cisgenesis and intragenesis

WELCOME

5/22/2015 1MAHESH R HAMPANNAVAR

MAHESH R HAMPANNAVARGenetics and Plant [email protected]

2

Farmers fate , hungers and malnutrition are barriers in developing nations

5/22/2015 MAHESH R HAMPANNAVAR

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Breeder who facing failure to come up with potential variety


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Respect the nature Nature for our desire not for grid ………


What is solution for all these?


MAHESH R HAMPANNAVAR 6

Sisters in Innovative Plant BreedingCisgenesis and Intragenesis ;

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Contents Introduction

Definition Pre-requisite for cis/intragenesis

Case study Comparison

Bio-safety measures Potential and disadvantage

Conclusion

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• Environmentally sound and efficient

production method

• Exclusive use genetic material from same species or related species

why it require?

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CISGENIC

Schouten et al. (2006)

“A cisgenic is a crop plant that has been genetically modified with one or more genes isolated from crop plant”

What is definition?

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the intragenic concept as the isolation of specific genetic elements from a plant, recombination of these elements in vitro and insertion of the resulting expression cassettes into a plant belonging to the same sexual compatibility group

INTRAGENIC

Rommens et.al.(2004)5/22/2015

MAHESH R HAMPANNAVAR 115/22/2015


(i). One or more identical

copy of the endogenous

gene including its promoter,

introns and the terminator.

(ii). No in vitro rearrangements

cisgenic can harbour-

What it contain ?

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(i). Combination of genetic elements

from sexually compatible gene

pool.(ii). Coding regions of 1 gene

can combine with the promoters and terminators from different genes of the

sexually compatible gene pool.

(iii). T-DNA border sequences for Agrobacterium

mediated transformation

isolated from sexually compatible DNA pool known as P- Borders.

Intragenic can harbour-

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. cisgenesis

intragenesis

Within species or related spice

Intragesis

P-DNA boarders

In vitro rearrangement

What is similarities and difference?

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Variations in definitions of coding-, regulatory-, border- and vector-backbone-sequences used for intragenesis and cisgenesis

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What is Sources of genes ?

l

Tertiary gene pool

Secondary gene pool

Primary gene pool

Breeder gene pool

Quaternary gene pool

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What are all Pre requisites of cis/intragenic plant?

• Sequence information of plant

• The isolation and characterization of gene of interest from crossable relatives

• Transformation technique• Marker free transformation• Intragenic vectors

development5/22/2015


Web site address for data base info..

• Database integrating genotypic and phenotypic data useful to finding candidate genomic regions involved in agronomic traits of interest

Crop plant world wide web address

Grass http://www.gramene.org/qtl/index.html

Grape http://www.vitaceae.org

Tomato http://164.107.85.47:8004/cgi-bin/_information.plhttp://zamir.sgn.cornell.edu/Qtl/Html/home.htm

Potato http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/potatoes/mappingqtls

Cucurbitaceous www.icugi.org

Rosaceous http://www.bioinfo.wsu.edu/gdr/

Various http://www.phenome-networks.com/

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http://www.gramene.org/qtl/index.html

http://www.vitaceae.org/

http://164.107.85.47:8004/cgi-bin/_information.pl

http://zamir.sgn.cornell.edu/Qtl/Html/home.htm

http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/potatoes/mappingqtls

http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/potatoes/mappingqtls

http://www.icugi.org/

http://www.bioinfo.wsu.edu/gdr

http://www.phenome-networks.com/


General scheme for the selection, confirmation and introduction of alleles from the breeder’s gene pool

• QTL mapping • Fine mapping • LD mapping • Transient Assays(VIGS)• New alleles TILLING/eco• Translational genomics

• Phenomics • synteny QTL maps• BIBAC libraries • transformation large

fragment • Validation of standard

transfer

Confirm gene of interest

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Table . Examples of Traits That Can Be Incorporated into a Plant by either Transferring or Modifying the Expression of Native Genes

(Rommens, 2004)

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Table . Examples of currently available native traits

(Rommens, 2004)

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Transformational technique

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Methods to produce the marker free

cis/intragenesis plant

Co-transformation

Site-specific recombinase-

mediated marker

Transposaon-based expelling

systems

Intrachromosomal

recombination based excision

Transformation without marker

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Co-transformation

Borys Chong.et.al5/22/2015

http://www.intechopen.com/source/html/44799/media/image1.png


Site-specific recombinase-mediated marker

Targeted site Recombinase source lox Cre Bacteriophase P1

FTR FLP Saccharomyces cerevisiae

RS RS Zygosaccharomyces rouxii

Excised and lost




Transposon-based expelling systems




Methods used to produce marker-free intragenic/cisgenic plants

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Development of Intragenic vector

• Intragenic vector system is an extension of the minimal T-DNA vector system.

Plasmid

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Intragenic vector

a plant derived T-DNA like region that should contain

2 or at least 1 T-DNA border like sequences in the correct orientation.

an origin of replication(ori)

a selectable antibiotic gene

segment

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The P-DNA Approach

• Rommens et al. in 2004.• A series of border specific degenerate primers, putative P-DNA’s

were isolated from pooled DNA’s of 66 genetically diverse potato accessions by PCR.

• The amplified fragment were sequenced. • And this information was used for inverse PCR with nested

primers to determine the sequence of the border like regions.

• This information allowed the identification of sequences with sufficient similarity to Agrobacterium T-DNA border sequences.

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A T-DNA-like region assembled from Petunia hybrida (petunia) ESTs

Source-Euphytica (2007) 154:341–3535/22/2015


• may horizontal gene transfer from bacteria

• T-DNA border-like sequences – rice, tomato, potato, Arabidopsis

• Replace - T-DNA for transformation

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Disadvantages P-DNA

• Found in some species only.

• P-DNA

• Probability of finding such features on a single relatively short fragment in a plant genome is extremely small.

• Reduced frequencies of gene transfer.(Rommens et al.2005)

Left boarder 1-2 kb apart / Restriction sites Right boarders

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Origin of replication

• The smallest known prokaryotic origins of replication are

the 32-33bp

• Helper plasmid-provide specific factor for replication . • The Col E- characterized by 2 direct repeat sequences of 7-

9bp separated by 5-8bp. • BLAST searches of plant ESTs with sequences similar to

ColE2 or ColE3 identified in numerous species

Bacteria Plasmid Ori of replicationE.coli Colicin E2 ColE2Shigella sp. Colicin E3 ColE3

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Selectable Marker

• Derived from plant sequences. • Mutant forms of the endogenous genes specific

herbicides resistance.• Over expression of the endogenous Atwbc19 ABC

transporter gene confers kanamycin resistance (Mentewab and Stewart 2005).

• For easy to transform crops such as potato, selectable marker genes are unnecessary (de Vetten et al.2003)

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Considerations for Proper Design of Intragenic Vectors

• T-DNA not from regulatory(promoter) elements of plants

• The DNA fragment should not derived from heterochromatic

region.

• Significant length of 1-2kb of intragenic DNA occurs outside the

left border.

• Small number of DNA fragments

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Objectives•Improve the existing varieties with disease resistance•Stacking of multiple R genes – broad spectrum resistance

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Materials and Methods

• Plant materials• Potato varieties –Atlantic , Bintje , potae9• Five Phytophthora infestans isolates and late

blight resistance test

IPO-C 90128EC1 Pic99189 DHD11

European American Korean 5/22/2015


Vector construction-• Resistance governing genesi. Rpi-vnt 1.1- S . venturiiii. Rpi-sto 1 - S. stoniliferum• pBINPLUS – binary vector• pBINAW2- modified form of pBINPLUS

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Potato transformation Explants – 4 week-old in vitro grown plants

Pre cultured on R3B medium supplemented with PACM- for two days

Explants were inoculated with agrobacterium strain AGL1+VirG+Binary plasmid –Two days

Explants transferred to GCVK medium for shoot regeneration

Shoot were transferred to CK medium for root regeneration

Three week later rooted plantlets were analyzed through PCR for desired R Gene5/22/2015


DNA extraction and polymerase chain reaction

• Total genomic DNA was isolated from young leaves

• PCR positive for both R genes , PCR negative for back bone integration

• Reaction performed using DreamTaqTM

• standard PCR program (94°C for 60 s followed by 30 cycles of 94°C for 30 s, 58°C for 60 s, 72°C for 90 s and a final extension time of 5 min at 72°C).

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Gene ID Sequences (5'-3')Fragment size (bp) Detection of

Rpi-vnt1.1 forward ATGAATTATTGTGTTTACAAGACTTG 1100 T-DNA

reverse AGCATTGGCCCAATTATCATTAAC

Rpi-sto1 forward ACCAAGGCCACAAGATTCTC 890 T-DNA

reverse CCTGCGGTTCGGTTAATACA

tetA forward CTGCTAGGTAGCCCGATACG 396 Vector backbone

reverse CCGAGAACATTGGTTCCTGT

trfA forward CGTCAACAAGGACGTGAAGA 146 Vector backbone

reverse CCTGGCAAAGCTCGTAGAAC

NPTIII forwardGAAAGCTGCCTGTTCCAAAG

162 Vector backbone

reverseGAAAGAGCCTGATGCACTCC

ColE1 forward ATAAGTGCCCTGCGGTATTG 246 Vector backbone

reverse GCAGCCCTGGTTAAAAACAA

oriV forward TGCGGCGAGCGGTATCAG 1045 Vector backbone

reverse CTTCTTGATGGAGCGCATGGG

traJ forwardACGAAGAGCGATTGAGGAAA

260 Vector backbone

reverseCAAGCTCGTCCTGCTTCTCT

Primers used for PCR analysis of transformants

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Results

Var/ strains IPO-C 90128 EC1 pic99189 DHD11

Atlantic S S S S S

Bintje S S S S S

potae9 S R R S S

Dethatched leaf assay conducted for testing varieties

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• Selection and validation of cisgenic potato plant with two late blight R genes

200 stem

explants from each

variety

1515 shoots were

collected and

screened by PCR with

Rpi-vnt1.1 , Rpi-sto 1 primers

27 PCR positive

But 25 containin

g both Rpi-

vnt1.1 , Rpi-sto 1

gene

19 events are

vector back

bone free transformation

14 plants were

tested for agroinfiltr

ation

8 plants responded for to both Avrvnt 1 , Avrsto1

infiltration

Cisgenic plant

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Detached leaf assays for cisgenic transformant H43-7.

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WHY APPRECIATE THIS TECHNIQUE?

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• Conclusion of case study

cisgenesis

C.M. ROMMENS 20075/22/2015


Comparable with traditional introgression resistance breeding using same gene pool.

Enhance the breeding speed to obtain durable multigenic resistance

How cis/intragenic can overcome problems of introgression breeding?

Cis/intrgenic

Conventional breeding

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How cis/intragenic plants can overcome problems of transgenic plants?

No change in fitness

No need sequence information of other species

No alter in gene pool

No additional traits in recipient spp.

Cisgenesis/ intragenic

Transgenesis

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Potential of these concept

• Improving traits with limited natural allelic variation

• Higher expression level of a trait• Hybrid gene & silencing constructs.• US and Europe, acceptable to a greater

number of people than transgenic crops

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Regulatory guidelines

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•Comparison hazards with other technique ( conventional, transgenic)

• The Panel concluded that-(i). Similar hazards can be associated with cisgenic and

conventionally bred plants while(ii). Novel hazards can be associated with intragenic and

transgenic plants.

(iii). No new guidelines for risk assessment.

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• All of these breeding methods can produce - unintended effects.

• Unintended effect assessed case by case

• The risk to human and animal health and the environment will depend on exposure factor(cultivation and consumption)

• For cis and intragenesis less event-specific data are needed for the risk assessment

EFSA journal 20125/22/2015


Global overview of consequences of different new plant breeding techniques for the environment and for food and

feed safety.

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Limitation of the two concepts

• Traits outside the sexually compatible gene pool cannot be introduced.

• Additional expertise and time • Not clearly define• Less transformation efficiency.

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Objection and clarification

ObjectionI. Random insertion of geneII. Mutation caused in plant

ClarificationIII. Translocation breeding , Transposable

elementIV. 2500 mutant varieties growing all around

world in different crops

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Current status of cis/intragenic crops

• In most countries, the release of cisgenic or intragenic crops currently falls under the same regulatory guidelines as transgenic crops

• The greatest expression of interest for less stringent regulations of these crops has been within the EU, the USA and New Zealand

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Intragenic/cisgenic crops developed or currently under development

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Field trials with intragenic/cisgenic crops

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Future trends

• generation and commercialization of intragenic and cisgenic crops will depend on willingness to apply less stringent regulation to these crops worldwide

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• Manipulate crop within gene of same species

• Overcome existing methods in some aspects

Strengths

• Low transformation efficiency

• Detail sequence study of crops

Weaknesses

• Transgenic opposition• consumer preference

Opportunities

• Use of biotechnical tools

• questionable biosafety measures

Threats

SWOTAnalysis of

Cis/intragnic

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CONCLUSION

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Discussion

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cisgenesis and intragenesis

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