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Professor of genetics and molecular biology, Faculty of Agriculture, Ain shams University, Cairo, Egypt. Professor of genetics, Director of Supreme Council.

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Presentation on theme: "Professor of genetics and molecular biology, Faculty of Agriculture, Ain shams University, Cairo, Egypt. Professor of genetics, Director of Supreme Council."— Presentation transcript:

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

2 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

3 Phylogenetic relationships in sugarcane

4 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.

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6 Fig. (1): Dendrogram showing genetic distances between 12 sugarcane genotypes based on 13 RAPD and 9 SSR markers combined.

7 Marker assisted selection in sugarcane

8 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 Commercial cultivar 12-15 years

9 Sugarcane breeding difficulties Saccharum spp. are genetically large genome size ( 3.05-8.91 pg). 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. Complexity levels in commercial cultivars (2n=99-168 chromosomes) are aneuploid with various ploidy levels. Occurrence of somaclonal variation. Occurrence of somaclonal variation. The challenge in plant breeding is identifying the The challenge in plant breeding is identifying the superior progeny Molecular markers are superior progeny Molecular markers are valuable tool in indirect and early selection. valuable tool in indirect and early selection.

10 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.

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14 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. 2- Molecular markers for sugar content(Brix):

15 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) high sugar content; 13 (A) & low sugar content; 9 (B). & 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

16 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.

17 Table(1): Means of Brix & some sugar content-related traits Clone No. Brix * No. of stalks / m 2 Stalk height (cm) Stalk diameter (cm 2 ) Cane yield (ton/fed) Sugar yield (ton/fed) Group (A) 190 22.5 A 14.1 A 298 D 2.78 A 52.500 AB 5.7 A 191 22.17 AB 14.3 A 290 F 2.86 A 53.000 A 5.68 A 189 21.75 ABC 15.6 ABCD 291 F 3.00 A 48.889 FGH 5.14 ABCD 209 21.67 ABC 16.1 AB 299 CD 2.45 A 51.150 BCD 5.38 AB 104 21.33 ABC 18.3 ABC 290 F 2.56 A 50.600 CDE 5.23 ABC 120 21.17 ABC 17.8 ABCDE 286 H 2.83 A 46.056 J 4.72 ABCDEF 194 21.08 ABC 16.0 AB 286 H 2.53 A 52.700 AB 5.38 AB 121 21 ABC 13.06 ABCD 280 I 3.06 A 50.000 DEF 5.09 ABCD 87 20.85 BC 8.55 ABCDE 275 K 3.11 A 48.734 FGH 4.93 ABCDE 198 20.83 BC 9.15 ABC 300 C 2.56 A 52.350 ABC 5.29 ABC 122 20.83 BC 14.32 ABC 299 CD 2.73 A 52.900 A 5.34 ABC 188 20.67 BC 10.32 ABCDE 288 G 2.92 A 48.740 FGH 4.89 ABCDE 193 20.33 C 8.00 ABCD 308 A 2.40 A 51.750 ABC 5.11 ABCD Group (B) 29 11.5 H 11.30 G 295 E 2.80 A 51.100 BCD 3.04 G 131 12.83 GH 9.61 FG 288 G 2.75 A 47.553 HIJ 3.12 FG 36 13.33 FG 7.13 FG 285 H 2.86 A 52.250 ABC 3.55 DEFG 97 13.67 EFG 13.61 FG 280 I 3.02 A 50.400 DEF 3.51 DEFG 133 14 EFG 10.16 FGH 270 M 3.01 A 47.000 IJ 3.34 EFG 94 14.67 EF 9.71 EFG 303 B 2.85 A 51.900 ABC 3.85 BCDEFG 160 15 DE 15.16 FGH 290 F 2.98 A 51.500 ABCD 3.9 BCDEFG 139 15 DE 12.30 FGHI 270 M 2.99 A 49.231 EFG 3.73 CDEFG 4 16.5 D 10.86 ABCDEFG 300 C 2.36 A 49.900 DEF 4.13 ABCDEFG

18 Marker type No. of primers or combinations +ve markers-ve Markers RAPD-PCR9 Primers227 ISSR-PCR5Primers85 R-ISSR-PCR20 Combinations2816 SSR-PCR6 Primers66 Table: Summary of molecular markers associated with sugar content (BRIX) (+ve) = Positive marker for high sugar content (-ve )= Positive marker for low sugar content

19 Functional genomic analysis for enhancement of sugar content by RNAi approaches

20 siRNA 5’ 3’5’ 3’

21 RISC RNA-Induced Silencing Complex RNA/DNA Helicase ( is required to unwind the dsRNA ) Translation Initiation Factor RNA-Dependent RNA Polymerase (RdRP) Transmembrane Protein

22 Effector Step siRNA binding siRNA unwinding RISC activation RISC (RNA-Induced Silencing Complex(

23 RNA interference is a powerful reverse genetic tool to study gene function by the interference with gene activity.

24 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.

25 Sucrose -6-phosphate synthetase(EC2.4.1.12) Sucrose synthase(EC2.4.1.13) Soluble acid invertase Sucrose synthesis

26 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) ) Steps of study

27 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

28 Candidate genes location and size from NCBI site

29 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 phosphate synthase

30 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 Sucrose synthase II

31 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

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34 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).

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36 heat- stable Diabetes -Zero glycemic 300 times sweeter non- caloric 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)

37 –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

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40 Means of some yield-related traits and stevioside content for the 15 stevia accessions

41 Molecular marker associated with some stevia traits. (+) = Positive marker, (-) = Negative marker

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43 Assessment of genotoxicity:

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53 Genetic activities of three different concentrations of stevioside in Saccharomyces cerevisiae strain D7. C= Control T= Treatment -=<2 control level.

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55 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

56 -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

57 Warts (Wts) phenotype Negative Control MMC 20μg/ml

58 Warts (Wts) phenotype MMC 20μg/ml post-treatments (MMC -Stevioside)

59 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.

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

61 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. Recommendations :

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