Barnase barstar system

Vikas kumar singh
Chaudhary charan singh university meerut

1. SUBMITTED BY VIKAS KUMAR SINGH M. SC. (AG), III rd SEM DEPARTMENT OF GENETICS & PLANT BREEDING, CHAUDHARY CHARAN SINGH UNIVERSITY, MEERUT SUBMITTED TO DR. S. S. GAURAV ASSO. PROFESSOR, DEPARTMENT OF GENETICS& PLANT BREEDING, CHAUDHARY CHARAN SINGH UNIVERSITY, MEERUT Heterosis and Its Exploitation Marker Assisted Selection Barnase Barstar system

2. MAS Contents Introduction Features Of Markers General Steps in MAS MAS Breeding Scheme Current status of molecular breeding Classification of Marker Achievements

3. Markers A trait, including a molecular and cytogenetic feature, that can be easily detected and whose inheritance can be easily fallowed.

4. Classification Of Markers Leaf tip necrosis seed coat color Isozymes SSR SNP RFLP RAPD etc.

5. Flanking marker Gene of interest

6. Features Markers must be tightly-linked to target loci. Marker A QTL 5 cM RELIABILITY FOR SELECTION Using marker A only: ~ 95% Marker A QTL Marker B 5 cM 5 cM Using markers A and B: ~ 99.5%

7. Marker should not affect the gene of interest. Marker should be co dominant. Markers must be polymorphic 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 RM84 RM296 P 1 P 2 P 1 P 2 Not polymorphic Polymorphic!

8. Introduction The use of genetic markers with the phenotypes in a process called Marker assisted selection. Marker assisted selection (MAS) refers to the use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening. or Marker assisted selection

9. Prerequisite for using MAS 1. High throughput DNA extraction. 6. Finally QTL. 4. Data management system. 5. Knowledge of associations between molecular markers and traits of interest. 3. Genetic maps. 2. Markers .

10. General Steps in MAS Selection of parents Development of Breeding Population Isolation of DNA Scoring Correlation with Morphological traits

11. LEAF TISSUE SAMPLING DNA EXTRACTION PCR GEL ELECTROPHORESIS MARKER ANALYSIS Overview of ‘marker genotyping’

12. F 2 P 2 F 1 P 1 x large populations consisting of thousands of plants Resistant Susceptible MARKER-ASSISTED BREEDING Method whereby phenotypic selection is based on DNA markers. MARKER-ASSISTED SELECTION (MAS)

13. F 2 P 2 F 1 P 1 x large populations consisting of thousands of plants PHENOTYPIC SELECTION Field trials Glasshouse trials Donor Recipient CONVENTIONAL PLANT BREEDING Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot

14. Variety of approaches MAS 1. MABC: MARKER-ASSISTED BACKCROSSING 3. MARS: MARKER-ASSISTED RECURRENT SELECTION 4. GWS: GENOME-WIDE SELECTION 2. GENE PYRAMIDING.

15. Marker-assisted backcrossing MAB Marker-assisted backcrossing is the simplest form of MAS, in which the goal is to incorporate a major gene from an agronomically inferior source (the donor parent) into an elite cultivar or breeding line (the recurrent parent). Foreground selection Background selection

16. 1 2 3 4 Target locus 1 2 3 4 1 2 3 4 BACKGROUND SELECTION TARGET LOCUS SELECTION Selection for target gene or QTL Accelerates the recovery of the recurrent parent genome FOREGROUND SELECTION BACKGROUND SELECTION Foreground Background Linkage drag : Co-inheritance of undesirable trait(s) with a gene of interest. Typically, this is a phenomenon that can be observed during backcross breeding using an exotic donor parent.

17. P 1 x F 1 P 1 x P 2 CONVENTIONAL BACKCROSSING BC1 VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT BC2 MARKER-ASSISTED BACKCROSSING P 1 x F 1 P 1 x P 2 BC1 USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME BC2

18. Pyramiding Widely used for combining multiple disease resistance genes for specific races of a pathogen Pyramiding is extremely difficult to achieve using conventional methods Consider: phenotyping a single plant for multiple forms of seedling resistance – almost impossible Important to develop ‘durable’ disease resistance against different races

19. 1. Rice-a.(PB1 x JRBB55)Improved. PB - 01 Pusa Basmati 1, IARI (1987) b. Improved Sambha Mahsuri (BPT5204), 4. Wheat- Patwin. Maize-Improved QPM-9, Downy mildew (HHB67- 2) 3. Pearlmillet-HHB67-2, ACHIEVEMENT OF MARKER ASSISTED SELECTION

20. Salt tolerant rice varieties developed by IRRI and released in Philippines CSR10, CSR13, CSR23, CSR27, CSR30, CSR36 and Lunishree, Vytilla 1, Vytilla 2, Vytilla 3, Vytilla 4, Panvel 1, Panvel 2, Sumati, Usar dhan 1, 2 & 3 (India); BRRI dhan 40, BRRI dhan 41 (Bangladesh); OM2717, OM2517, OM3242 (Vietnam) IRRI 112 - PSBRc48 (Hagonoy) IRRI 113 - PSBRc50 (Bicol) IRRI 124 - PSBRc84 (Sipocot) IRRI 125 - PSBRc86 (Matnog) IRRI 126 - PSBRc88 (Naga) IRRI 128 - NSICRc106 Other salt-tolerant rice varieties

21. REASON FOR LOW IMPACT AND FUTURE NEEDS FOR MAS Non-availability of robust markers. Epistasis , Background effect and G*E interaction. Cost-ineffective high throughput & low cost genotypic centers needed. Plant breeders-molecular biologist gap.

22. Fact About molecular breeding……. A literature review indicates thousands of QTL mapping studies but not many actual reports of the application of MAS in breeding Why is this the case?

23. Barnase-Barstar System Contents Introduction Mechanism of Barnase-Barstar system Fertility Restoration System Research articles on this topic…. Features Achievements

24. TRANSGENIC MALE-STERILITY For producing Dominant GMS. Need of effective fertility restoration systems. An effective restoration system is available in at least one case, Barnase/Barstar system Recombinant DNA techniques have made it possible to engineer new systems of male sterility by disturbing any or number of developmental steps specifically required for the production of functional pollen within the microspore or for the development of any somatic tissues supporting the microspores.

25. Tapetum Tissue

26. Bargene Mariani et al. in 1992 Successfully used a chimeric dominant gene construct having an anther specific promoter(from TA29gene of tobacco) and bacterial coding sequence for a ribonuclease (Barnase gene from Bacillus amyloliquefaciens ) for production of transgenic plant in B.napus. Barstar 110 bp long.

27. Induced GMS Barnase gene is cytotoxic killing the tapetul cells, thus preventing pollen development result transgenic male sterility.

28. Two-Component System Male sterility is generated by the combined action of two genes brought together into the same plant by crossing two different grandparental lines each expressing one of the genes. Each grandparent has each part of Barnase. Bn3 : 3’ portion of barnase gene. Bn5 : 5’ portion of barnase gene.

29. Two-Component System

30. Fertility restoration Barstar Mariani et al. in 1992 Mariani et al. used another gene construct the same anther specific promoter i.e.TA29 and the Barstar gene from B.amyloliquefaciens for production of transgenic plants in B.napus . Barstar 89 bp long.

31. Selected transgenes used for production of male sterility TRANSGENE SOURCE TRANSGENIC PLANT barnase Bacillus amyloliquefaciens Tobacco, B.napus Rnase T1 Aspergillus oryzae Tobacco, oilseed,rape Bcp1 Brassica campestris B.oleracea rolC A.rhizogenes Tobacco,potato ,

32. Achievements The transgenic hybrid rapeseed-mustard was developed by multinational Proagro Seed Company Ltd. located at Gurgaon (now a part of Belgium based Aventis crop science) large scale field trials in year 2001-2002 ,this hybrid mustard gave 25% yield advantage , but could not be cleared by GEAC for commercial sowing at the farmer fields in October 2002. Another effort by Delhi University South Campus.

33. Efficient fertility restorer system Easy maintenance of male sterile lines Easy elimination of a male fertile plants from male sterile lines Lack of adverse affects on other traits Stable male sterile phenotype over different environments Features

34. Research articles on this topic….

35. Weekly Science Research Journal Vol-1, Issue-16, 7 November 2013 Transgenic Male Sterility For Hybrid Seed Production. In Vegetables -A Review M.Ananthi , P.Selvaraju And P.Srimathi

36. 6. Engineering cytoplasmic male sterility via the chloroplast genome. They illustrated several approaches for inducing male sterility. like- 1. Cell cytotoxicity. 2. Male sterility through hormonal engineering. 3. Pollen self-destructive engineered male sterility. 4. Leading to male sterility by early degrading callose . 5. Male sterility through modification of biochemical pathways.

37. Transgenic Studies on the Involvement of Cytokine And Gibberellins in Male Sterility Development Shihshieh Huang, R. Eric Cerny, Youlin Qi, Deepti Bhat, Carrie M. Aydt, Doris D. Hanson, Kathleen P. Malloy, and Linda A. Ness Plant Physiol. Vol. 131, 2003

38. Hormone induciblemale sterility based on BCP1 Most attractive system of transgenic male sterility based on antisense construct of B. campestris gene BCP1 drawn by BCP1 promoter linked with a harmone inducible enhances sequence. This system is ready for use in hybrid seed production in Brassica oleracea . Restorer gene is not required. Most Attractive than Barnase Barstar system Banga et. al. B. oleracea

39. Gupta P.K., Langridge P, Mir R.R.(2010)Marker assisted wheat Breeding:present status and future possibilities, 2. Article shared by Sudhadip Mondal MAS meaning application step achievements, 3. Journal of Drug Metabolism&Toxicology (review), 4. Singh,B.D . Plant Breeding Principles and Methods, Marker-assisted selection:an approach for precision plant breeding in the twenty-first century-Bertrand C.Y Collard and David J Mackill , 6. Singhal.N.C,Hybrid Seed Production, 7. Internet&SlideShare etc References:-

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