1. INHERITANCE OF ANTHOCYANIN CONCENTRATION IN PURPLE WAXY CORN (Zea mays L.) KERNELS AND COB a Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand. b Plant Breeding Research Center for Sustainable Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand. c Department of Food Technology, Faculty of Technology. Khon Kaen University, Khon Kaen, 40002, Thailand. d USDA-ARS Corn Insects and Crop Genetics Research Unit, Iowa State University, Ames, 50011, USA. * Corresponding author e-mail: kamol9@gmail.com)kamol9@gmail.com 55 th ANNUAL MAIZE GENETICS CONFERENCE 2013 –St. CHARLES, ILLINOIS, USA. Bhornchai Harakotr a, Bhalang Suriharn a,b, Rutchada Tungwongchai c, Marvin Paul Scott d, Kamol Lertrat* a,b ACKNOWLEDGEMENTS; This research was supported financially by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (PHD/0028/2553). Part of this work was also fund by National Science and Technology Development Agency, and Plant Breeding Research Center for Sustainable Agriculture, Faculty of Agriculture, Khon Kaen University, Thailand. GenerationsC3GPg3GPn3Ganthocyaninsphenolics kernel P1P1 315.26+4.23 a 106.73+4.96 a 133.23+1.51 a 1556.41+7.51 a 5.71+0.26 a P2P2 0.54+0.02 c 0.49+0.12 c 0.27+0.03 d 2.62+0.13 d 2.00+0.20 d F1F1 114.20+3.05 b 37.63+2.75 b 34.66+2.33 bc 679.81+7.42 b 4.56+1.27 ab F2F2 118.41+24.78 b 30.78+25.30 bc 27.21+21.17 bcd 401.17+137.12 c 3.69+0.50 bc BC 11 109.69+63.83 b 37.11+34.40 b 50.97+32.19 b 431.09+262.57 c 4.26+0.94 b BC 12 31.77+36.56 c 8.49+2.86 bc 10.17+10.47 cd 135.15+72.83 d 2.83+0.53 cd MP157.953.6166.75779.5153.86 F-ratio b 35.79**13.61**25.68**59.37**9.87** Model R 2 0.910.790.880.940.72 cob P1P1 992.20+7.46 a 184.73+2.03 a 220.52+1.65 a 2881.68+4.71 a 27.54+1.24 a P2P2 0.79+0.07 e 0.33+0.07 c 0.61+0.05 d 3.85+0.68 f 8.04+0.37 d F1F1 902.85+8.54 a 164.08+8.84 ab 216.68+2.36 a 2340.23+6.22 b 19.11+1.26 b F2F2 404.85+115.94 c 118.98+34.18 b 134.64+27.17 b 1110.77+324.63 d 17.02+3.26 b BC 11 644.19+153.81 b 159.79+42.80 ab 163.41+16.28 b 1759.18+274.48 c 20.30+2.29 b BC 12 189.96+92.52 d 44.70+28.28 c 73.84+37.40 c 721.26+246.44 e 11.84+1.14 c MP496.592.53110.571442.7717.79 F-ratio b 61.07**25.34**54.67**84.56**40.99** Model R 2 0.950.880.940.960.92 ParametersC3GPg3GPn3GAnthocyanins kernel m157.90+2.02**53.61+1.05**66.75+2.01**779.52+1.95** [a]157.36+2.02**53.12+1.05**66.48+2.01**776.89+1.95** [d]-253.60+40.06**-97.65+14.52**-117.23+14.21**-1729.78+127.51** [ad]-137.20+45.35*-41.73+15.48**-54.77+16.41**-877.92+148.05** [dd]2091.90+39.88**81.67+14.51**85.05+14.28**1630.07+127.59** cob m496.49+1.59**92.53+1.65**110.56+1.11**1442.74+1.47** [a]495.71+1.59**92.20+1.65**109.95+1.11**1438.92+1.48** [d]-741.93+116.95**-9.32+34.46 ns -52.90+36.99 ns -1921.20+313.09** [ad]-90.09+124.43 ns 58.07+40.10 ns -35.06+44.37 ns - [dd]1148.30+117.06**80.87+34.74*159.03+37.01**2804.34+313.12** Analysis of variance was carried for anthocyanin concentration according to randomized complete block design using JMP software (version 10). The least significant difference (LSD) was calculated to identify significant differences (p<0.05) Calculated means and variances estimated the mid-parent (m), additive (a) and dominance (d) gene effects as described by Rowe and Alexander (1980) following the method three-parameter model of Mather and Jinks (1977). Adequacy of the additive-dominance model was determined by Chi-square (X 2 ) test with three degrees of freedom and was accepted if p<0.05. When the three-parameter model was inadequate (significant X 2 value), the interaction genetic parameters [additive x additive (aa), additive x dominance (ad) and dominance x dominance (dd)] were computed. The genetic parameters were tested for significance using an unpaired t-test. Environment variances (Ơ 2 E ) and broad-sense heritability (H 2 b) were estimated using the following formula (Ito and Brewbaker, 1991) H 2 b = (Ơ 2 F2 - Ơ 2 E )/ Ơ 2 F2 Where Ơ 2 E = ¼ (Ơ 2 P1 + Ơ 2 P2 +2 Ơ 2 F1 ) Ơ 2 F2 = variance of the F 2 populations Ơ 2 P1 = variance of the parental line 1 Ơ 2 P2 = variance of the parental line 2 Ơ 2 F1 = variance of the F 1 populations Total Anthocyanin contents Total Anthocyanin contents was determined according to the method described by Guiti and Wrulstad (2001) with minor modifications. Total phenolic compounds Total phenolic compounds was determined according to the method described by Xu and Hu (2011) with minor modifications. Anthocyanin profiles Anthocyanin profiles RP-HPLC analysis of individual anthocyanins was according to the method described by Kim et al. (2007) with minor modifications. III. Statistical analysis II. Biological markers determination Waxy corn is gaining popularity among Asian counties, mainly because of its good eating quality, ear shape and kernels color. There many reports reported that dark colored corn has high levels of beneficial bioactive compounds and antioxidants. Anthocyanins are especially abundant in dark-colored corn. The health benefit of anthocyanin was attributed to their high antioxidant and has potential anti- carcinogenic activity, cardiovascular disease prevention, obesity control, diabetes alleviation properties and anti-inflammatory capabilities (Jones, 2005; He and Giusti, 2010) The prime goal of corn breeders always yielded enhancement, but views of improving the livelihoods through an increase level of bioactive compounds, especially anthocyanins in food is highly warranted. Jones, K. 2005. Herbal Gram. 65: 46-49 He, J. and M.M. Giusti. 2010. Annu. Rev. Food Sci Technol. 1: 163-87. Yang, Z., et al. 2008. Eur Food Res Technol. 227: 409-15. Yang, Z. and W. Zhai. 2010. Inn Food Sci and Emer Technol. 11: 169-76. Table I Means and standard error of different generation for anthocyanin concentration in the kernels and cob of waxy corn cross. Table III Estimates of different gene effects for anthocyanin concentration in the kernels and cob of waxy corn cross. a m; mid-parent, [a]; additive, [d]; dominance, additive × dominance, and [dd]; dominance × dominance *, ** Significant from zero at p< 0.05 and 0.01 respectively, based on normal derivate table. ns non-significant. a P 1 ; KND10-4P, P 2 ; BW, F 1 ; First filial generation of crosses, F 2 ; Second filial generation of crosses, BC 12 ; First backcross generation with parental line 1 and BC 12 ; First backcross generation with parental line 2. C3G; Cyanidin 3-glucoside, Pg3G; Pelargonidin 3-glycoside, Pn3G; Peonidin 3-glucoside and anthocyanin contents expressed as micrograms of cyanidin 3-glucoside/g DW and phenolic contents was expressed as milligrams of GAE/g DW. b ** Significant at p< 0.01. Values in the same column sharing different letters are expressed as significantly different (p < 0.05). F 1, F 2, and backcross populations were derived from the parental inbreds KND10-4P and BW (Figure I), KND10-4P is a purple waxy corn variety. BW is a white waxy corn variety, has high general combining ability. The parents and all progenies were planted in a randomized complete block design with three replications on November, 2011 at Research Station, Plant Breeding Research Center for Sustainable Agriculture, Khon Kaen University, Thailand. The trials were planted in ear to row plots 5m long with 0.75m between -row spacing and 0.25m between plants within row. Hand-pollination of adjacent plants in each plot was practiced for parents, F 1 and backcross populations to avoid contamination from stray pollen and individual self-pollination of each plant was practiced for the F 2 -populations. Ears were picked by hand at the mature stage (35 Day after Pollination; DAP), the kernels and cob shelled by hand. Thereafter, samples were milled in to whole grain and cob flour, sieved through a 60 mesh screen, thoroughly mixed and stored at -20 ° C until analysis. I. Plant materials and Population development Anthocyanin concentrations were generally lower in the kernel than cob and this was consistent for all six generations. Anthocyanin concentrations of the parents differed significantly, as was expected, with KND10-4P having the highest, For the F 1 progeny, the mean of the phenolic contents in the kernel, while Cyanidin 3-glucoside (C3G), pelargonidin 3-glucoside (Pg3G), peonidin 3-glucoside (Pn3G), anthocyanin contents and phenolic contents in the cob were higher than mid-parent value, indicating partial dominance toward the highest parents. In contrast, the F 1 means for C3G, Pg3G, Pn3G and anthocyanin contents in the kernel were less than the mid-parent value indicating partial dominance toward the lowest parents. The values of the both backcross generations were shifted toward the recurrent parent. The additive and dominance components of the genetic models were significantly different from zero. All six- parameters were computed by the perfect fit method proposed by Mather and Jinks (1977). The test reveled that some genetic parameters were not significant (data not shown). No test of the adequacy of the six-parameter model was possible because the number of estimated components was equal to that of observed means, leaving no degrees of freedom for the test of goodness of fit. However, since there was no significant interaction, the non-significant component was omitted to test a three- to five-parameter model with three to one degree of freedom to test goodness of fit. This also improved the precision with which the remaining parameters were estimated (Ito and Brewbaker, 1991). Significant additive gene effects occurred more frequently than dominance gene effects. In most trait s of the kernels, dominance effects were important. Dominance effects were negative in most cases. The negative sign associated with the dominance components indicated that, in these hybrids, anthocyanin concentration could be decreased relative to the mid-parent. The dominance x dominance effects were important in C3G, Pn3G and anthocyanin contents of the cob, indicating that this components enhanced their concentration. Parameters C3GPg3GPn3Ganthocyaninsphenolics kernel m156.70+2.01**52.83+1.04**66.66+0.33**778.76+1.95**3.74+0.14** [a]156.16+2.01**52.35+1.04**66.39+0.33**776.13+1.95**1.74+0.13** [d]-43.00+2.48**-16.99+1.68**-32.17+0.96**-101.45+4.03**0.01+0.31ns X2X2 33.56**35.45**36.18**166.28**5.49 ns H2bH2b 0.570.660.680.620.71 cob m496.13+1.59**92.37+1.64**110.28+1.11**1442.62+1.48**17.56+0.44** [a]495.34+1.59**92.04+1.64**109.68+1.11**1438.78+1.48**9.56+0.44** [d]404.39+3.90**69.36+3.66**105.77+1.97**895.96+5.56**1.23+0.67** X2X2 103.64**13.96**19.17**84.79**5.26 ns H2bH2b 0.660.750.640.490.74 Table II Joint scaling test and estimates (+SE) with three-parameter model using six generations mean and broad-sense heritability for anthocyanin concentrations in the kernels and cob of waxy corn crosses. Generation mean analyses the additive-dominance model was adequate to explain the variation for only phenolic contents in the both kernels and cob, based on estimates of X 2. The result shown that phenolic contents governed by additive gene effect solely not epistatic gene effects in the kernels. broad-sense heritability estimates of C3G and Pg3G were same value in both the kernels and cob. H 2 b of Pg3G of the cob was the higher than others (0.75), while, H 2 b of anthocyanin contents in the cob has the lowest (0.49) Figure I. Kernels and cob color for parental lines [(A) KND10-4P, (B) BW]and F 2 (C). However, it has doubly beneficial, if corn has a high content of anthocyanin in the both kernels and cob. Yang and Zhai (2010) reported purple corn cob have anthocyanin concentration around 2 fold higher than kernels. The potential exists to breed corn with higher anthocyanin concentration. Choosing the most effective breeding strategies for this goal, however, can be facilitated by estimates of inheritance, heritability of the trait and the variance components. Among cereals, the inheritance of purple pigments in corn is well-studied by molecular genetics. The phenotype is affected by many genes, the sum of which is referred to as the genetic background (Ford, 2011). Thus, the aims of this study were to investigate the genetics of anthocyanin concentration in the kernel and cob of waxy corn. We did this by generation mean analysis (GMA). a m; mid-parent, [a]; additive, [d]; dominance ** Significant from zero at p< 0.01, based on normal derivate table. ns non-significant. Kim. et al. 2007. J. Agric. Food Chem. 55: 4802-09. Ford, R.H. 2000. The American biology Teacher. 63: 181-88. Hu, Q.-P.; Xu, J.-G.2011. J. Agric. Food Chem. 59: 2026–33. Ito, G.M. and J.L. Brewbaker. 1991. J. Amer Soc Hort Sci. 116: 1072-77. Mather, K and J.J. Jinks. 1977. Introduction to biometrical genetics. New York. (A) (B) (C) One consequence of the different gene effects on choice of a breeding strategy is that hybrids developed following crosses to the high parent would be expected to raise the concentration of anthocyanin in kernels due to the predominant dominance gene effects. while, line selection by repeated self-fertilization enhanced Pg3G levels of the cob due to the predominant additive gene action. The dominance x dominance epistatic components were present. These effects were positive and thus contributed to increased anthocyanin levels. Purple color is conditioned by the a1, c1, p and r genes of corn (Neuffer et al., 1997). These genes normally exhibit Medelian inheritance with the colored phenotype being dominant. We examined purple coloration in waxy corn and observed variation in the extent of pigmentation, suggesting a simple Mendelian model is not sufficient. We therefore used a quantitative genetic approach to examine coloration in this germplasm. Board-sense heritability estimates were moderate, indicating that these traits should respond well to selection. The effect of environment on these traits needs further research. Corn breeders may be able to exploit these genetic effects to developed hybrids with determined levels of anthocyanins.