Professor Dianna Bowles co-project leader

update on progress Professor Dianna Bowles co-project leader Department of Biology

our project is based at the Centre for Novel Agricultural Products (CNAP) at the 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 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 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 demand 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 Africa, India, China third phase hybrids hbid in/going i into field ti trials, dt data accumulating lti 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 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

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 in Switzerland and Madagascar these analyses enabled the performance of individual id parent lines to be assessed in each cross the general combining ability (GCA) was determined for each trait of interest

first phase hybrids examples of glass house data analysed in 2009 first examples of 2009 field data from Madagascar analysed in 2010 CNAP hybrids

a programme of field trials underway global and specific selection strategies defined target sites for hybrid evaluation working with field trial partners in the major production regions early experimental trials - Mediplant progressive testing of hybrids in multi-environment trials (experimental hybrid trials to advanced hybrid trials) subsequent trials in commercial regions common hybrids across the different environments in Africa, India and China Tackling malaria with fast track plant breeding

environments at commercial production/ field trial sites Kenya, India and Madagascar: relatively flat large fields which can accommodate large number of hybrids per trial Kenya India Madagascar Tackling malaria with fast track plant breeding

environments at commercial production/field trial sites China 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 vigour hybrid uniformity die-back or leaf senescence plant height at harvest artemisinin content at near maturity 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 (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

results to date: trials from Madagascar and India (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 India early flowering of many CNAP hybrids at Bangalore BUT two CNAP hybrids 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

first phase hybrids the time course of the diallel crossing strategy using the forward screen high-yielders CNAP hybrid seed available for demonstration 2011 seed available for commercial production 2012 June 10 June 10 March 09 2008 / 09 2008 / 09 field trial data accumulating April 2008 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 anessential requirement to trackfor 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 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 2 nd 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. Checker interface robust and comprehensive SOPs for genotyping, usage and maintenance of ABI3730xl are in place

comprehensive data sets for second and third phase hybrids High artemisinin Low levels of undesired compounds High QTL score for: At Artemisinin ii i yield ild Fresh weight Leaf area Trichome density Low heterozygosity Metabolite data Other characteristics Good hybrid parent Genotypic data Hybrid combination High biomass (leaf yield) Easy to propagate Low susceptibility to disease High genetic distance, heterozygosity and genetic uniformity High seed yield Goodresults from field trials

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

metabolites architecture leaf traits trichomes Tackling malaria with fast track plant breeding

artemisinin yield = fresh weight x artemisinin content Tackling malaria with fast track plant breeding

information released 360 Mb of sequence from 1,593,505 505 ESTs from five Artemis librariesi sequences for 447 SNP and 39 SSR markers and their map positions the map ppositions of 205 AFLP markers 134 QTL for 14 traits mapped over two field trials over two years Tackling malaria with fast track plant breeding

metabolite analysis of leaf and extractability interactions with commercial extractors to ensure equivalence of findings from laboratory to factory

Artemis amorpha-4,11-diene 411di HO O HO O P HO O P O farnesyl diphosphate H artemisinic alcohol H H dihydroartemisinic alcohol HO artemisinic aldehyde dihydroartemisinic aldehyde artemisinic acid dihydroartemisinic acid artemisinic acid hydroperoxide dihydroartemisinic acid hydroperoxide H? O O H H dihydro-epideoxyarteannuin B arteannuin B artemisitene artemisinin HO O O O deoxyartemisinin

Waters Acquity UPLC Thermo LTQ Orbitrap MS (sesquiterpenes) 2.5 min run separation and detection in the project at York 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/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

crosses selected based on QTL and genotyping data an example of parental combinations based on QTL scores P1_Genotype P2_Genotype Artemisinin score Biomass score Leaf area score Trichome score Heterozygosity score P1 Heterozygosity score P2 P1xP2 Population Uniformity C1 C4 1 0 0 0 0.407 0.686 0.578 0.528 Sd 023373 Sd 044065 1.25 1 0 2 0.291 0.305 0.387 0.719 Sd 023373 Sd 046062 225 2.25 2 1 1 0.291 0.427 0.333 0.713 Sd 023373 Sd 027746 2.75 1 1 1 0.291 0.314 0.402 0.723 Sd 023373 Sd 055366 2.25 1 1 1 0.291 0.513 0.404 0.656 Sd 023373 Sd 039990 1.25 1 1 1 0.291 0.308 0.379 0.725 Sd 023373 Sd 041060 1.25 1 1 1 0.291 0.314 0.378 0.745 Sd 023373 Sd 024448 2.25 1 0 1 0.291 0.373 0.382 0.724 Sd 023373 Sd 033267 2.25 1 0 1 0.291 0.345 0.483 0.704 Sd 023373 Sd 033745 2.25 1 0 1 0.291 0.263 0.397 0.757 Sd 023373 Sd 050309 2.25 1 0 1 0.291 0.368 0.472 0.704 Sd 023373 Sd 056205 2.25 1 0 1 0.291 0.576 0.502 0.625 Sd 023373 Sd 035279 1.75 1 0 1 0.291 0.385 0.463 0.707 Sd 023373 Sd 023567 1.25 1 0 1 0.291 0.310 0.424 0.737 Sd 023373 Sd 027453 1.25 1 0 1 0.291 0.513 0.491 0.662 Sd 023373 Sd 031295 1.25 1 0 1 0.291 0.195 0.318 0.779 Sd 023373 Sd 036384 1.25 1 0 1 0.291 0.364 0.436 0.716 Sd 023373 Sd 046155 1.25 1 0 1 0.291 0.410 0.422 0.693 Sd 023373 Sd 050839 1.25 1 0 1 0.291 0.405 0.378 0.693 Sd 023373 Sd 055961 1.25 1 0 1 0.291 0.373 0.402 0.735 Sd 023373 Sd 055974 1.25 1 0 1 0.291 0.347 0.415 0.741

phase 2 and phase 3 hybrids further crossing strategies deriving from FS high-yielders - heritability - backcross - biparental - line cross crossing strategies derived from the mapping populationp - F1 trait crosses - F2 crosses crossing strategies t using natural populations - forward screen high yielders - mapping F1

artemisinin is a crucially important product the science continually reveals new opportunities 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 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 and China partnerships are in place with a global network of contacts policy, pharma, extractors, growers, agricultural extension and seed producers / distributors

update on progress Professor Dianna Bowles co-project leader Department of Biology