genomics and crop improvement
TRANSCRIPT
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GENOMICS GENOMICS ANDAND
CROP CROP IMPROVEMENTIMPROVEMENT
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Genomics and crop improvementGenomics and crop improvement
Genome contains total genetic information Genome contains total genetic information carried by a single set of chromosomes in carried by a single set of chromosomes in a haploid nucleusa haploid nucleus
It is the unit of information transmission It is the unit of information transmission (DNA replication) whereas genes in an unit (DNA replication) whereas genes in an unit of expressionof expression
Genomics is the study of genes and their Genomics is the study of genes and their role in the structure, growth and role in the structure, growth and development, quality, health and diseases development, quality, health and diseases of plantsof plants
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Application of Genomics Application of Genomics Gene structureGene structure Gene identification and cloningGene identification and cloning Gene prediction/ discoveryGene prediction/ discovery Genetic mapping and locating genesGenetic mapping and locating genes Genome projectsGenome projects Genomic databaseGenomic database Genome manipulation (Mol. Biology)Genome manipulation (Mol. Biology) QTLsQTLs Molecular markers and MASMolecular markers and MAS Diagnostics on the basis of transcription to RNA Diagnostics on the basis of transcription to RNA
and translation to proteinand translation to protein
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Study of GenomicsStudy of Genomics Karyotype analysis/ chromosome profileKaryotype analysis/ chromosome profile Molecular cytogeneticsMolecular cytogenetics Molecular dissectionMolecular dissection DNA studies & DNA sequencingDNA studies & DNA sequencing Gene discoveryGene discovery Search for new genesSearch for new genes Exon PredictionExon Prediction Comparative genomics & orthologous lociComparative genomics & orthologous loci Gene expression and traitsGene expression and traits Genome projectsGenome projects
Genomic library: DNA library in which the Genomic library: DNA library in which the cloned DNA is from a genomic DNA of the cloned DNA is from a genomic DNA of the plantplant
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Application of genomics (Contd)Application of genomics (Contd)
Comparative genomicsComparative genomics Gene banks and chromosome stocksGene banks and chromosome stocks Expression (mRNA) profiles and Expression (mRNA) profiles and
responses and interactions responses and interactions Data bases and NetworkingData bases and Networking Genome projects Genome projects Crop improvementCrop improvement
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Plant Genomic StrategiesPlant Genomic Strategies
Diversity GenomicsDiversity Genomics Applied GenomicsApplied Genomics Genome VisionGenome Vision Structural GenomicsStructural Genomics Comparative GenomicsComparative Genomics Functional GenomicsFunctional Genomics Nutritional GenomicsNutritional Genomics Computational GenomicsComputational Genomics Integrative Bioinformatics plus K mgtIntegrative Bioinformatics plus K mgt
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Cell cycle / MitosisCell cycle / Mitosis
Life cycle of a cell marked by cell Life cycle of a cell marked by cell division. The four phases are:division. The four phases are: G1 (Gap 1)G1 (Gap 1) S (Synthesis) DNA replication’S (Synthesis) DNA replication’ G2 (Gap 2)G2 (Gap 2) M (Mitotic phase) Chromosome M (Mitotic phase) Chromosome
separationseparation Replication in a short time with precisionReplication in a short time with precision Somatic cellsSomatic cells
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WALTHER FLEMMING (1882)WALTHER FLEMMING (1882)
Describes “ MITOSIS”
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DESCRIBES STAGES OF MEIOSISDESCRIBES STAGES OF MEIOSIS
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Crossing OverCrossing Over
Crossing over (variation) Crossing over (variation) may occur may occur between nonsister between nonsister chromatidschromatids at the at the chiasmatachiasmata..
Crossing overCrossing over: segments of nonsister : segments of nonsister chromatidschromatids break and reattach to the break and reattach to the other other chromatidchromatid..
Chiasmata (chiasma) Chiasmata (chiasma) are the sites of are the sites of crossing overcrossing over..
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Crossing Over - variation Crossing Over - variation
nonsister chromatids
chiasmata: site of crossing over
variation
Tetrad
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Chromosome structureChromosome structure Cell cycle (replication)Cell cycle (replication) Interphase cells – DNA – Protein complexInterphase cells – DNA – Protein complex Euchromatin and HeterochromatinEuchromatin and Heterochromatin Euchromatin is the chromatin region relatively open, Euchromatin is the chromatin region relatively open,
less condensed where gene expression occursless condensed where gene expression occurs Heterochromatin are chromatin regions that are Heterochromatin are chromatin regions that are
always highly condensed and there is little gene always highly condensed and there is little gene expression in these regions. Heterochromatin takes expression in these regions. Heterochromatin takes stain during metaphasestain during metaphase
Heterochromatin is either constitutive or facultativeHeterochromatin is either constitutive or facultative Constitutive heterochromatin is the specific genome Constitutive heterochromatin is the specific genome
region often containing short repeated sequencesregion often containing short repeated sequences Facultative heterochromatin is the entire Facultative heterochromatin is the entire
chromosome that are transcriptionally inactivaed chromosome that are transcriptionally inactivaed (eg. One X chromosome in female)(eg. One X chromosome in female)
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Cytogenetics - applicationCytogenetics - application Genome manipulationGenome manipulation Polyploidy (Amplification)Polyploidy (Amplification) Chromosome number changesChromosome number changes Chromosome structure alterationsChromosome structure alterations Alien addition/ Transfer of chromosomesAlien addition/ Transfer of chromosomes Alien substitution of chromosomesAlien substitution of chromosomes Alien gene transferAlien gene transfer Genome amplification: Intrachromosomal or Genome amplification: Intrachromosomal or
extrachromosomal production of many DNA extrachromosomal production of many DNA copies from a certain region of DNA/ Chromosome copies from a certain region of DNA/ Chromosome occur spontaneouslyoccur spontaneously
Chromosome engineering 1960sChromosome engineering 1960s
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Organelle GenomesOrganelle Genomes (specialized functions)(specialized functions)
MitochondriaMitochondria Occurs in large numbers in the cytoplasm of Occurs in large numbers in the cytoplasm of
eukaryotic cellseukaryotic cells Double membrane bound structureDouble membrane bound structure Semi autonomous organelle containing its own Semi autonomous organelle containing its own
DNA (mt) and RibosomesDNA (mt) and Ribosomes Reproduces by binary fissionReproduces by binary fission
Chloroplast-semi autonomousChloroplast-semi autonomous Chloroplast DNA (Cp DNA)Chloroplast DNA (Cp DNA)
Responds to nuclear signals Responds to nuclear signals Inter genomic interactionsInter genomic interactions
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Why CGWhy CG
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Comparative genomicsComparative genomics CharactersCharacters
Identical
Similar
Analogous Homologous
Orthologous Paralogous
Homologous due to conserved function
Similar due to common ancestors
Homologous with divergent functions
Sharing the corresponding characters
Sharing identities
Similar due to convergence
Identical
Similar
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What to compareWhat to compare
ORTHOLOGUESORTHOLOGUES
Genes that share the same Genes that share the same ancestral gene performing same ancestral gene performing same biological function in different biological function in different species but have diverged in species but have diverged in
sequence due to selective sequence due to selective evolutionevolution
PARALOGUESPARALOGUES
Genes within the same genome Genes within the same genome that share an ancient gene that share an ancient gene
performing diverse biological performing diverse biological functionsfunctions
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Diversity genomicsDiversity genomics
Sequence polymorphismSequence polymorphism Heritable phenotypic differencesHeritable phenotypic differences Genetic diversityGenetic diversity Novel molecular markers/traitsNovel molecular markers/traits Linkage disequilibrium in natural and Linkage disequilibrium in natural and
domesticated crop plantsdomesticated crop plants Selection sweeps in genomics regionsSelection sweeps in genomics regions Introgression scalesIntrogression scales Mechanisms of crop diversityMechanisms of crop diversity
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Functional GenomicsFunctional Genomics
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Functional GenomicsFunctional GenomicsGenome sequenceGenome sequence
Gene identificationGene identification
Gene expressionGene expression Gene function Gene function
Genetics manipulation
cDNA expression
Protein expression
Co-expression
Gene trapping Mutagenesis
Protein modification Structure interaction
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SYSTEMS VIEW OF CENTRAL SYSTEMS VIEW OF CENTRAL DOGMADOGMA
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CENTRAL DOGMA OF CENTRAL DOGMA OF MOLECULAR BIOLOGYMOLECULAR BIOLOGY
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GENE STRUCTUREGENE STRUCTURE
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GENE EXPRESSIONGENE EXPRESSION
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GENE EXPRESSIONGENE EXPRESSION
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MOLECULES PARTICIPATING IN MOLECULES PARTICIPATING IN INFORMATION FLOW AND THE INFORMATION FLOW AND THE
FUNCTIONAL SITESFUNCTIONAL SITESMOLECMOLECULEULE
PROCESSINPROCESSINGG
FUNCTIONAL FUNCTIONAL SITESSITES
INTERACTING INTERACTING MOLECULESMOLECULES
DNADNA REPLICATIOREPLICATIONN
TRANSCRIPTTRANSCRIPTIONION
REPLICATION REPLICATION ORIGINORIGIN
PROMOTORPROMOTOR
ENHANCERENHANCER
OPERATOROPERATOR
OTHER OTHER PROKARYOTIC PROKARYOTIC REGULATORSREGULATORS
ORIGIN ORIGIN RECOGNITION RECOGNITION COMPLEX COMPLEX
RNA RNA POLYMERASEPOLYMERASE
TRANSCRIPTIOTRANSCRIPTION FACTORN FACTOR
REPRESSOR etcREPRESSOR etc
RNARNA POST-POST-TRANSCRIPTTRANSCRIPTIONAL IONAL PROCESSINGPROCESSING
TRANSLATIOTRANSLATIONN
SPLICE SITESPLICE SITE
TRANSLATION TRANSLATION INITIATION SITEINITIATION SITE
SPLICEOSOMESPLICEOSOME
RIBOSOMERIBOSOME
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MOLECULES PARTICIPATING IN MOLECULES PARTICIPATING IN INFORMATION FLOW AND THE INFORMATION FLOW AND THE
FUNCTIONAL SITESFUNCTIONAL SITES
CONT..
MOLEMOLECULECULE
PROCESSINPROCESSINGG
FUNCTIONAL FUNCTIONAL SITESSITES
INTERACTININTERACTING G MOLECULESMOLECULES
PROTEPROTEININ
POST-POST-TRANSLATITRANSLATIONAL ONAL PROCESSINPROCESSINGG
PROTEIN PROTEIN SORTINGSORTING
PROTEIN PROTEIN FUNCTIONFUNCTION
CLEAVAGE SITECLEAVAGE SITE
PHOSPHORYLATIPHOSPHORYLATION AND OTHER ON AND OTHER MODIFICATION MODIFICATION SITESSITES
ATP BINDING ATP BINDING SITESSITES
SIGNAL SIGNAL SEQUENCE, SEQUENCE, LOCALIZATION LOCALIZATION SIGNALSSIGNALS
DNA BINDING DNA BINDING SITESSITES
LIGAND BINDING LIGAND BINDING SITESSITES
CATALYTIC SITESCATALYTIC SITES
PROTEASEPROTEASE
PROTEIN PROTEIN KINASE etcKINASE etc
SIGNAL SIGNAL RECOGNITIRECOGNITION ON PARTICLEPARTICLE
DNADNA
LIGANDSLIGANDS
MANY MANY DIFFERENT DIFFERENT MOLECULESMOLECULES
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Functional GenomicsFunctional Genomics Ways of identifying gene function and assigning Ways of identifying gene function and assigning
functions to genes of unknown functions functions to genes of unknown functions
Gene familyGene family Group of similar or identical genes usually on the same Group of similar or identical genes usually on the same
chromosome, arising by gene duplication some of them chromosome, arising by gene duplication some of them work and some of them are switched off or silenced work and some of them are switched off or silenced (pseudo genes)(pseudo genes)
Gene(Genome) amplificationGene(Genome) amplification More or less specific production of multiple copiesMore or less specific production of multiple copies
Gene conversionGene conversion Process in which one member of a gene family acts as a Process in which one member of a gene family acts as a
blue print for the correction of the other - can result in blue print for the correction of the other - can result in either suppression of a new mutation or its lateral either suppression of a new mutation or its lateral spread in the genomespread in the genome
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Genomics – overview of general Genomics – overview of general functionsfunctions
Genome sequence gene Genome sequence gene identification identification gene gene expression gene functionexpression gene function
Gene identification – genomic motifs, Gene identification – genomic motifs, comparative genomics, cDNA & ESTs comparative genomics, cDNA & ESTs database, Protein identificationdatabase, Protein identification
Gene expression – cDNA expression, Gene expression – cDNA expression, Protein expressionProtein expression
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Gene expressionGene expression Gene activationGene activation Process in which information in a gene is Process in which information in a gene is
used to produce a protein and gene used to produce a protein and gene expression via transcription and expression via transcription and translation to produce protein and hence translation to produce protein and hence phenotypephenotype
Gene-phenotype relationship (functions) Gene-phenotype relationship (functions) understanding the field plot techniques, understanding the field plot techniques, stat.procedures, molecular markers, stat.procedures, molecular markers, information techniques and equipmentsinformation techniques and equipments
Gene transfer/ genetic engineering Gene transfer/ genetic engineering Chromosome engineeringChromosome engineering
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ExonExon
Exon prediction and Exon trappingExon prediction and Exon trapping Exon is the portion of the gene that is Exon is the portion of the gene that is
transmitted into the mRNA and is transmitted into the mRNA and is translated into proteintranslated into protein
Exon trapping is the method used to Exon trapping is the method used to isolate exons from new DNA. In exon isolate exons from new DNA. In exon trapping, an R fragment from a new trapping, an R fragment from a new DNA sequence is cloned into a cognate DNA sequence is cloned into a cognate R site in an intron of a cloned geneR site in an intron of a cloned gene
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Expressed sequence tagsExpressed sequence tags
DNA sequences derived by sequencung an DNA sequences derived by sequencung an end of a random cDNA clone from a library end of a random cDNA clone from a library of interest. Provides rapid ways of identifying of interest. Provides rapid ways of identifying cDNA of interest based on their sequence cDNA of interest based on their sequence EST is an exon specific sequence.50-500 bp EST is an exon specific sequence.50-500 bp from a cDNA. It represents a gene. Large from a cDNA. It represents a gene. Large sets of ESTs opens the door for studying sets of ESTs opens the door for studying gene expression on a large scale. ESTs gene expression on a large scale. ESTs represent tags of expression for a given represent tags of expression for a given cDNA library. (Rastogi cDNA library. (Rastogi et alet al ., 2004) ., 2004)
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Size of mRNASize of mRNA Gene size: Gene is sequence of chromosomal Gene size: Gene is sequence of chromosomal
DNA required for production of functional RNA DNA required for production of functional RNA molecule or functional protein. Range in size from molecule or functional protein. Range in size from 1.5Kb (globin gene) to 2000 Kb (muscular 1.5Kb (globin gene) to 2000 Kb (muscular distrophy gene)distrophy gene)
RNA: It is single stranded. RNAs are mRNA, tRNA, RNA: It is single stranded. RNAs are mRNA, tRNA, rRNArRNA
mRNA is about 1/10mRNA is about 1/10thth of the size of the gene from of the size of the gene from which it is transcribedwhich it is transcribed
mRNA levels define state of the cells. 5% mRNA, mRNA levels define state of the cells. 5% mRNA, 15% tRNA and 80% rRNA of total RNA. Other 15% tRNA and 80% rRNA of total RNA. Other minor RNAs are involved in splicing and telomere minor RNAs are involved in splicing and telomere synthesissynthesis
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mRNA contd….mRNA contd….
mRNAmRNA mRNA is template for protein synthesis mRNA is template for protein synthesis assume double helix or single stranded structureassume double helix or single stranded structure does not contain chemically modified basesdoes not contain chemically modified bases
tRNAtRNA Function in protein synthesis Function in protein synthesis Contain chemically modified basesContain chemically modified bases Single stranded DNA and RNA Single stranded DNA and RNA Adopt random coil structureAdopt random coil structure
RibosomesRibosomes Subcellular organelle involved in protein Subcellular organelle involved in protein
synthesissynthesis Made of large and small subunit and rRNA is Made of large and small subunit and rRNA is
integral part of itintegral part of it rRNA is single stranded and contains chemical rRNA is single stranded and contains chemical
modified basesmodified bases
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Markers are many..Markers are many.. RFLP – Restriction Fragment Length polymorphismRFLP – Restriction Fragment Length polymorphism RAPD – Randomly Amplified Polymorphic DNARAPD – Randomly Amplified Polymorphic DNA VNTRs – Variable Number Tandem Repeats VNTRs – Variable Number Tandem Repeats
(sat.DNA, omni sat, microsat)(sat.DNA, omni sat, microsat) SSR – Single Sequence Repeats or MicrosatellitesSSR – Single Sequence Repeats or Microsatellites DAF – DNA Amplified Finger PrintingDAF – DNA Amplified Finger Printing AP-PCR – Arbitarily primed PCRAP-PCR – Arbitarily primed PCR STS – Sequence Tagged SiteSTS – Sequence Tagged Site SCAR – Sequence Characterized Amplified RegionsSCAR – Sequence Characterized Amplified Regions CAPs – Cleaved Amplified Polymorphic SequenceCAPs – Cleaved Amplified Polymorphic Sequence SNP – Simple Nucleotide PolymorphismSNP – Simple Nucleotide Polymorphism
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TYPES OF MARKERS IN GENETICS
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MAPS are different kindsMAPS are different kinds Chromosome/ cytogenetic map in terms of Chromosome/ cytogenetic map in terms of
Chromosome BandingChromosome Banding Genetic mapping/ linkage mapsGenetic mapping/ linkage maps cDNA map showing locations of expressed DNA cDNA map showing locations of expressed DNA
regions (exons) on the chromosome mapregions (exons) on the chromosome map Cosmid contig map showing the order of Cosmid contig map showing the order of
overlapping DNA fragmentsoverlapping DNA fragments Macro restriction map – order of distance Macro restriction map – order of distance
between enzyme cutting cleavage sitebetween enzyme cutting cleavage site Highest restriction physical map showing the Highest restriction physical map showing the
complete elucidation of the DNA sequence of complete elucidation of the DNA sequence of each chromosome in the genomeeach chromosome in the genome
Physical mappingPhysical mapping
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MOLECULAR MARKERS AND GENETIC MARKERS
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Quantitative Trait Loci (QTL)Quantitative Trait Loci (QTL)
Linkage of trait (Quantitative) to marker Linkage of trait (Quantitative) to marker loci i.e. phenotype for the QT and loci i.e. phenotype for the QT and genotype for the marker loci are scored genotype for the marker loci are scored and if there are differences between mean and if there are differences between mean phenotype among the marker genotype phenotype among the marker genotype classes, then presence of QTL linked to the classes, then presence of QTL linked to the marker is inferred. The association marker is inferred. The association between quantitative trait variation and between quantitative trait variation and marker segregation pattern is worked out. marker segregation pattern is worked out. It is quantified linkage disequilibrium to It is quantified linkage disequilibrium to locate and clone gene responsible for QT. locate and clone gene responsible for QT. QTL is locus of DNA segments that carry QTL is locus of DNA segments that carry more genes coding for an agronomic trait more genes coding for an agronomic trait
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DNA Micro array – Recent DNA Micro array – Recent TechnologyTechnology
Measures relative to the number of copies Measures relative to the number of copies of a genetic message and thus levels of of a genetic message and thus levels of gene expression at different stages in gene expression at different stages in development and in different tissues. It can development and in different tissues. It can even measure poorly expressed geneseven measure poorly expressed genes
It is referred to as Reverse Northern. In It is referred to as Reverse Northern. In micro array experiment, (array micro array experiment, (array hybridization) cDNA are spotted onto a filter hybridization) cDNA are spotted onto a filter and hybridized with a probe made from and hybridized with a probe made from mRNA population. Probes are made by mRNA population. Probes are made by reverse transcribing mRNA into cDNAreverse transcribing mRNA into cDNA
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Microarray contd………Microarray contd……… The amount of hybridization to a given The amount of hybridization to a given
clone represents the amount of mRNA clone represents the amount of mRNA present to the corresponding genepresent to the corresponding gene
ApplicationsApplications Gene expression profilingGene expression profiling Identifying new targets for functional genomicsIdentifying new targets for functional genomics Single nucleotide polymorphism mappingSingle nucleotide polymorphism mapping GenotypingGenotyping
A A tool in proteomics to indicate protein tool in proteomics to indicate protein abundance. mRNA and protein levels do not abundance. mRNA and protein levels do not always correlate in the cellsalways correlate in the cells
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Why DNA Microchip Why DNA Microchip technology?technology?
DNA microarray gives snapshot of mRNA expression in a genome at a particular time
Can take multiple snapshots to watch changing patterns of mRNAs over time, space and in response
to stimuli E.g.: Developmental stages, Different tissues, Starvation, Disease invasion, etc.
Can give indirect indication about levels of specific protein by proxy
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Phenotypes and arraysPhenotypes and arrays
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Plant Breeding ApplicationPlant Breeding Application Gene discoveryGene discovery Molecular breeding – get into genesMolecular breeding – get into genes Diversity patterns and evaluationDiversity patterns and evaluation Molecular fingerprintingMolecular fingerprinting Marker Assisted/ Aided selectionMarker Assisted/ Aided selection Markers and genetic solutionsMarkers and genetic solutions Marker developmentMarker development Purity testing develop computational models for Purity testing develop computational models for
biological functions (for traits of interest/ target traits)biological functions (for traits of interest/ target traits) Manipulate value added traitsManipulate value added traits QTLs – pyramiding and interactionsQTLs – pyramiding and interactions Transformation and traitsTransformation and traits Biotic and abiotic stresses and diagnosisBiotic and abiotic stresses and diagnosis
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Research DirectionsResearch Directions
GENOME COMPARISONS
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COMPARITIVE GENOMICS OF TOMATO, POTATO AND PEPPER
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Comparative genomics of Comparative genomics of grassesgrasses
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Rice Genome UtilitiesRice Genome Utilities
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ProteomicsProteomics
PROTEins expressed by genOME is PROTEins expressed by genOME is proteomics (Wilkins, 1995). Genome is proteomics (Wilkins, 1995). Genome is entire set of genes and like that proteome is entire set of genes and like that proteome is inclusive of all proteins produced by a inclusive of all proteins produced by a species. Unlike genome, proteome varies species. Unlike genome, proteome varies with time and samplewith time and sample
Proteomics ApplicationProteomics Application1.1. Identifying diseases progressionIdentifying diseases progression2.2. Protein markers for diagnosticsProtein markers for diagnostics3.3. Information generated in proteomics is Information generated in proteomics is
complementary with the Genomics informationcomplementary with the Genomics information
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Component inter relationshipsComponent inter relationships
Genetics
Cytogenetics
Genomics
Proteomics
Plant Development
Plant Manipulations
Crop Improvement
BioinformaticSynthetic approach
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