Diversity and evolution of highland chile Capsicum pubescens

The American genus Capsicum L. (Solanaceae) is recognized for its economic value since it includes the sweet and hot chile peppers, vegetables and spices consumed worldwide. There are five cultivated species in this genus and one of them, C. pubescens Ruiz & Pav., is almost exclusively cultivated in the South American highlands, from Mexico to NW Argentina. The variation encountered in C. pubescens manifests itself mostly in fruit characters, but well-defined varieties are barely distinguishable. The extent of genetic diversity of C. pubescens is largely unknown and the origin and specific affinities of this species are still puzzling. Indeed, C. pubescens has been included, together with a few other species, in the so-called purple-flowered group of Capsicum, but it would form an independent lineage despite being genetically close to the other purple-flowered species, as evidenced by successful reciprocal interspecific crosses. Based on the current information, several hypotheses can be drawn: (1) C. pubescens and the other purple- flowered species do not share their most recent common ancestor, (2) there is high level of genetic variation among individuals/populations of C. pubescens which has been strongly influenced by human- assisted selection, but (3) it cannot be considered to be a domesticated crop per se, and finally that (4) the centre of origin of C. pubescens is in highlands of middle-western South America. Therefore, utilising phylogenetic analyses of DNA data this project aims at: (1) analyzing the evolutionary relationships and taxonomic affinities of C. pubescens which will allow to propose hypothesis of its evolution and geographic expansion, and (2) characterizing in detail the genetic diversity across the distribution range of C. pubescens following analyses of multiple populational samples. Restriction site-associated DNA sequencing (RAD-Seq), a method framed within the revolutionary Next-generation sequencing technologies, will be applied to obtain polymorphic genetic markers across the genome of the target species and its putative relatives. RAD-Seq allows rapid and cost-effective capture of (tens of) thousands of genetic markers, allowing to in depth analyses of genetic diversity and evolution. By applying this novel sequencing approach, the final goal of this proposal is to gain insight into C. pubescens origin, affinities, evolution, and genetic structure. This information is crucial for the management and conservation of C. pubescens, a popular cultivated chile in South America, as well as for assessment of the use of this species and its closest allies as genetic resources for breeding programmes.

The project had the objective of exploring the origin, affinities, evolution, and genetic structure of Capsicum pubescens, a hot chile pepper cultivated almost exclusively in the highlands of South America, from NW Argentina to Mexico. Genome-wide DNA data were obtained following the restriction site-associated DNA sequencing (RAD-Seq) methodology. Several dozen C. pubescens samples from the entire range of the species cultivation (i.e. Argentina, Bolivia, Peru, Ecuador, Costa Rica, Guatemala, and Mexico) were analyzed. More than 50 samples of the other species of Capsicum, including those considered more closely related to C. pubescens, were also included. Phylogenetic and population genetic analysis were followed using different datasets and diverse computer programs to address the objectives outlined. In addition, a pilot experiment was done to start studying the genetics of fruit pungency/hotness (the gene Pun1/AT3) in a phylogenetic context. The most relevant results and conclusions drawn can be summarized as follows: 1. The RAD-seq methodology was successfully applied for the first time to Capsicum, including almost all of its species. 2. A strongly supported resolution regarding C. pubescens phylogenetic origin and its closest allies was obtained. 3. The primary/secondary gene pool of C. pubescens (newly circumscribed Pubescens clade) could be reliably defined and a first overview of its genetic structure was obtained. 4. Time divergence dating for the origin of C. pubescens lineage and for the main clades of Capsicum was hypothesized for the first time. 5. A putative, more narrowly delimited geographic centre of origin for C. pubescens (Bolivian highlands NE from La Paz) was defined. 6. New evidence for the strong affinity between two of the closest allies to C. pubescens (C. eximium and C. eshbaughii) was presented, together with unprecedented original phenotypical data on C. eshbaughii. 7. The first comprehensive overview of the Pun1/AT3 gene sequence across clades encompassing the domesticated chiles (C. pubescens and closest relatives included) was achieved, meaning successful sequencing for all their species, both cultivated and wild. Three lines of research have also arisen from the present project, that will be continued onwards separately, namely: 1. Analysis of domestication traits and paths among C. pubescens and close allies. 2. Evolution and species identity among the closest wild relatives to C. pubescens. 3. Genetic and chemistry analyses of fruit pungency across all domesticated chile species and their closest wild allies.