Diversity and evolution of chili peppers, the genus Capsicum (Solanaceae)

Evolution, diversification and speciation of plants are usually accompanied by genomic, epigenetic, and chromosomal rearrangements, as well as genome size changes. Repetitive DNA is a major driver of genome evolution with most plant genomes containing large amounts of repetitive DNA. Despite enormous progress, numerous questions concerning the importance of chromosomal restructuring and changes of genome composition for the diversification and speciation remain. A suitable and attractive system for addressing aspects of genomic evolution in a group of closely related taxa is provided by the genus Capsicum (Solanaceae). This genus of c. 40 species is centred in Middle-South America and is of great economic significance, because it includes the sweet and hot chili peppers, vegetables and spices consumed worldwide. All of the species analyzed so far are diploids, with two polyphyletic base chromosome numbers (x = 12, 13) and significant variation in the heterochromatin content and genome size. Hypotheses on the species relationships exist, as well as hypotheses on the chromosomal and genome size evolution in the genus. Recent methodological advances, especially low-cost next generation sequencing (NGS) applied to repetitive genome fraction and implemented by molecular, bioinformatic, and cytogenetic techniques offer new tools to further advance our understanding of the evolutionary trajectories of this important genus in particular, and of genome evolution in general. In the proposed project a combination of methods and techniques will be used to analyze in detail the interspecific relationships and the evolution of genomes of the genus Capsicum. Phylogenetic approaches will allow inference of the evolutionary history of the genus, and will be complemented with in-depth analyzes of genome evolution accompanying species diversification and domestication, as well as analyses of genetic bases of self-incompatibility. The comparative study of this genus will address significant evolutionary questions concerning the impact and frequencies of genomic changes on diversification and speciation. Specifically, the molecular phylogeny of Capsicum will be completed and provide reliable hypothesis on the evolutionary differentiation of the genus. This in turn will allow inferrence of the ancrestral character states for various characters, partcilarly those related to genome composition and structure. It will allow hypotheses on the trends and mechanisms of chromosomal evolution in the genus (e.g., origin of two different chromosome base numbers, x = 12 13) and the origin and composition of heterochromatin to be proposed and tested. The repetitive genome fraction of all Capsicum species (wild and cultivated) will be specifically targeted to correlate the genome size variations with the origin and/or the amplification of various repeats. Finally, the genetics of different reproductive strategies among different strains of the cultivated chili C. pubescens will be addressed through S-genotyping and exploring self-incompatibility and its importance for species diversity.

ABSTRACT The genus Capsicum (Solanaceae) comprises about 40 species centred in Middle-South America and is of great economic significance, because it includes the sweet and hot chili peppers consumed worldwide. Genus Capsicum offers an excellent system in which to analyse the diversity and dynamics of evolution of repeats under natural and human-assisted selection, as domestication processes typically results in a reduction of genetic diversity. All of the species of Capsicum were diploids with either 2n = 24 or 2n = 26 chromosomes. Despite lack of polyploidy, significant variation in the repetitive DNA content and nearly 4.5-fold variation in genome size was found in this genus. Analyses of the evolutionary relationships among all species of peppers using genome-wide RAD sequencing analyses provided well resolved phylogeny of this group with nine lineages. The genus was inferred to be nearly 19 myo, with the lineages containing domesticated chiles only 2 myo. 2n = 24 was reconstructed to be an ancestral chromosome number and the derived number of 2n = 26 evolved independently in two unrelated lineages. Small ancestral genome size was reconstructed to evolve mostly via genome size increase independently and recurrently in several lineages. Changes of chromosome numbers and genome sizes were not correlated. Chromosomal and genome size changes are strongly dependent on the changes in content and localization of non-coding repetitive DNA, often referred to as junk DNA. Overall profiles of repetitive DNA fraction in all but two species of Capsicum have been obtained from analyses of several millions of DNA fragments sequenced using Next Generation Sequencing Illumina platform and clustered using bioinformatics pipeline RepeatExplorer. Repetitive DNA fraction of all pepper genomes was dominated by a dispersed repeat superfamily of gypsy retrotransposons. This superfamily has been responsible for recurrent increases of genome sizes in different lineages of peppers. Repeatomes of all five domesticated pepper species showed much lower overall diversity of repeats than wild relatives. Ribosomal RNA genes and satellite DNA families, although present in lower amounts than retrotransposons, contributed more significantly to the intra- and interspecific chromosomal variation within main lineages. Only three to five tandemly repeated satellite DNA families were found in cultivated taxa versus over 10 families in wild species, regardless of the lineage. Despite that, the levels of loci number and localization polymorphisms were much higher in domesticated species than in wild relatives. Newly identified tandem repeats were mapped in chromosomes of all species using fluorescence in situ hybridization (FISH), a method that allows localization of DNA sequences in chromosomes. Unique patterns of the loci number and distribution of individual satellite DNA families provided markers for identification of individual chromosome pairs in most species.