composition of carotenoids. The HPLC technique, although expensive, resulted in a sensitive, selective and reliable method for determining the qualitative and quantitative distribution of carotenoid compounds in cereals (Hentschel et al., 2002; Panfili et al., 2004). In view of the increasing importance of nutritional aspects in the definition of food quality, complete information on the estimation of colour and nutritional value of grain, semolina and end-products of durum and bread wheats may be obtained by combining the b* index and HPLC techniques. The present survey of 102 accessions of wild and cultivated tetraploid wheats demonstrated marked variations in the carotenoid composition and concentration. Such information is useful for plant breeders in screening and selecting cultivars with high yellow colour and enhanced phytochemical concentrations, and provides valuable information for evaluating potential contributions to health from the consumption of durum wheat end-products. Acknowledgements This research was supported by grants from the Ministero dell’Universita` e della Ricerca, Italy, projects ‘FISR’ and ‘AGROGEN’, and by the EU grant DEVELONUTRI. References AACC (American Association of Cereal Chemists), 2000. AACC Official Method 14-50. In: Approved Methods of the American Association of Cereal Chemists, tenth ed. St. Paul, MN, USA. Adom, K.K., Sorrells, M.E., Liu, R.H., 2003. Phytochemical profiles and antioxidant activity of wheat varieties. Journal of Agricultural and Food Chemistry 51, 7825–7834. Alvarez, J.B., Martin, L.M., Martin, A., 1999. Genetic variation for carotenoid pigment content in the amphiploid Hordeum chilense Triticum turgidum conv. durum. Plant Breeding 118, 187–189. Borrelli, G.M., De Leonardis, A.M., Fares, C., Platani, C., Di Fonzo, N., 2003. Effects of modified processing conditions on oxidative properties of semolina dough and pasta. Cereal Chemistry 80, 225–231. Burkhardt, S., Bo¨hm, V., 2007. Development of a new method for the complete extraction of carotenoids from cereals with special reference to durum wheat (Triticum durum Desf.). Journal of Agricultural and Food Chemistry 55, 8295– 8301. CIE (Commission Internationale de l’Eclairage), 1986. Publication 15.2. Colorimetry, second ed. CIE Central Bureau Kegelgasse, Wien, Austria. 27-A-1030. Clarke, F.R., Clarke, J.M., McCaig, T.N., Knox, R.E., DePauw, R.M., 2006. Inheritance of yellow pigment concentration in four durum wheat crosses. Canadian Journal of Plant Science 86, 133–141. Della Penna, D., Pogson, B.J., 2006. Vitamin synthesis in plants: tocopherols and carotenoids. Annual Review of Plant Biology 57, 711–738.
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