Genome relationships and LTR-retrotransposon diversity in three cultivated Capsicum L. (Solanaceae) species

Abstract

Background: Plant genomes are rich in repetitive sequences, and transposable elements (TEs) are the most accumulated of them. This mobile fraction can be distinguished as Class I (retrotransposons) and Class II (transposons). Retrotransposons that are transposed using an intermediate RNA and that accumulate in a "copy-and-paste" manner were screened in three genomes of peppers (Solanaceae). The present study aimed to understand the genome relationships among Capsicum annuum, C. chinense, and C. baccatum, based on a comparative analysis of the function, diversity and chromosome distribution of TE lineages in the Capsicum karyotypes. Due to the great commercial importance of pepper in natura, as a spice or as an ornamental plant, these genomes have been widely sequenced, and all of the assemblies are available in the SolGenomics group. These sequences were used to compare all repetitive fractions from a cytogenomic point of view.

Results: The qualification and quantification of LTR-retrotransposons (LTR-RT) families were contrasted with molecular cytogenetic data, and the results showed a strong genome similarity between C. annuum and C. chinense as compared to C. baccatum. The Gypsy superfamily is more abundant than Copia, especially for Tekay/Del lineage members, including a high representation in C. annuum and C. chinense. On the other hand, C. baccatum accumulates more Athila/Tat sequences. The FISH results showed retrotransposons differentially scattered along chromosomes, except for CRM lineage sequences, which mainly have a proximal accumulation associated with heterochromatin bands.

Conclusions: The results confirm a close genomic relationship between C. annuum and C. chinense in comparison to C. baccatum. Centromeric GC-rich bands may be associated with the accumulation regions of CRM elements, whereas terminal and subterminal AT- and GC-rich bands do not correspond to the accumulation of the retrotransposons in the three Capsicum species tested.

Keywords: Chili peppers Cytogenomics; FISH; Plant genome; Transposable elements.

Fig. 1

Comparison of the relative distribution (%) of repetitive DNA families among three Capsicum datasets. a Note that the Class I elements are more common than Class II and rDNA sequences in the three cases. b LTR-RT elements predominated over non-LTR and ERVs, but note that the C. baccatum genome exhibited 30% fewer sequences than other two datasets. Observe also that Copia superfamily elements were less accumulated (< 10%) than Gypsy ones. c Observe that the accumulated elements were Tekay/Del, Athila/Tat, and CRM, but, except for CRM lineage, there was a big difference in the quantity of the elements in each dataset

Fig. 2

Phylogenetic tree based on putative autonomous sequences of Capsicum using the maximum likelihood method with bootstrap 1000. CRM sequences organize four groups (grey, see also the Figure S2) and Athila sequences two groups (blue, see also the Figure S3). The Del sequences were organized in two well-supported groups (orange), corroborating with the MAUVE alignment (Figure S1), and the third group of sequences without well-supported values (green), except for the sequences Del_7 and Del_9 (Figure S1) corroborating with the MAUVE alignment

Fig. 3

Comparative distribution of the putative autonomous LTR-RTs and the reverse transcriptase sequences along the C. annuum, C. chinense, and C. baccatum datasets. a The Annuum clade, composed by C. annuum and C. chinense and the Baccatum clade (C. baccatum) can be distinguished by the dendrogram on top of the heatmap. In the heatmap, lower and higher accumulation (blue, intermediate and yellow, respectively) represent the amount of conserved sequences found in each dataset. Image shows that Athila/Tat elements accumulated more in C. annuum and C. chinense (marked in yellow and light-yellow), the CRM groups were differentially accumulated, highlighting the absence of CRM III and the predominance of Del I and II in C. baccatum, and bigger accumulation of Del III, IV, and V in C. annuum and C. chinense. The reverse transcriptase of these elements exhibits a similar pattern of distribution than the one observed for the complete elements, Athila/Tat I and II were more accumulate in C. annuum and C. chinense, respectively. CRM I and II were more accumulated in C. chinense, while the group CRM III was more accumulated in C. baccatum. Capsicum annuum and C. chinense had a bigger accumulation of Del I, II and II than C. baccatum, while the groups Del III and IV exhibited more accumulation in C. baccatum. b Graphical representation of LTR-RTs groups. LTR – long terminal repeat, GAG – nucleocapsid, RT – reverse transcriptase, RH – RNAse H, INT – integrase, ASP – aspartase. Asterisks present in Athila illustrations refer to the hallmark ORF for this lineage. Note a difference in extension (bp length) among elements, including GAG and POL positioning, and LTR sizes. Note also that only CRM II exhibits a chromodomain sequence and that all the Del elements present an additional aspartase locus

Fig. 4

FISH using LTR-RTs probes against metaphases and prometaphases of Capsicum species. Chromosomes were counter-stained with DAPI (blue), Copia probes with Cy3–11-dUTP (red) and Gypsy probes labelled with biotin-11-dUTP / avidin–FITC conjugate (green). The Copia Ivana/Oryco probe showed few hybridization signals scattered along chromosomes, with a low accumulated profile in both C. chinense (a) and C. baccatum (b). The Gypsy Tekay/Del probe exhibited hybridization signals dispersed along the chromosomes in the three species, but with a larger accumulation in C. annuum chromosomes (c) than the other two species, such as in C. baccatum (d). The Gypsy Athila/Tat probe showed brighter hybridization signals than Tekay/Del, accumulating in the pericentromeric to interstitial regions of all C. annuum chromosomes (e), differently of C. baccatum because some chromosomes accumulated many signals and others very few (h). The boxes i, ii, iii and iv are highlighting differences in the pericentromeric and interstitial Athila/Tat signals in two C. baccatum chromosomes. The Gypsy CRM probe showed FISH signals accumulated in the centromeric regions, but with two pairs in each species with much less intense signals. Note the arrows in C. baccatum (f) and C. chinense (g). The bar represents 10 μm

Fig. 5

Capsicum species show considerable diversity in the C-CMA/DAPI banding profiles. Observe that C. annuum presents four chromosomes without fluorescent signals. C-DAPI interstitial dots was detected in three chromosome pairs and terminal bands two pairs (a), all of them were co-located with C-CMA bands. Ten pairs exhibit C-CMA signals, being three pairs with stronger terminal and the other as the terminal to subterminal small signals (b). Capsicum chinense showed four pairs with strongest DAPI signals, besides minor centromeric, interstitial and terminal signals on a few chromosomes (c), while C-CMA bands were observed in all the chromosomes, varying as strongest terminal bands in seven pairs, minor centromeric bands in six pairs and as terminal and interstitial dots in ten pairs (d). Some of these bands have been evidenced by DAPI and CMA₃ (c, d). Observe that C. baccatum exhibits six pairs with minor terminal and six with centromeric and/or interstitial C-DAPI (e). C-CMA signals were detected in four pairs as strongest terminal bands, but minor terminal bands were observed in all chromosomes, as well as minor centromeric in almost all the chromosomes (f). The bar represents 10 μm