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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, Madurai

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

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Page 1: Genomics platform for agriculture-CAT lecture

Prof. Senthil NatesanDepartment of Biotechnology, AC&RI, Madurai

Bio-Technology…… Genomics platform for agriculture

www.tnau.ac.in Department of Biotechnology,Tamil Nadu Agricultural University AC&RI, Madurai

Page 2: Genomics platform for agriculture-CAT lecture

“….. 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

Page 3: Genomics platform for agriculture-CAT lecture

Genomics time line

Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com

Page 4: Genomics platform for agriculture-CAT lecture

Crop and plant genomes and their application. The figure gives the approximate timeline of when crop genomes were sequenced along with theunderlying techniques and sequencing strategy used. Hybrid strategies which use BAC by BAC and WGS are indicated by the placement ofa genome twice. Also note that the distinction between pure NGS and Hybrid sequencing is sometimes arbitrary as many genome projects rely onpreviously generated Sanger sequences. In addition, some major applications are marked by symbols: Grains for an improvement in grain quality, aflower for flowering time and a tomato for a tomato ripening trai

Department of Biotechnology, AC&RI, Madurai-www.tnaugenomics.com

Page 5: Genomics platform for agriculture-CAT lecture

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

Page 6: Genomics platform for agriculture-CAT lecture

Department of Biotechnology, AC&RI ,Madurai-www.tnaugenomics.com

Page 7: Genomics platform for agriculture-CAT lecture

Humans Have Limited Molecular Diversity

0.09%

Zhao et al, 2000, PNAS

1.34%

Department of Biotechnology, AC&RI, Madurai-www.tnaugenomics.com

Page 8: Genomics platform for agriculture-CAT lecture

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

Page 9: Genomics platform for agriculture-CAT lecture

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

Page 10: Genomics platform for agriculture-CAT lecture

The Human Genome ProjectThe 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

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Page 11: Genomics platform for agriculture-CAT lecture

Whole genome sequencingWhile 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

Page 12: Genomics platform for agriculture-CAT lecture

Automated sequencing reactions - each reaction can resolve 600 to 750 bp

(labeled with fluorescent dyes)

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Page 13: Genomics platform for agriculture-CAT lecture

FISH analysis of the centromeric core of chromosome 5 in Rice

The schema of constructing a physical map ofrice chromosome 5.

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Comparing genomes: Example from the grassesThis 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

Page 15: Genomics platform for agriculture-CAT lecture

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

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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

Page 17: Genomics platform for agriculture-CAT lecture

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

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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

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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 toflanking sequence of microsatellite

PCR analysis and analyze on 6 %denaturingpolyacrylamide gel with silver staining

A BSchematic of SSR assay

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Detection of PCR product

www.tnau.ac.in Department of Biotechnology,Tamil Nadu Agricultural University AC&RI, Madurai

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SSR

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Page 22: Genomics platform for agriculture-CAT lecture

Microsatellite markers polymorphism between parental lines and rice hybrids

Tamilkumar et al.,2009

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Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com

Page 24: Genomics platform for agriculture-CAT lecture

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

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Page 25: Genomics platform for agriculture-CAT lecture

Advantages of MAB: CostDepending 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

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Page 26: Genomics platform for agriculture-CAT lecture

Advantages of MAB: knowledgeUsing 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- www.tnaugenomics.com

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MAB: CostsUsing 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.

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Page 28: Genomics platform for agriculture-CAT lecture

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

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Page 29: Genomics platform for agriculture-CAT lecture

Crop DomesticationCrop 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)

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Page 30: Genomics platform for agriculture-CAT lecture

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.

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Page 31: Genomics platform for agriculture-CAT lecture

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, http://www.apsnet.org/education/feature/banana/; apple photo ourtesy of New York Apple Association

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Genetic diversity is available in genebanksFortunately, 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- www.tnaugenomics.com

Page 33: Genomics platform for agriculture-CAT lecture

Germplasm banksMost 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-grin.gov/npgs/).

The CGIAR system has a number of genebanks around the world: http://www.cgiar.org/impact/accessions.htm.

The International Rice Research Institute (IRRI) genebank in Los Banos, Philippines, has over 80,000 accessions of rice.

Department of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com

Page 34: Genomics platform for agriculture-CAT lecture

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

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Page 35: Genomics platform for agriculture-CAT lecture

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

Recombinant lines

X Elite inbred

Hybrid F1

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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

Particulars

UMI 1200

HP 467-15

Standard β-carotene -Type I

β-carotene (μg/g)

1.16 5.1 10.0

Peak area 85,959

1,58,628 3,83,815

Rt 23.50 23.50 23.50

Page 37: Genomics platform for agriculture-CAT lecture

CrtRB1 gene based marker screening

HPLC Screening- UMI 1200β-carotene 1.16 µg/g

UMI 1200 : Allele 2HP467-15 : Allele 1

UMI 176 : Yellow grainβ-carotene : 7.92 µg/g

crtRB1 3’TE gene Specific marker

296+875 bp

296+1221 bp

543 bp

HP 467-15 Yellow grainβ-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

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NutrigenomicsDepartment of Biotechnology,AC&RI,Madurai- www.tnaugenomics.com

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Metabolomics

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Page 41: Genomics platform for agriculture-CAT lecture

Ionomics

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Page 42: Genomics platform for agriculture-CAT lecture

Thank you

www.tnau.ac.in Department of Biotechnology,Tamil Nadu Agricultural University AC&RI, Madurai