Complete Chloroplast Genome Sequence of Medicago falcata: Comparative Analyses with Other Species of Medicago

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Since molecular and morphological evidence are insufficient to distinguish among M. sativa, M. falcata and their hybrid Medicago × varia, the authors studied the chloroplast genomes of Medicago falcata ecotypes, a perennial forage legume, from different regions and compared them with those of Medicago truncatula and Medicago sativa. Phylogenetic analysis showed that M. falcata had the closest relationship with M. hybrid and M. sativa.  

 

Abstract

 

The abstract is well written, presents the salient points and compares M. falcata to the closely related Medicago spp.

 

Introduction

 

The latest reference in this section is 2021. There is no information after this date. I would suggest the authors present the latest research findings on this subject.

Line 37: ‘evolutionary’ what? What comes after ‘evolutionary’?

Line 43: Explain what ‘IR’ is. Do you mean ‘inverted repeat’?

 

M & M

The authors used a published method for extracting total genomic DNA, used various software to assemble and annotate the chloroplast genome, to predict protein coding sequences and RNA and tRNA genes and a software to draw a circular diagram for the chloroplast genome. Chloroplast genome consistency, sequence repeats and nucleotide polymorphism were analyzed using various programs.  

I don’t see a reference for the phylogenetics analysis and description on how it was carried out in this subsection.

Line 77: Define ‘CDS’ as ‘CDS (Coding DNA Sequence)’

 

Results

 

Lines 146-147: Change ‘genes indicated genes position’ to ‘gene positions’….

Figure 2: Define ‘UTR’ and ‘CNS’ in the legend.

UTR: untranslated region; CNS: Conserved noncoding sequences

Line 139: The X axis in Figure 3 shows ‘rps18’ not ‘rps16’ as stated here.

Line 141: X axis in Figure 3 shows ‘rps18’ not ‘rps16’ and ‘ycf3’ not ‘ycf’.  

Lines 144 &149: Italicize ‘Medicago’

Line 165: The authors forgot to mention the ‘reverse repeats’

Line 185: …’during evolutionary’ what comes after this?  time? course? or just ‘during the evolution’

Lines 197-199: ‘By comparison, the complete chloroplast genome sequence of M. falcata MW271002 and M. falcata MW271003 are longer than those of M. sativa and M. truncatula.’ Describe the exact number of nucleotides that are bigger. In addition, mention that NC 32066.1 genome is 900 nucleotides less than that in M. sativa.  

 

Lines 199-201: ‘M. falcata (NC 032066.1) and M. sativa have two more genes (e.g. trnC-GCA and trnY-AUA) (Table 1) than the others, while M. truncatula lacks a protein-coding gene rps16 (Table 1).‘ Table 1 does not show this.

Lines 201-204: ‘M. falcata has a special chloroplast structure containing only one copy of the IR region, but lacks the quadripartite structure (Figure 1, Table 1), which is different from the chloroplast genomes of the majority of typical land plants that have two copies of IR region’ Table 1 shows none of the Medicago spp. have any IR region. Reconcile this!

 

Discussion

Line 228: Change comma to period after [41]

Line 230: Change comma to period after [33-34]

Line 237: Remove the second ‘our’

 

Conclusion

 

Line 249: Change ‘genes’ to ‘gene’

Line 251: Change ‘slightly’ to ‘slight’

Line 252: Place hyphen after ‘protein’: ‘protein-coding genes

Line 252: Remove ‘number’ after ‘genes’

Line 253: change ‘genes’ to ‘gene’

Line 253: Before ‘large’ add ‘and’

Line 253: Replace ‘between’ with ‘among’

Line 254: Add ‘s’ after ‘CDS’

Line 261: Replace ‘semicolon’ after ‘clustering’ with a period.

Line 262: Replace ‘for future research in’ with ‘regarding’  

Line 262: Replace ‘of’ with ‘on’

The manuscript needs revision.

 

I would suggest adding to and citing the following references in the manuscript and comparing and discussing their results to those obtained in this work.

 

Complete chloroplast genome sequence and characteristics analysis of Qingda no.1 alfalfa (Medicago sativa L. cv. Qingda no.1) (2022)

http://dx.doi.org/10.17221/71/2022-CJGPB

 

The complete chloroplast genome of Medicago sativa cv. Hangmu No.1, a plant of space

mutation breeding (2018)

https://doi.org/10.1080/23802359.2018.1561225

 

Complete sequencing of the chloroplast genomes of two Medicago species (2017)

http://dx.doi.org/10.1080/23802359.2017.1325336

 

 

Comments on the Quality of English Language

The quality of the English language needs to be improved. 

Author Response

Response to Review Comments on "Complete Chloroplast Genome Sequence of Medicago falcata and Comparative Analysis with Other Medicago Species"

Note: Revised parts are marked in blue

Responses to Reviewer 1's Comments

Introduction Section

Comment 1: The latest reference in the introduction is from 2021, lacking relevant information after 2021. It is recommended to supplement the latest research results in this field.

Response: Thank you for pointing out this issue; we agree with this comment. Therefore, we have supplemented the latest research findings in the field of Medicago chloroplast genomes published after 2021 in the introduction section. This revision is located in lines 42-46 of the revised manuscript. The updated text is: A recent study on the complete chloroplast genome of Medicago sativa L. cv. Qingda no.1 further confirmed this structural feature: the chloroplast genome of this alfalfa cultivar, which is adapted to high-altitude environments [16]. These findings provide additional evidence for the conservation and diversity of chloroplast genome structure in Medicago species.

Comment 2: The content after "evolutionary" in line 37 is missing. What is the complete expression?

Response: Thank you for pointing out this issue; we agree with this comment. Therefore, we have supplemented the missing content after "evolutionary" to complete the expression. This revision is located in lines 33-35 of the revised manuscript. The updated text is: The inheritance of the chloroplast genome with conserved gene content and order made it a valuable asset for studies in plant phylogenetic and evolutionary relationships [8-9].

Comment 3: Please explain the meaning of "IR" in line 43. Does it refer to "inverted repeat"?

Response: Thank you for pointing out this issue; we agree with this comment. We have added an explanation that "IR" refers to "inverted repeat" at its first occurrence. This revision is located in lines 36-39 of the revised manuscript. The updated text is: IIt has been reported that chloroplast genomes of some legume species have undergone rearrangements, including the loss of inverted repeats (IR) or genes (e.g. rpl22 and rps16) [10-11], as well as inversions of long fragments [12-13],

Materials and Methods

Comment 4: No references for phylogenetic analysis are provided in this subsection, and the specific implementation methods are not described.

Response: Thank you for pointing out this issue; we agree with this comment. We have supplemented references for phylogenetic analysis and detailed the specific implementation methods, including sequence alignment parameters, basis for model selection, and number of bootstrap replications. This revision is located in lines 116-131 of the revised manuscript. The updated text is: The sequences of CDS and the complete chloroplast genome sequences were retrieved using custom Python scripts, and homologous sequences from different species were aligned via a Python-based pipeline incorporating the clustalW2 program. Individual alignment outputs were concatenated sequentially. Phylogenetic trees were constructed from the concatenated CDS dataset and complete chloroplast genome sequences using both maximum likelihood (ML) and Bayesian inference (BI) methods. For ML analysis, RAxML-NG v1.0.1 was used with the GTR+G4+F+I substitution model, which was selected by ModelFinder integrated in Phylosuite v1.2.2 based on the corrected Akaike Information Criterion (AICc), with 1000 bootstrap replicates. For BI analysis, the mrbayes 3.2.6 program was run within Phylosuite v1.2.2 (citation) using the GTR+G4+F+I model as determined by the Bayesian Information Criterion (BIC). The Bayesian inference tree was constructed using MrBayes 3.2.7 [28]. The Markov chain Monte Carlo (MCMC) algorithm was executed for 1,000,000 generations, with trees sampled every 1000 generations until convergence (average standard deviation of split frequencies < 0.01). The initial 20% of trees were discarded as burn-in, and the remaining trees were used to generate a 50% majority-rule consensus tree.

Comment 5: Please define "CDS" in line 77, i.e., "Coding DNA Sequence".

Response: Thank you for pointing out this issue; we agree with this comment. Therefore, we have added the definition of "CDS" at its first occurrence. This revision is located in lines 84-86 of the revised manuscript. The updated text is: the total length of the chloroplast genome (bp), GC content, protein-coding gene number, CDS (Coding DNA Sequence) number

Results

Comment 6: In lines 146-147, change "genes indicated genes position" to "gene positions".

Response: Thank you for pointing out this issue; we agree with this comment. We have revised "genes indicated genes position" to "gene positions". This revision is located in line 173 of the revised manuscript. The updated text is: The Y-scale axis represents the percent identity between 50% and 100%. Gray arrows above the alignment indicate gene positions and their orientation. UTR: untranslated region；CNS: Conserved noncoding sequences.

Comment 7: Please define "UTR" and "CNS" in the legend of Figure 2. UTR: untranslated region; CNS: Conserved noncoding sequences.

Response: Thank you for pointing out this issue; we agree with this comment. We have added definitions of "UTR" and "CNS" in the legend of Figure 2. This revision is located in the legend of Figure 2 in the revised manuscript (line 171). The updated legend text is: Figure 2. Comparison of chloroplast genomes of Medicago species using the mVISTA program. A cut-off of 70% identity was used for the plots. The Y-scale axis represents the percent identity between 50% and 100%. Gray arrows above the alignment indicate gene positions and their orientation. UTR: untranslated region；CNS: Conserved noncoding sequences.

Comment 8: The X-axis of Figure 3 in line 139 is labeled "rps18" instead of "rps16" as described here. In line 141, the X-axis of Figure 3 is labeled "rps18" (not "rps16") and "ycf3" (not "ycf").

Response: Thank you for pointing out this issue; we agree with this comment. Therefore, we have revised the text description to be consistent with the actual labels on the X-axis of Figure 3. This revision is located in lines 160-166 of the revised manuscript. The updated text is: Compared with tRNA genes and rRNA genes, protein-coding genes had relatively higher nucleotide diversity (Figure 3, Table S1), and the highest nucleotide diversity was 0.486 for rps18, while the lowest value was 0.198 for psbE. The analyses on the nucleotide diversity revealed the variation size of the chloroplast genome in different Medicago species, and regions with high nucleotide diversity (e.g. rps18, rps3 and ycf3) may be developed as potential molecular markers for population genetics.

Comment 9: "Medicago" in lines 144 and 149 should be italicized.

Response: Thank you for pointing out this issue; we agree with this comment. We have italicized "Medicago" in the relevant positions. This revision is located in lines 168 and 173 of the revised manuscript. The updated texts are respectively: Figure 2. Comparison of chloroplast genomes of Medicago species using the mVISTA program. A cut-off of 70% identity was used for the plots. The Y-scale axis represents the percent identity between 50% and 100%. Gray arrows above the alignment indicate gene positions and their orientation. UTR: untranslated region；CNS: Conserved noncoding sequences. Figure 3. Nucleotide diversity values between five Medicago species determined using whole chloroplast genomes. The x-axis represents chloroplast genome genes, and the y-axis represents nucleotide diversity. Detailed Pi values were shown in Table S1.

Comment 10: The author omitted mention of "reverse repeats" in line 165.

Response: Thank you for pointing out this issue; we agree with this comment. We have added mention of "reverse repeats" in the relevant text. This revision is located in lines 186-189 of the revised manuscript. The updated text is: Four distinct types of long dispersed repeats (LDRs) were identified: forward (F), palindromic (P), reverse (R), and complementary (C) repeats. Among these repeats, forward repeats were the most abundant, with counts ranging from 47 to 182, followed by palindromic repeats (16 to 54) and reverse repeats (2 to 3) (Figure 5).

Comment 11: The content after "during evolutionary" in line 185 is missing; it should be "time", "course", or directly revised to "during the evolution".

Response: Thank you for pointing out this issue; we agree with this comment. We have revised the text to "during the evolution" to complete the expression. This revision is located in line 207 of the revised manuscript. The updated text is: In both phylogenetic trees, M. falcata MW271003 was closely related to M. hybrida and M. sativa, suggesting that M. falcata and M. sativa may have evolved from M. hybrida during the evolution.

Discussion

Comment 12: In lines 197-199: "In contrast, the complete chloroplast genome sequences of M. falcata MW271002 and MW271003 are longer than those of M. sativa and M. truncatula." It is necessary to specify the exact number of nucleotides by which they are longer. In addition, it should be mentioned that the NC 32066.1 genome is 900 nucleotides shorter than the genome of M. sativa.

Response: Thank you for pointing out this issue; we agree with this comment. We have supplemented the specific differences in nucleotide lengths and mentioned that the NC 32066.1 genome is shorter than that of M. sativa. This revision is located in lines 219-223 of the revised manuscript. The updated text is: In comparison, the complete chloroplast genomes of M. falcata MW271002 (125,657 bp) and M. falcata MW271003 (125,479 bp) are 327 bp and 149 bp longer than that of M. sativa (125,330 bp), respectively, and 1,624 bp and 1,446 bp longer than that of M. truncatula (124,033 bp), respectively.

Comment 13: Lines 199-201: "M. falcata (NC 032066.1) and M. sativa have two more genes (e.g., trnC-GCA and trnY-AUA) than other species (Table 1), while M. truncatula lacks the protein-coding gene rps16 (Table 1)." However, this content is not reflected in Table 1.

Response: Thank you for pointing out this issue; we agree with this comment. We have revised the relevant content in the text to ensure consistency. This revision is located in lines 239-241 of the revised manuscript. The updated text in the results section is: M. falcata (NC 032066.1) lacks two tRNA genes (e.g., trnC-GCA and trnY-AUA) compared to other species (Table 1), while M. truncatula is deficient in the protein-coding gene rps16 (Table 1).

Comment 14: Lines 201-204: "M. falcata has a unique chloroplast structure, containing only one copy of the IR region and lacking a quadripartite structure (Figure 1, Table 1), which differs from most typical land plants whose chloroplast genomes contain two copies of the IR region." However, Table 1 shows that all Medicago species lack the IR region. This needs to be checked and unified.

Response: Thank you for pointing out this issue; we agree with this comment. We have revised the text to clarify that all Medicago species lack the typical two copies of the IR region. This revision is located in lines 241-244 of the revised manuscript. The updated text is: M. falcata has a distinctive chloroplast structure that does not exhibit a typical quadripartite organization (Figure 1, Table 1), which differs from the chloroplast genomes of most typical land plants that contain two copies of the inverted repeat (IR) region[32].

Comment 15: Change the comma after [41] in line 228 to a period. Change the comma after [33-34] in line 230 to a period.

Response: Thank you for pointing out this issue; we agree with this comment. We have revised the punctuation marks after the references as required. This revision is located in lines 273 and 276 of the revised manuscript. The updated texts are respectively: our results showed that tetranucleotide repeats were more frequent than pentanucleotide repeats, which is consistent with findings in Quercus L. [43]. Hexanucleotide repeats were scarce across the five Medicago complete chloroplast genomes, a pattern that mirrors observations in Lilium L. and Allium L. [35-36].

Comment 16: Delete the second "our" in line 237.

Response: Thank you for pointing out this issue; we agree with this comment. We have deleted the second "our" in the text. This revision is located in line 284 of the revised manuscript. The updated text is: which is consistent with our phylogenetic analyses (Figure 6).

Conclusion

Comment 17: In line 249, change "genes" to "gene". In line 251, change "slightly" to "slight". In line 252, add a hyphen after "protein" to make "protein-coding genes". Delete "number" after "genes". In line 253, change "genes" to "gene". Add "and" before "large". Change "between" to "among". In line 254, add "s" after "CDS". In line 261, change the semicolon after "clustering" to a period. In line 262, change "for future research in" to "regarding". Change "of" to "on".

Response: Thank you for pointing out these issues; we agree with these comments. We have made all the required revisions to the conclusion section. These revisions are located in the conclusion section (320-336) of the revised manuscript. The updated conclusion text is: We determined the complete chloroplast genomes sequences of two M. falcata from Russia and Xinjiang in this study. The results revealed that gene orientation, genome structure, size, gene number, gene order, and GC content are conserved among the five Medicago accessions, including M. falcata NC 032066.1, M. falcata MW271002, M. falcata MW271003, M. truncatula NC 003119.6, and M. sativa NC 042841.1. However, there are slight differences in the number of protein-coding genes and tRNA gene number, comparative analysis of sequence differences, the protein-coding gene similarity was low, and large variation among CDSs. Further analysis of nucleotide polymorphisms showed that 87 out of 108 surveyed regions exhibit variations among the five Medicago species, with the nucleotide diversity of other coding genes being relatively high (exceeding 0.2). In our study, we observed that all rRNA and tRNA genes are highly conserved. The most abundant are mononucleotide repeats, followed by the di-, tri-, tetra-, and penta-, forward repeats were more abundant than reverse and palindrome repeats. Two phylogenetic tree analysis results are slightly different, the phylogenetic tree made from the full length is more accurate than the CDS phylogenetic tree clustering. These results offer valuable information regarding the identification on Medicago species and will benefit further investigations of these species.

Comment 18: It is recommended to supplement and cite the following references in the manuscript, and compare and discuss their research results with those of this study: "Complete Chloroplast Genome Sequence and Characterization of Medicago sativa L. cv. Qingda No.1" (2022) http://dx.doi.org/10.17221/71/2022-CJGPB

"Complete Plastome Sequence of Space-Mutated Alfalfa (Medicago sativa cv. Hangmu No.1) and Its Phylogenetic Analysis" (2018) https://doi.org/10.1080/23802359.2018.1561225

"Complete Chloroplast Genome Sequencing of Two Medicago Species" (2017) http://dx.doi.org/10.1080/23802359.2017.1325336

Response: Thank you for pointing out these issues; we agree with these comments. We have made all the required revisions to the discussion section and added the above references. These revisions are located in lines 224-236 and 306-309 of the revised manuscript. The updated texts are respectively: For example, a study on  M. sativa L. cv. Qingdao no.1 reported that its chloroplast genome has a length of 125,637 bp, lacks a distinct quadripartite structure, and contains 111 genes (77 protein-coding genes, 30 tRNA genes, and 4 rRNA genes) with a GC content of 38.33% [16]. While the gene composition (numbers of protein-coding genes, tRNA genes, and rRNA genes) in this study is generally consistent with that of M. falcata  in our research (78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes), there is a notable difference in GC content between the two—with the GC content of M. falcata in our study ranging from 33.84% to 33.85%. Another study on two Medicago species also confirmed the conservation of genome structure: the chloroplast genomes of M. sativa and M. falcata were 125,095 bp and 125,810 bp in length, respectively, and both contained 109 unique genes [31]. This further supports the conservation of gene composition and structural framework within the genus, while the difference in GC content may reflect adaptive divergence or genetic background variations among species.

Similarly, phylogenetic studies on Medicago sativa L. cv. Qingda no.1 and the space-mutated Medicago sativa cv. Hangmu No.1 also indicated close affiliations with M. sativa and M. falcata [16, 45], reinforcing the close evolutionary relationship within this complex.

 

References 16, 28, and 45 have been added.

Comment 19: The quality of English language needs improvement.

Response: Thank you for pointing out this issue; we agree with this comment. We have comprehensively optimized the English expression throughout the manuscript, corrected grammatical errors, adjusted sentence structures, and made the writing more fluent and accurate. These revisions are incorporated throughout the entire revised manuscript and are marked in blue.

 

Thank you again for your attention and suggestions on this study. We have improved the manuscript according to your comments and hope that the revised version meets the publication requirements.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed your manuscript describing two new plastomes for two samples of Medicago falcata. I found the manuscript well-written and the analysis well done, but you failed to include a detailed description of the methods used to construct the phylogenetic tree. Additionally, there are some minor issues that I have noted, which you can find in the attached PDF.

Comments for author File: Comments.pdf

Author Response

Response to Review Comments on "Complete Chloroplast Genome Sequence of Medicago falcata and Comparative Analysis with Other Medicago Species"

Note: Revised parts are marked in blue

Responses to Reviewer 2's Comments

Comment 1: Annotation location: Title (original line 2). Content: Complete plastome sequence…Plastome can substitute chloroplast genome.

Response: Thank you for pointing out this issue; we agree with this comment. We have replaced "chloroplast genome" with "plastome" in the title to make the expression more concise. This revision is located in line 2 of the revised manuscript. The updated texts are respectively: Complete plastome sequence of Medicago falcata: comparative analyses with other species of Medicago

Comment 2: Annotation location: Second paragraph of the introduction (original line 42). Content: Please add naming author for the four genera and Vicia faba.

Response: Thank you for pointing out this issue; we agree with this comment. We have added naming authors for the four genera and Vicia faba. This revision is located in lines 39-40 of the revised manuscript. The updated text is: which have been observed in genera such as Glycyrrhiza L., Astragalus L., Medicago L., Pisum L., and Vicia faba L. [14].

Comment 3: Annotation location: Section 2.3 (original line 73). Content: Can you provide those scripts in a Supplementary material?

Response: Thank you for pointing out this issue. The in-house Perl script cannot be provided.

Comment 4: Annotation location: Section 2.7 (original line 103). Content: Can you provide details about the software used to build the tree? Additionally please tell how you dealt with the molecular evolution model used and the bootstrapping.

Response: Thank you for pointing out this issue; we agree with this comment. We have supplemented relevant content in Section 2.7. This revision is located in lines 116-131 of the revised manuscript. The updated text is: The sequences of CDS and the complete chloroplast genome sequences were retrieved using custom Python scripts, and homologous sequences from different species were aligned via a Python-based pipeline incorporating the clustalW2 program. Individual alignment outputs were concatenated sequentially. Phylogenetic trees were constructed from the concatenated CDS dataset and complete chloroplast genome sequences using both maximum likelihood (ML) and Bayesian inference (BI) methods. For ML analysis, RAxML-NG v1.0.1 was used with the GTR+G4+F+I substitution model, which was selected by ModelFinder integrated in Phylosuite v1.2.2 based on the corrected Akaike Information Criterion (AICc), with 1000 bootstrap replicates. For BI analysis, the mrbayes 3.2.6 program was run within Phylosuite v1.2.2 (citation) using the GTR+G4+F+I model as determined by the Bayesian Information Criterion (BIC). The Bayesian inference tree was constructed using MrBayes 3.2.7 [28]. The Markov chain Monte Carlo (MCMC) algorithm was executed for 1,000,000 generations, with trees sampled every 1000 generations until convergence (average standard deviation of split frequencies < 0.01). The initial 20% of trees were discarded as burn-in, and the remaining trees were used to generate a 50% majority-rule consensus tree.

Comment 5: Annotation location: First sentence of Section 3.1 (original line 108). Content: Move to material and methods section.

Response: Thank you for pointing out this issue; we agree with this comment. We have moved the relevant content (We used NovaSeq 6000 sequencing platform to generate raw data (3.4G) from the two M. falcata chloroplast, and deposited them at the GenBank database, and MW271002 and SRR15182922 were for the one obtained from Russia, and MW271003 and SRR15182921 were for the other one collected from Xinjiang, China.) to the "Materials and Methods" section (2.1. Plant material, DNA extraction, and sequencing, lines 59-70). The specific content is: The plants of two Medicago falcata ecotypes were used in this study: one was obtained from the Federal Research Center of Russia Vavilov Institute of Plant Genetic Resources, and the other was collected in Xinjiang, China. Both ecotypes were cultivated in the greenhouse of the College of Grassland Science, Xinjiang Agricultural University, Urumqi, China. Total genomic DNA was extracted from fresh leaves using the modified CTAB method [17]. Raw sequencing data were generated using the NovaSeq 6000 platform, yielding 3.4G of raw data for the chloroplast genomes of the two M. falcata ecotypes. The sequencing data and assembled chloroplast genome sequences have been deposited in the GenBank database, with the following accessions: MW271002 (genome sequence) and SRR15182922 (raw data) for the ecotype from Russia; and MW271003 (genome sequence) and SRR15182921 (raw data) for the ecotype collected from Xinjiang, China.

Comment 6: Annotation location: Section 3.1 (original line 116). Content: four.

Response: Thank you for pointing out this issue; we agree with this comment. We have revised the text. This revision is located in line 139 of the revised manuscript. The updated text is: four rRNA genes.

Comment 7: Annotation location: First sentence of Section 3.4 (original line 173). Content: Move to material and methods section.

Response: Thank you for pointing out this issue; we agree with this comment. We have moved the relevant content (The phylogenetic trees were constructed based on both CDS (CDS regions of 78 protein-coding genes) and the complete chloroplast genome sequences of 36 leguminous species and one outgroup Arabidopsis thaliana L.) to the "Materials and Methods" section (2.7. Phylogenetic analysis, lines 110-115). The specific content is: A total of 37 chloroplast genome sequences were used for phylogenetic analysis, including 11 sequences of the Medicago genus and 25 other leguminous species, with the chloroplast genome of Arabidopsis thaliana L. as the outgroup. The phylogenetic trees were constructed based on two datasets: the CDS regions of 78 protein-coding genes and the complete chloroplast genome sequences of the 36 leguminous species and the outgroup Arabidopsis thaliana L.

Comment 8: Annotation location: First sentence of Section 3.4 (original line 174). Content: Please add naming author.

Response: Thank you for pointing out this issue; we agree with this comment. We have added the naming author for Arabidopsis thaliana. This revision is located in line 115 of the revised manuscript. The updated text is: A total of 37 chloroplast genome sequences were used for phylogenetic analysis, including 11 sequences of the Medicago genus and 25 other leguminous species, with the chloroplast genome of Arabidopsis thaliana L. as the outgroup. The phylogenetic trees were constructed based on two datasets: the CDS regions of 78 protein-coding genes and the complete chloroplast genome sequences of the 36 leguminous species and the outgroup Arabidopsis thaliana L.

Comment 9: Annotation location: Figure 6 (original line 188). Content: Please indicate what means branch numbers.

Response: Thank you for pointing out this issue; we agree with this comment. The branch numbers in Figure 6 represent bootstrap support values from maximum likelihood (ML) analysis and posterior probabilities from Bayesian inference (BI) analysis, respectively. For ML-derived branches, the numbers indicate the percentage of bootstrap support from 1000 replicate analyses, reflecting the statistical confidence in the branching pattern (values closer to 100 indicate stronger support). For BI-derived branches, the numbers represent posterior probabilities, which quantify the probability of the branching relationship given the data and model, with values closer to 1.0 indicating higher confidence. These support values were generated during the phylogenetic tree construction process described in Section 2.7, where ML analysis was performed with RAxML-NG ver 1.0.1 software (with 1000 bootstrap replications) and BI analysis was conducted using MrBayes 3.2.7 software (convergence was determined by an average standard deviation of split frequencies < 0.01).

Comment 10: Annotation location: Section 4.2 (original line 228). Content: Italicize and include naming author.

Response: Thank you for pointing out this issue; we agree with this comment. We have italicized Quercus and added its naming author. This revision is located in line 274 of the revised manuscript. The updated text is: Additionally, our results showed that tetranucleotide repeats were more frequent than pentanucleotide repeats, which is consistent with findings in Quercus L. [43].

Comment 11: Annotation location: Section 4.2 (original line 229). Content: Add naming author for both genera.

Response: Thank you for pointing out this issue; we agree with this comment. We have added naming authors for Medicago, Lilium, and Allium. This revision is located in lines 275-276 of the revised manuscript. Hexanucleotide repeats were scarce across the five Medicago complete chloroplast genomes, a pattern that mirrors observations in Lilium L. and Allium L. [35-36].

 

 

Thank you again for your attention and suggestions on this study. We have improved the manuscript according to your comments and hope that the revised version meets the publication requirements.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This study is devoted to phylogenetic analysis based on both coding sequences and complete chloroplast genome sequences of Medicago species. The authors  reported the complete chloroplast genome of two M. falcata ecotypes grown in different regions, and compare them with those of Medicago truncatula and Medicago sativa. The results demonstrate that M. falcata has closest relationship with M. hybrid and M. sativa. similar conclusion is stated in this paper. https://www.tandfonline.com/doi/full/10.1080/23802359.2018.1561225 Maybe the authors would find appropriate to discuss and compare the results. The reported results are convincing and well illustrated. It is worth to note that the figures are really good with all indications big enough and readable. The Authors use well accepted genetic methods and protocols, which are sufficiently described in the respective section. 

Would it be possible to discuss the notable morphological differences between M. falcata and M. hybrida  on the background of the recorded genetic similarity.

Please double check for misprints for example - line 237 "our" is duplicated and interval is missing. alos this sentence needs editing for clarity As reported previously, even grows in the same area, the phenotypic traits of M. falcata also
show considerable differences between individuals [2]. 

 

 

Author Response

Response to Review Comments on "Complete Chloroplast Genome Sequence of Medicago falcata and Comparative Analysis with Other Medicago Species"

Note: Revised parts are marked in blue

Responses to Reviewer 3's Comments

Comment 1: It is recommended to discuss and compare the results of this study with those of the literature (https://www.tandfonline.com/doi/full/10.1080/23802359.2018.1561225).

Response: Thank you for pointing out this issue; we agree with this comment. We have added a comparative analysis with this literature in the discussion section. This revision is located in lines 306-310 of the revised manuscript. The updated text is: Similarly, phylogenetic studies on Medicago sativa L. cv. Qingda no.1 and the space-mutated Medicago sativa cv. Hangmu No.1 also indicated close affiliations with M. sativa and M. falcata [16, 45], reinforcing the close evolutionary relationship within this complex.

Comment 2: It is recommended to discuss the significant morphological differences between M. falcata and M. hybrida in the context of genetic similarity.

Response: Thank you for pointing out this issue; we agree with this comment. We have added relevant content in the discussion section. This revision is located in lines 291-306 of the revised manuscript. The updated text is: Notably, although phylogenetic analyses based on plastid genomes indicate a close genetic relationship between M. falcataand M. hybrida, the two species exhibit marked morphological differences. M. falcata typically displays yellow flowers, linear-lanceolate leaflets, and a prostrate growth habit, which are adaptive traits to cold and arid environments [6, 7]. In contrast, M. hybrida (recognized as a natural hybrid of M. falcata and M. sativa) displays intermediate characteristics: including pale purple to white flowers, broader leaflets, and an erect stature, reflecting its adaptation to more diverse habitats [4, 17].

This paradox of high plastid genome similarity versus distinct morphology can be explained by two key factors. First, plastid genomes are maternally inherited and evolve slowly, retaining high sequence conservation even when nuclear genomes diverge [8, 31]. Morphological traits are more likely governed by nuclear genes (e.g., those controlling floral pigmentation or growth regulation), which are not captured by plastome analyses. Second, M. hybrida may have accumulated nuclear genetic variations through introgression between M. falcata and M. sativa, leading to phenotypic novelty while retaining the maternal plastid signature [2, 17].

Comment 3: Check for spelling errors (e.g., the repeated "our" in line 237, grammatical issues in sentences).

Response: Thank you for pointing out this issue; we agree with this comment. We have corrected spelling and grammar throughout the manuscript. The issue of repeated "our" in line 284 has been addressed by deleting the redundant word, revised to: which is consistent with our phylogenetic analyses (Figure 6).

We have comprehensively optimized the English expression throughout the manuscript, corrected grammatical errors, adjusted sentence structures, and made the writing more fluent and accurate. These revisions are incorporated throughout the entire revised manuscript and are marked in blue.

 

Thank you again for your attention and suggestions on this study. We have improved the manuscript according to your comments and hope that the revised version meets the publication requirements.

Author Response File: Author Response.docx