department of biology tackling malaria with fast-track ... · update on progresspdate on progress...

pdate on progressupdate on progressProfessor Dianna Bowles co-project leader

Tackling malaria with fast-track plant breeding

Department of Biology

our project is based at the Centre for Novel Agricultural Products (CNAP) at the University of York, UKthe University of York, UK

charitable funding is enabling us to develop new non-GM hybrid varieties of Artemisia annua that perform as a robust, uniform and readily extracted crop, high yielding in artemisininhigh yielding in artemisinin

our purpose is to help stabilise the supply of artemisinin for use in ACTs, through no-profit activities, providing seed at low cost for successful cultivation and extraction in the commercial production regions of Africa, India p g ,and China

the project is working with the international community , with the objective of providing a reliable source of artemisinin to underpin current and future


p g pdemand for ACTs to treat and ultimately eradicate malaria

a pipeline of new hybrids, increasingly building on new genetic, metabolic and agronomic data

first phase hybrids – first field trial data from the project – Africa, India

second phase hybrids – in field trials data accumulating during 2010/2011 –second phase hybrids – in field trials, data accumulating during 2010/2011 –Africa, India, China

thi d h h b id i / i i t fi ld t i l d t l ti d ithird phase hybrids – in/going into field trials, data accumulating during 2011/2012 – existing and additional sites in Africa, India and China

identify and verify the performance of robust elite CNAP hybrids in real commercial production environments – locally recommended from conditions in each of the major geographical regions of commercial production


first phase hybrids

forward screen of mutagenised Artemis, grown up, selfed and 23 000 individual lines screened under glass. Artemisinin content was measured in each line and the top 1% (10 individuals) from each batchmeasured in each line and the top 1% (10 individuals) from each batch of a 1000 was taken forward as the ‘high-yielders’ from the forward screen.

these forward screen high-yielders have been further analysed and used as parental lines in a variety of crossing strategies to give new hybrids.

one strategy will be described in detail


30 highly distinct high-yielding parental lines

first phase hybrids

30 highly distinct high yielding parental lines, identified, selected and used in the diallel cross

the hybrids from the cross were analysed under glass and also in experimental field trials inglass and also in experimental field trials in Switzerland and Madagascar

these analyses enabled the performance of i di id l li b d i hindividual parent lines to be assessed in each cross – the general combining ability (GCA) was determined for each trait of interest


first phase hybrids

first examples of 2009 field data from Madagascar analysed in 2010

examples of glass house data analysed in 2009

CNAP hybrids


a programme of field trials underway

global and specific selection strategies

y

defined target sites for hybrid evaluation working with field trial partners in the major production regions

progressive testing of hybrids in multi-environment trials

early experimental trials - Mediplant

progressive testing of hybrids in multi environment trials (experimental hybrid trials to advanced hybrid trials)

common hybrids across the different environments in Africa, India and China

subsequent trials in commercial regions

Tackling malaria with fast track plant breeding

environments at commercial production/ field trial sitesenvironments at commercial production/ field trial sites

Kenya, India and Madagascar: relatively flat large fields which can accommodate large number of hybrids per trialg y p

Kenya Indiay


Madagascar

environments at commercial production/field trial sitesenvironments at commercial production/field trial sitesChina and Uganda: mountainous, small plots, difficult terrain to accommodate large numbers of hybrids per trial

China

Uganda


data recorded and statistical analyses

weather data during the growing season

agronomic data– plant vigourplant vigour– hybrid uniformity– die-back or leaf senescence– plant height at harvest– artemisinin content at near maturitya e s co e a ea a u y– fresh weight per plot converted to tonnes/ha– fresh weight for each plant: plot weight/ number

of plants– dry leaf weight after sieving -saleable biomass

(k /h )(kg/ha)– artemisinin yield (kg/ha; % artemisinin content

near harvest x saleable biomass)

statistical analyses: REML approach in GENSTAT


statistical analyses: REML approach in GENSTAT

results to date: trials from Madagascar and India(China Kenya and Uganda trials at full plant development harvesting to(China, Kenya and Uganda trials at full plant development, harvesting to

be completed by mid Q4 2010, data analysed by mid Q1 2011)


main messages of first phase hybrids from these sites

Madagascar –Madagascar

India – early flowering of many CNAP hybrids at Bangalore – BUT two CNAP hybrids without this problem these are being further trialled as potential goodhybrids without this problem – these are being further trialled as potential good hybrids for cultivation in India

additional data from other sites available end of Q1 2011


additional data from other sites available end of Q1 2011

seed available for commercial

first phase hybrids

the time course of the diallel

2011

2012

seedavailable for

demonstration

production

CNAPhybrid

crossing strategy using the forward screen high-yielders

June 10

2011

2008 / 09

March 09

June 10

April 2008

2008 / 09

2008 / 09 field trial data accumulating…


2007 / 08

movement of plant material

earlier stages of the project readily controlled within York – LIMS, bar‐coding, QC

later stages of the project have increasing movements of plant material into and out of York involving contracted service providers

an essential requirement to track for QCan essential requirement to track for QC

stock plants, propagated cuttings of parental lines, hybrid seeds, bulked cuttings, field trials at multiple sites involving multiple stages of handling, seed production


QC genotyping

a core set of 9 ‐13 SSR markers (2‐3 PCR multiplexes) is routinely used to check the genetic

identities of vegetative propagated stock plants and the progenies of crosses

QC genotyping

identities of vegetative propagated stock plants and the progenies of crosses

ALL hybrid seed batches are quality controlled ‐ (before pooling ‐ with 11 individuals/ cross ‐ 10%

selfing/ out crossing detected with 70% confidence; after pooling ‐ with 32 individuals/ pool ‐

10% selfing/ out crossing detected with > 95% confidence);

ALL stock plants are QC’d every 2nd round of propagation;

ALL stock plants and 5‐10% cuttings used for seed production are genotyped, automated scripts

have been created for QC data analysis;

ALL plots from field trials ‐ protocol for QC checks is in place, data accumulating

Automated scripts have been created for QC data analysis in Revolution ™ software

robust and comprehensive SOPs for genotyping,

Automated scripts have been created for QC data analysis in Revolution ™ software.

Checker interface


usage and maintenance of ABI3730xl are in place

a pipeline of new hybrids, increasingly building on new genetic, metabolic and agronomic data

first phase hybrids – first field trial data from the project – Africa, India

second phase hybrids – in field trials data accumulating during 2010/2011 –second phase hybrids – in field trials, data accumulating during 2010/2011 –Africa, India, China

thi d h h b id i / i i t fi ld t i l d t l ti d ithird phase hybrids – in/going into field trials, data accumulating during 2011/2012 – existing and additional sites in Africa, India and China

identify and verify the performance of robust elite CNAP hybrids in real commercial production environments – locally recommended from conditions in each of the major geographical regions of commercial production


comprehensive data sets for second andcomprehensive data sets for second and third phase hybrids

High QTL score for:A t i i i i ld

High artemisinin

Low levels of undesiredcompounds

• Artemisinin yield• Fresh weight• Leaf area

• Trichome density

Low heterozygosity

Genotypic dataGood hybrid parent

Metabolite data

Hybrid combinationOther characteristics

High biomass (leaf yield) High genetic distance, h iEasy to propagate

Low susceptibilityto disease

heterozygosityand genetic uniformity

High seed yield

Good results from


Good results from field trials


15 January 2010: 327 Science. www.sciencemag.org

metabolitesarchitectureleaf traits


trichomes

artemisinin yield = fresh weight x artemisinin contentartemisinin yield = fresh weight x artemisinin content


information released

360 Mb f f 1 593 505 EST f fi A t i lib i360 Mb of sequence from 1,593,505 ESTs from five Artemis librariessequences for 447 SNP and 39 SSR markers and their map positionsthe map positions of 205 AFLP markersp p134 QTL for 14 traits mapped over two field trials over two years




High artemisinin




Low heterozygosity


Metabolite data





High seed yield

Good results from



metabolite analysis of leaf and extractability

interactions with commercial extractors to ensure equivalence of findings from laboratory to factory


h 4 11 diArtemis amorpha-4,11-diene

H

O

PO

HO

OP

OHO

HO farnesyl diphosphate

Artemis

artemisinic alcohol

artemisinic aldehyde dihydroartemisinic aldehyde

H H

HO

dihydroartemisinic alcohol

artemisinic acid dihydroartemisinic acid

dihydroartemisinic acid hydroperoxide

artemisinic acid hydroperoxide

H

O H

artemisininarteannuin B artemisitene

?

HO

O

O

HH

dihydro-epi-deoxyarteannuin B


artemisinin

deoxyartemisinin

O

O

O

HO

Waters Acquity UPLC‐

separation and detection in the project at York

Thermo LTQ‐ Orbitrap‐MS (sesquiterpenes) ‐2.5 min run

Combined data:

LECO Pegasus IV GC‐TOF‐MS (monoterpenes) – 25 min run

Combined data:

• Quantitative analysis of artemisinin and selected related compounds

• ID using de novo structural elucidation, comparison with authentic standards and/orcomparison with authentic standards and/or commercial data libraries

• UPLC‐MS returns ~100 unique compounds

• GC‐TOF‐MS returns ~200 unique compounds


multiple metabolite traits as input for parent selection / extractability impact




High artemisinin




Low heterozygosity


Metabolite data





High seed yield

Good results from



crosses selected based on QTL and genotyping data

an example of parental combinations based on QTL scores

crosses selected based on QTL and genotyping data

P1_Genotype P2_GenotypeArtemisinin

scoreBiomassscore

Leaf area score

Trichome score

Heterozygosity score P1

Heterozygosity score P2 P1xP2

PopulationUniformity

C1 C4 1 0 0 0 0.407 0.686 0.578 0.528Sd‐023373 Sd‐044065 1.25 1 0 2 0.291 0.305 0.387 0.719Sd 023373 Sd 046062 2 25 2 1 1 0 291 0 427 0 333 0 713

an example of parental combinations based on QTL scores

Sd‐023373 Sd‐046062 2.25 2 1 1 0.291 0.427 0.333 0.713Sd‐023373 Sd‐027746 2.75 1 1 1 0.291 0.314 0.402 0.723Sd‐023373 Sd‐055366 2.25 1 1 1 0.291 0.513 0.404 0.656Sd‐023373 Sd‐039990 1.25 1 1 1 0.291 0.308 0.379 0.725Sd‐023373 Sd‐041060 1.25 1 1 1 0.291 0.314 0.378 0.745Sd‐023373 Sd‐024448 2.25 1 0 1 0.291 0.373 0.382 0.724Sd‐023373 Sd‐033267 2.25 1 0 1 0.291 0.345 0.483 0.704Sd‐023373 Sd‐033745 2.25 1 0 1 0.291 0.263 0.397 0.757Sd‐023373 Sd‐050309 2.25 1 0 1 0.291 0.368 0.472 0.704Sd‐023373 Sd‐056205 2.25 1 0 1 0.291 0.576 0.502 0.625Sd‐023373 Sd‐035279 1.75 1 0 1 0.291 0.385 0.463 0.707Sd‐023373 Sd‐023567 1.25 1 0 1 0.291 0.310 0.424 0.737Sd‐023373 Sd‐027453 1.25 1 0 1 0.291 0.513 0.491 0.662Sd‐023373 Sd‐031295 1.25 1 0 1 0.291 0.195 0.318 0.779Sd‐023373 Sd‐036384 1.25 1 0 1 0.291 0.364 0.436 0.716Sd‐023373 Sd‐046155 1.25 1 0 1 0.291 0.410 0.422 0.693Sd‐023373 Sd‐050839 1.25 1 0 1 0.291 0.405 0.378 0.693Sd‐023373 Sd‐055961 1.25 1 0 1 0.291 0.373 0.402 0.735Sd‐023373 Sd‐055974 1.25 1 0 1 0.291 0.347 0.415 0.741


h 2 d h 3 h b id

further crossing strategies deriving from FS high-yielders

phase 2 and phase 3 hybrids

- heritability- backcross

biparental- biparental- line cross

crossing strategies derived from the mapping populationg g pp g p p- F1 trait crosses- F2 crosses

i t t i i t l l ticrossing strategies using natural populations- forward screen high yielders- mapping F1


mapping F1



High artemisinin




Low heterozygosity


Metabolite data





High seed yield

Good results from



artemisinin is a crucially important product

the science continually reveals new t iti f i d i ti f Aopportunities for improved varieties of A.annua


roll out of the new varieties

charitable funding is enabling us to develop new non-GM hybrid varieties of Artemisia annua that perform as a robust, uniform and readily extracted crop, high yielding in artemisining y g

our purpose is to help stabilise the supply of artemisinin for use in ACTs, through no-profit activities, providing seed at low cost for successful cultivation and extraction in the commercial production regions of Africa, India and Chinap g ,

partnerships are in place with a global network of contacts – policy, pharma, extractors, growers, agricultural extension and seed producers / distributors


pdate on progressupdate on progressProfessor Dianna Bowles co-project leader


Department of Biology

department of biology tackling malaria with fast-track ... · update on progresspdate on progress...

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