The Complete Plastome Sequences of Eleven Capsicum Genotypes: Insights into DNA Variation and Molecular Evolution

Abstract

Members of the genus Capsicum are of great economic importance, including both wild forms and cultivars of peppers and chilies. The high number of potentially informative characteristics that can be identified through next-generation sequencing technologies gave a huge boost to evolutionary and comparative genomic research in higher plants. Here, we determined the complete nucleotide sequences of the plastomes of eight Capsicum species (eleven genotypes), representing the three main taxonomic groups in the genus and estimated molecular diversity. Comparative analyses highlighted a wide spectrum of variation, ranging from point mutations to small/medium size insertions/deletions (InDels), with accD, ndhB, rpl20, ycf1, and ycf2 being the most variable genes. The global pattern of sequence variation is consistent with the phylogenetic signal. Maximum-likelihood tree estimation revealed that Capsicum chacoense is sister to the baccatum complex. Divergence and positive selection analyses unveiled that protein-coding genes were generally well conserved, but we identified 25 positive signatures distributed in six genes involved in different essential plastid functions, suggesting positive selection during evolution of Capsicum plastomes. Finally, the identified sequence variation allowed us to develop simple PCR-based markers useful in future work to discriminate species belonging to different Capsicum complexes.

Keywords: chloroplast genome; microsatellites; molecular markers; next-generation sequencing; pepper; perfect tandem repeats; sequence variability; simple sequence repeats; single-nucleotide polymorphism.

Figure 1

Map of the Capsicum pubescens chloroplast genome. Genes inside of the outer circle are transcribed in the clockwise direction, while those outside are transcribed in the counterclockwise direction. Different color codes represent genes belonging to various functional groups. The circle inside GC content graph marks the 50% threshold. The inverted repeat, large single-copy, and small single-copy regions are denoted by IR, LSC, and SSC, respectively.

Figure 2

Sliding window analysis of the multiple plastome sequence alignment within the Capsicum genus. The region with high nucleotide variability (Pi > 0.05), corresponding to the IR/SSC junction, is indicated. Window length = 200 base pairs (bp); step size = 50 bp.

Figure 3

Schematic representation of the five most variable genes (ndhB, accD, rpl20, ycf1 and ycf2) in the plastomes under investigation. Gray bars represent the multiple-sequence alignment (MSA) for each gene and are scaled according to the MSA length. Black boxes indicate highly variable regions in the MSA. Above each box, a snapshot of the MSA along with alignment positions is reported.

Figure 4

Phylogenetic tree of Capsicum genotypes. Phylogram of the best maximum-likelihood (ML) tree as determined using the RAxML software from the complete plastome dataset. Numbers associated with branches are ML bootstrap support values.

Figure 5

Results of molecular evolution analysis of plastid genes within the Capsicum genus. (A) Estimation of protein-coding gene divergence by the average branch length ± standard deviation for each gene tree; (B) number of putative sites under positive selection.

Figure 6

Examples of chloroplast molecular markers developed in this study. PCR markers based on the presence of perfect tandem repeats and insertions/deletions (InDels) able to discriminate C_B (A) and C_P (B) complexes. PCR results from representative genotypes in each complex are shown. C_B = C. baccatum; C_P = C. pubescens; C_A = C. annuum; 1 = bac.b; 2 = bac.p; 3 = bac.p2; 4 = bac.p3; 5 = bac.p4; 6 = pra; 7 = pub; 8 = pub2; 9 = pub3; 10 = cha; 11 = cha2; 12 = cha3; 13 = cha4; 14 = ann2; 15 = ann4; 16 = ann5; 17 = ann6; 18 = ann7; 19 = chi2; 20 = chi3; 21 = chi4; 22 = fru2; 23 = fru3; 24 = fru4.