gene pyramiding for biotic resistance through marker assisted selectio

Gene pyramiding for biotic resistance through marker assisted selection : 

Gene pyramiding for biotic resistance through marker assisted selection ABT 631 – SEMINAR – 0+1

Advent Of Markers : 

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

Contd.., : 

Contd.., MARKERS MAS

Marker Assisted Selection (MAS) : 

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”

Breeder’s perspective : 

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.

CONVENTIONAL PLANT BREEDING : 

CONVENTIONAL PLANT BREEDING

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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. Resistance Gene Susceptible The major goal is to reduce costs associated with screening for traits

MARKER-ASSISTED BREEDING : 

MARKER-ASSISTED BREEDING

MARKER ASSISTED PYRAMIDING : 

MARKER ASSISTED PYRAMIDING

Gene Pyramiding -Introduction : 

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

Contd.., : 

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.

Contd.., : 

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

Types of Gene Pyramiding : 

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

Gene-pyramiding scheme cumulating six target genes : 

Gene-pyramiding scheme cumulating six target genes

Fixation steps : 

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

Enhancing resistance level : 

Enhancing resistance level In Rice BPH resistance genes Bph1 and Bph2 were 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

Durable resistance : 

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) Multiple Pest Resistance :

Pyramiding of genes : 

Pyramiding of genes Rice plant Chitinase gene (Datta, et. al., 2002) Insect resistance Bacterial resistance Sheath blight

Iterative Strategies : 

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)

Co-transformation with Multiple Transgenes : 

Co-transformation with Multiple Transgenes Most promising approach for the introduction of multiple 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)

Expression Multiple Proteins From SinglePromoter : 

Expression Multiple Proteins From SinglePromoter (Baucher et al., 2003)

Contd.., : 

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

Chimeric Transgenes for Multiple Gene Suppression : 

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)

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CASE STUDY 1

Introduction : 

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.

Materials and Methods : 

Materials and Methods

RAPD to SCAR MARKER : 

RAPD to SCAR MARKER RAPD, OpY16 linked to Rkn-mn1 converted to SCAR (1060bp) ie SCAR Y16

RESULTS…. : 

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

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CASE STUDY 2

Introduction : 

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.

Contd.., : 

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.

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CASE STUDY 3

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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 Introduction

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CASE STUDY 4 TRANSGENIC APPROACH

Introduction : 

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.

GENE OF INTEREST : 

GENE OF INTEREST GENE SELECTION – RICE

GENE CONSTRUCT : 

GENE CONSTRUCT SELECTABLE MARKER – Hygromycin B

POLYMERASE CHAIN REACTION : 

POLYMERASE CHAIN REACTION

SOUTHERN BLOTTING : 

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.

SOUTHERN BLOT HYBRIDIZATION : 

SOUTHERN BLOT HYBRIDIZATION

WESTERN BLOT ANALYSIS : 

WESTERN BLOT ANALYSIS

PROGENY ANALYSIS : 

PROGENY ANALYSIS Pyramided line SM-PB1-1 did not segregate for Xa21 in both T1 and T2 progenies The influence of chi11 and/or tlp on Xa21-mediated bacterial blight resistance could not be assessed

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www.themegallery.com Company Logo Global Status of Approved GM Crops with Stacked Genes (Halpin, 2007)

21 st Century Product Developement : 

21 st Century Product Developement

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DISCUSSION

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Thank You !