Download Dr. Rajeshwari - IGMORIS - Indian GMO Research Information System

Plant Genomics : Molecular Farming
Dr Villoo Morawala Patell
(CEO, Avesthagen, Bangalore)
•Basis of all breeding is selection for traits of
interest.
•High yield, better quality, increased tolerance to
adverse environmental conditions, resistance to
pests and pathogens, etc.
Marker Assisted Selection
•Selection for a trait made on the basis of
presence or absence of a marker
•Marker could be a morphological marker
•Marker could be a DNA-based molecular
marker
Morphological Markers
Traditionally used for obtaining plants with desired traits
Eg. A gene conferring resistance to BPH is closely linked
to a gene for purple color of the coleoptile in rice
Purple X
BPH (R)
Green
BPH (s)
F1
F2
Purple: Resistant to BPH
Green: Susceptible to BPH
Morphological Markers : Limitations
•Very few morphological markers known
•Tend to be specific for particular varieties
•Most are mutations deleterious to the plant
•Approach limited to traits controlled by single genes
•Does not apply to traits governed by multiple unlinked
genes – QTLs.
Molecular marker based MAS
R
M
R
M
R
M
R
M
R
M
Marker present
Resistant
R
M
X mS
S
m
S
m
S
m
Marker present
Resistant
S
m
S
m
Marker absent
Susceptible
Advantages of MAS
•Selection at seedling stages
•Selection not subject to environmental constraints
•Selection for more than one trait in a breeding cycle; pyramiding of genes possible
•Selection for quantitative traits possible
•Enables breeders to distinguish heterozygotes from homozygotes in self-pollinated
crops.
•Non-destructive analysis – plants can be evaluated for other agronomic traits
•Offers opportunity to look for diversity in breeding populations.
•Smaller land, smaller maintenance field staff, scoring time identifying individuals with
desired traits greatly reduced.
•Whole operational time reduced by 2-3 years.
•Cost effective process
Uses in Breeding
•Pyramiding of genes
•Breeding for polygenic traits
•Removal of linkage drag
•Introgression from exotic germplasm
•Breeding by design
Pyramiding of Xa-4, xa-5, xa-13 and Xa-21 into rice (Huang et al., 1997)
Lines
Gene
Reaction
to races
of
BB
1
2
3
4
5
6
IRBB4
Xa-4
R
S
S
S
R
S
IRBB5
xa-5
R
R
R
MS
R
S
IRBB13 xa-13
S
S
S
S
S
R
IRBB21 Xa-21
R
R
R
R
R
R
Pyramiding of the four blight resistance genes would
provide durable resistance to all the 6 biotypes of
bacterial blight
Pyramiding of Xa-4, xa-5, xa-13 and Xa-21 into rice
Xa-4 X xa-13
Xa-4/xa-13
Xa-21 X xa-5
X
Xa-21/xa-5
Xa-21/Xa-4/xa-13/xa-5
Xa-4/xa-13
Four lines positive for markers for Xa-4 and xa-1
xa-5/Xa-21
Three lines positive for markers for xa-5 and Xa-2
Xa-4/xa-13/xa-5/Xa-21 Two lines positive for markers for all four genes
Linkage Drag
Development of lettuce resistant to the aphid Nasonovia ribisnigri
(Jansen 1996)
All
field grown lettuce susceptible to
aphid.
Wild relative resistant to aphid.
Crossing resulted in lettuce R to
aphid, but with poor quality, bearing
yellow leaves & reduced head.
Caused by a negative trait closely
linked to R gene.
Field Variety
Susceptible
Good Quality
Wild variety
Resistant
Poor Quality traits
F1
Susceptible
Good Quality
Resistant
Good Quality
Resistant
Poor Quality
Field Variety
Susceptible
Good Quality
Wild variety
Resistant
Poor Quality traits
F1
Susceptible
Good Quality
Resistant
Good Quality
Resistant
Poor Quality
Linkage Drag
Field Variety
Susceptible
Good Quality
Wild variety
Resistant
Poor Quality traits
F1
Susceptible
Good Quality
Resistant
Good Quality
Resistant
Resistant
Resistant
Poor Quality Good Quality Good Quality
1000 F2 plants screened to identify recombinants
Linkage Drag

Screen for recombinants with marker flanking the
gene. Only recombinants selected. Screening 1000 F2
led to selection of 100 individuals with recomb in this
region
F3
obtained from recombinants. One individual bearing
recomb events close to each side of the gene, thereby
removing linkage drag obtained
Introgression from exotic germplasm
X
Oryza sativa
(blight susceptible)
Oryza longistaminata
(blight resistant)
Introgression of small chromosome segment
including the Xa21 gene for bacterial blight
resistance
Breeding for Polygenic traits
Most agronomically important traits like nutritional quality, yield,
flowering time and durable resistance follow complex polygenic
inheritance patterns
Multiple genes have small effects on the trait value
Complex phenotype can be separated into separate genetic components
Yield: Root size, plant size, fruit number, size of fruit, fruit content, etc
Each component is itself affected by a number of loci
MAS is the only directed method of pyramiding the large no. of genes
for getting specific desired results
Breeding by design
•Choose traits in the crop of choice
•Breakdown complex traits into components
parts
•Pyramid all the loci using marker assisted
selection
•All available germplasm as well as wild species
can be used for the breeding
Benefits of Marker Aided Selection






Selection of traits expressed late in the growth season. Benefits
are multiplied when multiple traits are selected.
Eliminate unwanted traits from the exotic donor parent.
Pyramiding genes from diverse sources. MAS offers breeder an
opportunity to combine desirable genes into the individuals of the
same line.
MAS may be used to protect plant breeders’ rights.
High Levels of Similarity of Certain Genes of diverse groups
 Such genes can be isolated individually, characterized for
their function.
 Once their functions are determined, they can be
transferred across species and even genus barrier.
MAS allows breeders to access a large varied germplasm
collection/pools in their crops of interest.
Markers @ Avesthagen
•Marker Assisted Selection for breeding of varieties with
specific traits of interest
•MAS for pyramiding of genes
•Development of markers used for MAS
•Development of markers for predicting secondary
metabolite levels in medicinal plants
•Use of markers for contamination testing
•Use of markers for genetic diversity studies
Pyramiding of genes for resistance to pests and pathogen
204 parental varieties screened for 13 genes
2 varieties found to have right combination of genes
Donor X Recipient
F1 X Recipient
BCF1 X Recipient
2/50 BCF1 carried desired genes.
1500 BCF2 screened for genes
Expected time to market reduced by 3-4 years
Detection of adulterants of Basmati
RM9
RM55
Pure Basmati
112
164
Pusa Basmati
128
215
Tericot
128
225
Ratna
many
225
Kasturi
176
225
Microsatellite markers can be used for differentiating
different varieties
Detection of adulterants in Basmati.
25 microsatellite markers were used for screening Basmati
and its adulterants mixed in different ratios. The lowest
percentage of mixture that could be detected was determined.
•Mixtures of 4 different varieties can be detected.
•1% and more adulteration is very easily detected.
•The method is highly reliable and reproducible.
Seed Purity Testing
Microsatellite marker kits being developed for cost effective
seed purity testing.Cuts the time to test drastically.
Development of markers to be used in breeding
• Development of markers linked to specific traits limiting step in the production of value-added new
varieties




RFLP
RAPD
AFLP
Microsatellite
ISSR
SSCP
SNP
Comparison of Markers
DNA fingerprinting:
•
Genotype identification
•
Analysis of genetic diversity
•
Estimation of genetic relatedness
Genotype Identification
•Highly useful for the unequivocal identification and
discrimination of plant cultivars, micropropagated
plants, apomictic plants and clonally propagated plants.
•Currently used extensively for fingerprinting of
commercial seeds for protection
DNA Fingerprinting of Tomato samples: Avesthagen
OPC6 OPC19 OPD3 OPE11 OPE16 OPF2 OPF10 OPG18
1 2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
DNA Fingerprinting in Watermelon: Avesthagen
OPC6 OPC19 OPD3 OPE11 OPE16 OPF2 OPF10 OPG18
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
Estimation of genetic relatedness and genetic diversity
From the extent of band sharing between the
individuals of two species, it is possible to derive an
idea of the genetic relatedness between two samples.
Softwares for determining the genetic relatedness are
of high value.
By the same token, the amount of genetic diversity
available within a species can also be estimated from
an analysis of the fingerprints of individuals of each
species.
GMO Testing

Genetically modified (GM) crops are increasingly being
introduced into the world's food supply and concerns are raised
with regards to :




Potential gene flow to other organisms
Allerginicity
Antibiotic resistance
Gastrointestinal problems

Regimes are attempting to address issues like labeling,
disclosure and framing legislation.

Stringent Legislation enacted in Europe and Japan for trading of
GMO products
EU Regulations on GM food & Feed

On 2 July 2003, European Parliament adopted its
second reading opinion on two Commission
proposals on GMOs which establish a clear EU
system to trace and label GMOs & to regulate the
placing on the market and labelling of food and feed
products derived from GMOs.

According to Davis Byrne - Health and Consumer
protection Commissioner,EU will have the most
rigorous pre-marketing assessment of GM food and
feed in the world , which will give consumers greater
confidence that the safety of GM products will be
independently assessed by the European Food
Safety Authority.
GMO Testing Technique
Technique Applied : Polymerase Chain Reaction (PCR)
Sensitivity of PCR based GMO testing :
 GMO tests are capable of detecting as little as 10100
copies of GM DNA which is well below
0.001%.
 Sensitivity diminishes depending on the degree of
processing of the sample
 Limit of Detection (LOD) is 0.01%.
 Limit of Quantitation is 0.1%
What crops are GM?
Papaya
Rice
Cotton
Tomato
Soybean
Chicory
Rapeseed
Potato
Flax
Melon
Squash
Corn
Sugar beet
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employed
 Tests run in duplicate and compared to external standards.
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