Genetic Molecular tools for exploitation of genetic resource in wild species
Emmanuelle Dyrszka1, Moira Sheehan2, Steve Tanksley2, Alexandra Casa2, Vincent Petiard2, Marie
Coque1, Virginie Mirleau-Thebaud1, Stéphanie Bascouert1, Fabienne Gentou1, Denis Lespinasse 1,
Olivier Cottet1
Syngenta Seeds, 12 chemin de l’Hobit, 31790 Saint-Sauveur, France,
emmanuelle.dyrszka@syngenta.com
1
2
Nature Source Genetics, 33 Thornwood Dr., Suite 300, Ithaca, NY 14850-1275, USA,
MSheehan@naturesourcegenetics.com
ABSTRACT
 The process of domestication and breeding has narrowed the genetic base of many crop species.
Sustained genetic improvement of crops requires a continuous supply of useful genetic variation. The
current study has been initiated within a collaborative project between Nature Source Genetics (NSG) and
Syngenta Seeds with the objective to enhance Syngenta’s elite sunflower (Helianthus annuus L.) gene
pools using novel/unadapted germplasm sources.
 The approach consists on identifying a subset of wild sunflower accessions (land races and
related wild species) which represent the greatest degree of novel genetic diversity. Permanent Optimized
Discovery Populations (ODP) were generated by crossing elite lines and wild donor, and by applying
marker technology and using strategies/algorithms developed by NSG for optimization.
 The broad genetic diversity study in Helianthus genus allowed us to identify an optimized set of
30 wild accessions representing the broadest allelic diversity. The evaluation of ODP for targeted traits
(e.g. yield) allowed the identification of specific exotic QTLs that could be integrated into existing
programs. Through the current development of three ODP, several QTL linked to wild donor
introgression for traits such as flowering time and height and co-localisation of QTLs between traits were
reported.
 At the final step of ODP development with phenotypic screening in various environments, NSG
and Syngenta will work on exotic QTLs discovery and mining. This project is expected to yield a number
of tangible assets and outcomes, which will, in the medium and long term, directly and indirectly profit
Syngenta through enhanced and sustainable seed sales.
 The research program aims to generate elite QTL-inbreds (in original elite recipient variety
background) which can be used, both directly for new commercial varieties as well as source of exotic
QTLs for use in other Syngenta breeding material.
Key words: Sunflower - Wild Diversity – Optimization – Exotic QTL
INTRODUCTION
The genetic improvement of plants depends on a continuous supply of new genetic variation. For this
reason, commercial breeding programs spend significant resources monitoring and enhancing the
breeding pools from which they will select new varieties. Efforts in germplasm enhancement have
focused on existing elite breeding pools which do not contain the full range of potentially useful alleles
(Fick and Miller 1997). However interest on using wild introgression on breeding purpose increased, and
plays a key role (Jan 2008; Seiler et al. 2008).
Wild introgressions in cultivated pool represent a determinant potential for breeding program (Cuk, 1982;
Seiler 1992). Diverse original and targeted agronomic traits (e.g. disease, oil quality) can be introgressed
from diverse populations, including perennial (Dozet 1990; Seiler et al. 2008). Fifty-one species and 19
subspecies belong to Helianthus genus (Seiler and Reiseberg, 1997). Only a part of them are easily
crossable with cultivated Helianthus annus L. (Laferrière 1986). Among crop species, sunflower varieties
are based on a relatively narrow germplasm (Faure et al. 2002). Thus, sunflower is likely to be high
responder to programs aimed at selectively enhancing genetic variation for key economic traits.
This project is based on collaboration between Syngenta Seeds and Nature Source Genetics (NSG), a
computational genomics company dedicated to the development and application of new algorithms
designed to harness natural genetic variation in the improvement of plant and animal species for
agriculture and industry. The global project aims was to develop and implement a set of strategies and
Optimized Discovery Populations (ODP) that were used to enhance Syngenta’s elite sunflower breeding
pool, to provide permanent resources usable for the screening of biotic and abiotic stresses, and to
develop new elite commercial sunflower varieties.
The three objectives of the present study were i) to identify a subset of exotic sunflower accessions which
represent the greatest degree of novel genetic diversity, ii) to generate and genotype a set of Optimized
Discovery Populations (ODP), iii) to evaluate the ODP for target traits in order to identify specific
favorable exotic QTLs, and iiii) to develop new varieties from the ODP findings.
MATERIALS AND METHODS
Optimized donor set.
Exotic donors for sunflower were drawn from accessions/taxa with known cross compatibility with the
cultivated sunflower: H. annuus landraces, H. annuus wild accessions and accessions of other wild
species in the subsection Helianthus. Based on geographic and taxonomic data a subset of 300 potential
donors was identified. In addition, a set of elite sunflower inbreds from Syngenta were selected based on
phenotypes (such as plant height and flowering date) and marker information (from SNPs and SSRs).
This material was resequenced on 14 conserved orthologous sets of ESTs (Expressed Sequenced Tag) for
which sequence information were available in Chapman et al. (2007). Using specific algorithms
developed by NSG, a diversity study was carried out in order to identify an optimized donor set of
accessions with maximum unique genetic variation relative to i) elite sunflower and ii) to each other.
Optimized Discovery Populations construction.
From the previous optimized set of donors, four donors were chosen (based on their potential to enhance
Syngenta’s elite lines) in order to initiate the building of three different ODP. Permanent Optimized
Discovery Populations (ODP) of 200-500 inbred lines were then generated by crossing one or more wild
donors with an elite line using SNP markers information and computational optimization. An optimized
crossing schema based on NSG expertise was provided that significantly exceeds current methods (e.g.
introgression lines, RILs or Advanced Backcross). NSG performed optimization steps based on markers
data i) to maximize the portion of wild donor genome(s); and ii) to increase mean population performance
by addressing major negative traits, with the aim to increase statistical power in positive QTLs detection
and optimized genomic selection responding.
Optimized Discovery Populations phenotypic evaluation.
ODPs will be evaluated in appropriate environments with the aim to identify specific exotic QTLs, which
would improve the target traits (e.g. yield) in the elite recurrent parents. QTL-modified elite sunflower
lines will then be generated and used, either i) directly for new commercial varieties or ii) as source of
exotic QTLs for use in other Syngenta breeding material.
RESULTS
Optimized donor set.
From the broad genetic diversity study in Helianthus genus, the present work showed that the elite
sunflower germplasm contains only a small portion of the genetic variation available in wild sunflower
(Figure 1). Moreover evidence was made that H. argophyllus, H. bolanderi and H. exilis were closely
related to H. annuus than H. petiolaris and H. praecox (Figure 1). Based on these molecular data and
NSG optimization algorithm, a set of 30 wild accessions representing the broadest allelic diversity were
selected becoming the optimized donor set.
Optimized Discovery Populations evaluation.
A reference population facilitated the mapping of loci underlying key, negative domestication-related
traits coming from wild donors, which impact our ability to detect smaller positive QTLs for traits of
interest (yield). Experiments performed in controlled conditions and in field also highlighted the relevant
correspondence of QTL between the two cropping conditions for several targeted traits (Figure 2). The
phenotypic and genotypic screening of ODPs allowed the identification of QTLs with donor alleles
increasing or decreasing the value for a same trait (e.g. flowering time and height) compared to elite
alleles (Figure 3). Undesirable phenotypes were discarded at each generation with an optimized SNP set
by reducing the unfavourable allele frequency, but keeping as much as possible a large part of wild
genome.
Figure 1. Graph: multidimensional scaling plot based on DNA resequencing depicting relationships
among Helianthus accessions. Phylogenetic tree from Rieseberg 1991.
DISCUSSION
This study allowed a large access to new genetic variability from wild species. Wild sunflower genetic
variability makes difficult a rigorous classification (Seiler and Rieseberg, 1997). As observed by
Rieseberg (1991) H. argophyllus, H. bolanderi and H. exilis were closest to cultivated sunflower than H.
petiolaris and H. praecox. H. annuus genetic variation is greater than elite germplasm genetic variation,
and among H. annuus genetic variation only a small part is common with elite germplasm (Fick and
Miller 1997). This gives the range of improvement through wild introgression.
Figure 2. Localisation of QTL in field experiments and in controlled experiment. Dark stars: wild allele
decrease trait value. Light stars: wild donor increase trait value.
The experiments confirmed the localisation of a region influencing multiple traits (here plant height and
flowering; Burke et al. 2002). It also confirmed the reproducibility of QTL detection in controlled
condition as well as in field conditions.
At this stage of the project, the results also give insights into negative QTL coming from wild donor.
These results will allow the authors to deal with negative traits during the ODP development using
specific algorithms developed by NSG. The power of detecting true positive QTLs, the uniformity of
statistical detection throughout the genome, and the map resolution of QTLs had thus greatly enhanced in
comparison to screening a similar size population from a standard population (e.g. ILs or RILs).
The expectation is to increase the probability of finding useful QTLs from the wild with this approach
compared to traditional approaches. Based on phenotyping of ODP, the research program will generate
elite QTL-inbreds (in original elite recipient variety background) which can be used, both directly for new
commercial varieties as well as source of exotic QTLs for use in other Syngenta breeding material. In
addition, once generated and genetically mapped, these ODPs will represent a permanent resource that
can be continuously screened and re-screened for new QTLs for any trait that can be measured and might
become of importance for the market. More generally, this project is expected to increase the ability to
selectively and rapidly enrich the allelic diversity of elite breeding pools that is likely to become one of
the key factors in determining which commercial breeding programs continue to be competitive in the
future.
Figure 3. Localisation of QTL for plant height and flowering time in field experiments. Dark stars: wild
allele decrease trait value. Light stars: wild donor increase trait value.
CONCLUSION
This project is expected to yield a number of tangible assets and outcomes, which will, in the medium and
long term, directly and indirectly profit Syngenta through enhanced and sustainable seed sales. Indeed,
the ability to selectively and rapidly enrich the allelic diversity of elite breeding pools is likely to become
one of the key factors in determining which breeding programs continue to be competitive in the future.
AKNOWLEDGEMENTS
This work is the result of Syngenta Seeds and Nature Source Genetics partnership. The authors would like
to thank the operational genotyping and phenotyping teams for their involvement, reactivity and technical
support at each stage of this project. The authors would also like to thank Luka Cuk for his kind critics.
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