De Novo Hybrid Assembly of the Salvia miltiorrhiza Mitochondrial Genome Provides the First Evidence of the Multi-Chromosomal Mitochondrial DNA Structure of Salvia Species

Abstract

Salvia miltiorrhiza has been an economically important medicinal plant. Previously, an S. miltiorrhiza mitochondrial genome (mitogenome) assembled from Illumina short reads, appearing to be a single circular molecule, has been published. Based on the recent reports on the plant mitogenome structure, we suspected that this conformation does not accurately represent the complexity of the S. miltiorrhiza mitogenome. In the current study, we assembled the mitogenome of S. miltiorrhiza using the PacBio and Illumina sequencing technologies. The primary structure of the mitogenome contained two mitochondrial chromosomes (MC1 and MC2), which corresponded to two major conformations, namely, Mac1 and Mac2, respectively. Using two approaches, including (1) long reads mapping and (2) polymerase chain reaction amplification followed by Sanger sequencing, we observed nine repeats that can mediate recombination. We predicted 55 genes, including 33 mitochondrial protein-coding genes (PCGs), 3 rRNA genes, and 19 tRNA genes. Repeat analysis identified 112 microsatellite repeats and 3 long-tandem repeats. Phylogenetic analysis using the 26 shared PCGs resulted in a tree that was congruent with the phylogeny of Lamiales species in the APG IV system. The analysis of mitochondrial plastid DNA (MTPT) identified 16 MTPTs in the mitogenome. Moreover, the analysis of nucleotide substitution rates in Lamiales showed that the genes atp4, ccmB, ccmFc, and mttB might have been positively selected. The results lay the foundation for future studies on the evolution of the Salvia mitogenome and the molecular breeding of S. miltiorrhiza.

Mitochondrial Genome Sequence of Salvia officinalis (Lamiales: Lamiaceae) Suggests Diverse Genome Structures in Cogeneric Species and Finds the Stop Gain of Genes through RNA Editing Events

“…To determine the inter-specific variations of Salvia mitogenomes, we compared the mitogenome sequences of S. officinalis and S. miltiorrhiza [ 46 ] using Mummer (v3) [ 47 ] ( Figure 3 ). As shown, there were many rearrangements between the two genomes ( Figure 3 ).…”

“…They are vastly distributed among different plants and contribute to homologous recombination and variable mitogenomic structures [ 54 , 55 ]. To identify the repetitive sequences that might mediate homologous recombination in the S. officinalis mitogenome, we conducted a self-to-self sequence comparison using BLASTn with an e-value < 1 × 10 −6 and a word size of 7, as described previously [ 46 ]. The BLASTn results contained 52 high-scoring sequence pairs (HSPs).…”

“…MTPTs are mitochondrial DNA sequences that are derived from plastomes [ 61 ]. To identify MTPTs in the S. officinalis mitogenome, we compared the mitogenome and the plastome of S. officinalis using BLASTn with an e-value < 1 × 10 −6 and a word size of 7 [ 46 ]. We identified 23 MTPTs in the S. officinalis mitogenome, with a total length of 14,495 bp accounting for 4.70% and 9.59% of the S. officinalis mitogenome and plastome sequences, respectively.…”

“…Our long-term goal is to understand the diversity and evolution of Salvia mitogenomes, thereby ultimately providing valuable information for molecular breeding studies on these economically important Salvia species. Recently, we confirmed for the first time that the predominant conformations of Salvia mitogenomes contain two circular molecules [ 46 ]. In the present study, we sequenced the mitogenome of S. officinalis.…”

“…Recently, we reported on the mitogenome of S. miltiorrhiza on the basis of Pacbio long reads and Illumina short reads [ 46 ]. We were the first to confirm that the predominant conformations of the Salvia mitogenome are two circular chromosomes.…”