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2^ r
2
D
=^ r
2
A
p
were computed following the procedures
devised by Comstock and Robinson (1952) for Design
III. Also, the genetic variance ^ r
2
G
^ r
2
A
^ r
2
D
, the
genetic by location interaction variance ^ r
2
GL
^ r
2
AL
^ r
2
DL
, the phenotypic variance on a progeny
mean-basis ^ r
2
Ph
^ r
2
G
^ r
2
GL
=L ^ r
e
=RL and on a
plant-basis ^ r
2
PhP
^ r
2
G
^ r
2
GL
^ r
2
e
, and the herita-
bility coefcients on a broad-sense progeny mean-
basis (
^
h
b
^ r
2
G
=^ r
2
Ph
and strict-sense
^
h
2
s
^ r
2
A
=^ r
2
PhP
were computed. ^ r
e
, R, and L stand for the error
variance, number of replications, and number of
locations, respectively. Condence intervals at the
95 % probability level for the estimates of the genetic
and phenotypic variances, for the level of dominance,
and for the heritability coefcients were computed
following the procedures of Burdick and Graybill
(1992).
Genetic map
The genetic map used, and the procedures used to
develop it, was previously described by Sibov et al.
(2003). Briey, the F
2
plants that gave rise to the F
2:3
progenies were genotyped with microsatellite mark-
ers. The genetic map was developed using MAP-
MAKER/EXP version 3.0b (Lincoln et al. 1992) with
a LOD threshold of 3.0 and a maximum distance
between adjacent markers of 50 cM; i.e., 0.38 as the
maximum recombination frequency, to establish the
linkage groups, and the Kosambis (1944) mapping
function was used to convert recombination frequen-
cies into map distances. Sixty new microsatellite
markers were added to the map of Sibov et al. (2003),
for a total of 177 markers distributed along the 10
linkage groups. The genetic map spanned 2,052 cM in
length with an average interval of 11.60 cM between
adjacent markers.
QTL mapping
The composite interval mapping method (CIM)
extended to perform a joint analysis of multiple
locations (mCIM) was used to map QTL, since it is
more powerful than the CIM procedure. The least
square means of stay-green scores from each location
were used to perform the analysis. The underlying
mixture model for QTL mapping is (Jiang and Zeng
1995):
Y
jk
b
0k
a
k
x
j
d
k
z
j
X
t
l
a
lk
x
jl
d
lk
z
jl
e
jk
where Y
jk
is the phenotypic mean of the jth progeny at
the kth location; b
0k
is the mean effect of the model for
location k; a
k
is the additive effect of the putative QTL
for location k; x
j
counts the number of alleles from
parental inbred L-08-05F at the putative QTL taking
values of 2, 1, and 0 for genotypes QQ, Qq, and qq,
respectively, with probabilities depending on the
genotypes of the markers anking the putative QTL
and the recombination frequencies between the QTL
and the markers; d
k
is the dominance effect of the
putative QTL for location k; z
j
is an indicator variable
taking values of 1 for genotype Qq and 0 for genotypes
QQ and qq of the putative QTL, with probabilities
depending on the genotypes of the markers anking
the putative QTL and the recombination frequencies
between the QTL and the markers; x
jl
and z
jl
are
corresponding variables for marker l, with t markers
selected as cofactors for controlling residual variation;
a
lk
and d
lk
are the partial regression coefcients of Y
jk
on x
jl
and on z
jl
, respectively; and e
jk
is the residual of
the model. The cofactors were selected using the
stepwise regression procedure (P B 0.05) for each
location and combined for the joint QTL analysis.
QTL mapping was performed for each set of the
backcrossed progeny, and then two analyses were
performed: one for the progenies backcrossed to the
parental inbred L-14-04B (BC
1
), and one for those
backcrossed to the parental inbred L-08-05F (BC
2
).
The likelihood-ratio (LR) threshold used to declare the
presence of a QTL was computed following the
166 Euphytica (2014) 198:163173
1 3
procedure described by Vieira et al. (2000), which is
based on the number of independent tests and the
window size used (10 cM). The number of indepen-
dent tests was 74 and the genome-wide probability
level used was a = 0.05, so that for each test the
probability level was a
0
= 0.05/74 = 0.00067. The
threshold for the QTL mapping was then LR = 19.33
(LOD = 4.20) and for the interaction QTL by location
(QTL 9 L) the threshold was LR = 14.6
(LOD = 3.17). The LOD score used for the QTL
mapping was greater than that used in other studies for
this trait in maize (Beavis et al. 1994; Zheng et al.
2009). The QTL mapping was performed with the
software Windows QTL Cartographer, version 2.5,
JZmap procedure (Wang et al. 2005), using a window
size of 10 cM and a walking speed of 1 cM.
Since the QTL analyses were performed separately
for each of the backcrossed progenies, the mCIM
model did not return directly the values of the additive
and dominance effects, but they can be computed from
the pseudo-additive values (a) estimated from the
individual analyses as follows: the a value for a
QTL mapped in the BC
1
progenies is actually
a
1
= (a - d)/2, and for a QTL mapped in the
BC
2
progenies a
2
= (a ? d)/2, so that the additive
and dominance effects for each mapped QTL could be
computed as: a = (a
1
? a
2
) and d = (a
1
-
a
2
), respectively (Lu et al. 2003, Frascaroli et al.
2007). The phenotypic variances explained by each
QTL and by the set of all mapped QTL (R
2
Ph
) were also
computed in Windows QTL Cartographer. All the QTL
previously mapped using the mCIM procedure were
tted in a single model with multiple QTL, and then, the
software uses the EM algorithm to evaluate the likeli-
hoodof this mixture model with multiple QTL. Once the
model had been tted, the coefcients of phenotypic
determination of each QTL and of all QTL were
automatically computed by the software. The genotypic
coefcients of determination for each QTL and for the
set of all QTL mapped were computed by dividing the
respective phenotypic coefcients of determination
(R
2
Ph
) by the coefcient of heritability, i.e.
R
2
G
R
2
Ph
=h
2
. Parental inbred L-08-05F has slower
leaf senescence than the parental inbred L-14-04B.
Since the rating scale ranged from 1 for the highest
level of stay-green to 5 for no stay-green plants, a
negative sign for the additive effect of a QTL indicated
that the favorable allele (allele that increase stay-
green) was from the L-08-05F, while a positive sign
indicated that a favorable allele was from L-14-04B.
The level of dominance of a QTL was computed as the
ratio LD d j j= a j j, and the average level of dominance
(ALD) was computed as a sum of LD ratios, each
weighted by R
2
G
value of its respective QTL. The QTL
action for each QTL and for the set of all mapped QTL
were characterized as additive (A) for 0.00 B LD
B 0.20, partial dominance (PD) for 0.21 B LD B
0.80, dominance (D) for 0.81 B LD B 1.20, and
overdominance for LD C 1.21 (Lima et al. 2006).
Results
Means and variances
The Wtests performed for the distribution histograms of
the stay-green data and of the standardized residuals
showed that both histograms did not differ signicantly
from the normal distributions. The overall means of the
parental inbreds differed signicantly (P B 0.05) (L-
14-04B = 4.20 vs. L-08-05F = 2.20), their mean
(3.20) did not differ from the mean of the two
backcrosses (2.88), which in turn did not differ signif-
icantly between themselves (BC
1
= 3.05 vs.
BC
2
= 2.71). The means of the progenies assessed
ranged from 1.38 to 4.95 for BC
1
and from 1.31 to 4.58
for BC
2
, showing the presence of transgressive proge-
nies, with lower and higher stay-green means than
parental inbreds, for both backcrosses. The experimen-
tal coefcient of variation (CV %) was 18.01 %, which
is similar to other reported values (Costa et al. 2008;
Camara et al. 2007), showing that the data recorded
presented good experimental precision (Table 1).
Highly signicant differences (P B 0.01) were
detected in the combined analysis of variance of the
Design III (not shown) for progeny and for progeny by
parent interaction, indicating that both additive and
dominance effects were present in the variation for the
stay-green trait. Also, signicant (P B 0.05) progeny
by location interaction was detected, but the progeny
by parent by location interaction was not found to be
signicant, and therefore, the mean values of the
progenies from both backcrosses varied across loca-
tions whereas for the progeny by parent interaction did
not; then additive effects presented differential values
across locations but the dominance effects did not.
Euphytica (2014) 198:163173 167
1 3
The estimate of the genetic variance ( ^ r
2
G
, and its
components the additive ^ r
2
A
and the dominance ^ r
2
D
d
0.55 [0.34; 0.82]
a
Estimates of variances and their respective condence
intervals multiplied by 10
2
b
Condence intervals at the 95 % probability level
168 Euphytica (2014) 198:163173
1 3
statistically signicant. Nevertheless, the genetic
divergence of the parental inbreds allowed the devel-
opment of a population with high genetic variability
for the trait assessed, and the presence of transgressive
progenies for both backcrosses indicates that both
parental inbreds have loci with alleles to increase and
to decrease stay-green.
Both the additive and dominance variances were
signicant, with the additive variance exceeding the
dominance variance by a factor of 6.41. The additive
by location interaction variance was also signicant,
with a similar value to the additive variance, whereas
the dominance by location interaction variance was
not found to be different from zero. The average level
of dominance was classied as partial dominance.
Since the type of population used in this study presents
a high-level of linkage disequilibrium, the estimates of
the additive and dominance variances are biased.
Regardless of the phase of the linkage disequilibrium,
the dominance variance is always biased upward while
the additive variance is biased upward when the
linkage disequilibrium is in the association-phase and
downward for the repulsion-phase (Comstock and
Robinson 1952). The expectation is that the estimate
of the level of dominance will be biased upward, so
that the additive effects are probably even more
important compared to the dominance effects than
suggested by the estimate of the level of dominance.
The estimate of the broad-sense heritability coefcient
(0.63) was slightly lower than the estimates reported
by Bekavac et al. (2007) and Camara et al. (2007),
which ranged from 0.67 to 0.81, but the strict-sense
Table 3 Genomic positions, likelihood-ratio (LR) values, gene actions and average level of dominance (ALD), direction, and
phenotypic R
2
Ph
and genotypic R
2
G
coefcients of determination for the QTL mapped for stay-green in a maize population
QTL
a
QTL positions
b
LR
c
Genetic effect
d
Gene action
e
Direction
f
R
2
Ph
% R
2
G
%
Bin cM Flanking intervals a d LD Type
Stg1a 1.02/1.03 51.05 bnlg1083/umc1073 22.08 -1.29 0.71 0.55 PD L08-05 4.83 7.68
Stg1b 1.05/1.06 124.63 umc1601/bnlg2057 37.47 -2.80 1.93 0.69 PD L08-05 24.32 38.67
Stg1c 1.06/1.06 150.27 bnlg2057/umc1508 44.47 -1.52 -1.33 0.88 PD L08-05 8.00 12.72
Stg1d 1.10/1.11 274.12 umc1431/phi0120 21.76 0.59 0.38 0.64 PD L14-04 1.04 1.65
Stg2a 2.01/2.02 5.81 bnlg1338/umc1227 24.07 0.44 0.69 1.58 OD L14-04 1.08 1.72
Stg2b 2.02/2.02 12.72 bnlg1017/umc1265 19.58 0.27 0.51 1.91 OD L14-04 0.50 0.79
Stg2c 2.07/2.07 175.97 umc2129/umc1560 19.77 -0.84 1.29 1.53 OD L08-05 3.84 6.10
Stg2d 2.08/2.08 201.98 umc1464/umc1633 21.89 -0.86 1.05 1.22 OD L08-05 3.23 5.13
Stg2e 2.09/2.09 217.94 umc1230/bnlg1520 24.25 -1.14 0.71 0.62 PD L08-05 3.87 6.15
Stg3a 3.01/3.02 0.01 umc1394/bnlg1144 22.78 0.50 0.70 1.40 OD L14-04 1.23 1.95
Stg3b 3.07/3.08 147.40 umc1659/umc1320 22.27 -1.13 0.06 0.05 A L08-05 3.21 5.10
Stg3c 3.09/3.09 193.98 bnlg1754/bnlg1098 25.06 -0.25 -0.49 1.95 OD L08-05 0.47 0.74
Stg4a 4.01/4.01 0.01 umc1276/umc1757 25.49 -0.45 -0.32 0.71 PD L08-05 0.63 1.00
Stg4b 4.04/4.05 44.93 umc1652/phi0026 24.27 0.93 0.55 0.59 PD L14-04 2.56 4.07
Stg4c 4.05/4.06 56.85 bnlg0252/bnlg2291 23.72 1.82 0.15 0.08 A L14-04 8.36 13.29
Stg6 6.00/6.01 8.03 phi0126/bnlg1371 20.15 -1.22 -0.15 0.12 A L08-05 3.76 5.99
Stg9 9.03/9.04 67.67 bnlg0430/umc1107 27.13 -1.23 -0.09 0.07 A L08-05 3.80 6.04
ALD 0.65 PD Total 46.04 73.08
a
QTL names are indicated as Stg (stay-green) followed by the chromosome number and by a letter for more than one QTL on the
same chromosome
b
Position of QTL refers to the distance in centimorgans (cM) from the rst marker on the chromosome to the mapped QTL
c
Underlined LR (likelihood-ratio) values indicate that the QTL interacted signicantly with location
d
Additive and dominance effects were multiplied by 10
e
Gene action-type: A additive, PD partial dominance, OD overdominance, ALD average level of dominance
f
Direction indicates the parental inbred that increases the trait: negative and positive a values indicates that favorable alleles were
from L-08-05F and L-14-04B, respectively
Euphytica (2014) 198:163173 169
1 3
heritability coefcient on a plant-basis (0.23) was
slightly greater than those reported by Guei and
Wasson (1996) (0.12 and 0.15) for two tropical maize
populations.
QTL mapping
Fifteen out of the seventeen mapped QTL (88.23 %)
were found on just four chromosomes, and since the
set of mapped QTL accounted for a large portion of the
genetic variance (73.08 %) the results indicated that
the QTL underlying the stay-green trait were not
evenly distributed on the genome, but clustered in a
few chromosomes. Note that one of the QTL on
chromosome 1 (Stg1b) had a major effect accounting
for 38.67 % of the genetic variance.
The number of QTL mapped in the four previous
reported studies of the stay-green trait in maize varies
greatly, i.e., from eight (Beavis et al. 1994) to 33
(Camara 2006); and both Zheng et al. (2009) and
Wang et al. (2012) mapped 14 QTL for this trait.
Camara (2006) reported QTL mapping for two
tropical maize populations, and in one of these
populations the 20 QTL mapped were distributed
over the ten chromosomes, but in the other population
23 (76.67 %) of the 33 QTL mapped were clustered on
chromosomes 1, 2 and 5. Beavis et al. (1994) reported
that three out of their eight mapped QTL were on
chromosomes 1 and 2, in the study of Zheng et al.
(2009) the QTL were largely clustered on chromo-
somes 1, 2, and 5, and in the report of Wang et al.
(2012) they were largely clustered on chromosomes 1,
4, and 5. In our study no QTL were mapped on
chromosome 5; they were largely clustered on chro-
mosomes 1 and 2, and the major QTL (Stg1b) was on
chromosome 1. Thus, based on our results and on the
previously reported studies, it is likely that most of the
QTL underlying the stay-green trait are clustered on
chromosomes 1, 2, and 5.
Comparing our results with those of Beavis et al.
(1994), Zheng et al. (2009), and Wang et al. (2012), we
found only two (Stg2b and Stg2d), three (Stg1b, Stg1c,
and Stg3b), and three QTL (Stg1a, Stg1b, and Stg4b),
respectively, mapped in the same genomic regions.
However, 11 of the 17 QTL mapped in our study and
all the QTL reported by Beavis et al. (1994), Zheng
et al. (2009), and by Wang et al. (2012) were on the
same genomic regions of the QTL reported by Camara
(2006). Thus, six unreported QTL for stay-green were
mapped in this study, adding information on the
genetic architecture of this trait.
Of the QTL mapped, 11 displayed negative and six
displayed positive additive effects (a). Therefore,
alleles that increase stay-green, i.e., favorable alleles,
were present in both parental inbreds. As expected,
most of the QTL with favorable alleles (64.70 %) were
from the parental inbred L-08-05F. The presence of
QTL with favorable and unfavorable alleles in both
parental inbreds allowed the observed occurrence of
transgressive progenies as aforementioned. The dom-
inance effects d of the QTL were not unidirectional,
since they displayed positive and negative signs. The
average level of dominance was partial dominance
(ALD = 0.65), which is close to that computed from
the variance components (
^