Complete Plastid and Mitochondrial Genomes of Aeginetia indica Reveal Intracellular Gene Transfer (IGT), Horizontal Gene Transfer (HGT), and Cytoplasmic Male Sterility (CMS)

Round 1

Reviewer 1 Report

This article presents the plastid and mitochondrial genomes of Aeginetia indica, a holoparasitic Orobanchaceae. The plastid genome is compared to a previous database accession and evidence is presented that the latter may be faulty, or represent a divergent species. Intracellular gene transfer between organelles and horizontal gene transfer, in particular from hosts, are described. Cytoplasmic Male Sterility is proposed to rely on a Cox2 derived ORF in the mitogenome.

It is unfortunate that the Genbank entries MW851293 and MW851294 are not accessible. However, sequence analysis appears sound and thorough, and the conclusions will be taken at face value. The article is sound and the conclusions are interesting. However, the analysis is sometimes too superficial, and the conclusions could be strengthened if some questions were examined more in depth.

 

1) The data presented here for the Cp genome is in strong disagreement with that presented in a previous study by others (ref 31). Please be clearer in your conclusions whether this is the same or a different species. The notion of "different species A. indica" does not make sense. If you consider different species, discuss which sample is more likely to represent the true A. indica by taxonomic standards.

Please make it clearer what Illumina data was used to compute coverage over MN529629. Was it the one used to generate your genome, or the one in ref 31 (PRJDB5395) as should be? In the latter case, in view of the many drops in coverage, it would be hard to conceive how they could have assembled MN529629, so your mapping would need to be examined critically. In the former case, your mappings just shows how different the two genomes are. Indicate SRA IDs.

The fact that the CDS sequences appear more conserved than intergenic regions (S2) suggests that we are dealing with different species and not just a massive error in the prior assembly. Your assemblies must contain enough rDNA sequences to check whether they are identical with that of the previous study.

2) A major area of divergence is the atp gene cluster (atpH here, atpI/atpA in the prior version). Please examine the long regions between rps2 and trnS in detail in both assemblies, as they must be full of decaying pseudogenes. Look for an atpB pseudogene.

In particular, in view of the absence of other atp genes the remaining AtpH should be examined for functionality, for example by alignment with other Cp_AtpH proteins. Is it a pseudogene in this or the previous assembly ? Have you checked for its expression using the ample RNAseq data available for the species ? AtpH constitutes the rotor of the ATPase, its presence would have bioenergetic implications. Please indicate what is known about the presence of thylakoid membranes in the chloroplast of Orobanchaceae, in particular holoparasitic species.

3) Given the absence of IR in your Cp assembly (and not a single IR, as stated l.73), one aspect that needs to be clarified is whether your data is consistent with  a circular structure, in particular whether individual assemblies showed a join between rps15 and trnH. The absence of the essential infA in your but not the prior assembly is a concern: is it an annotation error in your or the prior assembly, has it been transferred to the nucleus? The availability of a transcriptome assembly (doi: 10.1093/gbe/evy219) must be taken advantage of.

3) The absence of genes for complex II and many ribosomal protein in the mitogenome should be discussed in terms of transfer to the nucleus rather than loss. The conclusions from the literature should be mentioned. Although it is quite clear that mitoribosomes must be present, loss of Complex II could in principle be considered.

4) The presence of a plastid-derived atpI gene in the mitochondrial genome should be discussed in terms of number of times this particular IGT independently occurred in Orobanchaceae. Cp_atpI should appear in Table S3 and its distribution correlated with the phylogeny. A short taxonomy should accompany the species names, or even better a phylogenetic tree should appear on top of the table. Is the predicted AtpI protein intact ? An alignment with diverse Cp-encoded AtpI might help.

5) The IGT of tRNA genes should be examined in greater detail and discussed in relationship to tRNA import from the cytoplasm. Please indicate the source of your phylogenetic affiliations in Table S4. A. indica is the only Lamiale with a Cp-derived trnN-GUU or trnM. How certain are you if their phylogeny ? Please show a bootstrapped tree. In the table, group trnM and trnfM and in the text discuss the absence of a mitochondrial initiator tRNA in Orobanchaceae.

The comparison of Tables S2 and S4 suggests that you consider some plastid IGTs as introducing functional tRNAs (trnM, trnN-GUU, trnI-CAU, trnW-CCA, trnD-GCU), but not trnS-GGA. Please explain and discuss how ancient these IGTs may be.

Is there a trnI-CAU lysidine synthase in the nuclear genome ? Is there a potential for dual targeting, that species that import this tRNA from the cytosol would not need ?

6) Regarding CMS genes: please detail your reasons to think that CMS should be present in Aeginetia. How widespread is it in Lamiales ? Doe the Aeginetia transcriptome contain PPR proteins similar to Restorers of fertility? Unless you can strengthen your case for the presence of CMS, the title should be changed.

You seem to consider only orf43 as potential CMS because of its containing TM segments. Figure 4 is unclear on which regions are supposed to be transmembrane. What is the meaning of the arrows and blue-shaded regions? The sequences shown do not seem to be full length. Specify if the part shown corresponds only to the region of homology.

Is there a reason for not considering the Cp_atpI as a potential CMS gene ?

 

Minor details and typos:

l15: missing word after gene

l19: "plastid-derived" rather than chloroplast

l21: intracellular

l23: in the plastid of

l24: angiosperm plastid

l25: sinensis

l26: unclear

l81: a pseudogene

l117: define IGS

l123: sdh3 and sdh4 do not code for mitochondrial proteins

l167: no need to mention Miscanthus sienesis (misspelled) as a possible source of the HGT, just because it is part of your tree: it could be any grass species

l174: remove "of"

l176: expression

l193: Plant sampling, DNA sequencing and genome assembly and annotation.

l200: do you mean using k-mers of 69 and 99 ? How many k-mers were used, how as the reconciliation done ?

l2020: do you mean "organellar contigs from multiple assemblies were aligned…" ?

l2221: 19 concatenated

 

Table S2: rather than identity, indicate number of gap and mismatched nt. What is DAN ? DNA? trnI has to be longer than 57 nt, so this must be a fragment. In the annotation column, add the coordinates on the query, to allow checking whether transfer involves neighboring sequences.

Supplemental figures: add the legends below he figures

l240: indicate meaning of the annotation tracks. What is the blue track with red and grey marks?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

I can't find table 1 in the manuscript.

Author Response

Thanks for comments.

We confirmed the figure 1.

Reviewer 3 Report

Authors have assembled the complete chloroplast and mitochondrial genomes of Aeginetia indica using next generation sequencing methods. They assembled the complete chloroplast and mitochondrial genomes with the size of 56,381 bp and 401,628 bp, respectively. Authors have annotated both the genomes and found the reported the gene transfer through IGT and HGT. Authors could address the following comments to improve the manuscript,

1. It is not clear in the manuscript if the authors have produced a circular genome of chloroplast and mitochondrial genomes. If so, please include the circular genome map of these genomes showing the gene structures and locations.
2. Authors could try to include the expansion and contraction analysis results for the chloroplast genome.

Author Response

Thanks for your comments.

We changed figure 1 like circular chloroplast genome.

And, we included sentence about contraction of chloroplast genome in Orobanchaceae.

Thank you.