GENOMICS AND
CROP IMPROVEMENT
Genomics and crop improvement
Genome contains total genetic information carried by a single set of chromosomes in a haploid nucleus It is the unit of information transmission (DNA replication) whereas genes in an unit of expression Genomics is the study of genes and their role in the structure, growth and development, quality, health and diseases of plants
Application of Genomics
Gene structure Gene identification and cloning Gene prediction/ discovery Genetic mapping and locating genes Genome projects Genomic database Genome manipulation (Mol. Biology) QTLs Molecular markers and MAS Diagnostics on the basis of transcription to RNA and translation to protein
Study of Genomics
Karyotype analysis/ chromosome profile Molecular cytogenetics Molecular dissection DNA studies & DNA sequencing Gene discovery Search for new genes Exon Prediction Comparative genomics & orthologous loci Gene expression and traits Genome projects
Genomic library: DNA library in which the cloned DNA is from a genomic DNA of the plant
Application of genomics (Contd)
Comparative genomics Gene banks and chromosome stocks Expression (mRNA) profiles and responses and interactions Data bases and Networking Genome projects Crop improvement
Plant Genomic Strategies
Diversity Genomics Applied Genomics Genome Vision Structural Genomics Comparative Genomics Functional Genomics Nutritional Genomics Computational Genomics Integrative Bioinformatics plus K mgt
Cell cycle / Mitosis
Life cycle of a cell marked by cell division. The four phases are:
G1 (Gap 1) S (Synthesis) DNA replication’ G2 (Gap 2) M (Mitotic phase) Chromosome separation Replication in a short time with precision Somatic cells
WALTHER FLEMMING (1882)
Describes MITOSIS”
“
DESCRIBES STAGES OF MEIOSIS
Crossing Over
Crossing over (variation) may occur between nonsister chromatids at the chiasmata.
Crossing over: segments of nonsister chromatids break and reattach to the other chromatid.
Chiasmata (chiasma) are the sites of crossing over.
Crossing Over - variation nonsister chromatids
chiasmata: site of crossing over
Tetrad
variation
Chromosome structure
Cell cycle (replication) Interphase cells – DNA – Protein complex Euchromatin and Heterochromatin Euchromatin is the chromatin region relatively open, less condensed where gene expression occurs Heterochromatin are chromatin regions that are always highly condensed and there is little gene expression in these regions. Heterochromatin takes stain during metaphase Heterochromatin is either constitutive or facultative Constitutive heterochromatin is the specific genome region often containing short repeated sequences Facultative heterochromatin is the entire chromosome that are transcriptionally inactivaed
Cytogenetics - application
Genome manipulation Polyploidy (Amplification) Chromosome number changes Chromosome structure alterations Alien addition/ Transfer of chromosomes Alien substitution of chromosomes Alien gene transfer Genome amplification: Intrachromosomal or extrachromosomal production of many DNA copies from a certain region of DNA/ Chromosome occur spontaneously Chromosome engineering 1960s
Organelle Genomes (specialized functions)
Mitochondria
Chloroplast-semi autonomous
Occurs in large numbers in the cytoplasm of eukaryotic cells Double membrane bound structure Semi autonomous organelle containing its own DNA (mt) and Ribosomes Reproduces by binary fission Chloroplast DNA (Cp DNA)
Responds to nuclear signals Inter genomic interactions
Why CG
Comparative genomics Characters Sharing the corresponding characters
Identical
Sharing identities
Similar
Homologous
Analogous Similar due to convergence
Similar due to common ancestors
Orthologous Homologous due to conserved function
Paralogous Homologous with divergent functions
What to compare
PARALOGUES
ORTHOLOGUES
Genes within the same genome that share an ancient gene performing diverse biological functions
Genes that share the same ancestral gene performing same biological function in different species but have diverged in sequence due to selective evolution
Diversity genomics
Sequence polymorphism Heritable phenotypic differences Genetic diversity Novel molecular markers/traits Linkage disequilibrium in natural and domesticated crop plants Selection sweeps in genomics regions Introgression scales Mechanisms of crop diversity
Functional Genomics
Functional Genomics Genome sequence Gene identification Gene expression cDNA expression
Protein expression
Gene function Co-expression
Protein modification Structure interaction
Gene trapping Mutagenesis Genetics manipulation
SY ST EMS VIEW OF C EN TRAL DOGMA
CE NTRAL DOGM A OF MO LECUL AR BI OLOGY
GENE S TRU CTU RE
GE NE EX PR ESS IO N
GE NE EX PR ESS IO N
MOLECULES PARTICIPATING IN INFORMATION FLOW AND THE FUNCTIONAL SITES MOL EC UL E
PRO CE SSI NG
FUN CT IO NAL SI TE S INT ER AC TIN G MOL EC UL ES
DNA
REP LI CAT IO N TRA NS CRI PT ION
REP LI CA TIO N O RI GIN PRO MO TO R ENH AN CE R OPE RA TO R OTH ER P ROK ARY OT IC REG UL AT ORS
RNA
POS TTRA NS CRI PT ION AL PR OCE SS ING TRA NS LAT IO N
SPL IC E SIT E TRA NS LA TIO N INI TI AT ION SI TE
ORI GI N REC OG NI TIO N COM PL EX RNA P OL YME RAS E TRA NS CR IPT ION FAC TO R REP RE SS OR etc SPL IC EO SOM E RIB OS OM E
CONT..
MOLEC ULES PARTI CIPAT ING IN INFO RMAT ION FL OW AN D TH E FUNC TION AL SIT ES
MOL EC UL PRO CE SS ING E
FUN CT ION AL SI TE S
INT ER ACT ING MOL EC ULE S
PRO TE IN POS TTRA NS LA TIO NA L P RO CE SSI NG
CLE AV AGE S ITE PHO SP HOR YL ATI ON AND O THE R MOD IF ICA TI ON SI TES ATP B IND IN G S IT ES SIG NA L S EQ UEN CE , LOC AL IZA TI ON SIG NA LS DNA B IND IN G S IT ES LIG AN D B IN DIN G SIT ES CAT AL YTI C SIT ES
PRO TE ASE PRO TE IN KIN AS E e tc
PRO TE IN SOR TI NG PRO TE IN FUN CT IO N
SIG NA L REC OG NIT ION PAR TI CLE DNA LIG AN DS MAN Y DIF FE REN T MOL EC ULE S
Functional Genomics
Gene family
Group of similar or identical genes usually on the same chromosome, arising by gene duplication some of them work and some of them are switched off or silenced (pseudo genes)
Gene(Genome) amplification
Ways of identifying gene function and assigning functions to genes of unknown functions
More or less specific production of multiple copies
Gene conversion
Process in which one member of a gene family acts as a blue print for the correction of the other - can result in either suppression of a new mutation or its lateral spread in the genome
Genomics – overview of general functions
Genome sequence gene identification gene expression gene function Gene identification – genomic motifs, comparative genomics, cDNA & ESTs database, Protein identification Gene expression – cDNA expression, Protein expression
Gene expression
Gene activation Process in which information in a gene is used to produce a protein and gene expression via transcription and translation to produce protein and hence phenotype Gene-phenotype relationship (functions) understanding the field plot techniques, stat.procedures, molecular markers, information techniques and equipments Gene transfer/ genetic engineering Chromosome engineering
Exon
Exon prediction and Exon trapping
Exon is the portion of the gene that is transmitted into the mRNA and is translated into protein Exon trapping is the method used to isolate exons from new DNA. In exon trapping, an R fragment from a new DNA sequence is cloned into a cognate R site in an intron of a cloned gene
Expressed sequence tags
DNA sequences derived by sequencung an end of a random cDNA clone from a library of interest. Provides rapid ways of identifying cDNA of interest based on their sequence EST is an exon specific sequence.50-500 bp from a cDNA. It represents a gene. Large sets of ESTs opens the door for studying gene expression on a large scale. ESTs represent tags of expression for a given cDNA library. (Rastogi et al ., 2004)
Size of mRNA
Gene size: Gene is sequence of chromosomal DNA required for production of functional RNA molecule or functional protein. Range in size from 1.5Kb (globin gene) to 2000 Kb (muscular distrophy gene) RNA: It is single stranded. RNAs are mRNA, tRNA, rRNA mRNA is about 1/10th of the size of the gene from which it is transcribed mRNA levels define state of the cells. 5% mRNA, 15% tRNA and 80% rRNA of total RNA. Other minor RNAs are involved in splicing and telomere synthesis
mRNA contd….
mRNA mRNA is template for protein synthesis assume double helix or single stranded structure does not contain chemically modified bases tRNA Function in protein synthesis Contain chemically modified bases Single stranded DNA and RNA Adopt random coil structure Ribosomes Subcellular organelle involved in protein synthesis Made of large and small subunit and rRNA is integral part of it rRNA is single stranded and contains chemical
Markers are many..
RFLP – Restriction Fragment Length polymorphism RAPD – Randomly Amplified Polymorphic DNA VNTRs – Variable Number Tandem Repeats (sat.DNA, omni sat, microsat) SSR – Single Sequence Repeats or Microsatellites DAF – DNA Amplified Finger Printing AP-PCR – Arbitarily primed PCR STS – Sequence Tagged Site SCAR – Sequence Characterized Amplified Regions CAPs – Cleaved Amplified Polymorphic Sequence
TYPES OF MARKERS IN GENETICS
MAPS are different kinds
Chromosome/ cytogenetic map in terms of Chromosome Banding Genetic mapping/ linkage maps cDNA map showing locations of expressed DNA regions (exons) on the chromosome map Cosmid contig map showing the order of overlapping DNA fragments Macro restriction map – order of distance between enzyme cutting cleavage site Highest restriction physical map showing the complete elucidation of the DNA sequence of each chromosome in the genome Physical mapping
MOLECULAR MARKERS AND GENETIC MARKERS
Quantitative Trait Loci (QTL)
Linkage of trait (Quantitative) to marker loci i.e. phenotype for the QT and genotype for the marker loci are scored and if there are differences between mean phenotype among the marker genotype classes, then presence of QTL linked to the marker is inferred. The association between quantitative trait variation and marker segregation pattern is worked out. It is quantified linkage disequilibrium to locate and clone gene responsible for QT. QTL is locus of DNA segments that carry more genes coding for an agronomic trait
DNA Micro array – Recent Technology
Measures relative to the number of copies of a genetic message and thus levels of gene expression at different stages in development and in different tissues. It can even measure poorly expressed genes It is referred to as Reverse Northern. In micro array experiment, (array hybridization) cDNA are spotted onto a filter and hybridized with a probe made from mRNA population. Probes are made by reverse transcribing mRNA into cDNA
Microarray contd……… The amount of hybridization to a given clone represents the amount of mRNA present to the corresponding gene Applications
Gene expression profiling Identifying new targets for functional genomics Single nucleotide polymorphism mapping Genotyping A tool in proteomics to indicate protein abundance. mRNA and protein levels do not always correlate in the cells
Why DNA Microchip 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
Phenotypes and arrays
Plant Breeding Application
Gene discovery Molecular breeding – get into genes Diversity patterns and evaluation Molecular fingerprinting Marker Assisted/ Aided selection Markers and genetic solutions Marker development Purity testing develop computational models for biological functions (for traits of interest/ target traits) Manipulate value added traits QTLs – pyramiding and interactions Transformation and traits Biotic and abiotic stresses and diagnosis
GENOME COMPARISONS
Research Directions
COMPARITIVE GENOMICS OF TOMATO, POTATO AND PEPPER
Comparative genomics of grasses
Rice Genome Utilities
Proteomics
PROTEins expressed by genOME is proteomics (Wilkins, 1995). Genome is entire set of genes and like that proteome is inclusive of all proteins produced by a species. Unlike genome, proteome varies with time and sample
Proteomics Application 1. 2. 3.
Identifying diseases progression Protein markers for diagnostics Information generated in proteomics is complementary with the Genomics information
Bioinformatics
Bioinformatics is management information system for molecular biology. It is the application of information techniques derived from disciplines such as applied mathematics, computer science and statistics to understand and organize the information associated with the molecules (molecular biology) and biological phenomena / functions It is the mathematical, statistics and computing methods that aim to solve biological problems using DNA and amino acid sequences and related information
Component inter relationships Genetics
Plant Development
Cytogenetics
Genomics
Proteomics
Bioinformatic Synthetic approach
Plant Manipulations
Crop Improvement