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

F1

Eg. A gene conferring resistance to BPH is closely linkedto a gene for purple color of the coleoptile in rice

Purple X GreenBPH (R) BPH (s)

F2

Purple: Resistant to BPHGreen: 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

RR

MMRR

MMSS

mmSS

mmX

SS

mmRR

MMRR

MMRR

MM

SS

mmSS

mm

SS

mmRR

MM

Marker presentResistant

Marker presentResistant

Marker absentSusceptible

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

  LinesLines GeneGene ReactionReaction to to races races of of BBBB

11 22 33 44 55 66  IRBB4IRBB4 Xa-4Xa-4 RR SS SS SS RR SS  IRBB5IRBB5 xa-5xa-5 RR RR RR MSMS RR SS  IRBB13IRBB13 xa-13xa-13 SS SS SS SS SS RR  IRBB21IRBB21 Xa-21Xa-21 RR RR RR RR RR RR        

        

Pyramiding of Xa-4, xa-5, xa-13 and Xa-21 into rice (Huang et al., 1997)Pyramiding of Xa-4, xa-5, xa-13 and Xa-21 into rice (Huang et al., 1997)

Pyramiding of the four blight resistance genes would provide durable resistance to all the 6 biotypes of bacterial blight

Xa-4/xa-13 Xa-4/xa-13 Four lines positive for markers for Xa-4 and xa-1 Four lines positive for markers for Xa-4 and xa-1xa-5/Xa-21 xa-5/Xa-21 Three lines positive for markers for xa-5 and Xa-2 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 genesXa-4/xa-13/xa-5/Xa-21 Two lines positive for markers for all four genes

Xa-4 X xa-13 Xa-21 X xa-5

Xa-4/xa-13 Xa-21/xa-5X

Xa-21/Xa-4/xa-13/xa-5

Pyramiding of Xa-4, xa-5, xa-13 and Pyramiding of Xa-4, xa-5, xa-13 and Xa-21 into rice Xa-21 into rice

Linkage DragDevelopment 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.

SELFING

  

Linkage Drag

Field VarietySusceptibleGood Quality

Wild varietyResistantPoor Quality traits

F1

SusceptibleGood Quality

Resistant Good Quality

Resistant Poor Quality

SELFING

  

Linkage Drag

Field VarietySusceptibleGood Quality

Wild varietyResistantPoor Quality traits

F1

SusceptibleGood Quality

Resistant Good Quality

Resistant Poor Quality

SELFING

  

Linkage Drag

Field VarietySusceptibleGood Quality

Wild varietyResistantPoor Quality traits

F1

SusceptibleGood Quality

Resistant Good Quality

Resistant Poor Quality

Resistant Good Quality

1000 F2 plants screened to identify recombinants

Resistant Good Quality

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

Linkage Drag

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

Oryza sativa

X

Oryza longistaminata(blight resistant)

Introgression of small chromosome segment including the Xa21 gene for bacterial blight resistance

(blight susceptible)

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 for Polygenic traits

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 Marker Assisted Selection for breeding of varieties with specific traits of interestspecific traits of interest

•MAS for pyramiding of genesMAS for pyramiding of genes

•Development of markers used for MASDevelopment of markers used for MAS

•Development of markers for predicting secondary Development of markers for predicting secondary metabolite levels in medicinal plantsmetabolite levels in medicinal plants

•Use of markers for contamination testingUse of markers for contamination testing

•Use of markers for genetic diversity studiesUse of markers for genetic diversity studies

Pyramiding of genes for resistance Pyramiding of genes for resistance to pests and pathogento 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

2/50 BCF1 carried desired genes.

1500 BCF2 screened for genes

Expected time to market reduced by 3-4 years

BCF1 X Recipient

RM9 RM55

Pure Basmati 112 164

Pusa Basmati 128 215

Tericot 128 225

Ratna many 225

Kasturi 176 225

Detection of adulterants of Basmati

Microsatellite markers can be used for differentiating different varieties

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.

Detection of adulterants in Basmati.Detection of adulterants in Basmati.

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

ISSRSSCPSNP

Comparison of Markers

• Genotype identification

• Analysis of genetic diversity

• Estimation of genetic relatedness

DNA DNA fingerprintingfingerprinting::

Genotype Genotype IdentificationIdentification

•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 DNA Fingerprinting of Tomato samples: Avesthagensamples: 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 diversityEstimation 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 GMO TestingTesting

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 & EU Regulations on GM food & FeedFeed

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 GMO Testing TechniqueTechnique

Technique Applied : Polymerase Chain Reaction (PCR)

Sensitivity of PCR based GMO testing :

GMO tests are capable of detecting as little as 10-100 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 What crops are GM?are GM?

Sugar beet

Cotton

Chicory

Corn

MelonPotato

Rice

Squash

Rapeseed

Papaya

Flax

Soybean Tomato

Why AQUAS ?Why AQUAS ?

Customized programs to meet your unique needs

Fully equipped laboratory & Personnel trained and certified

We will maintain an uncompromising commitment to quality and client service by providing accurate results, delivered on time, according to customer requirements

The most rigorous and internationally recognized techniques are employed

Tests run in duplicate and compared to external standards.

Rigourous Systems based approach yielding reliable results.

Responsive service, competitive prices

Comprehensive services - analytical, consulting and research services to protect your company’s assets so you can be assured

Thank You!!