Department of Biology Tackling malaria with fast-track ... · update on progresspdate on progress...
Transcript of 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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
first phase hybrids
first examples of 2009 field data from Madagascar analysed in 2010
examples of glass house data analysed in 2009
CNAP hybrids
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast track plant breeding
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
Tackling malaria with fast track plant breeding
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
Tackling malaria with fast track plant breeding
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)
Tackling malaria with fast track plant breeding
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
Tackling malaria with fast track plant breeding
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…
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
Good results from field trials
Tackling malaria with fast-track plant breeding
15 January 2010: 327 Science. www.sciencemag.org
metabolitesarchitectureleaf traits
Tackling malaria with fast track plant breeding
trichomes
artemisinin yield = fresh weight x artemisinin contentartemisinin yield = fresh weight x artemisinin content
Tackling malaria with fast track plant breeding
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
Tackling malaria with fast track plant breeding
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
Tackling malaria with fast track plant breeding
Good results from field trials
metabolite analysis of leaf and extractability
interactions with commercial extractors to ensure equivalence of findings from laboratory to factory
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
multiple metabolite traits as input for parent selection / extractability impact
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
Good results from field trials
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
mapping F1
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
Tackling malaria with fast-track plant breeding
Good results from field trials
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
Tackling malaria with fast-track plant breeding
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
Tackling malaria with fast-track plant breeding
pdate on progressupdate on progressProfessor Dianna Bowles co-project leader
Tackling malaria with fast-track plant breeding
Department of Biology