Mitogenome Assembly Reveals Gene Migration and RNA Editing Events in Plateau Hongliu (Myricaria elegans Royle.)
, Hao Wu 1,†, Xingyao Hu 1, Yunhua Wu 2, Feng Nie 2, Tao Su 1,*, Mei Han 1,* and Fuliang Cao 3
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors sequenced the Myricaria elegans Royle.-M. elegans mitogenome using the Illumina and PacBio technology. Obtained mitogenome sequence suggest the presence of two DNA molecules. Authors annotated 31 unique protein-encoding genes (PEGs), fifteen 20 tRNAs, and three rRNA genes to the obtained sequence. Moreover, the codon usage biases were revelaed. Authors described 350 potential C-U RNA editing sites and experimentally verified selected C-U RNA editing sites. Finally, Authors tested the gene transfers occurring between the plastome and mitogenome of M. elegans as well as evolutionary and colinearity relationships between M. elegans and other plant species.
Article provides novel and important results that could be interesting to researchers in the field. Research is properly planned and performed. Obtained results support conclusions. Article is well written and figures are of good quality.
Following comments could further improve the manuscript:
11. Provide the volume and concentration of libraries used for sequencing.
22. Provide name of manufacturer and country of origin of PCR equipment used in section 2.5. The same ofor Illumina and PacBio sequencing.
33. Codon usage biases are usually connected with the amount of available tRNA. Could it be possible to found relationship between presence of tRNA genes or their copy numbers in M. elegans mitogenome and observed codon usage biases? If yes, add several sentences describing it to the section 3.2 and Discussion.
44. Provide in one-two sentences the information of GenBank accession numbers for M. elegans mitogenome not only in Data Availability Statement but also at the end of Introduction and in the Results – section 3.1.
Comments on the Quality of English LanguageMinor editing of English language required.
Author Response
Reviewer 1
Authors sequenced the Myricaria elegans Royle.-M. elegans mitogenome using the Illumina and PacBio technology. Obtained mitogenome sequence suggest the presence of two DNA molecules. Authors annotated 31 unique protein-encoding genes (PEGs), fifteen 20 tRNAs, and three rRNA genes to the obtained sequence. Moreover, the codon usage biases were revelaed. Authors described 350 potential C-U RNA editing sites and experimentally verified selected C-U RNA editing sites. Finally, Authors tested the gene transfers occurring between the plastome and mitogenome of M. elegans as well as evolutionary and colinearity relationships between M. elegans and other plant species.
Article provides novel and important results that could be interesting to researchers in the field. Research is properly planned and performed. Obtained results support conclusions. Article is well written and figures are of good quality. Following comments could further improve the manuscript:
Q1: 11. Provide the volume and concentration of libraries used for sequencing.
R1: Thanks. The detailed volumes of the raw data using the hybrid sequencing platforms of Illumina, NovaSeq 6000 (Illumina, USA), and PacBio RS SMRT (Pacific Biosciences, USA) for sequencing and library construction have been provided in Lines 186-187 of the revised manuscript. The raw data have also been deposited in GenBank with available SRA IDs: SRR28705194 (Illumina) and SRR28705193 (PacBio).
Q2: 22. Provide name of manufacturer and country of origin of PCR equipment used in section 2.5. The same ofor Illumina and PacBio sequencing.
R2: Thanks for the comment. The equipment used for PCR and sequencing has been specified. Please check Lines 106-107 and 163-164 in the revised manuscript.
Q3: 33. Codon usage biases are usually connected with the amount of available tRNA. Could it be possible to found relationship between presence of tRNA genes or their copy numbers in M. elegans mitogenome and observed codon usage biases? If yes, add several sentences describing it to the section 3.2 and Discussion.
R3: Many thanks and this comment is critical. A total of 15 tRNA genes were annotated in the mitogenome, including three multi-copy genes (trnC-GCA (×2), trnM-CAU (×5), and trnN-GUU (×2)). These tRNA genes appeared to carry the codon only for seven amino acids, suggesting that the nuclear genes primarily regulate the mitochondria protein biosynthesis. Also, the calculation of codon usage preference (RSCU) revealed that alanine (GCU) has the highest values, followed by the termination codon (UAA), leucine (UUA), proline (CCU), and threonine (ACU). These findings might not be compatible with the tRNA genes with multi-copy numbers (e.g., trnM-CAU (×5) carrying the codon for histidine). Thus, unfortunately, the potential link between the codon usage bias and tRNA genes/copy numbers has not been set up.
Q4: 44. Provide in one-two sentences the information of GenBank accession numbers for M. elegans mitogenome not only in Data Availability Statement but also at the end of Introduction and in the Results – section 3.1.
R4: Thanks. The descriptions of the accession ID for the assembled mitogenome have been provided in Lines 91-92.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe presented manuscript is devoted to the study of the mitogenome of Myricaria elegans. This is a unique woody shrub halophyte that grows high in the mountains. This plant has a complex of interesting properties, the study of which can reveal the secrets of the mechanisms of resistance to critical climatic conditions. The determination of the genome and mitogenome of M. elegans contributes to the study of these mechanisms. The presented manuscript widely uses the most modern software methods for processing mitogenomes, which allowed the authors to identify interesting features of the structure of the M. elegans mitogenome. The manuscript is well organized, the results obtained are discussed in detail, and there is no doubt about the conclusions drawn.
There are some minor comments.
98 - Since the authors isolated total DNA, it is no longer integral, since it contains nuclear, mitochondrial and chloroplast DNA. Check DNA integrity.
120 - Apollo program cannot manually correct annotation errors
183 - Is it true that the size of the M. elegans mitogenome is more than 400 kbp, which is more than 20 times larger than that of humans? This is a giant mitogenome.
Figure 1 - indicate in the caption what the red and green inserts mean
Figure 5 - There is no data on chloroplast DNA in the manuscript. Why does Figure 5 show сpDNA and the connections between it and M1 and M2?
Figure 7 - how do you explain the difference in PCR products when using DNA and cDNA for nad1 and especially nad7?
Author Response
Reviewer 2
The presented manuscript is devoted to the study of the mitogenome of Myricaria elegans. This is a unique woody shrub halophyte that grows high in the mountains. This plant has a complex of interesting properties, the study of which can reveal the secrets of the mechanisms of resistance to critical climatic conditions. The determination of the genome and mitogenome of M. elegans contributes to the study of these mechanisms. The presented manuscript widely uses the most modern software methods for processing mitogenomes, which allowed the authors to identify interesting features of the structure of the M. elegans mitogenome. The manuscript is well organized, the results obtained are discussed in detail, and there is no doubt about the conclusions drawn.
There are some minor comments.
Q1: 98 - Since the authors isolated total DNA, it is no longer integral, since it contains nuclear, mitochondrial and chloroplast DNA. Check DNA integrity.
R1: Thanks. The isolated total DNA was divided into two proportions subjected to the Illumina and PacBio sequencing platforms. For the DNA quality and concentration assessment, the agarose gel electrophoresis and spectrophotometer were used subsequently. Please check the revised manuscript in Lines 102-105.
Q2: 120 - Apollo program cannot manually correct annotation errors
R2: Sorry for the typo. It has been corrected in Lines 124-125 in the resubmitted manuscript.
Q3: 183 - Is it true that the size of the M. elegans mitogenome is more than 400 kbp, which is more than 20 times larger than that of humans? This is a giant mitogenome.
R3: Thanks, this comment is quite interesting. The complete plant mitogenome sequences can be expanded from the minimal size (66 kb) in the phytoparasite V. scurruloideum, to a large size of (11.3 Mb) in the annual herb S. conica. The plant mitogenome undergoes genome replication, frequent homologous recombination, gene loss, or transfer to the plastome or nuclear genome, prompting that the metagenome’s size and conformation among different plant lineages were variable. That could be why a more marked mitogenomic architecture and length variations were commonly characterized in plants than in animals due to enormous sequence repeats.
Q4: Figure 1 - indicate in the caption what the red and green inserts mean
R4: Thanks. The descriptions of the red and green inserts in Figure 1 are specified in the legend.
Q5: Figure 5 - There is no data on chloroplast DNA in the manuscript. Why does Figure 5 show сpDNA and the connections between it and M1 and M2?
R5: This comment is significant. The additional assembled plastid genome has been provided in Figure S1. Please check the uploaded supplementary materials.
Q6: Figure 7 - how do you explain the difference in PCR products when using DNA and cDNA for nad1 and especially nad7?
R6: Thanks. RNA editing is an essential post-transcriptional modification process that can change the transcript sequences and associated gene expression. In the experiment, a total of 350 potential RNA editing sites were deduced among 31 mitochondrial PEGs, including experimentally verified cox2, nad1, nad7, and atp6. Interestingly, the C to U/T editing events occurred in all PEGs (see Table S14). In Figure 7a, the PCR-amplified products in the gel images were sent directly for the Sanger sequencing using DNA and cDNA as templates. The band size variations between DNA- and cDNA-PCR products were determined by the specific primers used, also for the Sanger sequencing. The comparisons of their sequencing data (colored peak code) could discriminate the C-U/T changes, as shown in Figure 7b.
