Genomics platform for agriculture-CAT lecture

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The popular lecture for the undergraduate students of agriculture to know about the application of biotechnology in agriculture science graduates. Some of the major break through inventions how it impact on agriculture research and development

Transcript of Genomics platform for agriculture-CAT lecture

  • Prof. Senthil Natesan Department of Biotechnology, AC&RI, Madurai Bio-Technology Genomics platform for agriculture www.tnau.ac.in Department of Biotechnology, Tamil Nadu Agricultural University AC&RI,
  • .. the best teaching can be done only when there is a direct .. situation in which the student discusses the ideas, thinks about the things, and talks about the things. Its impossible to learn very much by simply sitting in a lecture, or even by simply doing problems that are assigned Richard Feynman 1963
  • Genomics time line Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Crop and plant genomes and their application. The figure gives the approximate timeline of when crop genomes were sequenced along with the underlying techniques and sequencing strategy used. Hybrid strategies which use BAC by BAC and WGS are indicated by the placement of a genome twice. Also note that the distinction between pure NGS and Hybrid sequencing is sometimes arbitrary as many genome projects rely on previously generated Sanger sequences. In addition, some major applications are marked by symbols: Grains for an improvement in grain quality, a flower for flowering time and a tomato for a tomato ripening trai Department of Biotechnology, AC&RI, Madurai-www.tnaugenomics.com
  • Examples of the range of phenotypic variation in maize germplasm held in the CIMMYT genebank (Photo provided by Dr. Taba Suketoshi) Department of Biotechnology, AC&RI ,Madurai-www.tnaugenomics.com
  • Department of Biotechnology, AC&RI ,Madurai-www.tnaugenomics.com
  • Humans Have Limited Molecular Diversity 0.09% Zhao et al, 2000, PNAS 1.34% Department of Biotechnology, AC&RI, Madurai-www.tnaugenomics.com
  • Maize diversity is greater than the difference between human and chimps Tenallion et al, 2001, PNAS 1.42% Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Arabidopsis Sequencing Facts Arabidopsis has a small (125 Mb) sized-genome on 5 chromosomes -Human has 3,000 Mb on 23 chromosomes -Maize has 2,500 Mb on 10 chromosomes -Medicago has 520 Mb on 8 chromosomes -Rice has 430 Mb on 12 chromosomes -Lily has 50,000 Mb on 12 chromosomes Arabidopsis has approx. 25,500 genes - humans have slightly fewer, about 24,000 Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • The Human Genome Project The most public large-scale sequencing project has been the Human Genome Project. Started by the Department of Energy, who realized the possible implications on human health-related issues, it began in 1990, with collaborative funding from a number of sources. After much drama and bickering in the scientific community, the genome was actually sequenced twice by 2 different groups (the publicly funded group headed by Francis Collins and Craig Venters company Celera) and the completion announced simultaneously at a joint press conference*. *Published separately: International Human Genome Sequencing Consortium (2001) and Venter et al. (2001) J. Craig Venter (l) and Francis Collins (r) at the historic announcement June 26, 2000 Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Whole genome sequencing While we will not go into technical details or pros and cons here, you should be aware of the two main approaches to sequencing a whole genome. Top-down strategy: An anchored physical map is needed; overlapping clones (a minimal tiling path) are sequenced in order. Since the positions of the clones (and therefore the sequences) are already known, little post- sequencing work is needed. Images from The Creative Science Quarterly, Helmut Kae (2003) http://www.scq.ubc.ca/?p=392 Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Automated sequencing reactions - each reaction can resolve 600 to 750 bp (labeled with fluorescent dyes) Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • FISH analysis of the centromeric core of chromosome 5 in Rice The schema of constructing a physical map ofrice chromosome 5.
  • Comparing genomes: Example from the grasses This is now one of the most well-known figures in plant comparative genomics. This consensus comparative map of 7 grasses shows how the genomes can be aligned in terms of rice linkage blocks (Gale and Devos 1998). Any radial line starting at rice, the smallest genome and innermost circle, will pass through regions of similar gene content in each of the other species. Therefore a gene on the chromosome of one grass species can be anticipated to be present in a predicted location on a specific chromosome of a number of other grass family species. This has facilitated much sharing among researchers working on any of these species and others that may be also related (Phillips & Freeling 1998). Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • SNP discovery- Early methods Re-sequencing of PCR amplicons with or without pre-screening Direct sequencing of DNA segments amplified by PCR)from several individuals is the most direct way to identify SNP polymorphisms Alternatively, an allele-specific-PCR or primer-extension assay may be developed relatively straightforwardly. Rafalski 2002 Curr Opin Plant Biol 5 :94-100 Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • DNA sequencing output If you have DNA sequence produced from a PCR product or a library of ESTs, the sequence of your DNA segment(s) will be given to or, more usually, emailed or electronically transferred to you.. If the data is in the chromatogram form, you will need to manually generate a text file such as the one below (by reading the bases yourself) or, more typically, use one of the many software programs available to do this for you. If you retrieve a sequence from a public database, it will already be in this format for you. The first 480 bases of the DNA sequence of GAN, a drought tolerance related gene in Arabidopsis (GenBank Accession AY986818). Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • What are markers? Markers, in the context of breeding, are identifiers of characteristics of the phenotype and/or genotype of an individual; their inheritance can be followed through generations. Markers can be: Morphological: variation in traits which is scorable in single plants (eg flowering time) Biochemical: reflect variation at the protein or metabolite level (eg isozymes) Molecular: reflect variation at the DNA sequence level (eg microsatellites) In these beans, color could be a morphological marker, as could size, plant height, etc. The gel picture on the previous slide showed a molecular marker that identified differences between the various plant lines. Image: CGIAR Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Protein markers & quality of wheat 12 7 8 12 10 5 9 HMW glutenin -gliadins albumins globulins LMW glutenins (B subunits) , ,-gliadins LMW glutenins (C subunits) albumins Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Repetitive sequence primer I primer II plant A plant B microsatellite plant A plant B flanking region II flanking region I specific primers were designed corresponding to flanking sequence of microsatellite PCR analysis and analyze on 6 %denaturing polyacrylamide gel with silver staining A BSchematic of SSR assay Department of Biotechnology,AC&RI, Madurai- www.tnaugenomics.com
  • Detection of PCR product www.tnau.ac.in Department of Biotechnology, Tamil Nadu Agricultural University AC&RI,
  • SSR Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Microsatellite markers polymorphism between parental lines and rice hybrids Tamilkumar et al.,2009 Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Testing genetic purity of hybrid seeds of CORH3 using the SSR marker RM 234 Lane 2 = TNAUCMS2A (CMS line), Lane 3 = CB87R (restorer line). DNA was isolated from single seedlings of the CORH3 hybrid, PCR analysis was performed and genotype assessed (Lanes 412) Off type in Lanes 8. Tamilkumar et al.,2009 Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com
  • Advantages of MAB: Cost Depending on the trait and the cost of phenotyping, MAB may also cut down on costs. The costs of field plots, g