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Genetics of Resistance to Field Kernel

Infection by Aspergillus flavus in Maize
a a
Ruming Li & Manjit S. Kang
a
Department of Agronomy and Environmental Management,
Louisiana State University Argricultural Center, Baton Rouge, LA,
70803-2110, USA
Version of record first published: 23 Sep 2008.

To cite this article: Ruming Li & Manjit S. Kang (2006): Genetics of Resistance to Field Kernel
Infection by Aspergillus flavus in Maize, Journal of Crop Improvement, 15:1, 11-32

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 Genetics of Resistance
to Field Kernel Infection
by Aspergillus flavus in Maize
Ruming Li
Downloaded by [Dr Ruming Li] at 07:04 19 December 2012

Manjit S. Kang

ABSTRACT. To solve the chronic problem of maize (Zea mays L.) ker-
nel infection by Aspergillus flavus in the southeastern USA, genetic re-
sistance to A. flavus is needed. A 2-yr study of a diallel cross of five
parents containing the leafy gene (Lfy) was conducted to evaluate the
inhertance of resistance to A. flavus during 2003 and 2004. The maize
percent kernel infection (PKI) rates were determined via the media-free
and kernel-isolated incubation method. Highly significant general com-
bining ability (GCA), specific combining ability (SCA), and reciprocal
effects were found. Parents 914 and A619 had desirable (negative) GCA
effects to enhance the average performance of A. flavus resistance in hy-
brid progeny. The crosses 914 ⫻ A632, 914 ⫻ Wf9, and Hy ⫻ Wf9 had
consistently negative SCA effects across years. The reciprocal effects
were detected, indicating the presence of maternal effects in the maize
kernel and the importance of direction of a cross. For example, A632 ⫻
Hy had the largest positive significant reciprocal effect; the reversal of
this cross, i.e., Hy ⫻ A632, should impart resistance to progeny. A North
Carolina Design-II analysis of four female lines (914, A632, HY, and
Wf9) crossed with three male lines (A619, B73, and Mo17), was used
to estimate additive and dominance genetic variances. Narrow-sense
heritability for PKI was 37.6% and broad-sense heritability estimate was

Ruming Li and Manjit S. Kang are affiliated with the Department of Agronomy and
Environmental Management, Louisiana State University Agricultural Center, Baton
Rouge, LA 70803-2110.
Address correspondence to: Manjit S. Kang (E-mail: mkang1@lsu.edu).
Journal of Crop Improvement, Vol. 15(1) (#29) 2005
Available online at http://www.haworthpress.com/web/JCRIP
 2005 by The Haworth Press, Inc. All rights reserved.
doi:10.1300/J411v15n01_03 11
 12 JOURNAL OF CROP IMPROVEMENT

89.1%. Breeding procedures to handle both additive and dominance

variances would need to be used to improve resistance to PKI. [Article
copies available for a fee from The Haworth Document Delivery Service:
1-800-HAWORTH. E-mail address: <docdelivery@haworthpress.com> Web-
site: <http://www.HaworthPress.com>  2005 by The Haworth Press, Inc. All
rights reserved.]
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KEYWORDS. Aspergillus flavus, inheritance, kernel infection rate,

maize, resistance

INTRODUCTION

Maize (Zea mays L.) is an important feed and food crop around the
world. In the United States, maize production annually occurs on about
70 million acres; Louisiana’s share being about half a million acres
(Corn Refiners’ Association, 2004). More than half of the maize pro-
duced in the United States is marketed as commercial maize and makes
a major contribution to the US economy. Major yield and economic
losses in the southeastern United States, including Louisiana, however,
result frequently from field invasion by Aspergillus species and subse-
quent aflatoxin contamination of maize kernels (Anderson et al., 1975;
Zuber and Lillehoj, 1979; Kang et al., 1988). The aflatoxin contamina-
tion of maize kernels and use of maize in food are potential threats to
human and animal health. Therefore, it is necessary to develop effective
and efficient methods to prevent aflatoxin contamination in maize.
Although efforts have been made to prevent A. flavus infection in
preharvest maize and aflatoxin contamination via cultural practices,
such as adjusted planting date, pest and weed control, and crop rotation,
a viable solution has been difficult to find (Lillehoj, 1983; Zuber et al.,
1987; Widstrom, 1996; Kang, 1996; Kang and Moreno, 2002). For
postharvest storage of maize, some chemical practices, such as ammo-
nia detoxification of contaminated kernels in storage and processing,
were adopted to eliminate or reduce aflatoxin contamination, but the
antifungal compounds turned out to be hazardous and expensive (Park
et al., 1995).
Research efforts have also been focused on understanding the bio-
synthesis of aflatoxin by A. flavus. Molecular genetics of aflatoxin for-
mation and identification of biosynthetic pathway genes have improved
our understanding about aflatoxin production (Bhatnagar et al., 1991;
 Ruming Li and Manjit S. Kang 13

Brown et al., 1998). Ongoing attempts to block the biosynthetic path-

way of aflatoxin production in the fungus have had some success in
developing atoxigenic variants of A. flavus. However, biological
modification or genetic manipulation of A. flavus motabolites in the
laboratory may not be meaningfully applicable to the field-maize en-
vironment.
From practical standpoints, maize breeders and pathologists have
been engaged in preventing or alleviating aflatoxin contamination in
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maize by identifying host-plant control mechanisms (e.g., Zuber et al.,

1978; Gardner et al., 1987; Gorman et al., 1992a; Campbell and White,
1995; Zhang et al., 1997). Host-plant resistance studies have been the
hope for developing resistant genotypes (Gorman and Kang, 1991;
Moreno and Kang, 1999). Host resistance to kernel infection by A.
flavus and subsequent production of aflatoxin has been found to be
partly under genetic control (Zuber et al., 1978; Widstrom et al., 1984;
Darrah et al., 1987; Gorman et al., 1992a; Naidoo et al., 2002). Naidoo
et al. (2002) found GCA effects for ear rot and aflatoxin levels to be
highly significant among hybrids overall, and for the inbreds Tex6 and
Oh516. Disagreements about the relative importance of GCA and SCA
exist and have been discussed in Kang and Moreno (2002).
Resistance of maize to A. flavus and aflatoxin contamination has
been found to be associated with the outer integuments of developing
kernels and other ear components (Widstrom et al., 1984; Wallin, 1986;
Darrah et al., 1987; Guo et al., 1993; 1995). The kernel cuticle and
pericarp are thought to play a role in resistance to A. flavus. Thus, utili-
zation of their protection in developing resistant genotypes has been of
interest (Wicklow and Horn, 1984; Wallin, 1986; Guo et al., 1993,
1995; Brown et al., 1998).
Previously several sources of resistance to A. flavus infection and af-
latoxin accumulation have been identified (Scott and Zummo, 1988;
Kang et al., 1990; Campbell et al., 1993; Zhang et al., 1997; Li et al.,
2002). Nevertheless, additional genetic sources of potentially higher re-
sistance must be identified to enrich the resistance gene pool. Some
resistant germplasm sources are agronomically and economically unde-
sirable for commercial use and some reported resistance is not high
enough to be of use in breeding (Widstrom et al., 1978). One of the strat-
egies should be to continue to screen broad-based maize germplasm
with genetic diversity of pathogen responsiveness. Further elucidation
of the genetic mechanisms of maize resistance to kernel infection by A.
flavus and identification and development of sources of resistance to A.
flavus are still critically needed.
 14 JOURNAL OF CROP IMPROVEMENT

The progress in combating aflatoxin contamination in maize (Lillehoj

and Wall, 1987; Zuber et al., 1987; Brown et al., 1998; Kang and
Moreno, 2002) and in elucidating genetic mechanisms and identifying
sources of resistance to aflatoxin, however, has been slow. A major rea-
son for the slow progress might be that genotype evaluation for aflatoxin
resistance in the laboratory is laborious, expensive, and time-consum-
ing (Wallin et al., 1982).
Aflatoxin contamination can be controlled through the prevention of
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kernel infection by A. flavus. Reduced aflatoxin levels in the grain

might be achieved through selection for reduced kernel infection rates.
Although there are differences in opinion on this issue, positive correla-
tions between kernel infection rates and actual aflatoxin accumulation
in the kernel have been reported (Tucker et al., 1986), and hybrids that
exhibit lower infection levels have significantly lower concentrations of
aflatoxin in the kernel at harvest (Tucker et al., 1986). Quite logically, if
infection can be completely prevented, aflatoxin production should also
be completely prevented. Therefore, a genetic study focused on kernel
infection by A. flavus could provide genetic information on resistance to
aflatoxin contamination in maize.
The Czapek agar medium plating (CAMP) protocol was developed
to quantify incidence of A. flavus as percent kernel infection (PKI) to re-
duce/circumvent the cost associated with determining aflatoxin concen-
tration via densitometry and HPLC (King and Scott, 1982; Tucker et al.,
1986; Scott and Zummo, 1988; Zummo and Scott, 1989). Li et al.
(2002) further reduced the cost of determining PKI by inventing, and
showing the effectiveness of, a media-free, kernel-isolated incubation
(MIKI) method (Li and Kang, 2004). The MIKI protocol is much
cheaper and more accurate for evaluating maize resistance to A. flavus
than the CAMP method.
The current knowledge of genetic nature of resistance to A. flavus is
inadequate due to the limited study environments, and different materi-
als and methodologies used (Campbell et al., 1993; Zhang et al., 1997).
Maize is typically an open-pollinated species with tremendous genetic
diversity (Stoloff and Lillehoj 1981; Widstrom et al., 1986). The limited
germplasm used in previous studies was not informative enough to as-
certain the entire spectrum of inheritance of resistance to A. flavus. Also
because genetic mechanisms of resistance to A. flavus and aflatoxin ac-
cumulation in maize kernel are varied and inconclusive, additional in-
formation on genetics of resistance to A. flavus is warranted. Therefore,
a diallel cross among five maize lines containing the leafy (Lfy) gene
was used to obtain new information on the genetics of resistance to A.
 Ruming Li and Manjit S. Kang 15

flavus. Previous studies with the Lfy gene have reported much-needed
resistance to A. flavus (Gorman, 1991; Gorman and Kang, 1991;
Gorman et al., 1992). The specific objectives of this study were: (1) To
ascertain the inheritance of resistance to kernel infection by Aspergillus
flavus using a diallel-cross analysis of Lfy maize, and (2) To conduct a
North Carolina Design II analysis to determine variance components
and heritability for resistance to A. flavus kernel infection.
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MATERIALS AND METHODS

2003 Study

Field experiments were carried out at the Ben Hur Plant Science
Farm near Baton Rouge, LA. Seeds of 20 F1 crosses of maize were ma-
chine-planted into a Commerce silt loam (fine-silty, mixed, nonacid,
thermic Aeric Fluvaquent) in a randomized complete-block design with
two replications. Thirty seeds of each cross/entry were planted in a 4.6
m-long, single-row plot with interrow spacing of 1.02 m and interplant
spacing of 0.15 m.

Field Inoculation

Midsilk (50% plants in a plot with silks emerged) dates were re-
corded for each plot. Twenty-eight days after midsilk, six ears per plot
were randomly selected and inoculated with two drops of conidial sus-
pension of A. flavus isolate NRRL 3357 (1.0 ⫻ 106 conidia/ml), using
an eyedropper to replace the frequently used needle-in-silk-channel
technique. The eyedropper technique used a blunt tip of eyedropper that
prevented kernel damage and provided operational ease (especially
when plants were too tall for a relatively short operator). It also let
inoculum drip vertically into the silk channel. Thus, the inoculum could
gently and uniformly spread therein without any overpressure and spill
(Wicklow and Horn, 1984). Field inoculations were done within one
day or at most two days to achieve consistent timing of start of inocula-
tion stress. The plots that silked late were inoculated 3 to 5 days later.

Harvest and Laboratory Analysis

In September 2003, inoculated ears were hand-harvested and bagged

on a plot basis. The harvested ears were air-dried to 13-15% moisture
 16 JOURNAL OF CROP IMPROVEMENT

content at room temperature (25°C) in a laboratory with good ventila-

tion, and machine-shelled. The shelled kernels from each plot were
sifted using a sieve with a mesh diameter of 0.6 cm to cull out under-
sized and cracked kernels, and to ensure that the kernels were com-
pletely blended. The sifted and blended kernels from each plot were put
in a paper bag and placed at room temperature (below 10°C) for storage
(Vincelli et al., 1995). This prevented secondary infection of uninfected
kernels by the infected ones. For fungal infection assay, about 200 ker-
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nels per plot were randomly sampled (Davis et al., 1980; Widstrom et
al., 1982). The sampled kernels were washed in running cold tap water
(not in hot tap water to keep subepidermal A. flavus from being affected
by heat) with a lab brush swirling in a strainer (colander) and then
washed in running distilled water to remove other microorganisms to
prevent possible biocompetitive growth (this step of using distilled wa-
ter was optional for MIKI assay as other chemical treatments were used
to ensure sterilization). The kernels were soaked in 70% aqueous etha-
nol in a beaker for 1 min and rinsed by running distilled water to disin-
fect the kernel surface, followed by soaking in 0.25% NaOCl solution in
a beaker for 1 min, and rinsed with running distilled water for further
surface sterilization (Sauer and Burroughs, 1986). The purpose of this
protocol was to doubly ensure complete surface sterilization of kernels.
Then, water was drained and kernels were placed, using a sterile pair of
tweezers, in 48-well polystyrene tissue culture plates with 1.2 cm well
diameter (Becton Dickinson Labware, USA). Each well contained a
single kernel (no culture medium was used). The procedure provided an
equal chance for A. flavus to emerge from every infection site. Each
plate was covered with a lid (to avoid or minimize cross-infection be-
tween infected and uninfected kernels) (Li et al., 2002; Li and Kang,
2004). A total of 144 kernels (3 plates with 48 kernels each) per sample
were assayed. All kernels for each block or replication were incubated
together in an incubator (NAPCO Series 6500). All plates were stacked
or positioned such that they had a uniform spacing between them and
experienced homogeneous incubating conditions. Incubation tempera-
ture was 35°C (optimal for A. flavus growth) and relative humidity was
kept at 100 to 95% for the first day, 95 to 90% for the second day, and 90
to 85% from the third day on (to simulate the field humid process)
(Winn and Lane, 1978). On the 10th day of incubation, kernel-infection
levels were scored by recording the number of infected kernels under a
fume hood (to prevent spore dispersal). Counting was done with the aid
of a needle to probe if the kernel site facing the bottom of wells had A.
flavus growth. Data were collected from 3 plates (3 records) for each
 Ruming Li and Manjit S. Kang 17

sample and then converted automatically to percent kernel infection

(PKI) on a plot basis via Windows-based software ‘Diallel Analyzer’
(Li and Kang, unpublished) (lower PKI = higher A. flavus resistance).
Statistical and Genetical Analyses
Data were analyzed according to linear mathematic models (Liu et
al., 1984; Gao, 1986; Bogartz, 1994; Sahai and Ageel, 2000) using a
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general notation of y = number of years, r = number of replications or

blocks, and g = number of genotypes. Genotypes (F1 hybrids) were
treated as fixed effects unless explicitly specified otherwise. The diallel-
cross analysis was based on Griffing’s (1956) Method 3 and performed
on a plot basis with a fixed-effect model using ‘Diallel Analyzer’ (Li
and Kang, unpublished). The statistical and genetic analyses for the
North-Carolina-Design II (NCD-II) crosses were conducted via another
newly developed ‘Genetic Data Analyzer for Windows’ software (Li
and Kang, unpublished).
The general linear model for the pooled analyses of variance in the
diallel cross was:
Yijkl = µ + yi + r(y)j(i) + xkl + (xy)ikl + ejkl(i)
where Yijkl = observed kernel infection rate or PKI for the ith year, jth
replication within the ith year, and kth and lth parents,
µ = grand mean of the experiment,
yi = effect of the ith year,
r(y)j(i) = effect of the jth replication within the ith year,
xkl = genotypic effect of the cross between kth and lth par-
ents,
(xy)ikl = interaction of the cross between kth and lth parents with
the ith year, and
ejkl(i) = residual effect or random error of the experiment.

The linear mathematical model for combining ability analyses in the

diallel cross was:
Xij = µ + gi + gj + sij + rij + 1b eijl(k)

where Xij = observed kernel infection rate or PKI from the cross be-
tween ith and jth parents,
µ = overall mean of the experiment,
 18 JOURNAL OF CROP IMPROVEMENT

gi = genetic effect of general combining ability (gca) for the

ith parent,
gj = genetic effect of general combining ability (gca) for the
jth parent,
sij = genetic effect of specific combining ability (sca) for
cross between ith and jth parents,
rij = genetic effect of reciprocal cross between the ith and jth
parents, and
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1
e
b ijl(k)
= residual effect of combining ability analysis from ran-
dom error of the experiment,

b is # of blocks or replications, and eijl(k) is ejkl(i) in the foregoing general

linear model.
The linear mathematical model for genetic effect analyses in the
diallel cross across years was:
1
Xij = µ + gi + gj + sij + rij + yb eijl(k)

where Xij = observed kernel infection rate or PKI from the cross be-
tween ith and jth parents,
µ = overall mean of the experiment,
gi = genetic effect of general combining ability (gca) for the
ith parent,
gj = genetic effect of general combining ability (gca) for the
jth parent,
sij = genetic effect of specific combining ability (sca) for
cross between ith and jth parents,
rij = genetic effect of reciprocal cross between the ith and jth
parents, and
1
e
yb ijl(k)
= residual effect of combining ability analysis from ran-
dom error of the experiment,

y is # of years, b is # of blocks, and eijl(k) is ejkl(i) in the foregoing general

linear model.
The linear statistical model for the nested-within-year effects in the
NCD-II crosses was:

Yijkl = µ + yi + r(y)j(i) + fk(i) + ml(i) + (fm)kl(i) + ejkl(i)

 Ruming Li and Manjit S. Kang 19

where Yijkl = observed kernel infection rate or PKI for the ith year, jth
replication within the ith year, kth female, and lth male
parents,
µ = overall mean of the experiment,
yi = effect of the ith year,
r(y)j(i) = effect of the jth replication within the ith year,
fk(i) = genotypic effect of the kth female parent within the ith
year,
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ml(i) = genotypic effect of the lth male parent within the ith
year,
(fm)kl(i) = interaction between kth and lth parents within the ith
year, and
ejkl(i) = residual effect or random error of the experiment.

RESULTS AND DISCUSSION

Diallel Analyses for Maize Kernel Infection Rates: 2003-2004

Separate analyses of variance for PKI for the 20 F1s from the Lfy
maize diallel cross for the years 2003 and 2004 showed highly signifi-
cant differences among crosses (genotypes) (data not shown). This sup-
ported the earlier evidence for variability of maize resistance to A.
flavus infection (Zhang et al., 1997; Li et al., 2002) and also demon-
strated differential Lfy-based pathogen responsiveness. The differentia-
tion of resultant infection rates between the genotypes in response to the
challenge of field inoculation with A. flavus are shown in the Figures 1,
2, 3, 4, and 5.
A combined analysis of variance for PKI for the 20 F1s from Lfy
maize diallel crosses for the years 2003 and 2004 also showed highly
significant differences among crosses (genotypes) (Table 1). Variabil-
ity for maize resistance to A. flavus infection was confirmed.

Analysis of Combining Ability for Maize Kernel Infection Rates

Because of the presence of marked genotypic effects, the analysis of

combining ability (ACA) for fixed effects was conducted to further re-
veal their component genetic effects. The separate ACA analyses by
year as well as pooled ACA analysis (Table 2) for the years 2003 and
2004 indicated that there were highly significant general combining
ability (GCA) effects and reciprocal effects. These two sources of varia-
 20 JOURNAL OF CROP IMPROVEMENT

FIGURE 1. The 48-well tissue culture plates and differential A. flavus growth
on kernels.
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tion accounted for most of the genetic effects contained in the term ‘Ge-
notypes.’ The specific combining ability (SCA) effect was significant
for the year 2003 as well as for 2004. The performance of resistance to
A. flavus with such a pattern across years made it difficult to establish
that the SCA effect was as important as GCA effect. Nevertheless, we
were able to ascertain this genetic effect by integrating data across
years, after interfering source of variation or noise was partitioned out
from the total variation. This led to enhanced testing power, i.e., two
 Ruming Li and Manjit S. Kang 21

FIGURE 2. High level of resistance shown by maize kernels lightly infected

with A. flavus.
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FIGURE 3. Moderate level of resistance shown by maize kernels moderately

infected with A. flavus.
 22 JOURNAL OF CROP IMPROVEMENT

FIGURE 4. Susceptibility shown by maize kernels heavily infected with A.

flavus.
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FIGURE 5. High level susceptibility shown by maize kernels severely infected

with A. flavus.
 Ruming Li and Manjit S. Kang 23

TABLE 1. Analysis of variance for pooled PKIs in 2003 and 2004 with a fixed ef-
fect model.

Source of variation d.f. Mean squares F-value Expected mean squares

Years 1 1281.11** 31.18 s e2 + 40s y2
Blocks (Y) 2 2800.52** 68.17 s e2 + 20s r2( y )
Genotypes 19 224.03** 5.45 s e2 + 4s g2
G⫻Y s e2 + 2s gy
2
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19 185.76** 4.52
Error 38 41.08 s e2
Total 79
** Significant at p = 0.01 level.

TABLE 2. Analysis of combining ability for maize kernel infection rates in

2003-2004.

Source of variation d.f. Mean squares F-value

Genotypes 19 224.03** 5.45
GCA 4 155.86** 15.18
SCA 5 142.39** 13.86
Reciprocal 10 292.12** 28.44
ACA Error 38 10.27
ANOVA Error 38 41.08
** Significant at p = 0.01 level.

times the degrees of freedom (up from 19 to 38). The results better de-
termined the weight or relative importance of SCA effects among the
multiple genetic effects (Table 2). The highly significant reciprocal ef-
fects were consistently found across the two years as well as in the
pooled analyses. This result agreed with the previous studies (Zhang et
al., 1997) in that the reciprocal effects existed for this trait measured in
field trials.
Analysis of Parental GCA Effects for Maize Kernel
Infection Rates
For the year 2003, the component GCA effects were estimated be-
cause of the highly noticeable GCA effect; all of them were highly sig-
nificant with the exception of parent A619 (data not shown). These
parents demonstrated general combining ability effects in response to
the challenge of field A. flavus inoculation. However, only negative
 24 JOURNAL OF CROP IMPROVEMENT

GCAs that had statistical significance would be desirable for develop-

ing A. flavus-resistant maize. That is, the larger the negative value of ge-
netic effects, the higher the resistance to A. flavus the parents possessed.
The GCA effects for the parents 914 (⫺5.28**) and Wf9 (⫺3.89**)
were highly significantly negative, suggesting that the use of these par-
ents in crosses should be conducive to reducing PKI. For the year 2004,
the results rejected the prior ones as a reversal in sign for GCAs was ob-
served (data not shown). The integrated ACA analysis (Table 3) took
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the year and genotype-by-year interfering effects into account and took
these two sources of variation off the total variation. Without the con-
founded factors, the parents with negative GCAs were validated; 914
(⫺1.74**) and A619 (⫺1.59**) were highly significant. The phenom-
ena that the parent A619 turned out to have a significant negative GCA
and the parent Wf9 not to have a significant one resulted mainly from
year effects. It was also the year effects (with much larger mean squares
in ANOVA) that generated genotype-by-year interaction and compli-
cated evaluation of the parental GCAs. On the other hand, A632 showed
highly significant positive GCA effects for individual years (6.12** for
2003 and 2.67** for 2004) and in the combined ACA analysis (4.40**).
Thus, the use of A63 should be avoided in efforts to reduce PKI al-
though it had marked combining ability in crosses with other lines.
Whether this resistance was attributable to the Lfy morphological/phys-
ical protection or to biological/biochemical mechanisms remains a
question. With the desirable GCA effects for the parents 914 and A619,
the higher average performance of A. flavus resistance from their prog-
eny could be expected and thus bettering the chances of achieving success
in resistance breeding. Better-performing progeny relative to resistance
to A. flavus infection should be expected from crossing these two par-
ents, as both possessed the largest GCA effects (Kang, 1994).

TABLE 3. Parental GCA, SCA, and reciprocal (REC) effects for PKI from the
diallels in 2003-2004.

Parents (Ps) GCA Crosses SCA REC Crosses SCA REC

914 (P1) ⫺1.74** 914 ⫻ A619 1.37* 1.65 A619 ⫻ Hy ⫺1.49* 6.08**
A619 (P2) ⫺1.59** 914 ⫻ A632 ⫺3.40** ⫺5.64** A619 ⫻ Wf9 1.06 ⫺5.12**
A632 (P3) 4.40** 914 ⫻ Hy 5.16** 7.90** A632 ⫻ Hy ⫺0.71 ⫺11.28**
Hy (P4) ⫺1.04* 914 ⫻ Wf9 ⫺3.14** 3.91** A632 ⫻ Wf9 5.05** 5.38**
Wf9 (P5) ⫺0.03 A619 ⫻ A632 ⫺0.94 2.69* HY ⫻ Wf9 2.97** ⫺5.12**
*,** Significant at p = 0.05 and p = 0.01 level.
 Ruming Li and Manjit S. Kang 25

Analysis of Parental SCA Effects for Maize Kernel

Infection Rates
From the analyses of combining ability for the 10 pairs of possible
parental combinations, the specific crosses that had significant negative
SCA effects were: 914 ⫻ Wf9 (⫺4.51**) and A619 ⫻ Hy (⫺4.22**)
for 2003; and 914 ⫻ A632 (⫺4.77**), A632 ⫻ Hy (⫺3.44*), and Hy ⫻
Wf9 (⫺5.47**) for 2004. There were no specific crosses that had con-
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sistently significant negative SCA effects across the two years. The fol-
lowing four crosses had significant negative SCA effects when data
were analyzed across years (Table 3): 914 ⫻ A632, 914 ⫻ Wf9,
A619 ⫻ Hy, and Hy ⫻ Wf9. Thus, resistance to kernel infection by A.
flavus existed among the parents and some of the crosses.
Analysis of Parental Reciprocal Effects for Maize Kernel
Infection Rates
In 2003 and 2004, there were seven crosses and six crosses, respec-
tively, that exhibited statistically significant reciprocal effects. Across
years, nine of the 10 crosses exhibited statistically significant reciprocal
effects (Table 3). The only cross that showed a non-significant recipro-
cal effect was 914 ⫻ A619. Among the nine crosses with significant re-
ciprocal effects, five (914 ⫻ Hy, 914 ⫻ Wf9, A619 ⫻ A632, A619 ⫻
Hy, and A632 ⫻ Wf9) had positive effects. For these crosses to be use-
ful, the direction of the cross must be reversed, i.e., the parents used as
females should be used as males, and vice versa. The following three
crosses (Table 3) were in the correct direction since they had significant
negative effects: 914 ⫻ A632 (⫺5.64**), A619 ⫻ Wf9 (⫺5.12**),
A632 ⫻ Hy (⫺11.28**), and Hy ⫻ Wf9 (⫺5.12**).
GCA Effects for Maize Kernel Infection Rates
A combined analysis for GCA effects indicated that there were six
significant pairwise differences among parental GCA effects (Table 4).
These differences among Lfy-based parents implied that these GCAs
represented a broad range of choice for selecting desired parents to
reduce PKI.
SCA Effects for Maize Kernel Infection Rates
The pooled comparisons (Table 5) among SCA effects across years
gave 35 significant paired differences. This relatively high rate of no-
 26 JOURNAL OF CROP IMPROVEMENT

TABLE 4. Multiple comparisons of parental GCA effects for PKI from the
diallels in 2003-2004.

GCA2 (P2) GCA3 (P3) GCA4 (P4) GCA5 (P5)

GCA1 (P1) 0.14 6.13** 0.69 1.71*
GCA2 (P2) 5.99** 0.55 1.56*
GCA3 (P3) 5.44** 4.43**
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GCA4 (P4) 101

*,** Significant at p = 0.05 and p = 0.01 (LSD), respectively.

TABLE 5. Multiple comparisons of parental SCA effects for PKI from the
diallels in 2003-2004.

SCA13 SCA14 SCA15 SCA23 SCA24 SCA25 SCA34 SCA35 SCA45

SCA12 4.77** 3.79** 4.51** 2.31** 2.86** 0.32 2.08* 3.67** 4.34**
SCA13 8.56** 0.26 2.46** 1.91* 4.46** 2.69** 8.45** 0.43
SCA14 8.30** 6.11** 6.66** 4.11** 5.87** 0.12 8.13**
SCA15 2.20* 1.65* 4.20** 2.43** 8.19** 0.17
SCA23 0.55 2.00 0.23 5.99** 2.03*
SCA24 2.55* 0.78 6.54** 1.48
SCA25 1.77* 3.99** 4.02**
SCA34 5.76** 2.26**
SCA35 8.02**
*,** Significant at p = 0.05 and p = 0.01 (LSD), respectively.

ticeable differences found among the SCAs implied that there were
good possibilities of finding desirable parental combinations from the
specific crosses than from the average performance evaluation of parents.

North Carolina Design II (NCD-II) Analyses: 2003-2004

Twelve single crosses were made from a set of four female maize
lines (914, A632, Hy, and Wf9) a set of male maize lines (A619, B73,
and Mo17). The NCD-II results for Females, Males, and their interac-
tion with each other (M ⫻ F) are given in Table 6. For this nested-
within-year study, all three terms of interest were highly significant.
The term ‘Females’ had the largest mean square. This indicated that the
genetic differences resulting from between females were major ones
and should become a focus in the fungal resistance. This also partially
 Ruming Li and Manjit S. Kang 27

TABLE 6. Analysis of variance for percent kernel infection (PKI) in 2003-2004

NCD-II crosses with a nested-in-year model.

Source of
d.f. Mean squares F-value Expected mean squares
variation
Years --- 1 --- 564.33** 11.64 --------------------------
Blocks (Y) --- 2 --- 494.36** 10.19 --------------------------
Females (Y) df1 6 M1 645.91** 13.32 s e2 + 2s fm
2
(y ) + 6 f (y )
s2
Downloaded by [Dr Ruming Li] at 07:04 19 December 2012

Males (Y) df2 4 M2 236.44** 4.88 s e2 + 2s fm

2
( y ) + 8s m ( y )
2

F ⫻ M (Y) df3 12 M3 169.97** 3.51 s e2 + 2s fm

2
(y )
Error dfe 22 Me 48.49 s e2
Total 47
** Significant at p = 0.01 level.

accounted for reciprocal effect that was previously noted for A. flavus
resistance. Thus, breeders should use this information in devising breed-
ing strategies for developing genotypes with resistance to A. flavus. The
‘Males’ source of variation had smaller contribution to the total varia-
tion for PKI than Females. The PKI variation attributable to the interac-
tion between Males and Females was also noticeable, which suggested
that some directional dominance might play a role in maize response to
the fungal infection within the materials examined. To further obtain
specific genetic information, genetic variances were estimated. When
s 2m ≠ s 2f , because of possible maternal effects it would be advisable to
use s 2m to obtain an unbiased estimate of s 2A (Kang, 1994). This is be-
cause it would be robust for one to estimate the parental genetic vari-
ance that was generated by the difference controlled by nuclear genes
alone. This would help circumvent the bias. If they are equal, then, they
may be pooled. In present analysis, s 2m ≠ s 2f (see below). Since the s 2j
and s 2m were highly significant and unequal, the maternal effects may
not be ignored. To take this significant effect into account, it might be
appropriate to use the arithmetic mean of s 2A (f) and s 2A (m). The nar-
row-sense heritability of maize PKI for an additive-dominance model
was estimated to be 37.6%, and broad-sense heritability was estimated
to be 89.1%.
The results from these investigations can be summarized as follows:
 28 JOURNAL OF CROP IMPROVEMENT

1. The maize PKI relative to A. flavus was a quantitative trait.

2. The maize PKI was both macro- and micro-environment-sensi-
tive, as noted throughout this study as well as in the previous stud-
ies (Gorman and Kang, 1991; Zhang et al., 1997; Li et al., 2002,
2004; Li and Kang, 2004). The macro-environment effect resulted
from the differential year effect and the microenvironment effect
related to the block effect.
3. The maize PKI had moderate character transmissibility between
Downloaded by [Dr Ruming Li] at 07:04 19 December 2012

generations, as reflected by its remarkable genotypic effects and

the estimated broad-sense heritability of 89.1%. The maize PKI
had modest fixable character transmissibility between genera-
tions, as reflected by its relatively low narrow-sense heritability of
37.6%.
4. The maize PKI appeared to be under a multi-gene system control
and to be additively inherited, as suggested by its typical quantita-
tive nature and by the highly significant additive genetic variance
present in this study.
5. The maize PKI also appeared to have a large dominance or non-
additive variance, as reflected by the large difference between
broad- and narrow-sense heritabilities. The large non-additive
variance could not be fully explained by the additive-dominance
model alone and interaction between non-allelic genes or epistasis
might also be present. Since the genetic variability for resistance
of maize to A. flavus was mostly attributable to nuclear genes, it
would be governed by the additive, dominance, and/or epistatic
actions.
6. As suggested by the significant additive variance, it should be
possible to accumulate favorable additive genes by means of
breeding schemes such as recurrent selection within a population
derived from the Lfy material evaluated. It should be possible to
develop exceptionally elite maize. The directive role of s 2A and
s 2D applies even though more and more emphasis is currently be-
ing placed on molecular aspects. For instance, detection of signifi-
cant s 2A for A. flavus resistance would be extremely meaningful in
gene mapping, sequencing, and cloning of possible quantitative
trait loci (QTL). If a QTL cannot be reflected by significant s 2A for
A. flavus resistance, one may not be able to discover real resis-
tance. In that case, one may need to “borrow” or introduce the de-
sired gene from other species.
 Ruming Li and Manjit S. Kang 29

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