SNPs discovery for targeted genotyping in Jatropha curcas

The ability of species to adapt to different environments resides in their genetic diversity. This diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and population histories that have played roles in species` evolution. A number of different techniques for identifying SNPs have been developed but all have their limitations. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic technology that can identify polymorphisms in a target gene by heteroduplex analysis. A variation of this technique (EcoTILLING) represents a means to determine the extent of natural variation in selected genes. EcoTILLING and TILLING may be a cost effective approach for haplotyping and SNPs discovery. The objectives of the project are i) to assess genetic variation in J. curcas accessions and in closely related species. ii) to assess EcoTILLING and TILLING as a reliable cost effective method to detect SNPs in a large number of J. curcas accessions, related species and induced mutants. iii) to use EcoTILLING and TILLING as a reverse genetic tool for functional analysis of J. curcas genes. The foreseen activities for this project will be as follows: Selection of 12 candidate genes (genes involved in oil production, toxicity and adaptation to stress defence response) of J. curcas. Assessment of haplotype diversity detected through EcoTILLING and TILLING in a wide range of germplasm of Jatropha and in EMS induced mutants. Search for allelic diversity and assessment of EcoTILING and TILLING as methods to discover novel alleles.

Jatropha curcas L. is a perennial plant at a pre-domestication stage, widespread in tropical and subtropical areas, assumed to be native to Central America. It thrives on almost any soil and is therefore able to prevent soil erosion. This tree produces seeds rich in oil (27-40%), which are suitable for biofuel production. Further, several pharmaceutical compounds of industrial importance found in J. curcas could provide the basis for the economic and social development of regions where it grows. However, efficient industrial exploitation of the plant is hampered by a lack of knowledge on the genetic background of J. curcas and hence further studies are needed to draw conclusions regarding the nature of genetic differences among toxic and non-toxic accessions.In order to develop sustainable management strategies, a clear and detailed understanding of the extent and distribution of genetic variation within and among J. curcas accessions, a large living collection of 907 J. curcas accessions from 53 geographical regions covering 15 countries and 3 continents were used. The present study confirms that human activities had a major influence on the genetic diversity of J. curcas, not only because of domestication, but also because of biased selection.A major aim in Jatropha genetic improvement is the development of high yielding varieties in terms of seed yield, high oil content and low toxin amount. To accelerate this process, the identification of natural variation in genes of interest is important. Since most of the functional variation resides in the coding regions of the genome, the logical first step was to use coding regions for the discovery of the causative genetic variants of traits of interest. For this purpose, Eco-TILLING (Targeting Induced Local Lesions in Genomes) was performed to investigate the function of candidate genes by discovering new SNPs. The chosen strategy could separate Mexican (center of origin) from other J. curcas accessions and distinguish toxic from non-toxic ones.Inducing genetic diversity and application of mutagenesis to create novel variation are reliable strategies for crop improvement, especially in crops with narrow genetic variability, like Jatropha. Although mutations occur spontaneously in nature, their frequency is so rare that plant breeding cannot rely on them solely. Therefore, general methods for mutation induction (chemical and physical mutagenesis) by ethyl methanesulfonate (EMS) treatment, gamma irradiation were used, to generate large numbers of mutants in different tissues of J. curcas under in vitro and in vivo conditions. However, it required fast and effective strategies in order to select the best genotypes as an industrial crop. Therefore, TILLING by sequencing, genotyping by sequencing (GBS) as well as double digest GBS sequencing (ddGBS) were applied on different tissues of J. curcas to identify and to provide a range of allele types, including missense and knockout mutations, which are potentially useful in a variety of gene function and selection of the best phenotypes.Because this non-edible plant is at a non-domesticated level, it requires the development of tools for its breeding and selection. In fact, a lack of knowledge of the quantitative genetic variations make it difficult to predict oil and toxin levels and to understand the differences in gene expression under different environmental conditions. With the goal of better understanding the development of Jatropha seeds and to improve its agronomic performance, a two-step approach was performed: 1) generating the entire transcriptome of six different developmental stages of J. curcas seeds, 2) comparing of transcriptional expression levels in six different developmental stages of seeds and leaves of different genotypes (toxic and non-toxic). These datasets provide comprehensive expression profiles for seed maturation and toxin production in different genotypes of J. curcas, ultimately improving our understanding of Jatropha biology, and represent useful resources and references for other related species or oil-produced seeds.Since the discovery of virus encoded miRNAs plays a crucial role in pathogenesis, host-pathogen interaction and regulating endogenous gene expression, for the first time we could predict viral microRNAs of begomoviruses and host plants (Jatropha and cassava), which plays a crucial role in pathogenesis, host-pathogen interaction and regulating endogenous gene expression. This is the first assessment of predicted viral miRs/miRs* and host plant miRNAs, providing a reference point for miRNA identification in pathogens and their hosts. These findings will improve the understanding of host- pathogen interaction pathways and the function of viral miRNAs in Euphorbiaceous crop plants.