Euphytica
DOI 10.1007/s10681-015-1385-4
Inheritance of high iron and zinc concentration in selected
bean varieties
F. Mukamuhirwa • G. Tusiime • M. C. Mukankusi
Received: 5 January 2014 / Accepted: 30 January 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Micronutrient malnutrition especially iron
(Fe) and zinc (Zn) has disastrous consequences for the
more vulnerable members of the human society,
especially poor women and pre-school children in
developing countries. The common bean (Phaseolus
vulgaris L.), an important grain legume directly
consumed by humans provides cheap and important
quantities of protein and calories and is an excellent
source of some minerals (Fe and Zn) and vitamins
which qualify it as perfect food. Unfortunately, many
of the adapted bean market class varieties are low in
these nutrients. Therefore there is need for biofortifi-
cation of these varieties with Fe and Zn through plant
breeding and genetic engineering in order to con-
tribute to improved health of bean consumers. The
objective of this study was to determine the mode of
inheritance of high Fe and Zn concentration in selected
bean varieties. Six parents high and low in Fe and Zn
were crossed following a full diallel crossing design.
Populations were advanced from F1 to F2. F3 seed were
analyzed for Fe and Zn concentration using X-ray
fluorescence. The study showed that both additive and
F. Mukamuhirwa (&)
G. Tusiime
Department of Agricultural Production, Makerere
University, P. O. Box 7062, Kampala, Uganda
e-mail: fmukamuhirwa@yahoo.cm
M. C. Mukankusi
CIAT Africa, National Agricultural Research
Laboratories- Kawanda, P. O. Box 7065, Kampala,
Uganda
non-additive gene effects are important in determining
the expression of high Zn and Fe concentration even
though additive gene effects contributed more. Nar-
row sense heritability of Fe and Zn was high estimated
at 71 % for Fe and 83 % for Zn. Significant maternal
and reciprocal effects suggest also that cytoplasmic
inheritance is involved in Zn and Fe concentration.
Strong positive correlation between Fe and Zn
concentrations (r = 0.75) was observed suggesting
that these micronutrients are not independently inher-
ited. The positive relationship between Fe and Zn plus
the negative relationship observed between Zn and
seed size (r = 56) suggest that Zn and Fe accumula-
tion is controlled multigenically or oligogenically with
some genes affecting the concentration of both
minerals. The variability in seed mineral concentra-
tion among crosses was larger for Fe (47–77 ppm)
than for Zn (28–38 ppm).
Keywords Gene effects
General and specific
combining ability
Heritability
Introduction
Increasing the amount of Fe and Zn concentration in
common bean grain may contribute significantly to
improving the health status of individuals dependent
on beans as a staple food (House et al. 2002).
Significant increases in the amount of bioavailable
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Zn and Fe in beans can be made through traditional Materials and methods

plant breeding techniques (Nchimbi-Msolla and
Muhamba 2010) based on the existing wide variation Study location and plant materials
of Fe and Zn concentration among bean genotypes.
Therefore the knowledge of inheritance is critical in The study was carried out in the screen house at CIAT
designing appropriate breeding strategies for incorpo- Uganda based at National Agricultural Research
rating a particular trait into economically useful Laboratories (NARL) in Kawanda, Uganda from June
populations. The aim of this study was to contribute 2011 to August 2012. Six bean inbred lines were used
to the development of high Fe and Zn beans through in this study, they included, three high and three low
studying the mechanism of inheritance of iron and Fe and Zn concentration bean varieties based on
zinc. The objectives of this study were: HarvestPlus data. The high Fe lines included;
KAB06F2827- developed at Kabete-CIAT breeding
1. To study the gene action governing Fe and Zn
program, NUA99 which is among several lines
concentration in beans;
deployed in Africa from CIAT-Colombia as high Fe
2. To determine the combining ability among six
bush Andean beans, RWR 2154 a released variety in
common bean varieties for Fe and Zn
Rwanda developed by the Rwanda Agriculture Board
concentration.
(RAB) bean breeding program. The low Fe parents
3. To estimate the role of maternal effects control-
included, CAL96 a red mottled bean developed at
ling Fe and Zn concentration in beans;
CIAT and released in Uganda. It is highly susceptible
4. To estimate narrow sense heritability (h2) for Fe
to all major diseases and is used as a local disease and
and Zn concentration in common bean
high yield check and is low in Fe. DOR500 is a CIAT
populations;
line currently used as a universal low Fe check.
5. To estimate heterosis on Fe and Zn concentration
RWR2076 is a RAB bred line used as a local yield
in the F2 generation of common bean crosses;
check in Rwanda.
6. To estimate the magnitude of relationship be-
Characteristics of these entries are outlined in the
tween iron and zinc concentration in F2
figure below (Fig. 1).
populations.

NUA 99 RWR 2076 RWR 2154

KAB06F2.8-27 DOR 500 CAL 96

Fig. 1 Seed of parents used in the study of inheritance of high iron and zinc concentration

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Euphytica
Population development
The study germplasm was grown and crossed follow-
ing a full diallel design for testing maternal and
paternal effects (Hallauer and Miranda in Tadess et al.
2008; Jones 1965; Crusio 1987). Planting of the
crossing block was done on a mixture of forest black
soil, lake sand and decomposed farm yard manure in a
ratio of 3:1:1 (currently used by CIAT). A carefully
mixed composite sample was packed in new paper
bags, labelled twice and analyzed at Kawanda Agri-
cultural Research Laboratories (KARL). Soil analysis
was done following the protocol of Soils and Soil
Fertility Management Programme of National Agri-
cultural Research Laboratory, actually at Kawanda.
Soil was analyzed for iron (Fe), zinc (Zn), acidity
(pH), organic matter (OM), nitrogen (N), Phosphorus
(P), Potassium (K), Calcium (Ca), and Magnesium
(Mg). The experimental soil had medium to very high
nutrients. Apart from iron (140 ppm) that was very
high and sufficient in concentration, calcium
(1,720 ppm), magnesium (360 ppm), potassium
(425 ppm) and zinc (5 ppm) concentrations were very
high but not sufficient. Phosphorus (17 ppm) concen-
tration was high and not sufficient. Organic matter
(3.7 %), pH 5.1 and nitrogen (0.21 %) were medium
but not sufficient. Each parent per cross combination
was planted in five buckets and repeated weekly for
4 weeks. Four crossing blocks were set up. N.P.K 17:
16: 16 fertilizer was added to experimental soils on a
weekly basis from germination to pod formation.
Thirty grams of N.P.K was dissolved in 10 l of water,
from which 50 mls of the fertilizer solution was
applied to each pot weekly. The experiment was
watered in the morning; although in some cases,
watering was done in the afternoon when deemed
necessary. The crossings were done in 6 9 6 full
diallel after emasculating the female parents. The bulk
method was used to advance F1 to F2 and F2 to F3.
Evaluation of developed populations for Fe and Zn
concentration
At F3, seed was harvested by individual plant and put
in clean new paper envelopes to avoid contamination
with dust and dirt. These were hand threshed under
conditions that kept the seed as free of dirt and dust as
much as possible. Thereafter, five seeds per plant were
randomly selected for advancement to the next
generation. The remaining seed was bulked into four
replicates for iron and zinc analysis at Rubona
Agriculture Research Station, Rwanda for XRF
analysis (Paltridge et al. 2012). XRF uses X-rays to
energize atoms within sample, causing them to
fluoresce. Energy and intensity of fluorescence pro-
vides information about elemental make-up. The
machine scans each sample for 100 s with spinning
of sample cup (use 4 g of grain (7 ml) to analyze Fe
and Zn and records intensities of emitted X-rays.
(Pfeiffer and McClafferty 2007; Harvest Plus 2011).
Inheritance and gene action of Fe and Zn was studied
based on results obtained from the analysis of F3 seeds.
Data analysis
Analysis of variance was performed by the ANOVA
procedure of Genstat 14th Edition (VSN International
Ltd., 2012). Significant treatment effects were separat-
ed using the least significant difference (LSD).
Correlation and regression analysis were carried out
with linear regression analysis of Microsoft Excel.
Estimation of general and specific combining
ability
General and specific combining ability were deter-
mined following Model 1 of Griffing’s analysis
(Griffing 1956). General combining ability (GCA)
for male and female parents as well as the specific
combining ability (SCA) effect means were obtained
following the formula developed by Hallauer and
Miranda in Tadesse et al. (2008). GCAi = mean of
crosses of parent ‘‘I’’- grand mean, while SCAij = -
cross mean - (grand mean ? GCAf ? GCAm).
Estimation of heritability
Narrow sense heritability (NSH) was estimated from
variance components. Broad sense heritability, was
calculated as H = VG/VP, and narrow sense herit-
ability, h2 = VA/VP where H and h2 are broad sense
heritability and narrow sense heritability respectively,
VA is additive variance, VG is genotypic variance and
VP is phenotypic variance (Hill et al. 2008). Baker’s
ratios were used to predict perfomance of crosses
based on GCA values. The closer the ratio is to 1 the
greater the chances of predicting performance based
on GCA (Baker 1978). Table 1 below summarizes the
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Table 1 Formulae used for estimating heritability and Baker’s ratio
Baker’s ratio
NS-CGD (genotype mean basis)
BS-CGD (genotype mean basis)
NS-CGD (narrow sense coefficient of genetic determination) = estimate of narrow sense heritability (h2) BS-CGD (broad sense
coefficient of genetic determination) = estimate of broad sense heritability (H)
formulae used for heritability and Baker’s ratio based
on GCA and SCA values (Griffing 1956; Baker 1978).
When for a given trait the narrow sense heritability
is high, the trait is highly heritable and when is small it
is lowly heritable. High heritability estimates indicate
that additive gene action is more important for that
trait and selective breeding of the best to the best
should produce more desirable progeny. Low esti-
mates on the other hand indicate that probably non-
additive gene action such as overdominance, dom-
inance and epistasis is important. If heritability is high,
individual selection is recommended and if heritability
is low, family selection is recommended (Behera
2007). Narrow sense heritability was also estimated by
mid-parent offspring regression analysis. The regres-
sion coefficient ‘‘b’’ is an estimate of narrow sense
heritability (Falconer and Mackay 1996).
Estimation of heterosis effects for iron and zinc
concentration observed at the F2 generation
In this study, heterosis was determined for the F2
populations that involved six parents (KAB06F2.8-27,
NUA 99, RWR 2076, RWR 2154, CAL 96 and DOR
500) high and low in Fe and Zn concentration. The
amount of heterosis was calculated by comparing the
mean of F2 progeny over mid-parental value, over
better parental value and over the worse parental value
in respect of Fe and Zn concentration using the
formula of Falconer and Mackay (1996). Mid-parent
heterosis was calculated as: MPH = (F2 - MP)/
MP * 100 where F2 is the mean performance of the
F2 population and MP is the mean of two parents.
Similarly, heterobeltiosis was obtained as the differ-
ence in the mean performance of the mean of F2 to
their high or low iron/zinc parent, that is; BPH (better
parent heterosis) = (F2 - BP)/BP * 100; WPH
(worse parent heterosis) = (F2 - WP)/WP * 100
where BP is the mean of the better (high Fe/Zn
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Formulae
2*re2GCA/(2*re2GCA ? SCA re2)
2*re2GCA/(2*re2 GCA ? re2 SCA ? re2)
(2*re2GCA ? re2 SCA)/(2*re2 GCA ? re2 SCA ? re2)
concentration) parent and WP is the mean of worse
(low Fe/Zn concentration) parent.
Results
Gene action determining iron and zinc
concentration
Twenty seven successful crosses out of the planned 30
were evaluated. The analysis of variance showed
significant difference among crosses (P \ 0.001) for
both Fe and Zn concentration. General combining
ability effects were highly significant at P \ 0.001 for
both Fe and Zn concentration while the specific
combining ability effects were also significant at
P \ 0.001 and P \ 0.01 for Fe and Zn concentration,
respectively. GCA effects accounted for 64 and 82 %
for Fe and Zn, respectively, while SCA accounted for
36 and 18 % for Fe and Zn, respectively. This indicated
that additive gene action was more important than
non-additive gene action implying that the highest Fe
and Zn concentration progeny may be produced by
crossing the two parents with high GCA effects.
Reciprocal effects were also highly significant
(P \ 0.001) for all traits evaluated (Table 2). Highly
significant maternal and non-maternal reciprocal ef-
fects were observed for both Fe and Zn concentration
indicating the importance of cytoplasmic 9 nuclear
gene interaction effects in accumulating high Fe and
Zn concentration.
The variability in seed mineral concentration among
crosses was larger for Fe (47–77 ppm) than for Zn
(28–38 ppm). Transgressive segregation for higher Fe
and Zn was observed where some crosses (RWR
2076 9 KAB06F2.8-27 and RWR2076 9 DOR500
(one direction) showed higher Fe or Zn concentration
than the higher parent (Table 3). The crosses of
RWR2076 9 KAB06F2.8-27 and RWR2076 9
DOR500 performed well for both iron and zinc since
Euphytica

Table 2 Mean squares for Source of variation DF Iron (ppm) Zinc (ppm) 100 SW Seeds per plant
different traits evaluated
along iron and zinc Entry (Tot.) 32 46.23 7.273 384.44 61.77
concentration in crosses of
GCA 5 171.01*** 32.40*** 2,167.52*** 319.66***
bean varieties high and low
in seed iron and zinc SCA 15 19.64*** 1.73** 72.14*** 12.85***
concentration Reciprocals 12 27.48*** 3.73*** 31.85*** 19.02***
Crosses 27 23.13*** 2.62*** 54.24*** 15.71***
ns, **, *** = not Maternal effects 6 19.59** 4.86*** 51.82*** 28.93**
significant, significant at Non maternal effects 6 35.37*** 2.61** 11.88ns 10.53**
1 % and 1 % level Error 32 4.34 0.56 7.17 2.29
respectively

their parents showed good general combining ability
suggesting additive genetic effects (Fig. 2).
Estimation of combining ability effects
for developed crosses
General combining ability
Positive GCA effects were desirable in this study
because they indicated the bean line’s contribution to
high Fe and Zn concentration while the negative GCA
effects represent contribution towards low Fe and Zn
concentration. RWR 2076, KAB06F2.8-27 and DOR
500 had positive GCA effects suggesting that these
varieties contributed to high Fe and Zn concentration
in the crosses that involved them. CAL 96, NUA 99
and RWR 2154 had negative GCA effects suggesting
that they contributed to low Fe and Zn concentration in
the crosses that involved them.
Differences in GCA values (Fig. 2) are attributed to
additive gene effects. Positive GCA effects of DOR
500 and high Fe concentration in the crosses that
involved it yet it is a universal low Fe check suggests
that it might be having recessive genes for high Fe and

Table 3 Means of F3 seeds for iron and zinc concentration along their parents in ppm
Female parents Male parents
CAL96 NUA99 RWR2076 RWR2154 KAB06F2.8-27 DOR500

Iron concentration CAL960 46.79 58.29 54.29 54.29 65.29

NUA99 52.29 55.79 59.04 50.79 63.54 62.54

RWR2076 60.29 53.29 64.04 70.04 76.79

RWR2154 51.79 62.79 66.79 53.79 58.54

KAB06F2.8-27 58.29 63.29 75.79 65.04 66.04 68.29

DOR500 58.65 57.29 60.04 60.29 64.29 57.29
Zinc concentration CAL96 27.99 30.57 30.57 31.57 34.49

NUA99 30.57 32.24 33.99 27.57 32.24 35.74

RWR2076 31.99 31.99 35.24 34.74 37.99

RWR2154 28.57 32.99 30.74 31.24 34.24

KAB06F2.8-27 31.07 36.57 37.74 31.99 34.24 37.49

DOR500 34.70 33.99 35.74 34.74 34.49 35.24
Figures in the diagonal are parents while figures in bold are crosses with distinctly transgressive segregation with good performance
for Fe and Zn respectively

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impacting on high Fe concentration in these crosses.

SCA effects were positive for CAL 96 9 RWR 2154,
NUA 99 9 KAB06F2.8-27, RWR 2076 9 KAB06
F2.8-27, CAL 96 9 DOR 500, RWR 2076 9 DOR
500, RWR 2154 9 DOR 500 indicating the presence of
non-additive gene effects governing high Zn concen-
tration in these crosses. Differences in SCA are
attributed to non-additive effects including dominance
and epistasis gene effects (Table 4).

Estimation of the role of maternal effects on Fe

Fig. 2 Parental GCA effects of F3 seeds of crosses for high iron
and zinc concentration. Average SE GCA was 0.40 for iron
concentration and 0.15 for zinc concentration
Zn concentration. Similarly NUA 99and RWR 2154
contributed to low Fe concentration yet they are high
Fe parents suggesting epistasis and or overdominance
gene effects.
Specific combining ability
SCA effects were positive for CAL 96 9 NUA 99, CAL
96 9 DOR 500, RWR 2076 9 RWR 2154, RWR
2076 9 KAB06F2.8-27 and RWR 2076 9 DOR 500
indicating the presence of non- additive gene effects
Table 4 SCA effects in F3 seed of crosses between beans high in iron and zinc concentration in ppm
Trait Parent Genotypes Males parents
CAL96 NUA99
and Zn concentration in beans
Reciprocal crosses RWR 2076 9 CAL 96, RWR
2154 9 NUA 99, KAB06F2.8-27 9 RWR 2076,
had positive reciprocal effects in F3 generation
(Table 5) implying that iron concentration was only
higher when RWR 2076, RWR2154 and KAB06F2.8-
27 were maternal parents in these crosses.
Crosses CAL 96 9 NUA 99, CAL 96 9 DOR 500,
NUA 99 9 RWR 2076, NUA 99 9 DOR 500, RWR
2154 9 DOR 500 had high iron concentration when
CAL 96, NUA 99 and RWR 2154 were maternal
parents and lower Fe concentration when NUA 99,
DOR 500, RWR 2076 were maternal parents in these
crosses. On the other hand reciprocal crosses RWR
RWR2076 RWR2154 KAB06F2.8- DOR500
27

Iron Female CAL96 -3.86 2.37 -1.60 -0.58 -2.10 4.73

parents NUA99 0.59 -5.00 0.89 0.75 0.40
RWR2076 -3.08 4.93 4.28 2.93
RWR2154 -2.81 1.67 -0.80

KAB06F2.8- -4.11 -0.70

27
DOR500 -6.55
AVG SE SCA 1.27
Zinc Female CAL96 -0.90 0.13 -0.65 0.51 -0.94 1.37
parents NUA99 0.25 -0.49 -0.83 0.85 0.09
RWR2076 0.27 -1.86 1.19 0.60
RWR2154 1.01 -0.69 0.59

KAB06F2.8- -0.88 -0.35

27
DOR500 -2.32
AVG SE SCA 0.47

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Table 5 Reciprocal (maternal and non-maternal) effect values in crosses between beans high and low in iron and zinc concentration
Trait Parent Genotypes Male parents
CAL96 NUA99 RWR2076 RWR2154 KAB06F2.8-27 DOR500

Iron Female parents CAL96

NUA99 -3.00
RWR2076 ?3.00 -2.88
RWR2154 -1.25 ?6.00

KAB06F2.8-27 -0.12 ?2.88

DOR500 -3.32 -2.63 -8.38 ?0.88 -2.00

AVG SE RECIP 1.41
Zinc Female parents CAL96
NUA99 0.00
RWR2076 ?0.71 -1.00
RWR2154 -1.50 ?2.71

KAB06F2.8-27 ?2.16 ?1.50

DOR500 ?0.11 -0.87 -1.13 ?0.25 -1.50

AVG SE RECIP 0.53
?,2 : positive and negative reciprocal effects indicating the importance of cytoplasmic 9 nuclear gene interaction effects in
accumulating high Fe and Zn concentration

2076 9 CAL 96, RWR 2154 9 NUA 99, KAB06
F2.8-27 9 NUA 99 and KAB06F2.8-27 9 RWR
2076 had high Zn concentration when RWR 2076,
RWR 2154 and KAB06F2.8-27 are female parents and
low Zn concentration when CAL 96, NUA 99, and
RWR 2076 are maternal parents in these crosses.
Crosses involving CAL 96 9 RWR2154, NUA
99 9 RWR 2076, NUA 99 9 DOR 500, RWAR
2076 9 DOR 500 and KAB06F2.8-27 9 DOR 500
had high zinc concentration when CAL 96, NUA 99,
NUA 99, RWR 2076 and KAB06F2.8-27 are maternal
parents and low Zn concentration when RWR2154,
RWR 2076 and DOR 500 are maternal parents in these
crosses. The reciprocal effects are also explained by
the maternal and non-maternal effects generated by
diallel analysis. These indicated that the cytoplasm of
these varieties contributed to high Fe and Zn concen-
tration of the crosses involving these parents.
Estimation of narrow sense heritability (h2) for Fe
and Zn concentration in 27 common bean
populations
The narrow sense heritability (h2) was estimated by
narrow sense coefficient of genetic determination at
genotype mean basis. The h2 was estimated at 0.83 and
0.71 for zinc and iron respectively suggesting that Fe
and Zn concentration can be improved more rapidly
with less evaluations. High Baker’s ratio of 0.90 and
0.78 for zinc and iron respectively were observed
(Table 6).
Narrow sense heritability was also estimated by
mid-parent offspring regression analysis where the
regression coefficient b represents narrow sense
heritability (h2). The mid-parent offspring regression
analysis was also significant (P \ 0.001) with the
adjusted regression coefficient b of 1.05 and 1.17 for
iron and zinc, respectively. This was an indicator of a
very high narrow sense heritability suggesting that
genetic effects on iron and zinc concentration were
very high. Therefore high iron and zinc concentration
in the F2 plants (F3 seed) generation can be reliably
predicted based on parental performance. F2 plants (but
F3 seed) data indicated that 46 and 55 % of the total
variation in iron and zinc concentration respectively of
F3 seed populations was accounted for by the parental
iron and zinc concentration (Fig. 2). A relatively high
r2 indicates high predictability of crosses from parents
since the mid parent has relationship with the cross.
This is shown by the scatter plots below (Fig. 3).

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Table 6 Baker’s ratio and heritability estimates for Fe, Zn, 100 SW and seed per plant
Component Source of variation Iron Zinc 100 SW Seeds per plant

Variance components GCA 15.43 2.95 200.03 28.34

SCA 8.50 0.65 36.09 5.65
RECIPS 11.57 1.59 12.34 8.36
Heritability Baker’s ratio 0.78 0.90 0.92 0.91
NS-CGD (genotype mean basis) 0.71 0.83 0.90 0.88
BS-CGD (genotype mean basis) 0.90 0.92 0.98 0.96

Heterosis and heterobeltiosis for iron and zinc
concentration observed at the F2 plants generation
Positive heterosis is desirable as it indicates the
superiority of the F2 to either mid-parent, high Fe/Zn
concentration parent and low Fe/Zn concentration
parent. The heterosis was used to understand the
contribution of different parents to Fe/Zn concentra-
tion and may help in the selection of desirable crosses
(those with high positive heterosis) in a breeding
scheme. Thirteen out of 14 high 9 low iron concen-
tration F2 crosses had positive relative/mid-parent
heterosis while 9 out of 13 low 9 high Fe concentra-
tion F2 crosses had positive relative/mid-parent
heterosis. The positive relative heterosis varied from
1.33 to 26.58. High 9 low Fe crosses had higher
heterosis level compared to low 9 high crosses indi-
cating that high Fe concentration levels were obtained
when high Fe parents were used as mothers than vice
versa. Crosses with positive heterosis show the
presence of joint action of a favorable combination
of genes at different loci. Six out of 14 high 9 low Fe
concentration F2 crosses had positive heterobeltiosis
to high Fe parent while 8 out of 13 low 9 high Fe
concentration F2 crosses had positive heterobeltiosis
to high Fe parent. On the other hand, 12 out of 14
high 9 low Fe concentration F2 crosses had positive
heterobeltiosis to low Fe parent while 13 out of 13
low 9 high Fe concentration crosses had positive
heterobeltiosis to low Fe parent.
The negative heterobeltiosis observed for the
crosses involving NUA 99 as high Fe parent may
suggest the involvement of over-dominance effects in
these crosses.
The positive heterosis observed with crosses with
KAB06F2.8-27 indicated that this bean line may be a
good source of Fe concentration since it still resulted
in a better offspring. Crosses involving KAB06F2.8-
27, RWR 2076 resulted into high Fe concentration
offsprings when they were female parents than when
they were male parents, indicating possession of
cytoplasmic genes that contribute to Fe concentration.
Crosses involving DOR 500 (a low universal check) as
male parent resulted into high Fe concentration
offspring suggesting recessive genes for high Fe.
Even though complete dominance effects was ob-
served for some crosses (CAL 96 9 RWR2154, DOR
500 9 CAL 96, DOR 500 9 NUA 99, KAB06F2.8-

Fig. 3 Regression of iron/

zinc concentration, F2
plants progeny (F3 seed)
iron/zinc concentration on
mid-parent for iron/zinc
concentration for 27
populations

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27 9 RWR2154 and RWR 2154 9 DOR 500), the Fe
level of these crosses was low. Most of crosses
involving CAL 96, NUA 99, RWR 2154 had negative
heterosis suggesting that they contributed to low Zn
concentration in these crosses. Interestingly, crosses
involving parents KAB06F2.8-27and RWR2076 had
high positive heterosis for zinc as they had for iron.
Though high heterosis and high Zn concentration were
obtained by using each of the parents as a female, the
use of KAB06F2.8-27 as a female parent was more
beneficial than any other parent indicating that this
line possesses cytoplasmic genes that contribute to
high Zn concentration. The positive heterosis ob-
served with crosses with KAB06F2.8-27 indicated that
this bean line could also be a good source high Zn
concentration. Even if RWR 2076 9 DOR 500 had
the same high parental Zn concentration, high hetero-
sis and good performance were observed when RWR
2076 was the female parent and DOR 500 the male
parent. Eight out of 14 high 9 low Zn concentration
F2 crosses had positive relative heterosis while 10 out
of 13 low 9 high Zn concentration F2 crosses had
positive relative heterosis. The positive relative
heterosis varied from 0.00 to 10.01. Low 9 High Zn
crosses had higher heterosis level compared to
high 9 low crosses (Table 7). Two out of 14 9 low
Zn concentration F2 crosses had positive heterobel-
tiosis to high Zn parent while 6 out of 13 low 9 high
Zn concentration F2 crosses had positive heterobel-
tiosis to high Fe parent. On the other hand 12 out of 14
high 9 low Zn concentration F2 crosses had positive
heterobeltiosis to low Zn parent while 12 out of 13
low 9 high Zn concentration crosses had positive
heterobeltiosis to low Zn parent. The negative
heterobeltiosis observed in high 9 low Zn concentra-
tion crosses and low 9 high Zn concentration crosses
as well as the negative heterosis and heterobeltiosis to
low Zn concentration observed in the crosses involv-
ing NUA 99 as high parent may suggest the involve-
ment of over-dominance gene effects in these crosses.
Estimation of the magnitude of relationship
between iron and zinc concentration in F2
populations
Simple correlations were calculated among mineral
mean values to examine the relationship between the
concentrations of the two minerals. Significant and
positive correlations were found between Fe and Zn
concentration (r = 0.75; P B 0.001. The relationship
between Zn and 100 seed weight (r = -56;
P B 0.001) was unfortunately significant but negative.
The relationship between number of seeds per plant
and Zn concentration was also significant (r = 0.50;
P B 0.01 (Table 8).
Discussions
The variability in seed mineral concentration among
crosses was larger for iron (47–77 ppm) than for zinc
(28–38 ppm) with significant and strong positive
correlations between iron and zinc (r = 0.75) and a
strong negative correlation between zinc and seed size
(r = 0.56). Phenotypic variability in seed iron and
zinc concentration observed in this study depended on
the genotypes and environmental conditions under
which the study was carried out. Similar observations
were earlier reported by Cichy et al. (2009).
Both general combining ability (GCA) and specific
combining ability (SCA) were found to be important
in determining progeny performance, indicating that
both additive and non-additive effects were important.
Baker’s ratio (1978) for the relative importance of
GCA and SCA variance components was 0.90 for zinc
and 0.78 for iron, demonstrating that additive gene
effects were more important than non-additive ones in
determining the expression of high zinc and iron
concentration even though SCA effects were also
highly significant.
Data in this study support Hill et al. (2008) in their
evidence from empirical studies of genetic variance
components found that additive variance typically
counts for over half and often close to 100 % of the
total genetic variance. The results also support the
study of Blair et al. (2009) where they found that
inheritance of higher iron and zinc concentration was
mainly additive. Mostly additive gene action suggests
that selection in early segregating generations should
be effective for improving these traits. The use of non
destructive measurements for Fe and Zn such Marker
assisted selection is recommended. Based on these
research findings one should first select genotypes
with high zinc concentration and evaluate them for
high iron concentration and then discard those with
low iron since zinc is correlated to iron with higher
heritability. Narrow sense heritability of iron and zinc
was high according to Johnson et al. (1955) and was
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Table 7 Midi-parent heterosis and heterobeltiosis for zinc concentration observed on F2 plants (high Zn 9 low Zn) and their
reciprocals (low Zn 9 high Zn) crosses
Cross code Mean Zn concentration of parents F2’s Zn Mid-parent Mid-parent Heterobeltiosis (%)
concentration Zn heterosis
High Zn Low Zn High Zn Low Zn

NUA99 9 CAL96 32.24 27.99 30.57 30.11 1.51 -5.18 9.21

CAL96 9 NUA99 32.24 27.99 30.57 30.11 1.51 -5.18 9.21
RWR2076 9 CAL96 35.24 27.99 31.99 31.61 1.19 -9.22 14.29
CAL96 9 RWR2076 35.24 27.99 30.57 31.61 -3.31 -13.26 9.21
RWR2154 9 CAL96 31.24 27.99 28.57 29.61 -3.53 -8.55 2.07
CAL96 9 RWR2154 31.24 27.99 31.57 29.61 6.60 1.05 12.79
KAB06F2.8-27 9 CAL96 34.24 27.99 31.07 31.11 -0.15 -9.26 11.00
DOR500 9 CAL96 35.24 27.99 34.70 31.61 9.76 -1.53 23.98
CAL96 9 DOR500 35.24 27.99 34.49 31.61 9.09 -2.13 23.22
RWR2076 9 NUA99 35.24 32.24 31.99 33.74 -5.19 -9.22 -0.78
NUA99 9 RWR2076 35.24 32.24 33.99 33.74 0.74 -3.55 5.43
RWR2154 9 NUA99 32.24 31.24 32.99 31.74 3.94 2.33 5.60
NUA99 9 RWR2154 32.24 31.24 27.57 31.74 -13.14 -14.49 -11.75
KAB06F2.8-27 9 NUA99 34.24 32.24 36.57 33.24 10.01 6.80 13.43
NUA99 9 KAB06F2.8-27 34.24 32.24 32.24 33.24 -3.01 -5.84 0.00
DOR500 9 NUA99 35.24 32.24 33.99 33.74 0.74 -3.55 5.43
NUA99 9 DOR500 35.24 32.24 35.74 33.74 5.93 1.42 10.86
RWR2154 9 RWR2076 35.24 31.24 30.74 33.24 -7.52 -12.77 -1.60
KAB06F2.8-27 9 RWR2076 35.24 34.24 37.74 34.74 8.64 7.09 10.22
RWR2076 9 KAB06F2.8-27 35.24 34.24 34.74 34.74 0.00 -1.42 1.46
DOR500 9 RWR2076 35.24 35.24 35.74 35.24 1.42 1.42 1.42
RWR2076 9 DOR500 35.24 35.24 37.99 35.24 7.80 7.80 7.80
KAB06F2.8-27 9 RWR2154 34.24 31.24 31.99 32.74 -2.29 -6.57 2.40
DOR500 9 RWR2154 35.24 31.24 34.74 33.24 4.51 -1.42 11.20
RWR2154 9 DOR500 35.24 31.24 34.24 33.24 3.01 -2.84 9.60
DOR500 XKAB06F2.8-27 35.24 34.24 34.49 34.74 -0.72 -2.13 0.73
KAB06F2.8-27 9 DOR500 35.24 34.24 37.49 34.74 7.92 6.39 9.49

Table 8 Relationship of iron and zinc concentration along with seed size and number of seeds per plant (based on entry means)
Trait Fe Zn 100 SW Seeds per plant

Fe 0.75*** -0.12NS 0.04NS

Zn -0.56*** 0.50**
100 SW –0.82***
Seeds per plant
**, *** = significant at P B 0.01 and 0.001 respectively. Fe = Iron, Zn = Zinc, 100 SW = 100 seed weight

estimated at 71 % for iron and 83 % for zinc. The interactions are involved in zinc and iron concentra-
significant maternal and reciprocal effects suggest also tion. A maternal effect was also found for seed protein
that cytoplasmic inheritance and cytoplasmic-genic in beans (Noubissié et al. 2012). Maternal effects play

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Euphytica
an important role at most developmental stage
(Variath et al. 2009) and this may have a direct impact
on the selection process and the progression of
segregating population in genetic improvement pro-
grams. The diallel analysis revealed that additive
effects as well as non-additive genetic effects influ-
ence iron and zinc concentrations. Therefore the
progeny performance on iron and zinc may not be
fully predictable based on parent performance alone.
Selection of parents with high concentrations of Fe
and Zn, along with screening crosses in early
generations, especially for zinc (Baker’s ratio of 0.90
compared to 0.78 for iron) should be effective in
improving the concentrations of these minerals in bean
seed. Correlation in iron and zinc concentrations
(r = 0.75) observed in this study support the results of
Blair et al. (2009) which showed co-localization of
QTL responsible for high zinc and iron in beans. This
may be interpreted as evidence that accumulation of
both minerals are of similar inheritance. Cichy et al.
(2009) also reported correlations of Fe and Zn up to
0.53 and the co-localization of QTL for iron and zinc.
The co-localization of QTL for seed iron and zinc
(Blair et al. 2009) suggests that some QTL contribute
to both minerals. Therefore, phenotypic selection for
zinc and iron may be done simultaneously. The
observed correlation in seed iron and zinc concentra-
tion may be explained by the fact that iron and zinc
have similar mechanisms of loading into the grain of
beans as well as in wheat and in rice (Hacisalihoglu
and Kochian 2003; Peleg et al. 2008; Zhang et al.
2012). The two traits may have a physiological
relationship probably because they share the same
biochemical pathway for accumulation. This could be
also a function of some parents being high in both iron
and high zinc, or low in both iron and zinc. The
negative correlation between zinc and seed size
suggest that micronutrient concentration can be
improved in the Mesoamerican gene pool as reported
by Blair et al. (2010). Islam et al. (2002) reported that
Andean beans are high in iron concentration despite
the correlation of zinc, and the positive correlation of
iron with zinc. Therefore, it would seem that selection
for these nutrients could be effective in both gene
pools. KAB06F2.8-27 the high seed iron and zinc
parent contributed to the high iron and zinc concen-
tration. The observed superiority of the F2 mean of
some crosses relative to the best parent would suggest
a type of epistasis operating in those crosses. The
parents KAB06F2.8-27 and RWR2076 should be used
to maximize opportunities for improvement of iron
and zinc and they should be used as female parents.
The parent DOR 500, even though is a low parent
should be used as male parent because it should be
having recessive genes for high Fe and Zn concentra-
tion. F2 populations such as KAB06F2.8-27 9 RWR
2076, and RWR 2076 9 DOR 500 performed better
with significant heterosis for both iron and zinc
concentration. Significant heterosis was also observed
in cotton by Maria Khan Panni et al. (2012) and in
wheat by Khatun et al. (2010). Therefore crosses
involving KAB06F2.8-27 and RWR2076 as maternal
parents and DOR 500 as paternal parent performed
well for iron and zinc and should be selected and
advanced to enhance the iron and zinc concentration in
these crosses.
Acknowledgments We are very grateful to Makerere
University, AGRA, CIAT, Harvest plus project and RAB for
the support to this work.
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