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.
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

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%

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

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

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

Automated sequencing reactions - each reaction can resolve 600 to 750 bp
(labeled with fluorescent dyes)

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).

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

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).

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

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

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
Biotechnology,

SSR
•

Microsatellite markers polymorphism between
parental lines and rice hybrids
Tamilkumar et al.,2009

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

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

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).

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.

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

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)

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.

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

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

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-
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.

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

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

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

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

Nutrigenomics

Metabolomics

Ionomics

Thank you
Biotechnology,

Genomics platform for agriculture-CAT lecture

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Genomics platform for agriculture-CAT lecture