Tilling seminar ajk final 2007 format

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Targeting Induced Local Lesions IN Genomes (TILLING) is a combined tool of plant mutagenesis and DNA Biology to investigate useful mutations at Genomic level. First time used for cotton improvement.

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  • As you know almost half of the people in pakistan directly or indirectly depends on agriculture for their livelihood. Most of them are small scale farmers who mostly get their income from crop production. To ensure the continuty of their earnings and food availability it is necessary.
  • WeAs the resource persons engaged in the agriculture whatever the disciplines and occupation we have, either we are agriculture scientists or agriculture business managers, importers or exporters of agriculture products or even if we are large scale productive crop growers we have a common mission to achieve i.e. delivery of edible food to ensure the availability of food for the whole population to keep the alive , and to ensure the income for the farmers to improve their living standards so that they become able to perform efficiently in the field, and after all make our homeland self sufficient in food production. And recently with the changing global environment we have to achieve some additional objectives i.e.Growing the crops in their favorable ecosystemsAnd selection of such suitable crops and crop varieties which must be able to adapt this ecosystemAnd finally to ensure that these genotypes have some effective protection against insect pest attack.Being plant breeders and geneticists we have the utmost responsibility to ensure these two objectives
  • WeAs the resource persons engaged in the agriculture whatever the disciplines and occupation we have, either we are agriculture scientists or agriculture business managers, importers or exporters of agriculture products or even if we are large scale productive crop growers we have a common mission to achieve i.e. delivery of edible food to ensure the availability of food for the whole population to keep the alive , and to ensure the income for the farmers to improve their living standards so that they become able to perform efficiently in the field, and after all make our homeland self sufficient in food production. And recently with the changing global environment we have to achieve some additional objectives i.e.Growing the crops in their favorable ecosystemsAnd selection of such suitable crops and crop varieties which must be able to adapt this ecosystemAnd finally to ensure that these genotypes have some effective protection against insect pest attack.Being plant breeders and geneticists we have the utmost responsibility to ensure these two objectivesPlant breeders and geneticists are always in search of ways which are able to improve the plant genetic makeup and ultimately whenever they come up with new tools and technologies this is either based on the principle of forward genetic or reverse genetic.
  • Tilling seminar ajk final 2007 format

    1. 1.
    2. 2. TILLING: Mutant Genome Mining for Crop Improvement<br />Usman Aslam (M. Phil Plant Biotechnology)<br />Research Associate <br />Cotton TILLING Group<br />Dr. Asif Ali Khan (Professor)<br />Dr. Hafiza Masooma Naseer Cheema (Assistant Professor)<br />Department of Plant Breeding and Genetics<br />University of Agriculture, FaisalabadPakistan.<br />
    3. 3. Agricultural Missions<br />1) Traditionally delivering edible food to ensure <br /><ul><li>Availability of food for the whole population
    4. 4. Income for the farmer to improve their living standards
    5. 5. The country’s self sufficiency in food production</li></ul>2) And recently in addition with minimum impact on the environment<br /><ul><li>Growing crops in a favorable ecosystem
    6. 6. Selection of crops and varieties well adapted to this ecosystem
    7. 7. Effective protection against pests: (weeds, insects, fungi, viruses, nematodes, etc.)</li></li></ul><li>Agricultural Missions<br />1) Traditionally delivering edible food to ensure <br /><ul><li>Availability of food for the whole population
    8. 8. Income for the farmer to improve their living standards
    9. 9. The country’s self sufficiency in food production</li></ul>2) And recently in addition with minimum impact on the environment<br /><ul><li>Growing crops in a favorable ecosystem
    10. 10. Selection of crops and varieties well adapted to this ecosystem
    11. 11. Effective protection against pests: (weeds, insects, fungi, viruses, nematodes, etc.)</li></li></ul><li>Forward vs. Reverse Genetics<br />Forward Genetics<br /> Phenotype Genotype<br />Select a biological process<br />Generate mutant populations<br />Screen for mutants with a desired phenotype<br />Map and clone the gene responsible for the phenotype<br />Reverse Genetics<br />Genotype Phenotype<br />Select a gene or genes of interest<br />Generate mutant populations<br />Develop and conduct sequence-based mutant screens<br />Analyze the phenotype of the mutants<br />
    12. 12. Forward Genetics Approaches<br />Reverse Genetics Approaches<br /><ul><li>Selection procedures based on phenotypic traits/characters
    13. 13. Conventional mutagenesis for phenotypic screens by radiations/
    14. 14. Conventional breeding techniques e.g., hybridization,
    15. 15. Gene mapping
    16. 16. Gene silencing
    17. 17. Targeted gene disruption by homologous recombination
    18. 18. T-DNA or transposon mediated insertional mutagenesis
    19. 19. TILLING by Chemical mutagenesis</li></li></ul><li>Forward Genetics Approaches<br />Reverse Genetics Approaches<br /><ul><li>Selection procedures based on phenotypic traits/characters
    20. 20. Conventional mutagenesis for phenotypic screens by radiations/
    21. 21. Conventional breeding techniques e.g., hybridization,
    22. 22. Gene mapping
    23. 23. Gene silencing
    24. 24. Targeted gene disruption by homologous recombination
    25. 25. T-DNA or transposon mediated insertional mutagenesis
    26. 26. TILLING by Chemical mutagenesis</li></li></ul><li>Advantages of TILLING as an approach for Reverse Genetics <br /><ul><li>Both natural and mutagenized populations can be screened. However mutagenized populations have a large number of randomly distributed mutations per plant genome
    27. 27. Plant heterozygous for a mutation can be detected.
    28. 28. Both mis-sense and non-sense mutations can be recovered.
    29. 29. No transgenic manipulation required
    30. 30. Cost effective than genetic engineering
    31. 31. No associated bio safety issues
    32. 32. Bypass sophisticated tissue culture barriers </li></li></ul><li>TILLING (Targeting Induced Local Lesion INGenomes)<br /><ul><li>With the completion of genome sequencing projects of several species, emphasis in genomics has shifted from analyzing sequences to understanding gene function.
    33. 33. A method called TILLING was devised by Claire M. McCallum et al in 2000
    34. 34. Identification of target genes based on the functional annotation of its sequence
    35. 35. Mutagenesis of target species to saturate its genome with mutations using suitable chemical mutagen
    36. 36. Detection of mutant allele through PCR of target genes and mismatch cleavage strategy
    37. 37. Phenotypic characterization of identified mutant allele </li></li></ul><li>Successfully improved crop species through TILLING <br /><ul><li>Mouse ear cress (Arabidopsis thaliana) 2001
    38. 38. Potato (Solanumtuberosum) 2002
    39. 39. Maize (Zeamays) 2006
    40. 40. Rice (Oryza Sativa) 2007
    41. 41. Wheat (Triticumestivum) 2004, 2009
    42. 42. Tomato (Solanumlycopersicum) 2010
    43. 43. Brassica (Brassica rapa) 2010
    44. 44. etc</li></li></ul><li>Development and testing of cotton (Gossypium hirsutum L.) TILLING population <br /><ul><li>Cotton is the backbone of Pakistan economy
    45. 45. Stagnant yield due to insect pest and disease attack (CLCuV)
    46. 46. Low fiber quality (length, strength and fineness) which hindered its export in international market
    47. 47. Polyploidy nature of cotton make it hard to manipulate through classical breeding approaches.
    48. 48. Lack of sophisticated tissue culture technology in cotton species
    49. 49. Cost of transgenic production and associated bio safety issues of GMOs can be avoided by use of TILLING
    50. 50. Induced mutagenesis provide cost effective way to create genomic variations</li></li></ul><li>Determination of LD50 (Kill curve)of EMS and gamma radiations for 3 species of cotton<br />
    51. 51. Germination of EMS treated genotypes of three Gossypium Species <br />
    52. 52. EMS and Gamma<br />Irradiationdose <br />response relationships <br />forgermination <br />percentage in 12 genotypes of three<br />Gossypium species<br />
    53. 53. Species wise EMS and GammaIrradiationdose response<br />forgermination percentage in three Gossypium species<br />
    54. 54. EMS and Gamma<br />Irradiationdose <br />response relationships <br />forseed cotton yield in 12 genotypes of 3<br />Gossypium species<br />
    55. 55. Species wise EMS and GammaIrradiationdose response<br />forseed cotton yield in three Gossypium species<br />
    56. 56. Primer designing for target genes on genomic sequences <br />Target gene families: Actin, Sucrose Synthase, Pectin Methyl Esterase (PME), DGAs and RGAs<br />Data base screening for retrieval of desired genomic sequences of target genes.<br />Determining the Intron-Exon modal in the retrieved sequences and <br />Finding the best region to TILL the gene<br />Pick required number of primer sets<br />
    57. 57.
    58. 58.
    59. 59.
    60. 60.
    61. 61.
    62. 62.
    63. 63. Mode of action of EMS with repeated cycles of <br />DNA Replication <br />Replication <br />
    64. 64. Intron-Exon Modal<br />Mutation in these regions are likely to be damaged<br />Best region to TILL the gene<br />Mutations in these region are not likely to be damaged<br />CODDLE will also determine amino acid change for every G and C in the sequence<br />
    65. 65.
    66. 66.
    67. 67.
    68. 68. CEL 1 enzyme assay<br />Celery (Apium graveolens ) is a common vegetable used for extraction of CEL1 enzyme<br />Heteroduplex formed after re annealing of the PCR products treated with CEL1 enzyme<br />Single strand specific endonulease which cleave 3’ site of single base mismatch in the heteroduplexes<br />CEL 1 treated PCR products resolved on PAGE and screened for the presence of novel fragments<br />Sample detected having mutant DNA tracking back (deconvolute) to identify actual plant sample<br />
    69. 69. Strategy for TILLING a gene<br />
    70. 70. Schematic diagram of TILLING for a specific gene <br />EMS<br />Mutant DNA<br />Point mutation detection by PAGE<br />
    71. 71. Possible outcomes of the Cotton TILLING Program<br /><ul><li>Phenotypic characterization of identified mutants for target genes
    72. 72. Development of Mutation libraries for target genes
    73. 73. Creation of new Alleles
    74. 74. Utilization of novel mutants for a particular trait in the local breeding program</li></li></ul><li>Questions are welcomed<br />

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