Association mapping of drought tolerance in sunflower : phenotypic and genetic characterization of INTA inbred lines

 Association mapping is a powerful tool to identify genes or alleles that contribute to variation in complex traits using ensembles of unrelated individuals such as germplasm collections or natural populations. Genome-wide and candidate gene approaches have been applied in plants but, regardless of the genotyping strategy, one of the key aspects of the method is obtaining reliable phenotypic measurements. In candidate gene approaches, both phenotypic evaluation and the selection of regions for genetic analysis are major determinants of the power to identify QTL. Tolerance to water deficit is a highly complex trait for which phenotypic assessment can be difficult. Different quantitative variables may serve to describe a plant response to drought and these may vary along different developmental stages. Here we present a preliminary study aimed to develop an association mapping platform for drought tolerance in sunflower. The goals of this work were: a) to assess the suitability of mannitol assays for the evaluation of drought tolerance during seed germination, and b) to study nucleotide diversity, linkage disequilibrium and evolutionary patterns of seven candidate genes previously reported to be involved in the response to water deficit in sunflower and other species.  Thirty four sunflower inbred lines were evaluated under two water deficit regimes (200 mM and 400 mM mannitol) during 11 days. The percentage of germinating seeds was scored at the end of the experiment and results were analyzed under a logistic regression model. A set of 7 SSR loci was assayed to infer genetic relationships among the 34 inbred lines studied and seven candidate regions (Hahb4-p, Hahb4, Suntip, FB, HaL1L, HaDhn1 and CK) were screened for variation to identify SNP and InDels.  Susceptible and resistant genotypes were clearly identified in 200 mM mannitol assays, whereas no such distinction could be achieved under the more stringent water deficit regime. Analysis of nucleotide diversity revealed an average SNP frequency of 1/48.3 bp and an average nucleotide diversity (θW) of 0.00501. In agreement with previous reports, a high level of LD was observed for all the regions (r=0.88 at 900 bp) except for Suntip, for which LD decays to r=0.325 at 207 bp and HaDhn1, for which LD decays to r=0.249 at 78 bp. No deviations from neutrality were observed for Suntip and HaDhn1, whereas evidences of purifying selection were found for Hahb4-p, Hahb4, FB and HaL1L.  The mannitol test proved to be a reliable and potentially useful method for the screening of drought tolerance in sunflower. The assignment of inbred lines to the susceptible and resistant categories was highly concordant with previous field trials. All regions evaluated are interesting candidate genes to be used in association mapping for this trait.  The results presented here constitute the first steps towards identifying drought tolerance QTL in sunflower via the association mapping approach. Further efforts are currently being made to expand the association mapping population, the number of candidate genes and the phenological stages of the phenotypic evaluation.