Application of Biotechnology Tools in
Egyptian Bread Wheat Breeding
Khaled F M Salem
Wheat Science to textbooks, 5-10 December 2010, CIMMYT
Egypt Map
Development of wheat area, productivity and
production in Egypt (1981 – 2008)
Area
Year
Productivity
% Inc
Production
% Inc
Ard/
fad.
t/ha
% Inc
m. fad.
1000
ha
1981
1.40
588
---
9.20
3.30
---
12.90
1.94
---
1986
1.21
507
- 14
13.70
3.81
15
12.86
1.93
-1
1991
2.22
931
58
14.10
4.82
46
29.88
4.48
131
1996
2.42
1017
73
15.80
5.64
71
38.24
5.74
196
2001
2.34
984
67
17.80
6.35
92
41.70
6.26
223
2008
3.0
1.3
92
18.15
6.50
97
49.20
8.0
313
m. Ard m. Ton
Wheat in Egypt
 Food for man and fodder for animal.
 Production:
7.5 m. t. (2009).
 Area:
1.2 m.ha.
 Consumption: 15.0 m.t.
 Gap:
7.5 m.t.
Wheat Research Interest
 Breeding Program.
 Genetic Resources.
 Biotechnology Component.
 Wheat Biotic and Abiotic.
Genetic Engineering Research Institute
(GEBRI)
Specific objectives:
 Generation of useful somaclonal variation via tissue culture.
 Rapid fixation of useful genetic variation into homozygous lines
in one generation via anther culture-derived doubled haploids.
 Improving parental selection for crossing via molecular
genotyping and measuring genetic diversity.
 Identification of molecular markers linked to abiotic stress
tolerance, particularly heat, salinity and drought.
 Implementation of marker-assisted selection in breeding efforts
for durable rust resistance and better salinity and drought
tolerance.
Current activities
 Generation of useful somaclonal variation:
 Selected lines resistance to rusts diseases and tolerant to salinity
stress.
 Use somaclonal variation to produce rusts resistant plants and
drought tolerant.
 Molecular marker activities
• Optimization of DNA extraction methods, high yield, high
quality genomic DNA, without use of liquid Nitrogen.
• Optimization of PCR-based molecular marker systems:
 Random amplification markers;
RAPDs, ISSRs
 Specific amplification markers;
STS, SSRs, SNP and ESTs.
 Main Molecular Marker Activities
 Gene and Genome Mapping
Identification and mapping quantitative trait loci for biotic and abiotic
stresses in wheat (Triticum aestivum L.)
 Genetic Diversity
Evaluation of genetic diversity in Egyptian wheat varieties using
microsatellite markers.
Assessing wheat (Triticum aestivum L.) genetic diversity using
morphological characters and microsatellite markers.
 MAS
Allelic detection at the microsatellite Xgwm261 locus linked to the Rht8
dwarfing gene in wheat.
 Prediction of Heterosis and Combining Ability in Early Generation
Relationship between genetic diversity based on DNA markers with
heterosis and combining ability in diallel cross of bread wheat (Triticum
aestivum L.).
Assessing wheat (Triticum aestivum L.) genetic diversity using
morphological characters and microsatellite markers
 Wheat, a self-pollinating crop, has been bred for a wide array
of specific end-use quality traits and various adaptive
characteristics.
 Knowledge of genetic diversity in a crop species is
fundamental to its improvement.
 Evaluation of genetic diversity levels among adapted, elite
germplasm can provide predictive estimates of genetic
variation among segregating progeny for pure-line cultivar
development (Manjarrez-Sandoval et al. 1997).
 The use of molecular markers for the evaluation of genetic diversity is
receiving much attention.





RAPDs (Joshi and Nguyen 1993),
RFLPs (Siedler et al. 1994; Kim and Ward 2000)
AFLPs (Barrett and Kidwell 1998)
STS (Chen et al. 1994) and
ISSRs (Nagaoka and Ogihara 1997).
However, most of these marker systems show a low level of
polymorphism in wheat, especially among cultivated lines and/or
cultivars (Chao et al. 1989; Devos and Gale 1992).
 Microsatellites are one of the most promising molecular
marker types able to identify or differentiate genotypes within
a species.
 Narrow genetic diversity is problematic in breeding for
adaptation to biotic and abiotic stresses as well as increasing in
yield productivity.
 Therefore, it is necessary to investigate the genetic diversity in
wheat germplasm in order to broaden the genetic variation in
future wheat breeding programme.
The objectives of this study were to
 (i) use SSRs to assess levels and patterns of genetic variability
among wheat genotypes.
 (ii) use wheat microsatellite markers for the characterization
and assessment of the genetic diversity of wheat genotypes.
Table : Name, origin and pedigree of the wheat genotypes used in this study.
No.
Variety Name
Origin
Pedigree
1
Sakha 69
Egypt
Inia-RL4220 x 7C/yr’S’ CM1540-25.65.0S
2
Sakha 93
Egypt
Sakha 92/TR 810328S 8871-1S-2S-1S-0S
3
Gemmiza 3
Egypt
Bb/7C*2//Y50/Kal*3//Sakha8/4/Prv/WW/5/3/B
g”s”//On CGM.4024-1GM-13 GM-2GM
4
Gemmiza 7
Egypt
CMH74 A. 630/5x//Seri 82/3/Agent
5
Sids 4
Egypt
Maya“ S“/Mon“ S“/CM1174.A592/3/Giza157*
6
Baviacora
Europe
///
7
Miriam
///
///
Table: Gene diversity for fifteen microsatellite loci
Microsatellite
marker name
Chromosomal
location
No. Of
Alleles
Fragment size
in CS (bp)
Range of allele
size (bp)
Gene Diversity
Xtaglgap
1B
3
282
219-266
0,625
Xgwm18
1B
2
182
188-192
0,489
Xgwm458
1D
3
112
112-122
0,529
Xgwm95
2A
2
122
109-131
0,277
Xgwm155
3A
2
143
145-147
0,444
Xgwm389
3B
4
131
119-136
0,617
Xgwm3
3D
2
80
77-84
0,408
Xgwm165
4A
6
--
187-202
0,778
Xgwm513
4B
3
144
145-149
0,620
Xgwm186
5A
2
135
122-130
0,408
Xgwm408
5B
2
180
180-184
0,494
Xgwm190
5D
3
204-212
0,612
Xgwm631
7A
3
196
190-202
0,406
Xgwm46
7B
4
180
147-187
0,700
Xgwm437
7D
7
108
91-123
0,816
Total
48
Mean
3,2
0,548
Sakha69
Sakha93
Gemmiza3
BaviacoraMW
Gemmiza7
Miriam
Seds4
Baviacora
0.42
0.47
0.53
0.58
0.63
Coefficient
Fig. Dendrogam analysis of genetic relationships based on gSSRs diversity
Table: Similarity matrix based on microsatellite markers.
Genotypes
Gemmiza 3 Gemmiza 7
Sids 4
Sakha 69
Sakha 93
Mariam
Gemmiza 3
-----------
Gemmiza 7
0.375
---------
Sids 4
0.325
0.478
--------
Sakha 69
0.540
0.395
0.390
---------
Sakha 93
0.562
0.443
0.406
0.688
-------
Mariam
0.434
0.612
0.481
0.416
0.405
---------
Baviacora
0.412
0.472
0.603
0.422
0.408
0.477
summary
 This study using wheat microsatellite markers revealed considerable
amount of genetic diversity among seven wheat genotypes.
 The WMS data can be used in selecting diverse parents in breeding
programme and in maintaining genetic variation.
 Also, this study also shows that analyzing higher numbers of genotypes
may not add much practical value to a general plant improvement program,
unless a specific crossing program is aimed towards the improvement of
specific traits.
 It is therefore suggested that a focused breeding scheme should be adopted.
ALLELIC DETECTION AT THE MICROSATELLITE Xgwm261 LOCUS
LINKED TO THE Rht8 DWARFING GENE IN WHEAT
 Plant height reduction is one of the single most important adaptations introduced
into cereals by breeders over the past century (Reynolds and Borlaug 2006).
 Tall wheat cultivars are more prone to lodging, particularly when grown in
favorable environments, whereas semi-dwarf cultivars are shorter, less prone to
lodging and usually partition more dry matter to the grain (Waddington et al.,
1986).
 The use of dwarfing genes to reduce plant height and improve yield potential has
been one of the major strategies in breeding program.
 Related to their response to exogenously applied gibberellins (GAs), dwarf genes
can be classified into two categories insensitive or sensitive to exogenous
gibberellic acid (GA) (Gale and Youssefian, 1985; Slafer et al., 1994; Calderini et
al., 1995, Borner et al., 1996).
 Plant carrying GA insensitive dwarfing genes can be recognized by
applying a weak solution of GA to germinating seedlings (Gale and
Gregory, 1977).
The aims of this work were
•
(i) to analysis the allelic variation in Xgwm261 microsatellite locus and
•
(ii) to detect the Rht8 gene in some Egyptian and exotic wheat varieties.
Marker assisted selection MAS
Table (1): Name and origin of the wheat genotypes used in Rht8.
No
Variety
1
Cheinese Spring
2
Tri 11712
3
Origin/Country
Continent
China
Asia
Afghanistan
''
Sakha 8
Egypt
Africa
4
Giza 155
Egypt
''
5
Seds 1
Egypt
''
6
Rialto
UK
Europe
7
Spark
UK
''
8
Soissons
UK
''
9
Dwarf
UK
''
10
Tri 4745
UK
''
11
Apollo
Germany
''
12
Capelle-Desprez
France
''
13
Tri 637
Greece
''
14
Bezostaya-1
Russia
''
15
Synthetic
USA
North America
16
Fiorello
Argentina
South America
Table ( 3): Allele size, CL and SL under control and GA3 test
Varieties
No
Allele size
(bp)
Coleoptile length (cm)
Seedling length (cm)
Control
Control
GA3
GA3
1
Cheinese Spring
189
2.78
3.00
6.06
12.67
2
Capelle-Desprez
174
2.32
2.92
5.06
12.43
3
Rialto
192
2.04
2.13
4.78
5.37
4
Spark
196
2.33
2.67
6.58
11.41
5
Soissons
192
1.64
1.88
4.16
5.23
6
Dwarf
192
1.57
1.95
3.65
5.20
7
Tri 4745
174
3.04
4.30
6.71
15.92
8
Bezostaya-1
192
2.12
2.40
4.92
5.75
9
Fiorello
174
2.33
3.11
5.95
13.70
10
Synthetic
186
2.64
2.78
6.18
10.72
11
Apollo
174
2.40
3.00
5.25
10.78
12
Tri 11712
174
2.46
2.78
5.14
11.90
13
Tri 637
176
2.78
3.40
6.93
14.40
14
Sakha 8
192
2.39
2.40
4.56
5.78
15
Giza 155
192
2.56
2.61
5.49
5.83
16
Seds 1
192
2.52
2.60
4.79
6.24
Table (3): Coleoptile length and seedling length mean performance under control and GA3 test for alleles at Xgwm261 locus.
No
Xgwm261
allele (bp)
No
of
Varieties
Mean coleoptile length
Mean seedling length
Control
GA3
Control
GA3
1
174
5
2.51
3.22
5.62
12.95
2
176
1
2.78
3.40
6.93
14.40
3
186
1
2.64
2.78
6.18
10.72
4
189
1
2.78
3.00
6.06
12.67
5
192
7
2.12
2.28
4.62
5.63
6
196
1
2.33
2.67
6.58
11.41
196 bp
6%
174 bp
31%
192 bp
45%
176 bp
6%
186 bp
6%
189 bp
6%
Figure (1): Percentage distribution of 6 alleles for the microsatellite locus Xgwm261 in the wheat varieties.
Conclusions
•
It was found that, a 192-bp allele at microsatellite locus Xgwm261 is associated
with reduction in plant height.
•
Therefore, a 192-bp allele at this locus is always diagnostic for the height
reducing gene Rht8 and its presence is sufficient to determine whether a
particular cultivar carries Rht8 or not.
•
In addition, the Egyptian wheat varieties were has 192-bp allele and carry
Rht8.
•
So this will reflects the importance of Rht8 and involve it in Egyptian breeding
programs.