Novel QTLs for growth angle of seminal roots in wheat (Triticum aestivum L.). Dr. Alhosein Hamada, Associate Professor (Email: a.hamada@aun.edu.eg) Department of Agronomy, Faculty of Agriculture, Assiut University This lecuter was presented in Plant Genetics Seminars. The content of this lecture was publised in Plant and Soil 2012

2. 2

3. Novel QTLs for growth angle of seminal
roots in wheat (Triticum aestivum L.)
Alhosein Hamada, Miyuki Nitta, Shuhei Nasuda, Kenji
Kato, Masaya Fujita, Hitoshi Matsunaka and Yutaka
Okumoto
Plant Soil (2012) 354:395–405
3

4. General introduction
• Stress conditions, such as drought, heat and salinity,
are major problems that adversely affect cereal crop
production.
Wheat
Rice
Maize
Barley
Sorghum
4

5. • By 2025, more than 2.8 billion people in 48 countries will
face water stress or water scarcity conditions.
United Nations Economic Commission for Africa (UNECA)
5

6. EgyptMap
6

7. • We must increase the area of cultivated land
because our population is increasing rapidly, but
we must know that our part of River Nile water
cannot be increased.
• As the Nile Water Agreement
1929, Egypt part of water is
calculated about 55.5 Milliard
m3 / year.
How can we get water for new
reclaimed land?
The question now is:
7

8. Scientists tried to solve this problem through
Decrease the amount
of water
used by crops
Improve crop
productivity with the
same amount of water
Improving irrigation methods
Creating new varieties
Escaping irrigation
The best distribution of water
on different growth stages
8

9. • Improvement of drought stress tolerance through
classical breeding approaches is a historically
difficult task.
• Progress is shown to be relatively slow in most
cases.
Creating new varieties
9

10. Among cereal crops I selected wheat as my plant
material.
Why wheat?
• Wheat is a main source of carbohydrates in the
developing countries.
• Besides being a high carbohydrate food, Wheat
contains valuable protein, minerals and vitamins.
10

11. In food industry, wheat is the major ingredient
in many products as
• breads, rolls, crackers, cookies, biscuits, cakes, doughnuts,
muffins, pancakes, waffles, noodles, piecrusts, ice cream cones,
macaroni, spaghetti, puddings, pizza, and many prepared hot and
cold breakfast foods.

12. Wheat straw is used for livestock bedding.
• The green forage may be grazed by livestock or used as hay
or silage.
• In many areas of the southern Great Plains, wheat serves a
dual purpose by being grazed in the fall and early spring and
then harvested as a grain crop .

13. Non-Food Wheat Products
• Wallboard, cosmetics, pet foods, newsprint, paperboard, and other
paper products, soap, trash bags, concrete, paste, alcohol, oil and
gluten.

14. • In Egypt, there is a big gap between needs and
production in wheat, as the import amount is about
52% of wheat needs (FAO, 2013).
8.14
8.27
7.38
7.98
8.52
7.18
8.41
8.80
9.46
13.83
16.28
15.61
16.30
17.64
17.77
18.20
20.22
19.75
2005 2006 2007 2008 2009 2010 2011 2012 2013
WHEAT PRODUCTION AND CONSUMPTION IN EGYPT
2005:2013 (FAO 2017)
Production Consumption
14

15. • Wheat production is adversely affected by drought
in 50% of the area under production in the
developed and 70% in the developing countries
(Trethowan and Pfeiffer, 2000).
15

16. Objectives
Evaluate genotypic difference in morphological traits
related to drought tolerance in wheat.
Identify genetic factors controlling these traits and their
molecular markers.
16

17. 1
Experiments outline
2
3
Genetic variation in seminal root growth
angle (GA)
QTL analysis of seminal root growth angle
using doubled haploid lines
QTL analysis of hydrotropism in root
growth
17

18. 1. Genetic variation in seminal root growth angle (GA)
1.1 Introduction
18
• Roots are playing an important role not only for
plant nutrition, but also have essential effect on
drought resistant.

19. 19
• Distribution of roots in the field condition is very
difficult to be observed, so that genetic analyses of
genetic factors contributing to root system
architecture are still very limited.

20. • To overcome these difficulties, some researchers
have been working on the relationship between
root characteristics at seedling stage and root
system architecture in the field.
20

21. 21

22. 8.5 cm
Wagner 1/5000a
16 cm
6 cm
18-19
cm
light-colored andosol
Nutrient solution
consisting of 1/1000
hyponex
Approximately
450 μ mol/m2/s
lamps for 12
hours a day
growth chamber
air temperature
20ºC.
• Oyanagi et al. (1994)

23. • Cultivars which show large
root growth angle in
seedlings have deep root
systems at the stem
elongation stage.

24. 24
• Wataru Tsuji et al. 2005, information on such
nodal root emergence would facilitate breeding
programs for improving drought tolerance in
sorghum.

25. • GA value was larger in the northern Japanese
cultivars with deep rooting system than the
southern Japanese cultivars with shallow rooting
system.
• In this experiment, genetic diversity and regional
differentiation among landraces were examined for
the seminal root growth angle.
25

26. • Root angle of 194 lines mainly from different
Asian regions had been measured using the basket
method described by Oyanagi et al., 1993.
1.2 Materials and Methods
1.2.1 Plant materials
26

27. Egypt
AustriaFrance
Morocco
India
Nepal
China
Japan
N. Korea
Russia
Turkmenistan
Tajikistan
KyrgyzstanUzbekistan
Azerbaijan
Poland
Ukraine
Germany
Czechoslovakia
Bulgaria
Afghanistan
Pakistan
Bhutan
27

28. 1.2.2 Experimental condition
Prepare Wagner pots and fill it with fine sand.
Put the baskets in the pots and fill it with sand.
28

29. Primary germination of seeds in the lab.
3-10 mm
29

30. Transplanting the seeds to the baskets in 1 cm depth.
Root direction in the basket is horizontal (3 seeds
per basket in the middle)
30

31. Nutrient solution (1/1000 Hyponex/water) will be
added in the container to the level 15:16 cm from
the ground level.
15:16 cm
• This experiment was
established under green
house with controlled
conditions (22 ºC night and
28 ºC day). 31

32. After 7 days check the roots which penetrated the sides of
the basket and measure the angle from the top of the
basket.
32

33. 33

34. 2/1w2/2w

C
D
E F
G
K




a
h
l
1l 2/1w
2/2w
s
d
Calculation method
for measuring root
angle
34

35. • For each line I used 2 replications.
• Root growth angle (GA)
• Deep root ratio (DRR)
DRR = No. of roots > 45° / No. of all roots
GA = average of all root angles for the penetrated roots
from the basket side
RN = number of roots which penetrated the basket
side
• Number of root/seedling (RN)
35

36. 1.3 Results
Frequency distribution of seminal GA, DRR and RN among 194 landraces
0
20
40
60
80
20 25 30 35 40 45 50 55 60 65
Numberoflines
Root angle (GA)
0
5
10
15
20
25
30
35
40
45
50
10 20 30 40 50 60 70 80 90 100
Numberoflines
Deep root ratio % (DRR)
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10
Numberoflines
Number of roots per seedling (RN)
36

37. Trait Source of variation Degree of freedom Mean square
GA Genotype 193 79.36**
Origin 3 201.65**
Growth habit 1 78.09*
Error 190 16.56
DRR Genotype 193 0.05**
Origin 3 0.04*
Growth habit 1 0.01ns
Error 190 0.01
RN Genotype 193 1.49**
Origin 3 7.11**
Growth habit 1 5.38**
Error 190 0.36
Analysis of variance of the effect of lines, origin and
growth habit on seminal root traits.
ns: not significant; *, **: significant at the 5% and 1% levels respectively 37

38. Region
Growth
habit
RN GA DRR
Number of
lines
Eastern Asia
Spring 3.58 35.87 0.35 16
Winter 3.15 40.01 0.46 20
Central Asia
Spring 3.94 41.19 0.45 76
Winter 3.83 41.64 0.45 30
Middle East
Spring 4.09 41.97 0.48 19
Winter - - - -
Europe
Spring 3.56 38.76 0.42 12
Winter 3.80 42.07 0.46 21
Means of seminal root traits of spring and winter wheat
landraces originate from four different regions
38

39. 2 QTL analysis of seminal root growth angle using
doubled haploid lines
2.1 Introduction
• QTL analysis is particularly helpful, bridging the gap
between genes and the phenotypic traits that result from
them.
39
• Once QTL have been identified, molecular
techniques can be employed to narrow
the QTL down to candidate genes.

40. • From the previous experiment, I observed a significant
difference between “Ayahikari” (a Japanese commercial
variety with shallow root architecture) and “U24” (a
Chinese variety with deep root architecture, originating
in the Shinjang district) in root growth angle.
2.2 Materials and Methods
2.2.1 Plant materials
Aayahikari
U24
40

41. • 103 doubled haploid lines (DHLs) were developed by
crossing maize pollen with the F1 plants of the cross
between “U24” and “Ayahikari” in 2004 and 2006.
These DHLs were kindly provided by Dr. Matsunaka from
National Institute of Crop Science, NARO, Tsukuba.
A doubled haploid (DH) is a genotype formed
when haploid cells undergo chromosome doubling. Artificial
production of doubled haploids is important in plant
breeding.
41

42. U24 AyahikariX
F1 X Maize pollen
1n
Colchicine
• The advantage of using DHLs is the ability to produce homozygous
lines after a single round recombination, saving a lot of time for the
plant breeders.
1n
Haploid Plants
2n 2n
Doubled haploid Plants
42

43. 2.2.2 Phenotyping of the DHLs
• GA, RN and DRR were measured for the 103
DHLs derived from the cross between Ayahikari
and U24 according to the basket method
described in exp.1
• we used a randomized block design with three
replications (three pots per line).
43

44. 2.2.3 Genotyping
• Total genomic DNA was extracted from young
leaves of the 103 DHLs and their parental
varieties.
• A total of 827 simple sequence repeat (SSR)
markers were examined for polymorphism
between “U24” and “Ayahikari”.
44

45. • The PCR products were detected using a capillary gel-
electrophoresis system (HDA-GT12, eGene Inc., Irvine,
CA, USA) with a GCK-5000 gel cartridge.
45

46. • Chromosome assignment was performed on the basis of
the map location of the SSR markers and their
corresponding chromosomes, previously established in the
linkage map of wheat (Somers et al., 2004). 46
• we constructed a genetic linkage map for the 103 DHLs
consisting of 281 SSR markers using Antmap ver. 1.2
(Iwata and Ninomiya, 2006) and MapMaker ver. 3.0
(Lincoln et al., 1993).

47. 2.2.4 QTL analysis
• The QTL analysis was conducted with WinQTL
Cartographer Version 2.5 (Wang et al., 2007) with
threshold LOD score 2.5.
• Initially, single marker analysis was used to identify
genetic markers significantly associated with phenotypic
traits. Then Composite interval mapping (CIM) was used
to determine likely QTL positions, and the work speed of
2 cM was chosen.
47

48. 2.3 Results
2.3.1 Phenotyping
• For GA, DRR and RN, DHLs exhibited broad and
continuous distribution of values ranging between the
values of the parental varieties.
• This indicates that these traits are controlled by multiple
loci and that “U24” and “Ayahikari” are representative
varieties with deep and shallow root architecture,
respectively. 48

49. Frequency distribution of GA, DRR and RN among the DHLs
( : Ayahikari; : U24).
0
20
40
60
80
20 25 30 35 40 45 50 55 60 65
Numberoflines
Root angle (GA)
0
5
10
15
20
25
30
35
40
45
50
10 20 30 40 50 60 70 80 90 100
Numberoflines
Deep root ratio % (DRR)
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7
Numberoflines
Number of roots per seedling (RN)
49

50. Analysis of variance of GA, DRR and RN among DHLs
Trait
Source of
variation
Degree of
freedom
Mean square
GA Replication 2 9.80ns
Line 102 454.15**
Error 2948 207.08
DRR Replication 2 0.02 ns
Line 102 0.08**
Error 2948 0.04
RN Replication 2 9.88**
Line 102 1.93**
Error 199 0.56
ns: not significant; **: significant at 1% levels respectively.
50

51. • I constructed a genetic linkage map for the DHLs consisting of
276 markers comprising 36 linkage groups.
• There were 5 markers unlinked to any other markers and 15
gaps in our map.
• The total map coverage was estimated to be greater than 70%
for most wheat chromosomes through comparison with
previously constructed linkage map (Somers et al., 2004).
2.3.2 Genotyping
51

52. 52

53. 2.3.3 QTL analysis
1. Deep root ratio (DRR)
• Two QTLs controlling DRR (qDRR-1, qDRR-2) were
detected on chromosome 1B and 5D, respectively. Each
QTL explained 9-11% of the total variation. However, we
could not detect a significant QTL (LOD > 2.5)
controlling GA.
53

54. 2. Number of roots/seedling (RN)
• Single QTL controlling RN (qRN) was detected on
chromosome 5A with LOD score 3.51 explained 11.63%
of the total variation.
54

55. • Comparing “Ayahikari” and “U24”, we found a
significant difference of root growth angle in response to
humidity.
3 QTL analysis of hydrotropism in root growth
U24 Ayahikari 55

56. • We used the same DHLs, which had been used in the
geotropic experiment.
3.1Materials and Methods
3.1.1 Plant materials
• we modified the method which used by Takahashi
et al. (2002) for measuring the root angle in
response to hydrotropic effect as follows:
3.1.2 phenotyping
56

57. • This system utilizes a saturated solution of salts and a
wet substrate placed in a closed chamber to make humid
gradient.
• we used acrylic jars with diameter 9 cm
and height 15.5 cm to establish
humidity gradient inside them
Lid
57

58. • 1% agar was made in a small plastic
quadrangular box (W×D×H = 1.5 cm × 1.0
cm × 9.0 cm).
Agar
• The bottom of each plastic box (2 cm high)
was removed to expose the agar prism. Agar
2 cm
58

59. • After 36 hours of germination, germinated wheat seed
with a straight primary seminal root was placed on
the surface of the agar as the root tip was about 1-2
mm from the agar edge.
Agar
• The top of the plastic box containing
the agar block was firmly glued to the
inner side of the lid of the acrylic jar
using doubled side tape.
Agar
Lid
2 mm
59

60. • To establish a humidity gradient inside jars,
100 ml of saturated K2CO3 solution was
placed in the jar bottom
100 ml K2CO3
Saturated solution
• The plastic box and germinated seed
were then placed inside the jar as
the primary root was positioned
vertically and the jar lid were firmly
closed
100 ml K2CO3
Saturated solution
Agar
Lid
4.5 cm
60

61. • After their lids were firmly closed, the jars were
kept at 25 ºC for 10 hours.
• The average angle of four seeds was used to
estimate the hydrotropic response (HR) of each
DHL.
• In addition to HR, elongation rate (ER) of the primary
seminal root was estimated by measuring the root length
36 hours after the germination.
61

62. 3.1.3 QTL analysis
• The QTL analysis was conducted with WinQTL
Cartographer Version 2.5 (Wang et al., 2007) with
threshold LOD score 2.5. with the same conditions of the
previous experiment.
62

63. 3.2 Results
• we found significant difference in root growth angle in
response to gradient humidity (HR).
63

64. • In addition to HR, I found significant difference in root
elongation rate (ER) after 36 hours from germination.
64

65. • Two QTLs controlling HR were detected on 1A (qHR-1)
and 2B (qHR-2).
65

66. • Finally two QTLs controlling ER were detected on 5D
(qER-1) and 7D (qER-2).
66

67. Root experiment conclusions
• we have confirmed the presence of significant
genotypic differences in the root traits among 194
wheat genotypes originating in diverse regions.
• The shallow root architecture specific to the East
Asian varieties was also observed in our results.
This might be an adaptation to the high moisture
content of the soil due to the East Asian monsoon.
67

68. • Winter-type varieties exhibited deeper root architecture
than spring-type varieties did. In wheat, the spring habit
is controlled by Vrn genes, including Vrn-D4 which is
located on 5D chromosome which qDRR-2 is also found
to be located.
68
• It is interesting that the two QTLs related to the
geotropism are not located on homologous
chromosomes.

69. • From our knowledge this is the first report of QTL
related to hydrotropism in crops as will as in
wheat.
• The QTL results clearly indicated that gravitropic
response and hydrotropic response are controlled
by genetically independent factors.
69

70. Thank you for your attention
Alhosein Hamada
‫استماعكم‬ ‫لحسن‬ ‫شكرا‬
70

71. Next presentation:
25.10.2017
Identification and verification
of QTL associated with frost
tolerance using linkage
mapping and genome wide
association study in winter
faba bean

72. • I did not found any deferent between the
reciprocal crosses so I will complete with one
hybrid for each character.
72

73. Trait QTL
Linkage
Group
Closest
Marker
Position
(cM)
Start End LOD Score
Additive
Effect 1
Contribution
%
RN qRN 5A wmc150a 29.02 18.69 41.06 3.51 0.28 11.63
DRR
qDRR-1 1B gwm140 166.95 162.35 166.95 3.00 -0.04 9.59
qDRR-2 5D wmc97 51.97 41.78 57.97 2.87 -0.04 11.49
ER
qER-1 5D cfd266 76.65 64.65 88.05 3.53 0.08 12.53
qER-2 7D wmc405 66.55 60.55 76.55 2.83 -0.08 14.73
HR
qHR-1 1A wmc278 51.34 45.34 59.21 3.10 -5.07 13.44
qHR-2 2B wmc764 12.05 10.05 14.05 2.60 -4.24 9.87
• QTLs for number of roots per seedling (RN), deep root ratio (DRR),
root elongation rate (ER), and root angle in response to hydrotropism
(HR).
73

74. • SSR marker analysis was conducted according to
the following method.
For one Sample
Name Volume Final conc.
Template DNA (5ng/ μl) 5 μl 5ng/μl
10x ExTaq Buffer (Mg2+ free) 1.25 μl 1x
MgCl2(25mM) 0.75 μl 1.5mM
dNTP Mixture (2.5 mM each) 1 μl 0.2mM
Working Primer* (20 pmol/ μl) 0.31 μl 0.5μlM
DMSO 0.38 μl 0.03
Betaine (5 M) 2.5 μl 1M
TaKaRa ExTaq (5 U/ μl) 0.1 μl 0.04U/μl
dH2O 1.21 μl 1.21
Total volume 12.5
74

75. • PCR conditions were as follows:
PCR Conditions
1 Cycle 1 step 98° C 5 Min
40 Cycle 1 st step 98° C 30 sec
2 nd step ?° C 30 sec
3 rd step 72° C 30 sec
1 Cycle 1 step 72° C 10 Min
1 Cycle 1 step 10° C ∞
75

76. • 103 doubled haploid lines (DHLs) were developed by crossing
maize pollen with the F1 plants of the cross between “U24”
and “Ayahikari” (Suenaga and Nakajima 1993) in years 2004
and 2006 by the following steps:
1. Emasculation and pollination
Spikes were emasculated by cutting the top portion of the
first two florets. Anthers were then removed with forceps and
the spikes covered with glassine bags.
One to three days after emasculation, feathery, receptive
stigmas were pollinated with fresh maize pollen.
76

77. 2. Treatment of spikes with 2,4-D
Twenty four hours after pollination, 5 ml of a solution of 2,4-D
(10 mg/l) was injected into the stems of the donor plants and
a drop of the same solution was also placed into each
pollinated floret.
Treatment of spikes with 2,4-D enabled the embryos to
remain alive until they were large enough to survive the
shock of the conventional embryo rescue procedures.
77

78. 3. Embryo rescue
The excised embryos were transferred to vials containing
MS (Murashige and Skoog, 1962) basal medium
supplemented with 20g sucrose/l and 8g agar /l (Zhang et
al., 1996; Inagaki, 1997).
78

79. Embryos that developed into plantlets with 10 cm long stems
were transferred to small pots.
Ten days after transfer to the pots, chromosomes were
counted in root tips.
79

80. 4. Colchicine treatment
Roots of the haploid plants with 3–4 tillers were treated with
0.05% w/v colchicine solution for 5.5 h. The roots were
washed after treatment and the plants replanted in pots till
getting seeds.
80