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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 …

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

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  • 1. Prof. Senthil Natesan Department of Biotechnology, AC&RI, Madurai Bio-Technology…… Genomics platform for agriculture Department of Biotechnology, Tamil Nadu Agricultural University AC&RI,
  • 2. “….. 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. It’s impossible to learn very much by simply sitting in a lecture, or even by simply doing problems that are assigned ……” Richard Feynman 1963
  • 3. Genomics time line Department of Biotechnology,AC&RI,Madurai-
  • 4. 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,
  • 5. 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 ,
  • 6. Department of Biotechnology, AC&RI ,
  • 7. Humans Have Limited Molecular Diversity 0.09% Zhao et al, 2000, PNAS 1.34% Department of Biotechnology, AC&RI,
  • 8. Maize diversity is greater than the difference between human and chimps Tenallion et al, 2001, PNAS 1.42% Department of Biotechnology,AC&RI,Madurai-
  • 9. 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-
  • 10. 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 Venter’s 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-
  • 11. 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) Department of Biotechnology,AC&RI,Madurai-
  • 12. Automated sequencing reactions - each reaction can resolve 600 to 750 bp (labeled with fluorescent dyes) Department of Biotechnology,AC&RI,Madurai-
  • 13. FISH analysis of the centromeric core of chromosome 5 in Rice The schema of constructing a physical map ofrice chromosome 5.
  • 14. 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-
  • 15. 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-
  • 16. 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-
  • 17. 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-
  • 18. 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-
  • 19. 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-
  • 20. Detection of PCR product Department of Biotechnology, Tamil Nadu Agricultural University AC&RI,
  • 21. SSR • Department of Biotechnology,AC&RI,Madurai-
  • 22. Microsatellite markers polymorphism between parental lines and rice hybrids Tamilkumar et al.,2009 Department of Biotechnology,AC&RI,Madurai-
  • 23. Department of Biotechnology,AC&RI,Madurai-
  • 24. 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 4–12) Off type in Lanes 8. Tamilkumar et al.,2009 Department of Biotechnology,AC&RI,Madurai-
  • 25. 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, greenhouse space, labour, and the measuring of some traits can be expensive, or in the case of certain diseases, impossible. Of course, some phenotyping will always be required to confirm results, but MAB can decrease the amount of phenotyping in many situations. The ability to test for the presence of a certain allele rather than waiting until the associated trait can be seen can decrease the amount of phenotyping that is necessary. Products such as the FTA cards shown at left can make DNA extractions, and therefore marker work, easier. Image: TM Fulton Department of Biotechnology,AC&RI,Madurai-
  • 26. Advantages of MAB: knowledge Using markers can also give us a deeper understanding of the traits we are selecting for and HOW they work. This could allow for more efficient selection in the future. For example, once a marker – trait correlation is established, the marker can be used to clone the gene, and more thoroughly study its action. In tomato, a major QTL affecting fruit weight was cloned and found to control carpel cell number early in fruit development (Frary et al. 2000). Department of Biotechnology,AC&RI,Madurai-
  • 27. MAB: Costs Using molecular markers requires the use of specific laboratory equipment, at the very least a PCR (polymerase chain reaction) thermalcycler and electrophoresis and visualization equipment. So start-up costs can be high (although these may be compensated for by later savings). A PCR machine and a basic agarose electrophoresis apparatus. Department of Biotechnology,AC&RI,Madurai-
  • 28. Crop Domestication: From plants in the wild to our kitchen Over time, humans have selected those plants that exhibited traits that are in OUR (humans) interests: larger fruit, more kernels. Examples of cultivated varieties and their wild relatives. Images: Steven Tanksley, John Doebley Department of Biotechnology,AC&RI,Madurai-
  • 29. Crop Domestication Crop domestication inherently decreases genetic variation, by the selection of just a few of the available lines (those with traits seen as desirable by the selectors, ie. humans) Department of Biotechnology,AC&RI,Madurai-
  • 30. Traits selected for by humans Traits that have been selected for by humans include: • Determinate growth habit (flowering occurs at the top of the plant, preventing further growth) • Retention of mature seed on the plant (loss of grain shattering) • Synchronous ripening, shorter maturity • Lower content of bitter tasting and harmful compounds • Reduced sprouting, higher seed dormancy • improved harvest index (the proportion of the plant which is used); larger seed or fruit size • elimination of seeds, such as in banana Many of these trait changes reduce the ability for the plant to compete in the wild, and also decrease the genetic variability remaining in the crop. Department of Biotechnology,AC&RI,Madurai-
  • 31. Consequences of loss of genetic diversity: One result of less diversity is that consumers and farmers are now accustomed to, and demand, uniformity: round red apples, plants all the same height in the field. But the loss of genetic diversity can have devastating consequences, such as the Irish potato blight of 1850, the Southern corn leaf blight of 1970, and the current crisis in banana, Black Sigatoka disease, shown above. Banana image Copyright 2001 by The American Phytopathological Society,; apple photo ourtesy of New York Apple Association
  • 32. Genetic diversity is available in genebanks Fortunately, many wild relatives of our crops have have been saved in genebanks around the world. Alleles that can be naturally introgressed in from wild relatives of crop plants can not only increase their genetic diversity but improve them for traits that would not be predicted by looking at their phenotypes (Tanksley and McCouch 1997). As of 2006, the CGIAR centers (Consultative Group on International Agricultural Research) together contain more than 650,000 accessions of crop, forage and agroforestry species (2006 Bioversity International). Photo: CGIAR-IRRI Department of Biotechnology,AC&RI,Madurai-
  • 33. Germplasm banks Most crops have many accessions stored in genebanks, or germplasm banks, that are available free of charge or with a shipping and handling fee, for example, the USDA-ARS National Plant Germplasm System (http://www.ars- The CGIAR system has a number of genebanks around the world: The International Rice Research Institute (IRRI) genebank in Los Banos, Philippines, has over 80,000 accessions of rice. Department of Biotechnology,AC&RI,Madurai-
  • 34. Science 20 :November 2009: The B73 Maize Genome: Complexity, Diversity, and Dynamics Nature 457, 551-556 (29 January 2009) The Sorghum bicolor genome Nature Biotechnology 30, 549–554 (13 May 2012) Genome sequence of foxtail millet Staking of key traits through marker assisted breeding Department of Biotechnology,AC&RI,Madurai-
  • 35. UMI 79 UMI 936(W)X F1 F2 F6 Molecular tagging of downy mildew resistance in maize and introgression into elite inbred lines phi053(21.3) bnlg420(0) dup23(80.4) bnlg1185(141.7) umc1223(53.8) umc1594(0) bnlg420(61.2) bnlg197(111.4) phi053(45.7) umc1594(828.9) phi088(596.49) phi046(605.44) bnlg197(511.5) bnlg420(318.4) phi073(344) bnlg1035(313.4) phi053(297.9) umc1223(234.4) phi029(168.08) phi099(159.0) phi104127(38.0) IBM2 2008 neighbors2 24 recombinant Nair et al.,2005 Kashmiri ,2010 Figure 12 a. Genetic linkage map showing location of SDM QTL on chromosome 3 on different mapping population CM139 XNAI116 UMI 79XUMI936(w) - SDM QTL Screening of RILs in artificial epiphytotic condition for sorghum downy mildew (P. sorghi) reaction Recom binant lines X Elite inbred Hybrid F1
  • 36. Marker assisted introgression of LycE /CrtRB1 gene for enhanced Pro VitA in maize The back cross progenies of UMI 1200 ( 1.16 μg/g β-carotene and popular inbred) x HP467-15 (5.10 μg/g β-carotene and CIMMYT donor) are under evaluation. HPLC analysis also revealed a considerable improvement in the β-carotene of selected F1 (1.50 μg/g ) and BC1F1 progenies ( 2.2 μg/g) as compared to the well-adapted low β-carotene inbred (UMI 1200). BC2 F2 progenies UMI 1200 HP467-15 Particula rs UMI 1200 HP 467- 15 Standard β- carotene -Type I β- carotene (μg/g) 1.16 5.1 10.0 Peak area 85,95 9 1,58,628 3,83,815 Rt 23.50 23.50 23.50
  • 37. CrtRB1 gene based marker screening HPLC Screening- UMI 1200 β-carotene 1.16 µg/g UMI 1200 : Allele 2 HP467-15 : Allele 1 UMI 176 : Yellow grain β-carotene : 7.92 µg/g crtRB1 3’TE gene Specific marker 296+875 bp 296+1221 bp 543 bp HP 467-15 Yellow grain β-carotene : 5.10 µg/g Co-dominant PCR assays for analyzing allelic variations at 3’TE site of crtRB1 gene among the maize inbreds. : Lane 1-UMI 936(O); Lane2-UMI 112; Lane 3- UMI 101; Lane 4-UMI 80; Lane 5-UMI 61; Lane6-UMI 176; Lane 7-UMI 1230; Lane 8-UMI 551; Lane 9- HP467-15; Lane 10-UMI 190; Lane 11-UMI 285; Lane 12-UMI 1200; Lane 13-UMI 69; Lane 14- UMI 395, Lane M: 100 bp DNA ladder. Thirusenthura selvi et al. 2014 Food Biotechnology 28:41-49
  • 38. Nutrigenomics Department of Biotechnology,AC&RI,Madurai-
  • 39. Department of Biotechnology,AC&RI,Madurai-
  • 40. Metabolomics Department of Biotechnology,AC&RI,Madurai-
  • 41. Ionomics Department of Biotechnology,AC&RI,Madurai-
  • 42. Thank you Department of Biotechnology, Tamil Nadu Agricultural University AC&RI,