Genepyramiding for biotic resistance


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Within the last twenty years, molecular biology has revolutionized conventional breeding techniques in all areas. Biochemical and Molecular techniques have shortened the duration of breeding programs from years to months, weeks, or eliminated the need for them all together. The use of molecular markers in conventional breeding techniques has also improved the accuracy of crosses and allowed breeders to produce strains with combined traits that were impossible before the advent of DNA technology

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Genepyramiding for biotic resistance

  1. 1. LOGOGene pyr amiding for biotic resistance through marker assisted selection
  2. 2. ADVENT OF MARKERS Molecular biology has revolutionized conventional breeding techniques Molecular markers in conventional breeding has improved the accuracy of crosses and allowed breeders to produce strains with combined traits that were impossible before the advent of DNA technology
  3. 3. Contd.., MARKERS MAS
  4. 4. Marker Assisted Selection (MAS) Exploitation of molecular markers linked to genetic factors are useful in assessing the genotype which is the genetic material for the development of new improved varieties. This led to the development (MAS) MAS– improves the efficiency with which breeders can select plants with desirable combinations of genes. A marker is a “genetic tag”
  5. 5. BREEDER’S PERSPECTIVE Efficient and effective crop trait selection Avoid genotype-environment interaction Shorter time to bring new varieties to market The discovery of molecular markers associated with many important agronomic triats has made marker-assisted selection (MAS) reality for several crops.
  6. 6. CONVENTIONAL PLANT BREEDING P1 x P2 Recipient Donor F1 large populations consisting F2 of thousands of plants PHENOTYPIC SELECTION Bacterial blight screening Phosphorus deficiency plotSalinity screening in phytotronGlasshouse trials Field trials
  7. 7. Resistance Gene Susceptible Marker - What for? Example: A breeder aims to improve the resistance of a cultivated form. Therefore, he/she performs a cross between the susceptible culitivated form with a wild form that possess the required resistance. However, at least 6 backcrossing steps are necessary and the resistance is difficult to detect. The major goal is to reduce costs associated with screening for traits
  8. 8. MARKER-ASSISTED BREEDING P1 x P2Susceptible Resistant F1 large populations consisting of F2 thousands of plants MARKER-ASSISTED SELECTION (MAS)
  9. 9. MARKER ASSISTED PYRAMIDING Breeding plan Genotypes P1 P1 P1: AAbb x P2: aaBB Gene A Gene B F1 F1: AaBb Gene A + B F2 MASSelect F2 plants that have Gene A and Gene B Fig: A simple scheme for marker assisted pyramiding of two different traits (Hittalmani et al., 2000; Liu et al., 2000)
  10. 10. GENE PYRAMIDING -INTRODUCTION Pyramiding is the accumulation of genes into a single line or cultivar. A pyramid could be constructed with major genes, minor genes, defeated genes, effective genes, ineffective genes, race-specific genes, non race-specific genes, or any other type of host gene that confers resistance
  11. 11. Contd.., It includes Stacking of traits Stacking of events Stacking of genes A genetically modified organism (GMO) and all subsequent identical clones resulting from a transformation process are called collectively a transformation event. If more than one gene from another organism has been transferred, the created GMO has stacked genes (or stacked traits), and is called a gene stacked event.
  12. 12. Contd.., 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 Main use- to improve existing elite cultivar Eliminates extensive phenotyping Control linkage drag Reduces breeding duration
  13. 13. TYPES OF GENE PYRAMIDING Conventional technique Serial gene pyramiding : Genes are deployed in same plant one after other Molecular technique Simultaneous gene pyramiding : Genes are deployed at a time in a single plant
  14. 14. Gene-pyramiding scheme cumulating six target genes
  15. 15. FIXATION STEPS Production of doubled haploids from root genotype Crossing the root genotype with a blank parent and selfing the offspring Crossing the root genotype with one of the founding parent Selfing the root genotype
  16. 16. ENHANCING RESISTANCE In Rice BPH resistance genes Bph1 and Bph2 were LEVEL pyramided Pyramided line shows resistance greater than the Bph2 single introgression line (Sharma et al., 2004) Gall midge resistant genes Gm2 and Gm6 was pyramided which shows non overlapping resistance to allopatric biotypes (Katiyar et al., 2001) In wheat, Russian wheat aphid resistant genes Dn2 and Dn4 were pyramided
  17. 17. Durable resistance In cotton, Bt genes (Cry1Ab)+high terpenoid plant trait pyramided line shows durable resistance to H. virescens (Sachs et al., 1996) In soybean, two QTLs + Cry 1Ac increased resistance to soybean looper and corn ear worm (Walker et al., 2004) In rice, three BLB resistance genes Xa 5, Xa13 and Xa21 have been pyramided. (Joseph et al., 2004) In rice, transgenic pyramided IR 72 lines showed increased resistance to BB (Xa21),yellow stem borer(Bt fusion gene) and sheath blight( chitinase gene) (Datta et al., 2004)
  18. 18. Pyramiding of genes RICE PLANT Xa 21 gene Chitinase gene B.t. geneBacterial resistance Sheath blight Insect resistance (Datta, et. al., 2002)
  19. 19. Iterative Strategies Two or more transgenes can be sequentially introduced into a plant by conventional iterative procedures Pyramided cry1Ac and cry1C Bt genes in broccoli controlled diamond back moth (Cao et al., 2002)
  20. 20. Co-transformation with Multiple Most promising approach for the introduction of multiple Transgenes genes into plants Quick & Simple Tend to co-integrate at the same chromosomal position in a high proportion of transgenics In Arabidopsis, six genes (including two selectable marker genes) on two T-DNAs were deployed to produce copolymer, The availability of metabolic precursors had to be increased by redirecting intermediates, manipulating Protein expressing and gene suppressing transgenes (Baucher et al., 2003)
  21. 21. Expression Multiple Proteins From Single Promoter (Baucher et al., 2003)
  22. 22. Contd.., Chimeric polycistronic constructs that incorporate internal ribosome entry sites (IRESs) from different viruses Different protein sequences are connected in a single open reading frame via short linker sequences A polyprotein incorporating coat proteins of tobacco mosaic tobamovirus and soybean mosaic potyvirus was expressed in tobacco to yield plants resistant to multiple viruses
  23. 23. Chimeric Transgenes for Multiple Gene Suppression Single transgenes can also be used to simultaneously suppress multiple genes Containing fused sequences of several target genes under the control of a single promoter But in most cases resulted in Co-suppression or post- transcriptional gene silencing (Baucher et al., 2003)
  24. 24. UD Y1 STCA SE
  25. 25. Introduction Cereal Cyst Nematode (CCN)-Heterodera avenae Two CCN genes (CreX and CreY) from Ae. variabilis in a wheat background Employing MAS – comparison of two pyramided CCN resistance level with parental single-gene recombinant lines. The development of markers is the best way to achieve the pyramiding of genes conferring partial resistance in a single recombinant line.
  26. 26. Materials and Methods 32 32 6 7
  27. 27. RAPD to SCAR MARKER RAPD, OpY16 linked to Rkn-mn1 converted to SCAR (1060bp) ie SCAR Y16
  28. 28. RESULTS…. The level of resistance observed suggested an additive effect of the two genes in the pyramided line The identification of codominant markers linked to these two genes facilitate gene pyramiding and rapid screening of different genotype SCAR Y16 and OpR4-1600, the latter after transformation into a SCAR, could be used as introgression markers to pyramid CreY and CreX genes in different backgrounds
  29. 29. UD Y2 STCA SE
  30. 30. Introduction Soybean mosaic virus (SMV) Single-dominant resistance genes Rsv genes against strains of SMV. Pyramiding respective Rsv genes from different loci (Rsv1, Rsv3, and Rsv4) MAS - ideal -creating durable and wide spectrum resistance to all strains of SMV.
  31. 31. Contd.., Two-gene and three-gene isolines of Rsv1Rsv3, Rsv1Rsv4 and Rsv1Rsv3Rsv4, acted in a complementary manner, conferring resistance against all strains of SMV, whereas isolines of Rsv3Rsv4 displayed a late susceptible reaction to selected SMV strains. We demonstrate with MAS and three near-isogenic lines, each containing a different SMV-resistance gene, that pyramided lines can be generated in a straightforward manner into two- or three- gene–containing lines with high levels of resistance to SMV.
  32. 32. UD Y3 STCA SE
  33. 33. Introduction Barley Yellow Mosaic Virus disease caused by different strains of BaYMV and BaMMV For pyramiding of resistance genes rym4, rym5, rym9 and rym11, located on chromosomes3Hand4Hof barley using two approaches doubled haploid lines (DHs) and marker assisted selection procedures
  35. 35. Introduction Indica rice cultivars - cotransformed with genes Rice chitinase (chi11) Thaumatin-like protein (tlp) (fungal pathogens) Serine-threonine kinase (Xa21) (bacterial blight resistance) Particle bombardment (Callus and imature embryo) Varieties – PB1, ASD16, ADT 38 , IR72 AND WHITE PONNI Putative transgenic lines analysed PCR, Southern Blot hybridization and Western Blotting showed stable integration and expression of the transgenes in a few independent transgenic lines.
  36. 36. GENE OF INTERESTChi 11 t lp Xa 21 3 genes pyramided Se Fu Sh Sh teria ng r- bac ea thr eat al lth pat eo h b bligh blig ho kin ligh t gen l ht ase s t& PUTATIVE GENE SELECTION – RICE TRANSGENICGENE 1 GENE 2 GENE 3 PLANT
  37. 37. GENE CONSTRUCT 1 2 3 p MKU-RF2 p GL2-ubi-tlp p C822 3.2kb cassette 3.1kb cassette 9.6kb cassette 1.1kbp 1.1kbp rice chil 1 gene tlp geneUbiquitin promoter Ubiquitin promoter NOS terminator NOS terminator SELECTABLE MARKER – Hygromycin B
  39. 39. SOUTHERN BLOTTING Chi11 - genomic DNA - HindIII -to release 3.2 kbp chitinase expression cassette and blotted onto the membrane  α-32P dCTPlabeled 1.08 kbp chi11 coding sequence by digesting pMKU-RF2 with PstI. tlp gene- the genomic DNA-HindIII -to release the 3.1 kbp TLP expression cassette  α-32P dCTP-labeled 1.1 kbp TLP coding sequence by digesting 3.1 kbp TLP expression cassette with BamHI. Xa21 -genomic DNA-EcoRV -to release 3.8 kbp Xa21  Hybridized with α-32P dCTP-labeled 3.8 kbp Xa21 coding sequence by digesting pC822 with EcoRV.
  42. 42. PROGENY ANALYSISPyramided line SM-PB1-1 did not segregate for Xa21 in both T1and T2 progeniesThe influence of chi11 and/or tlp on Xa21-mediated bacterialblight resistance could not be assessed
  43. 43. Global Status of Approved GM Crops with Stacked Genes (Halpin,Company Logo 2007)
  44. 44. 21 st Century Product Developement
  45. 45. LOGO
  46. 46. LOGO