GENOMICS GENOMICS ANDAND

CROP CROP IMPROVEMENTIMPROVEMENT

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

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

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

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

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

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

WALTHER FLEMMING (1882)WALTHER FLEMMING (1882)

Describes “ MITOSIS”

DESCRIBES STAGES OF MEIOSISDESCRIBES STAGES OF MEIOSIS

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..

Crossing Over - variation Crossing Over - variation

nonsister chromatids

chiasmata: site of crossing over

variation

Tetrad

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)

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

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

Why CGWhy CG

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

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

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

Functional GenomicsFunctional Genomics

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

SYSTEMS VIEW OF CENTRAL SYSTEMS VIEW OF CENTRAL DOGMADOGMA

CENTRAL DOGMA OF CENTRAL DOGMA OF MOLECULAR BIOLOGYMOLECULAR BIOLOGY

GENE STRUCTUREGENE STRUCTURE

GENE EXPRESSIONGENE EXPRESSION

GENE EXPRESSIONGENE EXPRESSION

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

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

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

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

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

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

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)

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

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

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

TYPES OF MARKERS IN GENETICS

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

MOLECULAR MARKERS AND GENETIC MARKERS

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

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

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

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

Phenotypes and arraysPhenotypes and arrays

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

Research DirectionsResearch Directions

GENOME COMPARISONS

COMPARITIVE GENOMICS OF TOMATO, POTATO AND PEPPER

Comparative genomics of Comparative genomics of grassesgrasses

Rice Genome UtilitiesRice Genome Utilities

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

Component inter relationshipsComponent inter relationships

Genetics

Cytogenetics

Genomics

Proteomics

Plant Development

Plant Manipulations

Crop Improvement

BioinformaticSynthetic approach