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Investigation of Chocolate Spot and Rust Resistance in Egyptian Faba Bean Population Using Morphological Traits and Molecular Markers

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  • Food Legumes Research Department, Field Crops Research Institute, Agricultural Research Center

Abstract and Figures

Two faba bean cultivars; "Sakha 3" and "Misr 1", their F1 and F2 populations were evaluated for foliar disease resistance (Choclate spot and rust). Analysis of variance of the 11 morphological traits showed highly significant differences among genotypes in all recorded traits. Genetic parameters of the studied traits showed little difference between GCV and PCV for some traits indicating a negligible environmental effect but PCV was higher than GCV for other traits suggesting a significant environmental effect on the expression of these characters. For disease reaction of rust showed moderate heritability coupled with low genetic advance suggested the involvement of epistatic interactions. While, chocolate spot disease reaction showed that the moderate heritability coupled with high genetic advance. Heterotic effects for seed yield/plant were associated with other yield components and significant positive inbreeding depression values were detected for number of seed/plants, seed yield/plant and 100-seed weight. SSR cluster analysis showed that cross F1 "Sakha3 X Misr1" was separated lonely apart of all other genotype. Moreover, some F2 plants were clustered along with P1 and some others were clustered along with P2 parent indicating distribution of genetic material among F2 plants. The results of molecular markers were in discontiguous with morphological results which may be attributed to the low number of morphological traits used and the habit of the morphological traits that affected by the environmental conditions. The results of both morphological and molecular markers proposed that selection in these particular populations should be effective and satisfactory in the successful breeding purposes.
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Investigation of Chocolate Spot and Rust Resistance in Egyptian Faba Bean
Population Using Morphological Traits and Molecular Markers
Walid M. El-Rodeny (1), Samah M. M. Eldemery (2), Alaa A. Soliman (1) and
Kamal F. Abdellatif (3)*
(1) Food Leg. Dept., Sakha Agric. Res. Station, Field Crop Res. Inst., Agric. Res. Center, Egypt;
(2) Molec. Biol. Dept., Genet. Eng. Biotech. Res. Inst., Univ. Sadat City, Egypt;
(3) Plant Biotech. Dept., Genet. Eng. Biotech. Res. Inst., Univ. Sadat City, Egypt.
* Corresponding Author: kamal.abdellatif@gebri.usc.edu.eg, kamal2004gr@yahoo.com
ABSTRACT
Two faba bean cultivars; Sakha 3 and Misr 1, their F1 and F2
populations were evaluated for foliar disease resistance (Choclate spot and rust).
Analysis of variance of the 11 morphological traits showed highly significant
differences among genotypes in all recorded traits. Genetic parameters of the
studied traits showed little difference between GCV and PCV for some traits
indicating a negligible environmental effect but PCV was higher than GCV for
other traits suggesting a significant environmental effect on the expression of these
characters. For disease reaction of rust showed moderate heritability coupled with
low genetic advance suggested the involvement of epistatic interactions. While,
chocolate spot disease reaction showed that the moderate heritability coupled with
high genetic advance. Heterotic effects for seed yield/plant were associated with
other yield components and significant positive inbreeding depression values were
detected for number of seed/plants, seed yield/plant and 100- seed weight. SSR
cluster analysis showed that cross F1 “Sakha3 X Misr1was separated lonely apart
of all other genotype. Moreover, some F2 plants were clustered along with P1 and
some others were clustered along with P2 parent indicating distribution of genetic
material among F2 plants. The results of molecular markers were in discontiguous
with morphological results which may be attributed to the low number of
morphological traits used and the habit of the morphological traits that affected by
the environmental conditions. The results of both morphological and molecular
markers proposed that selection in these particular populations should be effective
and satisfactory in the successful breeding purposes.
Keywords: Vicia faba, Genetic parameters, Heterosis, Heritability, Genetic
advance, Disease resistance, molecular marker.
INTRODUCTION
Faba bean is a major food and feed grain legume owing to the high nutritional
value of its seeds, which are rich in protein 27-34% (Link et al., 1995). It is
considered as one of the oldest grain legumes and is grown in many countries for
both human consumption and animal feed. Faba beans are affected by many fungal
diseases, which vary in incidence and severity from one region to another. The most
relevant ones are ascochyta blight (caused by Ascochyta fabae Speg.), rust
(Uromyces Vicia faba (Pers.) J. Schro¨ t.), chocolate spot (Botrytis fabae Sard.),
downy mildew (Peronospora viciae Berk.) and foot rots (Fusarium spp.). The
pathogen of Chocolate spot attacks all of the aboveground parts of the faba bean
plant, thereby causing chocolate-colored lesions that may spread quickly around the
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infection site, killing the tissue above the lesion. In Egypt, yield losses exceed 20-
25% (Khalil et al., 1993) and may reach 100% under severe epidemic conditions
(Bouhassan et al., 2004; Torres et al., 2004). In addition, faba bean rust, is a
serious disease of faba beans in the subtropical agricultural region of North Delta
(Emeran et al., 2005). The deployment of resistant faba bean varieties is an
efficient strategy for controlling the disease and promoting the development of
sustainable agriculture (Bouhassan et al., 2004; Rhaïem et al., 2002). In the
Mediterranean basin, the Nile valley, Ethiopia, expertise in novel molecular
breeding approaches is limited. However, significant advances have been made
towards understanding the faba bean genome (2n = 12; x = 6), one of the largest
among legumes (1C = 13.33 pg 13.000 Mbp, Bennett and Smith, 1976; Johnston
et al., 1999). Thus, the use of molecular markers and the development of suitable
F2 and advanced inbred populations have allowed significant progress in mapping
to enhance breeding strategies in the species. This investigation provides an
overview of the genomic resources currently available in faba bean, with an
emphasis on development and application of MAS for genetic improvement of the
crop. The aims of this work are to: i) measure variability and genetic parameters (ii)
identify and test SSR PCR markers for screening genetic diversity in the two
commonly cultivated faba bean genotypes (iii) Estimate the morphological
diversity and relationships among these genotypes for disease resistance. The
following experiments were designed to confirm varietal differences in resistance
to chocolate spot and rust infection and determinate the genetic variability of faba
bean genotypes, and molecular breeding of faba bean for disease resistance, by
using Simple Sequence Repeats (SSRs) for analyzing F1, F2 populations and their
parents.
MATERIALS AND METHODS
Field experiment:
The field experiment was carried out at Food Legumes Department, Sakha
Agricultural Research Station, Agriculture Research Center, Egypt, during three
growing seasons 2015/2016, 2016/2017 and 2017/2018. Two faba bean genotypes
(Vicia faba L.) were chosen for this study on the basis of their genetic diversity and
origin (Table 1). These genotypes were Sakha3 and Misr1.
Table 1: Pedigree and reaction to foliar disease of faba bean cultivars under
study.
Genotype
Pedigree
Reaction to foliar
disease
Sakha 3
Promising line 716/402/2001 derived
from cross716 (Giza461 X 503/453/83)
Resistant
Misr1
Giza 3 x 123A/45/76
Susceptible
In 2015/2016 season, the two faba bean genotypes were crossed under insect
free cage to obtain F1 hybrid seeds. In 2016/2017 season, the parents and their F1
hybrid seeds were sown in a Randomized Complete Block Design (RCBD) with
three replications under insect free cage, and the F1 plants were self-pollinated to
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obtain the F2 seeds. In 2017/2018 season, field experiment was conducted to
evaluate the parents and their F1s and F2s generations for disease resistance.
F1, F2 generations and their parental genotypes were sown in RCBD design
with three replications. Each entry was represented by one ridge for parents and
F1s and four ridges for F2s, each ridge was 3m long and 60cm apart. Seeds were
planted on one side of the ridge at 20cm hill spacing with one seed per hill. Plots
varied in size; for segregation generation 12 rows F2 and three rows for non-
segregation generation P1, P2 and F1. Data were recorded as an average of 10, 10
and 50 individual guarded plants chosen at random from each plot for the parents,
F1 and F2 generations, respectively. The data were recorded on flowering date
(days) (FD), maturity date (days) (MD), plant height (cm) (PH), branches number
(NB), number of pods/plants(NPP), number of seeds/plants(NSP), number
seeds/pod(NSP), seed yield/plant (g) (SY), 100 seed weight (g) (100SW), chocolate
spot and rust reaction (Table 2).
Table 2: Rating scale for chocolate spot and rust diseases according to Bernier
et al. (1993).
Chocolate spot scale
1
No disease symptoms or very small specks (highly resistance)
3
Few small disease lesions (resistant)
5
Some coalesced lesions, with some defoliation (moderately resistant)
7
Large coalesced sporulating lesions, 50% defoliation and some dead
plants (susceptible)
9
Extensive, heavy sporulation, stem girdling, blackening and death of
more than 80% of plants (highlysusceptible)
Rust scale
1
No pustules or very small non-sporulating flecks (highly resistant)
3
Few scattered pustules covering less than 1% of the leaf area, and few or
no pustules on stem (resistant)
5
Pustules common on leaves covering 1-4% of leaf area, little defoliation
and some pustules on stem (moderately resistant).
7
Pustules very common on leaves covering 4-8% of leaf area, some
defoliation and many pustules on stem (susceptible).
9
Extensive pustules on leave, petioles and stem covering 8-10% of leaf
area, many dead leaves and several defoliations (highly susceptible).
Statistical and genetic analysis: -
The ordinary analysis of variance for the RCBD was done according to
Steel and Torrie (1980) using JMP JMP® 7.0 software (Sall et al., 2007).
Heritability and genetic advance estimates were calculated using the standard
methods given by Steel and Torrie (1980). Genotypic and phenotypic coefficients
of variability (GCV and PCV %), heritability estimates in broad-sense and genetic
advance as percentage of mean were estimated following Allard, 1960. The amount
of heterosis was expressed as the percentage deviation of F1 mean performance
from the better-parent values. Inbreeding depression was calculated as the
difference between the F1 and F2 means expressed as a percentage of the F1 mean
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(Wynn et al 1970). The nature and type of dominance were determined by means
of potence ratio method (P) which can be defined according to Smith (1952).
Heritability in broad sense was estimated according to Mather (1949). Data
regarding the above-mentioned traits were averaged and subjected to analysis of
variance. According to Sivasubramanian and Menon (1973), the value of PCV
and GCV were classified as low (< 10%), moderate (1020%) and high (> 20%).
According to Robinson et al. (1949) that the value of the heritability (h2 (b))
percentage was categorized as low (< 30%), moderate (3060%) and high (> 60%).
In addition, Johnson et al. (1955) categorized that the value of genetic advance as
percent of the mean is categorized as low (< 10%), moderate (1020%) and high (>
20%). Predicted genetic gain as mean percent from selection (∆g %) was calculated
according to Johnson et al. (1955). Prediction genetic advance as percent of the F2
mean (GS %) was calculated as given by Miller et al, (1958).
Molecular markers experiment:
The molecular experiment was carried out at the Plant Molecular Biology
Laboratory (PMBL), Plant Biotechnology Department, Genetic Engineering and
Biotechnology Research Institute (GEBRI), University of Sadat City (USC), Egypt
to study the genetic diversity of the cross Sakha3 X Misr1 and the parents in
addition to F2’s plant regarding to their response towards chocolate spot and rust
diseases. To achieve this goal, seven SSR primers pairs were selected to test F2
plants and their parents as well as F1 generation.
Plant material and DNA extraction:
DNA was extracted from young, healthy leaf tissue of each genotype using
i-genomic Plant DNA Extraction Mini Kit (iNtRON Biotechnology, Korea)
according to their manufacturer instructions. DNA quality was tested using 1%
agarose gel electrophoresis and DNA quantity was estimated using Nanodrop
device at 260nm in order to determine DNA concentration. The final concentration
of DNA was adjusted to 25 ng/μl.
SSR-PCR analyses:
A total of seven SSR primer pairs have been selected to investigate the
genetic diversity of the F2 population of the cross Sakha3 X Misr1in relation to
their rust and spot diseases resistance; as well as their parents (Table 3). SSR
primers pair have been selected according to literature (El-Absawy et al., 2012).
The PCR reactions were made up of 15 μl reaction volume using 50 ng template
DNA, 7.5 μl of 2X PCR Master Mix (TIANGEN) and 0.5 μmoles of both primers
(forward and reverse). Touchdown PCR program was carried out using six cycles
with touchdown of the annealing temperature from 45°C for 50 sec. decreasing 1oC
in every cycle until 40°C for 50 sec. The last cycle was repeated 28 times and
finalized with an extension step at 72°C for 5 minutes. The PCR products were
separated on 1.5% agarose gel electrophoresis. The gels were scored as (1) or (0)
for band presence or absence, respectively. Total number of bands generated as well
as the number of polymorphic bands and their percentages were calculated for each
primer pair. The polymorphic information content (PIC) was calculated according
to Anderson et al. (1993) using the following simplified formula: PICi = 1- Σp2ij;
Where pij is the frequency of the jth allele for marker ith summed across all alleles
for the locus. Similarity coefficient matrix was calculated using the simple matching
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algorithm and dendrogram was generated using UPGMA method in NTSYS PC 2.0
software (Rohlf, 1998).
Table 3: Primers names, sequences, total number and polymorphic number of
bands and PIC of SSR markers for Vicia faba genotyps.
Prim
er
Pair
Bands number
PIC
Forward
Reverse
Total
Polymo
rphic
%Poly
morph.
SSR1
AGCGATGGTGCTCATGCTTA
TCTCTCACGGAATCACATCTT
4
3
75
75
SSR2
TTTCAGCAAACTAGAACCAA
GGCATTCAGTTTTTACCTTGT
3
0
0
0
SSR3
GCACTCGAAGGAATTAATTTT
GAACAGTTGTTTCGTGTCGTA
3
2
66.7
66.7
SSR4
GATGTTGTTGGTGTTGTTTA
CAATTAGGAGCAAAATCAGA
4
4
100
75
SSR5
GGTTTTGAATAGAAATGCAA
AAGATGTGTCAATATTGTTTT
3
2
66.7
66.7
SSR6
GGCTATTGTCACGAACAAAT
GATTCAGACCCGGATACATT
8
8
100
81
SSR7
AGAGTCCCAAAGAGTGGGTT
CCAAAGGCAAAAATGAGGGCTT
9
4
44.4
64.2
RESULTS AND DISCUSSION
Morphological analysis of faba bean traits:
Analysis of variance of the 11 faba bean traits showed that there are highly
significant differences among genotypes in all recorded traits (Table 4). This means
that all genotypes including parents and the F1 cross are differed in relation to the
morphological traits. The genotypes were significantly differed for growth related
traits and yield traits as well as disease resistance traits. Similar results have been
reported for growth related traits and yield and its components in faba bean (El-
Absawy et al., 2012 and Abdellatif et al., 2012) as well as for disease resistance
traits (Zakaria et al., 2015; Eldemery et al., 2016 and Belal et al., 2018).
Table 4: Analysis of variance of 11 morphological traits for Vicia faba F2
plants and their parents.
S.O.V.
FD
(day)
MD
(day)
PH
(cm)
NB
NPP
NSP
NSP
SYP
(g)
100 SW
(g)
Spot
Rust
Genotype
147.5**
13.21**
156.4**
7.82**
146.2**
1710.3**
1.0**
1435.3**
266.7**
2.6**
1.12**
Error
7.07e-13
2.28e-12
2.19e-12
1.64e-15
3.7e-13
1.76e-12
0.000
0.00
0.000
1.12e-
5.16e-
** highly significant differences
I) Growth related traits:
The F2 population plants of the cross “Sakha3 X Misr1 significantly
varied in flowering date from very early (43 days such as F2-2 and F2-20 plants) to
very late in flowering date (70 days such as F2-10, F2-16, F2-17 and F2-25 plants),
while the parents and F1 cross gave intermediate record for this trait (Table 5). On
the other hand, the parents Sakha3 and Misr1 were the latest genotypes in
maturity date (156, 154 days, respectively) and the earliest genotypes in this trait
were F2-16 and F2-24 plants which recorded 147 days for maturity. The shortest
significant plant height was recorded for the parent “Sakha3” (104.2 cm) while the
longest significant plant height was recorded for F2-11 plant (140 cm). About 3.5
branches were recorded for the cross and its parents while the highest number of
branches was obtained from the F2-5 plant (five branches) and only two branches
were recorded from the plants F2-6, F2-9 and F2-10 (Table 5). It seems that the
genotypes differed in the growth-related traits. The most important note is that there
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was transgressive segregation in these traits. For example, while the parents of the
cross were the latest genotypes in maturity date, F2-16 and F2-24 plants recorded
147 days for maturity which means transgressive segregation or early maturity.
Abdalla et al., (2015) also reported that transgressive segregation occurred in seed
index, seed length, seed width, seed thickness and seed shape index of faba bean
and thus useful transgressive segregates may be selected.
II)Yield and its components traits:
Among the yield and its components traits, the number of seeds per plant
showed highly significant differences among the cross Sakha3 X Misr1and their
parents; whereas, the cross recorded the highest number of seeds per plant (154)
and the parents recorded the lowest number (33 and 28for P1 and P2, respectively).
This result could be related to the growth vigor of the cross resulted from
aggregation of different alleles (from both parents) for this trait. For traits number
of pods per plant, number of seeds per pod, seed yield for plant and 100 seeds
weight, the F2 population plants surpassed the cross and its parents (negatively or
positively). For example, F2-25 plant gave average of 105.6 grams as a weight for
the 100 seeds while the cross and parents did not exceed 78 grams and F2-16 plant
produced average of 5.6 seeds per pod while it was 3.4 seeds per plant for P1
Sakha3 and less for the other parent and the cross (Table 5). In the yield and its
components traits the growth vigor as a result of the cross could be obtained
specially in the number of seeds per plant where the cross gave more than five times
of the best parent in this trait. Growth vigor in seed yield of faba bean crosses was
reported by Ghassemi and Hosseinzadeh (2009) whereas they reported that high
vigor seeds of faba bean cultivars can be produced under both well and limited
irrigation conditions.
III) Disease resistance traits:
Data recorded for resistance of both chocolate spot and rust diseases showed
that P2 Misr1 was susceptible variety for both diseases (6 and 7 for chocolate
and rust resistance, respectively), while P1 Sakha3 was resistant variety (3 and 4
for chocolate and rust resistance, respectively) and the cross was intermediate (5
and 6 for chocolate and rust resistance, respectively, Table 5). The F2 population
plants showed different responses toward both diseases. For example, F2-6, F2-8,
F2-9, F2-13, F2-15 and F2-25 plants behaved like P2 Misr1 concerning the
chocolate spot disease (6, resistance) while the plants F2-16, F2-17, F2-18, F2-19,
F2-21, F2-22 and F2-23 behaved like P1 Sakha3 regarding the same disease (3,
susceptible). On the other hand, most of F2 plants behaved intermediately regarding
the rust resistance disease except F2-10 plant which behaved like P2 (resistance).
The plants F2-18 and F2-21 showed susceptibility against both diseases like P1
parent (Table 5). It can be noted that some F2 plants behaved similar to the resistant
parent, some others behaved similar to the susceptible parent but most of the F2
plants behaved intermediately regarding the rust resistance disease, similar results
were obtained by El-Rodeny et al., (2017). Elsawy et al., (2015) found different
responses of wheat genotypes against leaf rust disease.
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Table 5: LSD values of 11 morphological traits for Vicia faba F2 plants and
their parents.
Genotype
Growth related traits
Yield and its components traits
Resistance
traits
FD
(day)
MD
(day)
PH
(cm)
NB
NPP
NSP
NSP
SYP
(g)
100 SW
(g)
Spot
Rust
P1 (Sakha3)
56.8E
156.0A
104.2K
3.5H
21.6J
3.4P
33.0Z
66.9I
90.9B
3D
4D
P2 (Misr1)
52.4H
154.7C
116.7G
3.3I
16.9O
2.9W
28.0[
35.6U
71.7Q
6A
7A
F1
56.1F
155.0B
120.9E
3.5G
26.5E
3.3R
154.0A
78.0G
88.7D
5B
6B
F2 (1)
46.0M
155.0B
105.0J
5.0E
18.0M
2.3\
127.0B
29.3Y
69.7S
5B
5C
F2 (2)
43.0N
153.0D
120.0F
5.0E
36.0C
2.5X
73.0M
81.4F
90.4C
5B
6B
F2 (3)
52.0I
155.0B
125.0D
8.0B
40.0B
3.2T
55.0S
103.7B
81.7J
5B
6B
F2 (4)
52.0I
155.0B
130.0C
4.0F
12.0S
2.3[
53.0T
17.9\
63.8W
5B
6B
F2 (5)
49.0K
153.0D
130.0C
12 A
48.0A
3.2S
49.6U
101.0C
65.6U
5B
6B
F2 (6)
49.0K
153.0D
130.0C
2.0K
20.0K
3.7K
47.0V
57.7K
79.1M
6A
6B
F2 (7)
52.0I
155.0B
120.0F
7.0C
24.0G
3.8H
46.0W
46.2O
50.7\
4C
6B
F2 (8)
46.0M
150.0F
120.0F
6.0D
13.0R
4.2C
43.0X
33.8W
61.4Y
6A
6B
F2 (9)
58.0D
155.0B
125.0D
2.0K
12.0S
3.9G
42.0Y
33.8X
71.8P
6A
6B
F2 (10)
70.0A
153.0D
115.0H
2.0K
17.0N
3.6M
123.0C
41.5P
68.0T
5B
7A
F2 (11)
67.0B
150.0F
140.0A
4.0F
19.0L
3.2U
107.0D
38.4Q
64.1V
5B
6B
F2 (12)
55.0G
152.0E
125.0D
5.0E
23.0H
4.3B
99.0E
100.4D
87.3E
5B
5C
F2 (13)
49.0K
153.0D
115.0H
3.0J
15.0P
3.53N
91.0F
37.1S
69.9R
6A
6B
F2 (14)
64.0C
150.0F
120.0F
4.0F
9.0T
3.7J
90.0G
18.6[
56.3[
5B
6B
F2 (15)
46.0M
150.0F
110.0I
5.0E
17.0N
3.4Q
88.0H
46.9N
81.4K
6A
6B
F2 (16)
70.0A
147.0G
115.0H
4.0F
19.0L
5.6A
81.0I
90.4E
84.4H
3D
6B
F2 (17)
70.0A
155.0B
110.0I
4.0F
19.0L
4.2D
80.0J
72.1H
91.2A
3D
6B
F2 (18)
46.0M
155.0B
130.0C
4.0F
14.0Q
3.1V
79.0K
34.6V
80.4L
3D
4D
F2 (19)
46.0M
150.0F
115.0H
6.0D
22.0I
3.6L
73.6L
49.1M
62.3X
3D
5C
F2 (20)
43.0N
155.0B
130.0C
6.0D
22.0I
3.7I
67.0N
62.9J
77.7O
4C
6B
F2 (21)
50.0J
153.0D
130.0C
4.0F
19.0L
2.4Y
61.0O
35.8T
77.8N
3D
4D
F2 (22)
48.0L
150.0F
125.0D
4.0F
25.0F
2.4Z
60.0P
18.9Z
82.3I
3D
6B
F2 (23)
49.0K
155.0B
135.0B
4.0F
15.0P
3.9F
59.0Q
51.4L
87.1F
3D
6B
F2 (24)
50.0J
147.0G
125.0D
4.0F
19.0L
3.5O
59.0Q
37.8R
56.4Z
5B
6B
F2 (25)
70.0A
150.0F
110.0I
4.0F
31.0D
3.9E
57.0R
105.6A
85.9G
6A
5C
Levels not connected by same letter are significantly different.
IV) Morphological Hierarchical Cluster analysis:
The combined data of the morphological traits were used to construct two-
way hierarchical cluster analysis using Ward’s method in order to monitor the
relationship among the F2 population plants and their related parents depending
upon the morphological traits. The second way of the analysis has been made to
determine the relationships among the measured morphological traits and if there
any traits are related to each other. According to this analysis, the F2 plants along
with the cross “Sakha3 X Misr1and the parents were distributed into three
clusters. The first cluster contained the first parent “Sakha3and five F2 population
plants (F2-3, F2-12, F2-16, F2-17andF2-25). The second cluster included the
second parent “Misr1along with 13 F2 population plants (F2-1, F2-4, F2-5, F2-6,
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F2-7, F2-8, F2-9, F2-10, F2-11, F2-13, F2-14, F2-15 andF2-19, Figure 1). The third
cluster contained the cross Sakha3 X Misr1and the six F2 population plants (F2-
2, F2-18, F2-20, F2-21, F2-22 andF2-23). It seems that some F2 plants were
clustered along with each parent and some others were clustered along with the F1
cross. This means that the genetic background played significant role in the
inheritance of the morphological traits and there is significant genetic diversity in
the plant material in relation to the recorded morphological traits. Belal et al.,
(2018) found enrich genetic diversity in faba bean genotypes in relation to heat and
drought tolerance traits. Similar results were reported also by Abdellatif et al.,
(2012) and Zakaria et al., (2015).
In the second way of clustering, the 11 morphological traits clustered in two
main groups. The first group included five traits (flowering date, number of seeds
per pod, plant height, resistance to chocolate spot disease and resistance to rust
disease). The second group contained six traits (maturity date, weight of 100 seeds,
number of branches, and number of pods per plant, number of seeds per plant and
seed yield per plant, Figure 1. The most related to each other traits were number of
seeds per plant and seed yield per plant which means measuring any of them is
index to the other trait. It can be seen that some morphological traits could be used
as indicator to the other such as number of seeds per plant trait which could be used
as indicator for the yield seed per plant. Similar results were obtained by Abdellatif
et al., (2012), Zakaria et al., (2015) and El-Rodeny et al., (2017).
Figure 1: two-way hierarchical cluster analysis of 25 F2 population plants of
the faba bean cross “Sakha3 X Misr1” depending upon 11
morphological traits.
P1 (Sakha3)
P2 (Misr1)
F1 (Sakha3*Misr1)
F2 (1)
F2 (2)
F2 (3)
F2 (4)
F2 (5)
F2 (6)
F2 (7)
F2 (8)
F2 (9)
F2 (10)
F2 (11)
F2 (12)
F2 (13)
F2 (14)
F2 (15)
F2 (16)
F2 (17)
F2 (18)
F2 (19)
F2 (20)
F2 (21)
F2 (22)
F2 (23)
F2 (24)
F2 (25)
Flowering Date
Maturity Date
Plant Height
Branches No#
No# Pods/Plant
No# Seeds/Plant
No# Seeds/Pod
Seed Yield/Plant
100 Seed Weight
Spot
Rust
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V) Genetic parameters of the studied traits: -
a- Heritability and genetic advance:
Heterosis over mid- parent and better parent values, potence ratio, genetic
variability, heritability and genetic advance for 11 agronomic traits of faba bean
genotypes are presented in Table 6.
Table 6. Gene action parameters of the studied traits in one faba bean cross.
Cros
s
Trait
Heterosis
Inbreeding
depression
H
PCV
GCV
Δg%
Mp%
PR
Bp%
Sakha 3 x Misr 1
Flowering date
-6.37
1.61
-2.50*
-3.99
73.09
27.41
6.14
10.82
Maturity date
-3.50*
6.89
-3.01*
-1.67
63.32
4.39
1.35
2.21
Plant height
12.97**
2.30
19.72**
2.44
61.55
12.89
2.55
4.13
Branches / plant
54.73
7.36
44.03
7.15
79.17
91.40
39.13
71.73
pods/ plant
37.29**
3.07
22.40**
22.36
91.72
187.27
28.91
57.04
seeds /plant
44.35**
1.97
17.85**
17.69**
70.42
40.27
6.27
10.84
Seed yield
50.81**
1.61
14.72**
43.42**
61.65
43.33
7.85
12.70
No. of seed/pod
24.22
3.09
15.20
11.66
45.98
15.63
14.33
20.02
100- seed weight
8.99*
0.78
-2.33
21.51**
78.14
46.63
7.24
13.18
Chocolate spot
-0.98
0.03
46.38
-0.80
49.24
29.70
17.98
25.99
Rust
-10.66
0.39
22.65
-21.79
25.44
16.36
8.32
8.65
(H) = heritability, PCV= phenotypic coefficient of variation and GCV= Genotypic coefficient
of variation and GA= Genetic Advance. * & ** significant at the 0.05 & significant at
the 0.01 probability levels, respectively.
The analyses of parameters of variability showed little difference between
GCV and PCV for number of seed/pod and maturity date, indicating a negligible
environmental effect on these characters. These results are in agreement with Salwa
et al., 2017 when estimated of genetic variability, heritability and genetic advance
in three segregating generations (F2, F3 and F4 generations) of three faba bean
crosses. However, PCV was higher than GCV for the traits flowering date, plant
height, number of seed/plants, seed yield /plant and 100 seed weight, suggesting a
significant environmental effect and indicating marked influence of environment
on the expression of this characters. High heritability degree was observed for most
of studied traits, while moderate heritability was observed for number of seed/pod
and for chocolate spot disease reaction, and low heritability was observed for rust
disease reaction. According to Johnson et al., (1955) high heritability estimates
along with the high genetic advance as per mean is usually more helpful in
predicting gain under selection than heritability alone. High heritability estimates
(H2bs=91%) along with high genetic advance (GA=57%) for number of pod/plant
and number of branches (H2bs=79%) with high genetic advance (GA=71%),
reflecting the presence of additive gene action for the expression of these traits
which is fixable for next generations, and selection in next population based on this
character would be ideal, similar results were also reported by Abbas and El-
Rodeny (2009) and Abou- Zaid et al., (2017). Moreover, the 100 seed weight and
flowering date having high values of heritability coupled with moderate genetic
advance, suggest that selection for improvement of these characters may be
rewarding. It also indicates greater role of non-additive gen action in their
2020 54(2 )
24
inheritance. The high heritability along with low genetic advance indicates non
additive type of gene action where genotype environment interaction plays a
significant role in the expression of the traits flowering time and maturity date, these
characters could be improved through hybridization, these findings are in
agreement with those of Dawwam and Abdel-Aal (1991). Moderate heritability
coupled with low genetic advance suggested the involvement of epistatic
interactions; whereas, for rust disease reaction (25.44%, 8.65%), both heritability
and genetic advance were low. and Moderate heritability coupled with high genetic
advance for chocolate spot disease reaction (49.24%, 25.99%), Similar results were
obtained by Abou- Zaid and Sarhan (2017). These findings could be useful to
select high yielding materials to exploit in the breeding program.
b- Heterosis
Heterosis percentage relative to mid parent (M.P), potence ratio and better
parent (B.P) are given in Table 6. Plant breeders have been investigated the
possibility of developing hybrid cultivars. Thus, the utilization of heterosis in
various crops through the world has tremendously increased the production.
Heterosis is a complex phenomenon which depends on the balance of different
combinations of genotypic effect as well as the distribution of plus and minus alleles
in the parents. Heterosis is expressed as the percentage deviation of F1 mean
performance from the better or mid parent of the traits. As it will be expected, better-
parent for seed yield was the highest one and heterosis relative to the mid- parent
value may be also effective. In this concern, percentage of heterosis over mid-
parent and better parent values is presented in the cross Sakha3 X Misr1are
presented in Table (6). Heterosis relative to the mid- parent value was significantly,
or highly significantly, positive in five traits, for plant height, number of
pods/plants, number of seed/plants, seed yield/plant and 100-seed weight, 12.97,
37.29, 44.35, 50.81 and 8.99 respectively. Significant and highly Heterosis was
obtained for most studied traits, except flowering date and number of branches and
chocolate spot and rust disease reaction. Negative significant Heterosis value was
found in to the mid- parent and better parent for maturity date (-3.5&-3.0). Positive
and significant heterosis percentages over MP or BP were reported by several
researchers for faba bean characters which varied according to the cross
combinations and traits such as Abou- Zaid et al., (2017) and El-Rodeny et al.,
(2017). Positive significant heterosis to the better- parent was obtained for plant
height, number of pods/plants, number of seed/plant and seed yield/plant.
Meanwhile, Negative significant heterosis to the better- parent was obtained for
flowering date and maturity date (-2.5&-3.0). The differences in heterosis percent
might be due to genetic variability of the parents and for non-allelic interactions,
which can either increase or decrease the expression of heterosis. Even in the
absence of epistasis, multiple alleles at a locus could lead to either positive or
negative heterosis (Cress 1966). These data suggest that heterotic effects for seed
yield/plant were associated with other yield components, such as 100-seed weight
and number of pods per plant. In addition, the heterosis estimates, compared to
either MP or BP, for seed yield per plant and its major yield components indicated
that there was sufficient genetic variability among the assessed parents to favor
efficient breeding for these characters.
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25
c- Inbreeding depression
The estimates of the inbreeding depression from F1 to F2 in cross Sakha3
X Misr1for 11 characters are presented in the Table 6. Inbreeding depression
measured the extent of reduction of the F2 generation due to inbreeding. Significant
positive values were obtained for number of seed/plants, seed yield/plant and 100-
seed weight traits. Significant effects for both heterosis and inbreeding depression
seem logic since the expression of heterosis in F1’s was followed by considerable
reduction in the F2 performance. Also, reduction in values of non- additive genetic
components is expected caused by means of inbreeding depression. Similar
conclusions were reviewed by Zeinab et al., (2013). In addition, the conflicting
estimates heterosis and inbreeding depression were associated in most traits. The
results revealed also that, the genetic variance was mostly attributed to the additive
effects of genes for the other studied traits. This confirms the previous results that
found by means of gene action estimates of additive genetic portion, which was
mostly predominant. Similar findings were reported by Gasim and Link (2007).
Genetic Diversity by Molecular marker
a-SSR polymorphism:
The PCR banding patterns of the SSR analysis is illustrated in the Figure 2.
The SSR banding polymorphisms results revealed that the total amplified bands for
each primer pair ranged from three bands for the primer pairs SSR2, SSR3 and
SSR5 to nine bands for the primer pair SSR7; while the number of polymorphic
bands ranged from zero for the primer pair SSR2 to eight bands for the primer pair
SSR6 (Table 3). The percentage of polymorphism of the primer pairs varied from
zero for the primer pair SSR2 to 100% for the primer pair SSR6 and the
polymorphic information content (PIC) ranged from zero to 81% for the same
primer pairs, respectively (Table 3). SSR banding pattern differed among the
primers as well as the percentage of polymorphisms among the primer pairs. The
polymorphic information content (PIC) was differed as well which means that these
primer pairs are informative to detect the genetic diversity of the selected genotypes
and differentiate among the F2 plants of the cross “Sakha3 X Misr1”. The same
group of SSR primer pairs have been proven to be informative in other crop plants
such as Maize (Abdellatif and Khidr, 2010) and Ficus (Esmaiel et al., 2014).
Similar conclusions were reviewed by El-Rodeny et al., (2014), in a polymorphic
analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES
markers showed polymorphisms
2020 54(2 )
26
Figure 2: PCR banding pattern of the SSR analysis of the F2 population plants
generated from the faba bean cross “Sakha3 X Misr1” and its
parents.
b-SSR Dendrogram:
According to the cluster analysis of the SSR data that obtained from the
scoring of the SSR gels, the F2 population plants of the faba bean cross Sakha3 X
Misr1 were distributed into three clusters at similarity coefficient 0.77. The first
cluster included the first parent P1 Sakha3” along with the F2 population plants
F2-1, F2-5,F2-6,F2-7,F2-9,F2-10, F2-11, F2-14, F2-15,F2-16,F2-18,F2-19, F2-
21,F2-22,F2-23,F2-24 and F2-25 (Figure 3). The second cluster contained P2
Misr1” and eight F2 population plants F2-2, F2-3, F2-4, F2-8, F2-12, F2-13, F2-
17 and F2-20. The third cluster included only the cross Sakha3 X Misr1(Figure
3). Thus, it seems that according to the SSR cluster analysis the cross “Sakha3 X
Misr1” was separated lonely apart of all other genotype.
Thus, some F2 plants were clustered along with P1 and some others were
clustered along with P2 parent. This distribution of the F2 plants in the cluster
analysis indicates distribution of genetic material (gathered from both parents)
among F2 plants, while F1 separated apart of all other genotypes. This could be
explained as the genetic material of the two parents were distributed among the F2
plants but not in F1 generation which remains differed from parents as well as F2
plants. El-Absawy et al., (2012) revealed that molecular markers were efficient to
study genetic diversity in faba bean. They obtained three clusters form using
molecular markers in faba bean. Duc et al., (2010) reported that large genetic
variability has already been identified in V. faba in terms of floral biology, seed size
and composition. Our results are in agreement with their results hence they reported
that the use of simple sequence repeat (SSR) markers has allowed genetic resources
to be distinguished according to their geographic origin and the structuring of
collections. The results of molecular markers were also somehow in discontiguous
with morphological results and this may be attributed to the low number of
morphological traits that was used to determine the genetic diversity and the habit
of the morphological traits that affected by the environmental conditions. One
another reason may affect the disagreement of both results is the low number of the
primers used for the molecular markers. Black-Samouelsson et al., (1997) exposed
2020 54(2 )
27
results agree with this suggestion when studied the genetic variation in two Swedish
and three Czech populations of the plant Vicia pisiformis. Their results did not
reveal any significant correlation between morphological and molecular matrices.
Similar results were obtained by El-Rodeny et al., 2017 when studied SSR markers
to evaluating germplasm and assessing the genetic diversity in Egyptian faba bean
populations
Figure 3: Dendrogram of cluster analysis of the F2 population plants generated
from the faba bean cross “Sakha3 X Misr1” and its parents.
ACKNOWLEDGMENT
The authors acknowledge Prof. Donal OSolivan, University of Reading, UK, the
PI of Project entitled Traits and technologies to boost North Africa protein self-
sufficiency (BEANS4NAFRICA) for the financial support of the publication of
this paper.
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