Study On Chloroplast And Mitochondrial Genomes Of Salvia Miltiorrhiza

Plant organellar genomes (chloroplasts and mitochondria) are important components of plant genomes, which play a significant role in phylogenesis, species identification, intra-cellular interactions and chloroplast engineering. Salvia miltiorrhiza Bunge (Danshen in Chinese) is a deciduous perennial flowering plant in the family Lamiaceae. It is a significant traditional Chinese medicinal herb with great economic and medicinal value. However, little is known regarding the organellar genomes of Salvia miltiorrhiza to date, which hampers its releated studies.Currently, there are still some difficulties in obtaining the organellar genomes particularly the mitochondrial genomes of high plants. This work concentrated on the plant organellar genome assembly and sequence analysis based on whole genome sequencing data.We present a new chloroplast assembly strategy in this study. Fisrt, the chloroplast genome backbone was constructed by extending the seed sequences of two conserved genes on both the5’ and3’ ends in a step-by-step manner until they overlapped at both the IRa and IRb regions. Then all of the nucleotides were checked and corrected to generate the complete chloroplast genome. This strategy did not need to isolate the chloroplast DNA prior to sequencing and only produced one contig, which reduced the assembly complexity. Based on this strategy, we reported the complete chloroplast genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family. The chloroplast genome is151,328bp in length, and exhibits a typical quadripartite structure of the large (LSC,82,695bp) and small (SSC,17,555bp) single-copy regions, separated by a pair of inverted repeats (IRs,25,539bp). It encodes131predicted functional genes, of which114are unique, including80protein-coding genes,30tRNA genes and four rRNA genes. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the30asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum.We also present a hybrid assembly strategy based on second generation and third generation sequencing to de novo determine the plant mitochondrial genome sequences. This strategy benefits from the higher relative abundance of DNA derived from organellar genomes compared to the nuclear genome in the same living plant tissue. First, small-scale454sequecing data were introduced for the enrichment of organellar continuous long reads (CLR) and for error correction. Then the chloroplast CLR were removed by fully mapping to the chloroplast genome. Finally, the remaining CLR were assembled into the mitochondrial genome. Based on this strategy, we obtained the complete mitochondrial genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family. This is also the first application of SMRT sequencing on the complete mitochondrial genome assembly of land plants. The499,236bp circular molecule exhibits features typical of angiosperm mitochondrial genomes, including69genes (44proteins,22tRNAs, and three rRNAs) and22introns (18cis-and four trans-splicing). Repeat-mediated intra-genomic recombination analysis was documented using corrected CLR and was confirmed by SOLiD mate-paired reads with two insert sizes. A total of18identified repeats showed evidence of recombination, with the three largest repeats showing roughly equivalent frequencies of the reference and recombinant conformations. Although fairly consistent results were observed in the three sequencing datasets, we noticed that the proportion of recombinants of the674bp forward repeat detected by the SOLiD reads was more than double that by CLR, which indicates a potential tissue-specific recombination event.The sequence transfer between the two organellar genomes of Salvia miltiorrhiza was also conducted here. Only the sequence transfer from the chloroplast to the mitochondrial genome was returned. These transfer fragments totalled37,612bp in length, accounting for7.5%of the entire mitochondrial genome. Twenty-six chloroplast-derived fragments≥100bp were detected, of which the largest single fragment, comprising16,684bp, is one of the largest contiguous, chloroplast-derived sequences characterized to date in plant mitochondria, second only to a18,534bp region from Cucurbita pepo.