Assembly of the Complete Mitochondrial Genome of Gelsemium elegans Revealed the Existence of Homologous Conformations Generated by a Repeat Mediated Recombination

Abstract

Gelsemium elegans (G. elegans) is a Chinese medicinal plant with substantial economic and feeding values. There is a lack of detailed studies on the mitochondrial genome of G. elegans. In this study, the mitochondrial genome of G. elegans was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of G. elegans is represented by two circular chromosomes of 406,009 bp in length with 33 annotated protein-coding genes, 15 tRNA genes, and three rRNA genes. We detected 145 pairs of repeats and found that four pairs of repeats could mediate the homologous recombination into one major conformation and five minor conformations, and the presence of conformations was verified by PCR amplification and Sanger sequencing. A total of 124 SSRs were identified in the G. elegans mitochondrial genome. The homologous segments between the chloroplast and mitochondrial genomes accounted for 5.85% of the mitochondrial genome. We also predicted 477 RNA potential editing sites and found that the nad4 gene was edited 38 times, which was the most frequent occurrence. Taken together, the mitochondrial genome of G. elegans was assembled and annotated. We gained a more comprehensive understanding on the genome of this medicinal plant, which is vital for its effective utilization and genetic improvement, especially for cytoplasmic male sterility breeding and evolution analysis in G. elegans.

Keywords: Gelsemium elegans; RNA editing events; homologous recombination; mitochondrial genome; phylogenetic analysis.

Figure 1

The mitochondrial genome of G. elegans according to a graph model. The purple contigs belong to chromosome 1. The yellow contigs belong to chromosome 2. The green contigs are the mitochondrial plastid sequences (MTPTs), in which contigs 26, 39, 49, 75, and 84 belong to chromosome 1, and contigs 15, 28, 38, 45, and 77 belong to chromosome 2. The red contigs are the repeat regions, in which contig 82 belongs to chromosome 1, and contigs 13, 14, 53, 61, 77, 79, 80, and 83 belong to both chromosomes.

Figure 2

The circular maps of the mitochondrial genomes of G. elegans. Genomic features transcribed clockwise and counterclockwise are drawn on the inside and outside of the circle, respectively. Color-coding is used to distinguish genes of different functional groups.

Figure 3

Repeat analysis of the mitochondrial genome in G. elegans. (A,B) Repeated sequences were analyzed for each of the two chromosomes. The colored lines on the innermost circle connect the two repetitive sequences of the scattered repeats, with the yellow line representing palindromic match and the green line representing forward match. The black line on the second circle represents the tandem repeat sequence, and the black line on the outermost circle represents the microsatellite or simple sequence repeat (SSR) sequence.

Figure 4

Validation of the homologous recombination mediated by different pair repeats. (A,B) The schematic diagram of primer design and experimental design after reverse primer exchange of direct repeat sequences that can mediate homologous recombination in representative circular molecules. (C) PCR validation results of the existence of various conformations of mitochondrial DNA. From left to right, each of the five lanes represents a set of three experiments, namely, repeat2, repeat3, and repeat4. The experiments are as follows, from left to right: marker, major conformation 1, major conformation 2, minor conformation 1, and minor conformation 2.

Figure 5

Hypothetical products generated by recombinations mediated by R1, R2, R3, and R4. R1, R2, R3, and R4 in the picture represent repeats 1–4. The black arrows on the circular chromosome represent the repeat sequences, and the colored lines represent the DNA fragment between the repeats. The circles represent the mitochondrial genome conformations. Minor conformations 1, 2, 4, and 5 contain one circular chromosome, and the major conformation contains two circular chromosomes, while minor conformation 3 contains three circular chromosomes.

Figure 6

Homologous fragments and phylogenetic analysis of G. elegans. (A) The blue arcs represent the mitochondrial genome, the green arcs represent the chloroplast genome, and the red lines between the arcs correspond to homologous genomic fragments. (B) The evolutionary relationship between G. elegans and homologous species.

Figure 7

Prediction of RNA editing events in G. elegans mitochondrial genome. The horizontal and vertical coordinates indicate the gene name and the number of RNA edits, respectively.