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 , R ISSR 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.

two groups 4 these clones were (according to Brix): divided into 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

Brix * Clone No.

No. of stalks / m 2 Stalk height (cm) Stalk diameter (cm 2 ) Cane yield (ton/fed) Sugar yield (ton/fed) 190 191 22.5

A 22.17

AB 14.1

A 14.3

A Group (A) 298 D 290 F 2.78

A 2.86

A 52.500

AB 53.000

A 5.7

A 5.68

A 189 209 104 120 194 121 87 198 122 188 193 29 131 36 97 133 94 160 139 4 21.75

ABC 21.67

ABC 21.33

ABC 21.17

ABC 21.08

ABC 21 ABC 20.85

BC 20.83

BC 20.83

BC 20.67

BC 20.33

C 11.5

H 12.83

GH 13.33

FG 13.67

EFG 14 EFG 14.67

EF 15 DE 15 DE 16.5

D 15.6

ABCD 16.1

AB 18.3

ABC 17.8

ABCDE 16.0

AB 13.06

ABCD 8.55

ABCDE 9.15

ABC 14.32

ABC 10.32

ABCDE 8.00

ABCD 11.30

G 9.61

FG 7.13

FG 13.61

FG 10.16

FGH 9.71

EFG 15.16

FGH 12.30

FGHI 10.86

ABCDEFG 291 F 299 CD 290 F 286 H 286 H 280 I 275 K 300 C 299 CD 288 G 308 A Group (B) 295 E 288 G 285 H 280 I 270 M 303 B 290 F 270 M 300 C 3.00

A 2.45

A 2.56

A 2.83

A 2.53

A 3.06

A 3.11

A 2.56

A 2.73

A 2.92

A 2.40

A 2.80

A 2.75

A 2.86

A 3.02

A 3.01

A 2.85

A 2.98

A 2.99

A 2.36

A 48.889

FGH 51.150

BCD 50.600

CDE 46.056

J 52.700

AB 50.000

DEF 48.734

FGH 52.350

ABC 52.900

A 48.740

FGH 51.750

ABC 51.100

BCD 47.553

HIJ 52.250

ABC 50.400

DEF 47.000

IJ 51.900

ABC 51.500

ABCD 49.231

EFG 49.900

DEF 5.14

ABCD 5.38

AB 5.23

ABC 4.72

ABCDEF 5.38

AB 5.09

ABCD 4.93

ABCDE 5.29

ABC 5.34

ABC 4.89

ABCDE 5.11

ABCD 3.04

G 3.12

FG 3.55

DEFG 3.51

DEFG 3.34

EFG 3.85

BCDEFG 3.9

BCDEFG 3.73

CDEFG 4.13

ABCDEFG

Table: Summary of molecular markers associated with sugar content (BRIX)

Marker type

RAPD-PCR

No. of primers or combinations

9 Primers ISSR-PCR R-ISSR-PCR SSR-PCR 5Primers 20 Combinations 6 Primers (+ve) = Positive marker for high sugar content (-ve )= Positive marker for low sugar content

+ve markers

22 8 28 6

-ve Markers

7 5 16 6

Functional genomic analysis for enhancement of sugar content by RNAi approaches

3’ 5’

siRNA

5’ 3’

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 candidate genes and sequencing of the 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

 1-

, 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 gluco se molecules .

Stevioside has the chemical formula of a diterpene glycoside (C38H60O18) non caloric heat stable Diabetes -Zero glycemic

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

(2000)

In a study on reported stevioside in ,

Allam et al.,

and 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 for accessions with used to assist 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 A. , stevioside and rebaudioside 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).

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 systems for testing different genetic end points.

in vivo

biological The yeast strain was treated with three different concentrations of stevioside ( genotoxic effect.

The survival rate was increased with the increasing of stevioside concentration than the concurrent negative control. The assay using

S. cereviciae

) to evaluate its 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 levels. than the

Genetic activities of three different Saccharomyces cerevisiae strain D7. concentrations of stevioside in

C= Control T= Treatment -=<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 experim ent, 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

Negative Control

MMC 20μg/ml

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.

anti obesity Antioxidant activity Antimicrobial activity antineoplastic effect Antihyper glycemic antidiabetic anti human rota-virus activities improves cell regeneration

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.