55th ANNUAL MAIZE GENETICS CONFERENCE 2013 –St. CHARLES, ILLINOIS, USA.
INHERITANCE OF ANTHOCYANIN CONCENTRATION IN PURPLE WAXY CORN (Zea mays L.) KERNELS AND COB
Bhornchai Harakotra, Bhalang Suriharna,b, Rutchada Tungwongchaic, Marvin Paul Scottd, Kamol Lertrat*a,b
a Department
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 anticarcinogenic 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.
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).
I. Plant materials and Population development
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)
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.
Parameters
Table I Means and standard error of different generation for anthocyanin
concentration in the kernels and cob of waxy corn cross.
Generations
C3G
Pg3G
Pn3G
anthocyanins
phenolics
phenolics
m
156.70+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**
106.73+4.96a
133.23+1.51a
1556.41+7.51a
5.71+0.26a
[d]
-43.00+2.48**
-16.99+1.68**
-32.17+0.96**
-101.45+4.03**
0.01+0.31ns
P2
0.54+0.02c
0.49+0.12c
0.27+0.03d
2.62+0.13d
2.00+0.20d
X2
33.56**
35.45**
36.18**
166.28**
5.49ns
F1
114.20+3.05b
37.63+2.75b
34.66+2.33bc
679.81+7.42b
4.56+1.27ab
H2b
0.57
0.66
0.68
0.62
0.71
F2
118.41+24.78b
30.78+25.30bc
27.21+21.17bcd
401.17+137.12c
3.69+0.50bc
cob
BC11
109.69+63.83b
37.11+34.40b
50.97+32.19b
431.09+262.57c
4.26+0.94b
m
496.13+1.59**
92.37+1.64**
110.28+1.11**
1442.62+1.48**
17.56+0.44**
BC12
31.77+36.56c
8.49+2.86bc
10.17+10.47cd
135.15+72.83d
2.83+0.53cd
[a]
495.34+1.59**
92.04+1.64**
109.68+1.11**
1438.78+1.48**
9.56+0.44**
MP
157.9
53.61
66.75
779.515
3.86
[d]
404.39+3.90**
69.36+3.66**
105.77+1.97**
895.96+5.56**
1.23+0.67**
F-ratio b
35.79**
13.61**
25.68**
59.37**
9.87**
X2
103.64**
13.96**
19.17**
84.79**
5.26ns
Model R2
0.91
0.79
0.88
0.94
0.72
H2b
0.66
0.75
0.64
0.49
0.74
m; mid-parent, [a]; additive, [d]; dominance
** Significant from zero at p< 0.01, based on normal derivate table. ns non-significant.
a
cob
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 X2.
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. H2b of Pg3G of the cob was the higher than
others (0.75), while, H2b of anthocyanin contents in the cob has the lowest (0.49)
P1
992.20+7.46a
184.73+2.03a
220.52+1.65a
2881.68+4.71a
27.54+1.24a
P2
0.79+0.07e
0.33+0.07c
0.61+0.05d
3.85+0.68f
8.04+0.37d
F1
902.85+8.54a
164.08+8.84ab
216.68+2.36a
2340.23+6.22b
19.11+1.26b
F2
404.85+115.94c
118.98+34.18b
134.64+27.17b
1110.77+324.63d
17.02+3.26b
BC11
644.19+153.81b
159.79+42.80ab
163.41+16.28b
1759.18+274.48c
20.30+2.29b
BC12
189.96+92.52d
44.70+28.28c
73.84+37.40c
721.26+246.44e
11.84+1.14c
MP
496.5
92.53
110.57
1442.77
17.79
F-ratio b
61.07**
25.34**
54.67**
84.56**
40.99**
kernel
Model R2
0.95
0.88
0.94
0.96
0.92
Table III Estimates of different gene effects for anthocyanin concentration in
the kernels and cob of waxy corn cross.
Parameters
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 F1 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 F1 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.
Figure I. Kernels and cob color for parental lines [(A) KND10-4P, (B) BW]and F2 (C).
II. Biological markers determination
• Total Anthocyanin contents was determined according to the method described by Guiti and Wrulstad (2001) with minor modifications.
• Total phenolic compounds was determined according to the method described by Xu and Hu (2011) with minor modifications.
• 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
 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 (X2)
test with three degrees of freedom and was accepted if p<0.05. When the three-parameter model was inadequate (significant X2 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 (Ơ2E) and broad-sense heritability (H2b) were estimated using the following formula (Ito and Brewbaker, 1991)
H2b = (Ơ2F2- Ơ2E)/ Ơ2F2
Where
Ơ2E = ¼ (Ơ2P1 + Ơ2P2 +2 Ơ2F1)
Ơ2F2 = variance of the F2 populations
Ơ2P1 = variance of the parental line 1
Ơ2P2 = variance of the parental line 2
Ơ2F1 = variance of the F1 populations
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.
anthocyanins
315.26+4.23a
KND10-4P, P2; BW, F1; First filial generation of crosses, F2; Second filial generation of crosses, BC12; First backcross generation with
parental line 1 and BC12; 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).
(C)
Pn3G
P1
aP ;
1
(B)
Pg3G
kernel
kernel
F1, F2, 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, F1 and backcross
populations to avoid contamination from stray pollen and individual self-pollination of each
plant was practiced for the F2-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.
(A)
C3G
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.
C3G
Pg3G
Pn3G
Anthocyanins
m
157.90+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**
m
496.49+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+34.46ns
-52.90+36.99ns
-1921.20+313.09**
[ad]
-90.09+124.43ns
58.07+40.10ns
-35.06+44.37ns
-
[dd]
1148.30+117.06**
80.87+34.74*
159.03+37.01**
2804.34+313.12**
cob
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
The additive and dominance components of the genetic models were significantly different from zero. All sixparameters 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.
 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.
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.