Transcript Slide 1

Professor of genetics and molecular biology, Faculty of
Agriculture, Ain shams University, Cairo, Egypt.
Professor of genetics, Director of Supreme
Council of Sugar Crops, Cairo, Egypt
List of contents
1- Sugarcane
1-1 Phylogenetic relationships in sugarcane
1-2 Marker assisted selection in sugarcane
1-2-1 Molecular markers for smut resistance
1-2-2 Molecular markers for sugar content (Brix)
1-2-3 Functional genomic analysis for enhancement of sugar content by
RNAi approaches
2-Stevia
2-1 MAS
2-2 Genotoxity
3- Recommendations
Phylogenetic relationships in
sugarcane
• The phylogenetic relationships between twelve
sugarcane genotypes belonging to three different
Saccharum spp. were elucidated based on RAPD and SSR
molecular markers.
• The combined marker analysis (RAPD and SSR) revealed
some closely vs. distantly related taxa with respect to
phylogenetic relationships .
• Microsatellites are valuable tools, not only for their
rapidity to generate markers, but also for their high
polymorphism. This indicated that markers specific to a
genotypes could be easily identified with SSR markers.
Therefore, such markers seem to be an appropriate tool
to follow the efficiency of introgression programs in
sugarcane.
• Fig. (1): Dendrogram showing genetic distances between 12 sugarcane
genotypes based on 13 RAPD and 9 SSR markers combined.
Marker assisted selection in
sugarcane
Traditional sugarcane breeding steps
1- Parental selection from a source population.
2- Hybridization using bi-parental crosses and
polycrosses.
3- Progeny selection at several stages.
Commercial cultivar
12-15 years
Sugarcane breeding difficulties
• Saccharum spp. are genetically large genome size (
3.05-8.91 pg) .
• Complexity levels in commercial cultivars (2n=99-168
chromosomes) are aneuploid with various ploidy levels.
• Occurrence of somaclonal variation.
• The challenge in plant breeding is identifying the
superior progeny
Molecular markers are
valuable tool in indirect and early selection.
1- Molecular markers for smut resistance:
-Ten cultivars were used in this study including seven promising
cultivars, i.e., G99/165, G95/19, G95/21, G98/28, G98/24, G84/47,
G85/37, one susceptible cultivar NCo310 and the commercial
cultivars; GT54/9 and Ph8013.
-The performance of the ten cultivars which were artificially infected
with teliospores suspension was assessed under greenhouse
conditions.
-The results revealed that nine cultivars were relatively resistant
(R). Some molecular markers, using RAPD-PCR and ISSR-PCR
techniques were positively associated with smut resistance.
- The molecular markers identified in this study could be used to
accelerate selection programs for smut resistance in cost-effective
way.
2- Molecular markers for sugar content(Brix):
1- Evaluation of sugarcane
progeny from different crosses
for sugar content and some
sugar-related traits.
2- Development of molecular
markers associated with sugar
content using RAPD, ISSR, RISSR and SSR-PCR techniques.
1- Twenty two clones were chosen from 4000 clones
resulted from crosses in green house of SCRI
according to some vegetative traits.
2- Differences between means were compared using
Duncan’s Multiple range test (Duncan 1955).
3-Brix values were used as an indicator of sugar
content.
4- these clones were divided into two groups
(according to Brix):
high sugar content; 13 (A)
& low sugar content; 9 (B).
Formula to calculate percent pol (sucrose) in juice:
% pol = {-6.517 + (25.3 X PR*) – 0.X (PR x PR) + 2.37 X brix) – 0.207 x (brix x brix) } / 100
*PR = correction indices of brix from specific table
1- Stalk diameter had no significant differences while
the other traits showed significant differences
between individuals.
2-The two groups showed significant differences
regarding Brix, sugar yield and number of stalks
traits.
Table(1): Means of Brix & some sugar content-related traits
Clone No.
Brix*
No. of stalks / m2
Stalk height
(cm)
Stalk diameter
(cm2)
Cane yield (ton/fed)
Sugar yield (ton/fed)
Group (A)
190
22.5A
14.1A
298D
2.78A
52.500AB
5.7A
191
22.17AB
14.3A
290 F
2.86 A
53.000 A
5.68 A
189
21.75ABC
15.6 ABCD
291 F
3.00 A
48.889FGH
5.14 ABCD
209
21.67ABC
16.1 AB
299 CD
2.45 A
51.150BCD
5.38 AB
104
21.33ABC
18.3 ABC
290 F
2.56 A
50.600CDE
5.23 ABC
120
21.17ABC
17.8ABCDE
286 H
2.83 A
46.056J
4.72 ABCDEF
194
21.08ABC
16.0 AB
286 H
2.53 A
52.700AB
5.38 AB
121
21 ABC
13.06ABCD
280 I
3.06 A
50.000DEF
5.09 ABCD
87
20.85BC
8.55ABCDE
275 K
3.11 A
48.734FGH
4.93 ABCDE
198
20.83BC
9.15ABC
300 C
2.56 A
52.350ABC
5.29 ABC
122
20.83 BC
14.32ABC
299 CD
2.73 A
52.900 A
5.34 ABC
188
20.67 BC
10.32ABCDE
288 G
2.92 A
48.740FGH
4.89 ABCDE
193
20.33 C
8.00ABCD
308 A
2.40 A
51.750ABC
5.11 ABCD
29
11.5H
11.30G
295 E
2.80 A
51.100BCD
3.04G
131
12.83 GH
9.61FG
288 G
2.75 A
47.553HIJ
3.12FG
36
13.33 FG
7.13FG
285 H
2.86 A
52.250ABC
3.55DEFG
97
13.67EFG
13.61FG
280 I
3.02 A
50.400DEF
3.51DEFG
133
14 EFG
10.16FGH
270 M
3.01 A
47.000IJ
3.34EFG
94
14.67 EF
9.71EFG
303 B
2.85 A
51.900ABC
3.85BCDEFG
160
15 DE
15.16FGH
290 F
2.98 A
51.500ABCD
3.9BCDEFG
139
15 DE
12.30FGHI
270 M
2.99 A
49.231EFG
3.73CDEFG
4
16.5 D
10.86ABCDEFG
300 C
2.36 A
49.900DEF
4.13ABCDEFG
Group (B)
Table: Summary of molecular markers associated with sugar
content (BRIX)
Marker type
No. of primers or
combinations
+ve markers
-ve Markers
RAPD-PCR
9 Primers
22
7
ISSR-PCR
5Primers
8
5
R-ISSR-PCR
20 Combinations
28
16
SSR-PCR
6 Primers
6
6
(+ve) = Positive marker for high sugar content
(-ve )= Positive marker for low sugar content
Functional genomic analysis for enhancement of sugar
content by RNAi approaches
5’
3’
siRNA
3’
5’
RISC
RNA-Induced Silencing Complex
Translation Initiation
Factor
RNA/DNA Helicase
(is required to unwind the dsRNA)
RNA-Dependent RNA
Polymerase (RdRP)
Transmembrane
Protein
Effector Step
RISC (RNA-Induced
Silencing Complex(
• siRNA binding
• siRNA unwinding
• RISC activation
RNA interference is a powerful reverse genetic tool to study gene function by
the interference with gene activity.
• Three major enzymes, soluble acid invertase ﴾SAI﴿, sucrose
synthase(SUC SYN) and sucrose phosphate synthase ﴾SPS﴿ are
involved in regulation of accumulation and / or breakdown of
sucrose.
• Both SAI and SUC SYN are implicated in the degradation of sucrose
while SPS is involved in sucrose biosynthesis and accumulation
(Chandra et al., 2012 ﴿.
• Down - regulation of SAI gene expression can be effectively
achieved by RNAi approach to minimize its role of inversion of
sucrose into glucose and fructose which represents a major
problem due to significant loss of sucrose content.
• On the other hand ,Up - regulation of SPS gene expression by
introducing one copy of that gene by the appropriate
transformation procedure with efficient promoter may lead to
significant accumulation of sucrose in the plant.
• Our on – going research has been exploring this approach and
some promising progress is anticipated.
Sucrose synthesis
• Sucrose -6-phosphate synthetase(EC2.4.1.12)
• Sucrose synthase(EC2.4.1.13)
• Soluble acid invertase
Steps of study
• Isolation of some genes responsible for sucrose
content in sugarcane.
• Down regulation of genes responsible of sucrose
breakdown in sugarcane. (invertases)
• Up regulation of genes which increase sucrose
percentage in sugarcane. ( sucrose phosphate
synthase) )
• Using databases to detect the sequence of genes
affecting sucrose content.
• Isolation, cloning and sequencing of the
candidate genes
• Comparing the obtained sequences with the
related genes using bioinformatics approaches.
• Designing SiRNA sequence for targeted genes
and insert it in suitable expression vector
• Transform it in sugarcane plant callus
• Evaluating the transformed plants for the sucrose
content trait in GM and control plants
Candidate genes location
and size from NCBI site
Sucrose phosphate synthase
1) LOCUS: HQ117935
SIZE: 3252 bp mRNA linear
PLN 02-SEP-2011
2) LOCUS: JN584485
Size: 3481 bp mRNA linear
PLN 19-SEP-2011
3) LOCUS: AB001338
Size: 3287 bp RNA linear
PLN 17-OCT-2008
4) LOCUS: EU278617
Size: 6493 bp DNA linear
PLN 11-DEC-2007
5) LOCUS: EU278618
Size :7382 bp DNA linear
PLN 11-DEC-2007
6) LOCUS: EU269038
Size: 3186 bp mRNA linear
PLN 03-DEC-2007
7) LOCUS :AB001337
Size : 3322 bp mRNA linear
PLN 13-FEB-1999
Sucrose synthase II
1) LOCUS: AY670701
Size: 3632 bp DNA linear
PLN 15-MAR-2005
2) LOCUS: AY670699
Size: 3857 bp DNA linear
PLN 15-MAR-2005
3) LOCUS: AY670702
Size: 3857 bp DNA linear
PLN 15-MAR-2005
4) LOCUS: AY670700
Size:3867 bp DNA linear
PLN 15-MAR-2005
5) LOCUS: AF263384
Size: 2717 bp mRNA linear
PLN 03-SEP-2003
6) LOCUS : AY118266
Size: 7771 bp DNA linear
PLN 15-MAR-2005
7) LOCUS: AY670698
Size: 3634 bp DNA linear
PLN 15-MAR-2005
1) LOCUS :AF083855
Size: 494 bp mRNA linear
PLN 17-SEP-1998
2) LOCUS: AF062734
Size :1808 bp mRNA linear
PLN 18-MAY-1998
3) LOCUS: AF062735
Size: 1808 bp mRNA linear
PLN 18-MAY-1998
4) LOCUS : AF083856
Size: 1402 bp mRNA linear
PLN 17-SEP-1998
5) LOCUS : AY302083
Size: 2274 bp mRNA linear
PLN 12-JAN-2010
Stevia is a branched bushy shrub of the Asteraceae
(Compositae) family, native to the
region
in the north east of
.
Known as a ‘‘
’’ or called ‘‘
’’
Source of a high-potency
It is 1safe for
, as it does not affect blood
sugar levels.
Used in Paraguay for centuries,
for decades
Discovered centuries ago,
approval in
There are many advantages of using Stevia :
-
• Steviol Glycosides
• Variety of sweet-flavored
molecules within the leaf
• 9 Steviol glycosides recognized by
Joint FAO/WHO Expert Committee
on Food Additives (JECFA).
Structure of the major glycosides of Stevia
rebaudiana leaves. Glc, Xyl, and Rha represent,
respectively, glucose, xylose, and rhamnose
sugar moieties (Geuns, 2003).
Stevioside, the major sweet
substance of stevia plant (5-10%
of dry weight), is 300 times as
sweet as sucrose, having steviol
as its aglycone and attached to
three glucose molecules .
Stevioside has the chemical
formula of a diterpene glycoside
(C38H60O18)
heatstable
Diabetes
-Zero
glycemic
noncaloric
300
times
sweeter
– Soft drinks, teas, fruit juices
– Table top sweeteners
– Hot and cold cereals
– Granola and snack bars
– Yogurt
– Flavored milk
– Ice cream
– Salad dressing
– Baked goods
– Chewing gum
– Canned fruit and jams
– Desserts
– Alcoholic beverages
In a study on
, Allam et al.,
(2000) reported
in
and
stevioside
which allowed selection to make
substantial improvements in this natural sweetner.
The stevioside content were highly associated with
dry weight, leaves / stem ratio and plant vigor
(visual ranking).
markers for some stevia yield components
were detected using
,
,
isoenzymes and randomly amplified polymorphic DNA (
).
These
could be
used to assist
for accessions with
content .
Means of some yield-related traits and stevioside content
for the 15 stevia accessions
Molecular marker associated with some stevia traits.
(+) = Positive marker, (-) = Negative marker
Assessment of genotoxicity:
Stevia rebaudiana Bertoni, a plant originated from
,
contains the
, stevioside and rebaudioside
A.
Stevioside is 300 times sweeter than sugar. Therefore,
stevioside is considering a good resource as a non-caloric
sweetener in human foods for different proposes.
has been subjected to
critical
Assessment of genotoxicity:
of stevioside was studied in different biological
systems e.g.,
,
a, and
.
In vivo study on
(both sexes) revealed that it had
on bone marrow cells or lower weight.
In vivo study on
melanogaster showed
effect since there were no significant differences in mutation
frequencies between the treated and the control insects .
In vitro study on
revealed
differences between the treated cells and controls ones.
In general,
revealed
effect of stevioside, which makes it safe for human consumption.
(Abdel – Tawab et al., 2000).
Female
Male
Liver %
Kidneys %
Heart %
Spleen %
Lung %
Testes %
Control
4.99 ± 0.26
1.62 ± 0.06 0.54 ± 0.02 0.68 ± 0.43 1.22 ± 0.07 0.49 ± 0.07
Treated
5.10 ± 0.30
1.81 ± 0.07 0.62 ± 0.04 0.26 ± 0.04 1.27 ± 0.05 0.41 ± 0.05
Control
6.18 ± 0.30
1.37 ± 0.05 0.60 ± 0.04 0.36 ± 0.06 1.23 ± 0.07
-
Treated
5.55 ± 0.14
1.47 ± 0.05 0.59 ± 0.02 0.35 ± 0.05 1.18 ± 0.04
-
(7)
The possible mutagenic hazardous of stevioside has been
investigated by two efficient mutagenicity systems (
cereviciae and
melanogaster) as an in vivo biological
systems for testing different genetic end points.
The yeast
strain was treated with three different
concentrations of stevioside (
) to evaluate its
genotoxic effect.
The survival rate was increased with the increasing of stevioside
concentration than the concurrent negative control.
The
assay using S. cereviciae D7 strain revealed that
including the induction of
mitotic gene conversion, mitotic crossing-over and reversion.
Instead, the frequencies of the three end points were
than the
levels.
Genetic activities of three different
Saccharomyces cerevisiae strain D7.
C= Control
T= Treatment
concentrations of stevioside in
-=<2 control level.
No mutagenic activities including the induction of mitotic gene conversion, mitotic
crossing-over and reversion were obtained. Instead, the frequencies of the three end
points were lower than the spontaneous levels.
In addition the obtained results revealed that stevioside
has no mutagenic effects in all tested genetic end points
on
; moreover, it decreased the spontaneous
mutation rate than the concurrent negative control .
Therefore, the possible
effect of
stevioside has been tested against the mutagenic
activities of
using the well
defined antimutagenicity assays on Drosophila. The
reduction of mutagenic activity of MMC indicated that,
stevioside has a
activity on
Drosophila. (Abdel – Tawab et al., 2009).
Diagram represents the frequencies of spontaneous and induced warts epithelial tumors
in wts/+ flies after treatments with Mitomycin C (MMC), stevioside (St) and combinations
of MMC and stevioside (St) in post and pre treatments
-The
frequency
of
induced tumor after MMC
treatment of pretreated
larvae with stevioside (5
mg/ml)
was
highly
significant reduced (0.81
per fly), which showed
64% reduction of induced
tumors.
in post-treatment experiment, larvae exposed to
stevioside (5 mg/ml) after
MMC treatment showed
highly significant reduction
of induced tumors (66 %)
with a tumor average of
0.77 tumor/fly
Fig. (7): Diagram represents the frequencies of spontaneous and induced warts
epithelial tumors in wts/+ flies after treatments with Mitomycin C (MMC), stevioside
(St) and combinations of MMC and stevioside (St) in post and pre treatments in
addition to the reduction rate of induced tumor frequencies due to antimutagenic activity
of stevioside
Warts (Wts) phenotype
MMC 20μg/ml
Negative Control
Warts (Wts) phenotype
MMC 20μg/ml
post-treatments (MMC -Stevioside)
Abdel-Tawab et al., (2008) reported that Stevioside
has no mutagenic effect in all tested genetic end points in
Drosophila. PCR-based RAPD analysis was used to assess
possibility of detecting molecular markers associated with
genotoxic effect.
In summation, it is evident from the aforementioned
discussion that from the stand points of both the
cytogenetic analysis (chromosomal aberrations) and
molecular analysis (RAPD) that the biomarkers obtained
in this study indicated that we can get reliable
evidences regarding the biosafety of this world wide
uses of sweetener indicated no hazards to the health and
welfare of the consumers.
antiobesity
antineoplastic
effect
Antioxidant
activity
Antihyperglycemic
Antimicrobial
activity
antidiabetic
improves cell
regeneration
anti human
rota-virus
activities
Recommendations :
It is evident from the aforementioned discussion that
there are good opportunities for improvement of sugarcane
biomass and sucrose content as well as enhancement of smut
tolerance by modern molecular breeding methods (MAS).
In addition RNA interference is a powerful reverse genetics
tool to study gene function by the interference with gene
activity. Our on – going research has been exploring this
approach and some promising progress is anticipated which
enable the breeder to achieve substantial improvement in fast,
reliable and cost- effective way.
Furthermore, introducing new unconventional natural
sweetners such as stevia can contribute to filling the gap
between supply and demand. As for the debate about the
safety of stevia for human consumption, it is evident from our
extensive tests on several biological systems that no risks on
human health were encountered.