Combined Analysis of SRAP and SSR Markers Reveals Genetic Diversity and Phylogenetic Relationships in Raspberry (Rubus idaeus L.)

Response to Reviewer 2 Comments

1. Summary

We sincerely appreciate the constructive comments from you and the reviewers regarding our manuscript “Genetic Diversity and Phylogenetic Relationships in Raspberry (Rubus idaeus L.) Assisted by SRAP and SSR Markers”. (Manuscript number: agronomy-3675872). These suggestions have significantly enhanced the quality of our work. In this new version, we have taken into account all the comments raised by the reviewers, as described in the list of detailed responses provided.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

We agree with your evaluation.

Are all the cited references relevant to the research?

Yes

We agree with your evaluation.

Is the research design appropriate?

Can be improved

We agree with your evaluation.

Are the methods adequately described?

Yes

We agree with your evaluation.

Are the results clearly presented?

Yes

We agree with your evaluation.

Are the conclusions supported by the results?

Yes

We agree with your evaluation.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: If there are any outstanding traits in any of the material tested, please highlight them. Also, please provide a reference for information discussed in conclusion.  Linking the SSR data to any traits would really strengthen this paper.

Response 1: Thank you for your kind suggestion. We have added it in red.

Line 375-387: In detailed cluster analysis, we observed distinct patterns: the majority of Guizhou cultivars co-clustered with a few common cultivars (e.g., 'Polka') in the SSR dataset (Figure 1A yellow group), whereas SRAP analysis revealed complete segregation of Guizhou cultivars into an independent cluster (Figure 1B green group). This divergence highlights geography as a critical factor in clustering outcomes. Notably, 'Heritage' and 'Heritage Seeding', along with 'Fertod Zamatos' and 'Fertod Zamatos Seeding', failed to cluster together under either method, indicating pronounced trait segregation in raspberry progeny. Simultaneously, 'Autumn Miss' consistently clustered with 'Autumn Bliss', and 'Yellow Raspberry' with 'Yellow Raspberry2' across both techniques, confirming these nominally distinct accessions represent identical genotypes. Furthermore, despite including numer-ous Russian cultivars cultivated in Heilongjiang Province, we detected no significant clustering distinction between Russian and local Heilongjiang germplasm—strongly suggesting historical introduction of northeastern China's raspberries from Russia.

Comments 2: Introduction-please provide number of chromosomes in raspberry and ploidy level.

Response 2: Thank you for your kind suggestion. We have added it in red.

Line 37-38: Raspberry is a diploid species with a chromosome number of 2n = 14.

Comments 3: Also please explain why a SNP based approach was not used instead of SNP and SRAP.

Response 3: Thank you for your kind suggestion. SNP genotyping was not adopted in this study due to the unavailability of a reference genome for raspberry and our laboratory's lack of resource-intensive infrastructure required for high-throughput SNP analysis.

Comments 4: Lines 63-65: SNP markers are very common now, inexpensive and lend themselves to high throughput processing through systems like KASP.

Response 4: Thank you for your kind suggestion. We have revised it in red.

Line 65-69: Despite the technical sophistication of third-generation sequencing-derived markers such as SNPs, their implementation necessitates substantial infrastructure investment, requires reference genome dependency, and lacks real-time monitoring capabilities during detection processes [14].

Comments 5: Lines 66-68: Define acronyms SSR and SRAP when first using them.

Response 5: Thank you for your kind suggestion. We have added it in red.

Line 69-72: Consequently, among second-generation markers, simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) markers (both requiring specific primer designs) have emerged as mainstream technologies due to their operational simplicity, cost-effectiveness, and technical reliability [15].

Comments 6: Line 78-Combined should not be capitalized if preceded by semicolon.

Response 6: Thank you for your kind suggestion. We have revised it in red.

Line 83: …; combined SSR-morphometric analyses …

Comments 7: Line 84-no dash needed between berries and are.

Response 7: Thank you for your kind suggestion. We have revised it in red.

Line 90: …polymorphism in berries are constrained…

Comments 8: Line 119-leaves from mature plants or seedings?, any replication used within accessions?

Response 8: Thank you for your kind suggestion. We have revised it in red.

Line 139-143: Mature plant leaf samples (0.1 g) were ground to powder in liquid nitrogen-cooled mortars, transferred to pre-chilled centrifuge tubes, and homogenized with 1–1.5 mL of carbohydrate-depleted buffer. After centrifugation at 5,000 × g for 10 min, supernatants were assessed for viscosity and color transition to white. Subsequent steps followed the modified cetyltrimethylammonium bromide (CTAB) protocol [22].

Comments 9: Lines 122-132- Only 10 pairs of primers seems like a very limited look into the genome, same for SRAP markers.

Response 9: Thank you for your kind suggestion. During initial screening: For SSR markers, 30 primer pairs were screened, with the 10 optimal primer pairs advanced to subsequent experiments and the remaining 20 pairs documented in Supplementary Material (Table S1); For SRAP markers, 5 forward and 5 reverse primers were cross-combined to yield 25 combinatorial sequences, from which the 10 optimal primer pairs were selected for downstream analysis.

Line 146-148: Thirty pairs of SSR primers (BGI Genomics, Shenzhen, China) were screened preliminary through evaluation with amplification effects…

Comments 10: Table 2: Explain how Nei’s gene diversity index and Shannon’s information index were calculated, how are these numbers interpreted-provide minimum and maximum values for each.

Response 10: Thank you for your kind suggestion. We have added it in red.

Line 212-216: Note: Primer nomenclature followed user-defined conventions, with full sequences provided in Appendix (Tables S1 and S2). Population genetic parameters—including Percentage of Polymorphic Bands (PPB), Nei's Gene Diversity (H), and Shannon's Information Index (I)—were computed using POPGENE version 1.32. Higher values of Nei's H indicate greater genetic divergence among cultivars, while elevated Shannon's I values denote increased overall genetic diversity.

Comments 11: Lines 349-358: Not clear where this fruit size and fruiting timing data is coming from. No citation provided.

Response 11: Thank you for your kind suggestion. These field-collected temporal data are currently unpublished in study.

Comments 12: Lines 332-345: Do the accessions in each cluster share any common features? Any speculation as to why they clustered together?

Response 12: Thank you for your kind suggestion. We have added it in red.

Line 375-387: In detailed cluster analysis, we observed distinct patterns: the majority of Guizhou cultivars co-clustered with a few common cultivars (e.g., 'Polka') in the SSR dataset (Figure 1A yellow group), whereas SRAP analysis revealed complete segregation of Guizhou cultivars into an independent cluster (Figure 1B green group). This divergence highlights geography as a critical factor in clustering outcomes. Notably, 'Heritage' and 'Heritage Seeding', along with 'Fertod Zamatos' and 'Fertod Zamatos Seeding', failed to cluster together under either method, indicating pronounced trait segregation in raspberry progeny. Simultaneously, 'Autumn Miss' consistently clustered with 'Autumn Bliss', and 'Yellow Raspberry' with 'Yellow Raspberry2' across both techniques, confirming these nominally distinct accessions represent identical genotypes. Furthermore, despite including numer-ous Russian cultivars cultivated in Heilongjiang Province, we detected no significant clustering distinction between Russian and local Heilongjiang germplasm—strongly suggesting historical introduction of northeastern China's raspberries from Russia.

Comments 13: Lines 344-346-Could you provide any examples of what “ecologically resistant alleles may provide? Disease resistance, climate tolerance, please be specific.

Response 13: Thank you for your kind suggestion. We have added it in red.

Line 368-372: During our germplasm collection efforts, certain cultivars from Guizhou Province demonstrated enhanced cold tolerance by achieving typical growth performance in open-field conditions of Northeast China without protective measures. Consequently, for breeding programs aiming to develop cultivars with enhanced environmental adaptability (e.g., cold tolerance), we propose…

Comments 14: Line 376- Please explain how this research provides precision tools. Polymorphims are not linked to any traits so how would a breeder use this information to improve cultivars of raspberry?

Response 14: Thank you for your kind suggestion. We have added it in red.

Line 95-106: Integrated application of SSR and SRAP marker systems offers significant advantages in raspberry breeding programs. SSR markers exhibit high codominance and stability, enabling precise assessment of genetic diversity and relatedness, thereby optimizing parental selection. SRAP markers are operationally simple, cost-effective, and efficiently screen for polymorphisms in functional genomic regions, making them highly suitable for initial screening of large populations. Both systems facilitate the localization of genes underlying key agronomic traits. This enables genotype-assisted selection at the seedling or even seed stage, substantially accelerating the breeding cycle and improving selection efficiency. Furthermore, these markers synergistically enhance the accuracy of cultivar identification and the precision of genetic map construction. Collectively, they advance raspberry breeding towards a more precise and efficient paradigm, expediting the development of new cultivars.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your suggestion. We have carefully revised the manuscript to enhance the clarity, grammar, and overall readability of the English. All changes are marked in red in the revised version for your reference.

5. Additional clarifications

No

Response to Reviewer 3 Comments

1. Summary

We sincerely appreciate the constructive comments from you and the reviewers regarding our manuscript “Genetic Diversity and Phylogenetic Relationships in Raspberry (Rubus idaeus L.) Assisted by SRAP and SSR Markers”. (Manuscript number: agronomy-3675872). These suggestions have significantly enhanced the quality of our work. In this new version, we have taken into account all the comments raised by the reviewers, as described in the list of detailed responses provided.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

We agree with your evaluation.

Are all the cited references relevant to the research?

Yes

We agree with your evaluation.

Is the research design appropriate?

Must be improved

We agree with your evaluation.

Are the methods adequately described?

Must be improved

We agree with your evaluation.

Are the results clearly presented?

Must be improved

We agree with your evaluation.

Are the conclusions supported by the results?

Must be improved

We agree with your evaluation.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: L16 It’s hardly the first standardized method, unless you can say that if you use any raspberry accession for other part of the world it will give you its origin. It’s the first approach using this methodology.

Response 1: Thank you for your kind suggestion. We have deleted it.

Line 16-17: …and established the standardized SRAP system for this species.

Comments 2: L36 Reference 1 does not correspond to this sentence and is repeated.

Response 2: Thank you for your kind suggestion. We have deleted it.

Comments 3: L50 Ref 11 is redundant here, use only ref 10.

Response 3: Thank you for your kind suggestion. We have deleted it.

Comments 4: L61 Ref 16 is too unrelated to the subjet of this study.

Response 4: Thank you for your kind suggestion. We have deleted it.

Comments 5: L64 You mention 3rd generation sequencing derived markers without explaining what they are.

Response 5: Thank you for your kind suggestion. We have revised it in red.

Line 65-69: Despite the technical sophistication of third-generation sequencing-derived markers such as SNPs, their implementation necessitates substantial infrastructure investment, requires reference genome dependency, and lacks real-time monitoring capabilities during detection processes [14].

Comments 6: L68 Ref 19 is too unrelated to the subject of this study and redundant.

Response 6: Thank you for your kind suggestion. We have deleted it.

Comments 7: L70 SSR are not low throughput specially when compared to SRAP.

Response 7: Thank you for your kind suggestion. We have revised it in red.

Line 75-76: However, their application incurs significant costs and is constrained by a limited number of available loci.

Comments 8: L74 explain better what are SSR and SRAP.

Response 8: Thank you for your kind suggestion. We have revised it in red.

Line 73-80: SSR markers, which detect polymorphism in short tandemly repeated DNA sequences (1-6 bp), exhibit high polymorphism, multi-allelic variation, and codominant inheritance as well-established molecular tools [16]. However, their application incurs significant costs and is constrained by a limited number of available loci. In contrast, SRAP technology, targeting open reading frames (ORFs) through primers amplifying exonic and intronic regions, provides a cost-effective approach with broad genomic coverage de novo [17]. Nevertheless, SRAP analysis faces challenges in analytical complexity and reproducibility.

Comments 9: L86 consider using this for the study’s title.

Response 9: Thank you for your kind suggestion. We have revised it in red.

Line 2-4: Combined Analysis of SRAP and SSR Markers Reveals Genetic Diversity and Phylogenetic Relationships in Raspberry (Rubus idaeus L.)

Comments 10: L124 Reference for the 30 pairs of primers? All primers should be disclosed, not only the ones that worked.

Response 10: Thank you for your kind suggestion. We have added it in appendix.

Comments 11: L130 Capillary eletrophoresis is made in a sequencer. Reference for the equipment and overall reaction conditions are mising.

Response 11: Thank you for your kind suggestion. Our study employed agarose gel electrophoresis for band detection. We sincerely appreciate your valuable suggestions and have revised the manuscript by removing the erroneous methodology description.

Line 149-152: Ten pairs of primers (Table S1) were demonstrated consistent amplification efficiency across all specimens and produced distinct agarose gel electrophoretic profiles that satisfied the predefined quality criteria (Figure S1). Therefore, the validated primers were utilized for subsequent analysis.

Comments 12: L136 you mean SSR primers?

Response 12: Thank you for your kind suggestion. We have revised it in red.

Line 157: …1U Taq polymerase and SSR forward and reverse primers (0.4 mmol/L).

Comments 13: L144 Reference for the 25 pairs of primers? All primers should be disclosed, not only the ones that worked.

Response 13: For SRAP markers, 5 forward and 5 reverse primers were cross-combined to yield 25 combinatorial sequences, from which the 10 optimal primer pairs were selected for downstream analysis.

Comments 14: L167 SSR analysis by capilary eletrophoresis produces peaks, not bands. The criteria for chosing the peaks should be disclosed. Did you use all the peaks from every run? Did you use all peaks between 100-400, or only the highest peaks?

Response 14: Thank you for your kind suggestion. Our study employed agarose gel electrophoresis for band detection. We sincerely appreciate your valuable suggestions and have revised the manuscript by removing the erroneous methodology description.

Line 152: Ten pairs of primers (Table S1) were demonstrated consistent amplification efficiency across all specimens and produced distinct agarose gel electrophoretic profiles that satisfied the predefined quality criteria (Figure S1). Therefore, the validated primers were utilized for subsequent analysis.

Comments 15: L176 results from SSR are not clear. Criteria to choose peaks, statistics on peaks per sample?

Response 15: Thank you for your kind suggestion. Thank you for your kind suggestion. Our study employed agarose gel electrophoresis for band detection. We sincerely appreciate your valuable suggestions and have revised the manuscript by removing the erroneous methodology description.

Line 152: Ten pairs of primers (Table S1) were demonstrated consistent amplification efficiency across all specimens and produced distinct agarose gel electrophoretic profiles that satisfied the predefined quality criteria (Figure S1). Therefore, the validated primers were utilized for subsequent analysis.

Comments 16: L201 Criteria to choose bands is SRAP is not clear. What was the maximum distance (bp) you considered to assign bands to a locus? Also, results from SRAP are not clear. Statistics for bands, including max and minimum number of bands per accession, should be disclosed.

Response 16: Thank you for your kind suggestion. Thank you for your kind suggestion. Our study employed agarose gel electrophoresis for band detection. We sincerely appreciate your valuable suggestions and have revised the manuscript by removing the erroneous methodology description.

Line 152: Ten pairs of primers (Table S1) were demonstrated consistent amplification efficiency across all specimens and produced distinct agarose gel electrophoretic profiles that satisfied the predefined quality criteria (Figure S1). Therefore, the validated primers were utilized for subsequent analysis.

Comments 17: L296 this should be explained in the introduction.

Response 17: Thank you for your kind suggestion. We have revised it in red.

Line 325-326: Compared with traditional molecular markers, the SRAP system demonstrates superior efficiency in gene mapping, genetic diversity assessment, and DNA fingerprinting [37].

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1: Thank you for your suggestion. We have carefully revised the manuscript to enhance the clarity, grammar, and overall readability of the English. All changes are marked in red in the revised version for your reference.

5. Additional clarifications

No

Response to Reviewer 1 Comments

1. Summary

We sincerely appreciate the constructive comments from you and the reviewers regarding our manuscript “Genetic Diversity and Phylogenetic Relationships in Raspberry (Rubus idaeus L.) Assisted by SRAP and SSR Markers”. (Manuscript number: agronomy-3675872). These suggestions have significantly enhanced the quality of our work. In this new version, we have taken into account all the comments raised by the reviewers, as described in the list of detailed responses provided.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

We agree with your evaluation.

Are all the cited references relevant to the research?

Can be improved

We agree with your evaluation.

Is the research design appropriate?

Yes

We agree with your evaluation.

Are the methods adequately described?

Yes

We agree with your evaluation.

Are the results clearly presented?

Yes

We agree with your evaluation.

Are the conclusions supported by the results?

Yes

We agree with your evaluation.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The authors have made notable progress to improve the manuscript. However, I think that adding phenotypic data and evaluating the relationship between phenotypic and genotyping will add value to the manuscript. I would also recommend submitting all SSR and SRAP gel images as supplemental, and outlying the limitations of this study.

Response 1: Thank you for your kind suggestion. We strongly agree with your suggestion and acknowledge that our study has some deficiencies in investigating the association between genotype and phenotype across different raspberry varieties. In future research, we will refine the entire experimental design and incorporate additional experimental components related to this aspect. Furthermore, we will add partial gel images in the appendix, with the relevant sections highlighted in red.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your suggestion. We have carefully revised the manuscript to enhance the clarity, grammar, and overall readability of the English. All changes are marked in red in the revised version for your reference.

5. Additional clarifications

Response to Reviewer 3 Comments

1. Summary

We sincerely appreciate the constructive comments from you and the reviewers regarding our manuscript “Genetic Diversity and Phylogenetic Relationships in Raspberry (Rubus idaeus L.) Assisted by SRAP and SSR Markers”. (Manuscript number: agronomy-3675872). These suggestions have significantly enhanced the quality of our work. In this new version, we have taken into account all the comments raised by the reviewers, as described in the list of detailed responses provided.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

We agree with your evaluation.

Are all the cited references relevant to the research?

Yes

We agree with your evaluation.

Is the research design appropriate?

Can be improved

We agree with your evaluation.

Are the methods adequately described?

Must be improved

We agree with your evaluation.

Are the results clearly presented?

Can be improved

We agree with your evaluation.

Are the conclusions supported by the results?

Can be improved

We agree with your evaluation.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: L15 abstract: The first sentence is too generalistic. Consider something more informative about the crop, like Raspberry (Rubus idaeus L.) is a high-value horticultural crop recognized for its significant economic importance and exceptional nutritional profile.”

Response 1: Thank you for your kind suggestion. We have revised it in red.

Line 15-16: Raspberry (Rubus idaeus L.) is a high-value horticultural crop recognized for its significant economic importance and exceptional nutritional profile.

Comments2: L18 do you mean “established *a* standardized SRAP system for this species”?

Response 2: Thank you for your kind suggestion. You are absolutely right. We have revised it in red.

Line 18: …and established a standardized SRAP system for this species.

Comments 3: L20 cultivar = cultivars

Response 3: Thank you for your kind suggestion. We have revised it in red.

Line 21: Cultivars exhibited higher intra-group homogeneity than wild…

Comments 4: L21 showing = showed

Response 4: Thank you for your kind suggestion. We have revised it in red.

Line 22: …showed more stable similarity metrics…

Comments 5: L29 mechanisms = information

Response 5: Thank you for your kind suggestion. We have revised it in red.

Line 30: This dual-marker approach revealed complementary information…

Comments 6: L29 applications = application

Response 6: Thank you for your kind suggestion. We have revised it in red.

Line 31: Their integrated application enhance…

Comments 7: L44 established = was established

Response 7: Thank you for your kind suggestion. We have revised it in red.

Line 45: Raspberry cultivation program in China was established…

Comments 8: L146: “Thirty pairs of SSR primers (BGI Genomics, Shenzhen, China) were screened preliminary through evaluation with amplification effects of three randomly”

This sentence is confusing Do you mean: “Thirty pairs of SSR primers (BGI Genomics, Shenzhen, China) were screened prelim- inary for their efficiency to amplify R.idaeus using three randomly selected wild genotypes…” etc. ?

Response 8: Thank you for your kind suggestion. We fully endorse your perspective. We have revised it in red.

Line 147-150: Thirty pairs of SSR primers (BGI Genomics, Shenzhen, China) were screened preliminary for their efficiency to amplify raspberry using three randomly (Fanjingshan Mountain) and three cultivated accessions (Northeast Agricultural University Experimental Station) selected wild genotypes.

Comments 9: L149 “Ten pairs of primers (Table S1) were demonstrated consistent amplification efficiency across all specimens and produced distinct agarose gel electrophoretic profiles”

Do you mean “Ten pairs of primers (Table S1) showed consistent amplification across all specimens and produced distinct agarose gel electrophoretic profiles”?

Also, agarose gel electrophoresis conditions must be disclosed, as you did for SRAP analysis

Also, as pointed out in the first round of revision, you do not clarify what are the “predefined quality criteria” for chosing the primer pairs. Visible bands between xx and yy bp?

Response 9: Thank you for your kind suggestion. We fully endorse your perspective. Both SSR and SRAP analyses employed identical agarose gel electrophoresis conditions, as detailed in Section 2.5. We have revised it in red.

Line 150: …showed consistent amplification…

Line 151-152: …produced distinct, well-separated bands in agarose gel electrophoresis (Figure S1).

Line 185-187: The amplification products were separated on a 6% polyacrylamide gel using 0.5× TBE buffer as the electrophoresis medium, with electrophoresis performed at a constant current of 40 mA for 60–80 min, visualized by silver staining, and documented for band pattern analysis.

Comments 10: L149-151 states that “Ten pairs of primers (Table S1) were demonstrated consistent amplification efficiency across all specimens and produced distinct agarose gel electrophoretic profiles that satisfied the predefined quality criteria (Figure S1). ”However, Figure S1 doesn’t show the primer screening described in the text. Figure S1 shows the screening for a single primer pair (Rubus24a), which is not referred elsewhere in the article. There’s a primer Rubus24 in the SSR primer table but that one is listed as ‘Eliminate’.

I advise that you either:

- Replace figure S1 with the images of the gels for primers SSR Rubus1 to Rubus10; or

- Correct the caption of Figure S1, stating that it is an example of the preliminary screening and state the correct primer used to generate those bands

Response 10: Thank you for your kind suggestion. We wholeheartedly concur with your observation. The designation "Rubus24a" was an interim identifier used during our experimental phase. Subsequently, the nomenclature for all primers was standardized for publication, with this amplification primer finalized as Rubus9. We sincerely apologize for any confusion caused by the oversight in updating the title of Figure S1. We have revised it in red.

Comments 11: L187 2.6. Data Statistics and Analysis

Comment16 from the first round of revision is not addressed. Criteria to choose bands is SRAP is not clear. Criteria to choose bands is SRAP is not clear. What was the maximum and minimum bp you considered?

Table 2. Analysis the number of site and gene polymorphism. Has 2 columns named “Number of ploymorphic bands”, it’s a typo, correct to polymorphic

Response 11: Thank you for your kind suggestion. We fully concur with your suggestion; however, our experimental protocol involved recording only the presence or absence of bands, which were compiled into a binary (0/1) matrix. Consequently, parameters such as maximum or minimum base pair (bp) values were not incorporated into our analysis. We sincerely apologize for any inconvenience this may have caused. We have revised it in red.

Line 185-188: The amplification products were separated on a 6% polyacrylamide gel using 0.5× TBE buffer as the electrophoresis medium, with electrophoresis performed at a constant current of 40 mA for 60–80 min, visualized by silver staining, and documented for band pattern analysis.

Comments 12: L217 Overall, it is not clear which samples integrate groups A1, A2 and A3

Response 12: Thank you for your kind suggestion. We have revised it in red.

Line 235-237: Note: A1: Wild accessions from Guizhou Province (The nine accessions labeled as 3 in Table 1), China; A2: Common cultivated varieties (The five accessions labeled as 2 in Table 1—where sample correspond precisely to their respective Latin name);

Comments 13: L227 “All three metrics were significantly higher than those in A1 (Ne = 1.2100, H = 0.1800, I = 0.2800) and A3 (Ne = 1.1500, H = 0.1600, I = 0.2500) (Table 3).” Well yes, considering the difference in group size, these groups are way samller than A2! All your results have to be discussed in the light of group size: – it’s quite probable that the difference in these metrics is due to specimens from origins A1 and A3 being underrepresented in your set.

Response 13: Thank you for your kind suggestion. We have revised it.

Line 224-227: Population A2 demonstrated the highest genetic diversity using SSR markers, with the greatest effective allele count, Nei’s gene diversity index, and Shannon’s information index. All three metrics were significantly higher than those in A1 and A3 (Table 3).

Comments 14: L375 and onwards: Several of the accessions you used are from the same breeding programs, which can explain their grouping, but you didn’t mention it for all of them which have that information available. For example, Erika was produced from an open pollination of the female parent ‘Autumn Bliss’ (U.S. Plant Pat. No. 6,597) (https://patents.google.com/patent/USPP20841P3/en). For most commercial varieties the breeding program name is available, and often you can check their pedigree and find at least one of the parent varieties. Please check if accessions are grouping with those that are reported in their pedigree. This can actually help you validate your marker system and support it, or do the opposite. Either way, it should be included in the result’s analysis. Also, a table with the accessions known pedigree in the supplementary files may be appropriate, if you find enough data on them to justify it.

Response 14: Thank you for your kind suggestion. We fully agree with your suggestion. Regarding the 76 raspberry accessions, the parentage of some can be traced, while that of many others can no longer be determined. Should you require, we will supplement information on accessions with documented parentage; however, for materials with unclear parentage, specific details cannot be provided. We sincerely apologize for any inconvenience this may cause.

Comments 15: L236 sentence doesn’t start with caps lock

Response 15: Thank you for your kind suggestion. We have revised it in red.

Line 239: Phylogenetic analysis revealed…

Comments 16: Table 4 jumps a page…format it so it doesn’t do that

Response 16: Thank you for your kind suggestion. We have revised it.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response 1: Thank you for your suggestion. We have carefully revised the manuscript to enhance the clarity, grammar, and overall readability of the English. All changes are marked in red in the revised version for your reference.

5. Additional clarifications