Construction of an SNP Fingerprinting Database and Population Genetic Analysis of Auricularia heimuer
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this manuscript, the authors perform whole-genome resequencing analysis of 150 A. heimuer germplasm samples, resulting in the identification of a high proportion of high-quality SNP loci in order to create a DNA fingerprint constructed based on core KASP markers.
Line 23. KASP is mentioned at the beginning of the text, and the meaning of the abbreviation is explained later on. I think “competitive allele-specific PCR (KASP)” should be: “kompetitive allele-specific PCR”
Line 42. Does the A. heimuer germplasm refer to natural variants or strains? Please provide more background about the existence of different germplasm of this fungus that motivates the research. Is it only based on different geographical distributions?
Figure 2: Improve the labels in the figure; the letters are blurred. In the text, mention the number and length of A. heimuer's chromosomes.
Line 218. The term CV is mentioned first, and the meaning is specified later (line 219). Check through the text. Indicate what is the relevance of CV for the population structure
Figure 3. Improve the labels in the figure; the letters are unclear and small. In the phylogenetic tree indicate the support values.
Figure 4. The caption of figure 4 is missing
Table 1. In the table´s title Indicate the meaning of Ref, Alt, PIC and MAF
Figure 6. The image is in low resolution; please improve it
Line 312 I don’t understand why you mention the varieties of Auricularia auricula and their relation to A. heimuer.
Line 319. PIC, MAF were defined before
Author Response
Comments1：Line 23. KASP is mentioned at the beginning of the text, and the meaning of the abbreviation is explained later on. I think “competitive allele-specific PCR (KASP)” should be: “kompetitive allele-specific PCR”
Response1：Thank you for pointing out the error. We have made the necessary corrections in the manuscript. For details, please refer to line 24.
Comments2：Line 42. Does the A. heimuer germplasm refer to natural variants or strains? Please provide more background about the existence of different germplasm of this fungus that motivates the research. Is it only based on different geographical distributions?
Response2：The A. heimuer germplasms used in this study include both wild and cultivated types, and they originate from a wide range of geographical locations. Detailed information on these strains is provided in Supplementary Table 1.
Comments3：Figure 2: Improve the labels in the figure; the letters are blurred. In the text, mention the number and length of A. heimuer's chromosomes.
Response3：Thank you for your suggestion. We have made the corresponding revisions in the manuscript. Please refer to Figure 2 for details.
Comments4：Line 218. The term CV is mentioned first, and the meaning is specified later (line 219). Check through the text. Indicate what is the relevance of CV for the population structure.
Response4：Thank you for your valuable suggestion. The issue has been addressed and revised in the manuscript, as indicated in line 214. The explanation regarding the correlation between the coefficient of variation (CV) and population structure is as follows: The magnitude and variation trend of the coefficient of variation (CV) directly reflect the scientific validity of population structure delineation and the degree of genetic differentiation among populations. Therefore, CV is closely associated with population structure both statistically and biologically.
Comments5：Figure 3. Improve the labels in the figure; the letters are unclear and small. In the phylogenetic tree indicate the support values.
Response5：Thank you for identifying the issue. We have uploaded a high-resolution version in the manuscript, and the corresponding revisions have been made. Please refer to Figure 3 for details.
Comments6：Figure 4. The caption of figure 4 is missing.
Response6：Thank you for pointing out the error. We have made the necessary correction, which can be found in Figure 4.
Comments7：Table 1. In the table´s title Indicate the meaning of Ref, Alt, PIC and MAF
Response7：Thank you for your suggestion. We have made the corresponding revisions in the manuscript, as shown in Table 1.
Comments8：Figure 6. The image is in low resolution; please improve it
Response8：Thank you for your suggestion. The figure has been re-uploaded in high resolution in the manuscript. Please refer to Figure 6 for details.
Comments9：Line 312 I don’t understand why you mention the varieties of Auricularia auricula and their relation to A. heimuer.
Response9：Thank you for pointing out the issue. Auricularia auricula and A. heimuer are both Latin names for black fungus; however, A. heimuer is the more widely accepted nomenclature. We have made the corresponding revision in the manuscript, as indicated in line 297.
Comments10：Line 319. PIC, MAF were defined before.
Response10：Thank you for identifying the issue. We have made the necessary revision in the manuscript, as indicated in line 307.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors
In the attached document I have some recommendation for you.
Comments for author File: 
Comments.pdf
Please improve the English to make some expressions in the manuscript clearer. In the attached manuscript I have recommended a few corrections.
Author Response
Comments1：Why is genomic resequencing of 150 A. heimuer germplasms necessary for identifying high-quality SNP sites?
Response1：Genomic resequencing of 150 A. heimuer germplasms is necessary for identifying high-quality SNP (Single Nucleotide Polymorphism) sites due to several important reasons:1. Genetic Diversity Capture: Resequencing a large and diverse set of 150 germplasms allows comprehensive sampling of the genetic variation present in the A. heimuer population. This ensures that both common and rare SNPs across different strains or cultivars are detected. 2. Accurate SNP Discovery: High-depth resequencing across many individuals improves the accuracy of SNP calling. It helps differentiate true SNPs from sequencing errors and reduces the chance of false positives. 3. Filtering and Quality Control: With sufficient sample size, stringent filtering criteria (e.g., minor allele frequency, call rate, sequencing depth) can be applied to select only high-confidence SNPs that are informative and reproducible across individuals. 4. Support for Breeding and Conservation: High-quality SNP markers identified from diverse germplasms provide a valuable resource for genetic improvement, trait mapping, and conservation of A. heimuer genetic resources.
Comments2：Line 88: “All wild germplasm…” – germplasms
Response2：Thank you for pointing out the error. We have made the necessary correction, which can be found in line 84 of the manuscript.
Comments3：“Genomic DNA was extracted from A. heimuer mycelia using the CTAB method.” - Please specify the exact protocol you followed and cite the author.
Response3：Thank you for your suggestion. The author’s information for the referenced method has been added to the manuscript, as indicated in line 92.
Comments4：Please specify the methods you used for collecting A. heimuer from different regions, because in the paper you mention that they are difficult to identify phenotypically.
Response4：You have raised a very important point. When collecting A. heimuer specimens in the field, we first perform a preliminary identification based on morphological characteristics. Subsequently, tissue isolation is conducted promptly, followed by DNA extraction. We then carry out ITS sequencing and compare the obtained sequences with those of published A. heimuer germplasms in the NCBI database to confirm whether the collected specimen is indeed A. heimuer.
Comments5： In the results section, it is stated that there is a correlation between genetic diversity and geographical distribution, but the statistical tests used to support this claim are not mentioned..
Response5：Thank you to the reviewer for pointing out this important issue. In the original manuscript, our discussion of the correlation between genetic diversity and geographical distribution was primarily based on observations from the phylogenetic tree and genetic distances among populations. However, we acknowledge that no formal statistical tests were conducted to support this correlation.
In response to the reviewer’s suggestion, we plan to carry out such statistical analyses in the follow-up work to more rigorously evaluate the relationship between geographic distance and genetic diversity. If the analysis yields significant results, we will incorporate the findings into the Results and Discussion sections accordingly. If the results are not statistically significant, we will report them transparently and explore possible explanations.
Once again, we sincerely thank the reviewer for this valuable suggestion, which will help enhance the scientific rigor and credibility of our study.
Comments6：PCA shows that pop-1 and pop-4 are grouped together; could you clarify why this is the case?.
Response6：Thank you for your question. In the PCA results, Pop-1 and Pop-4 clustered together in the principal component space, indicating a high degree of similarity in the genetic variation captured by the principal components. This clustering phenomenon may be attributed to the following factors:
- Geographical proximity: The strains in Pop-1 and Pop-4 are from relatively close geographic locations, which may facilitate gene flow and lead to similar genetic structures. This pattern is also evident in the population structure plot.
- Similar environmental selection pressures: The two regions may share similar ecological or environmental conditions, resulting in the retention of alleles with comparable adaptive value in both populations, thereby producing a clustering trend in the PCA.
We have added this analysis and explanation to the “Discussion” section of the manuscript, as indicated in line 331.
Comments7：In figure 3D, are the colors of the populations mentioned in figure 3A followed? If yes, please mention in explanation of the figure.
Response7：Thank you for the reviewer’s detailed suggestion. We confirm that the color coding of each population in Figure 3D is consistent with that used in Figure 3A, allowing readers to intuitively compare the performance of each population across different analyses. To avoid ambiguity, we have added the following note to the legend of Figure 3:“The population colors in Figures 3A and 3D are consistent to facilitate comparison of population performance across different analyses”.
Comments8：The discussions are well-founded, but they could provide more explanations regarding the applicability of the discussed technologies in the context of variety protection and marker-assisted selection..
Response8：Thank you for the reviewer’s valuable suggestion. We have added a discussion on the application value of the techniques used in this study for variety protection and marker-assisted selection (MAS). The corresponding content has been added and can be found in line 344 of the manuscript.
Variety protection: Genetic markers can be used to construct “molecular fingerprint maps” of cultivars, effectively distinguishing different varieties or germplasm resources. This helps establish the distinctness and uniqueness of a cultivar, providing crucial support for the identification of new varieties and the application and protection of plant variety rights.
Marker-assisted selection: By identifying molecular markers associated with target traits, early selection of superior individuals becomes possible, thereby improving breeding efficiency. The genetic information obtained in this study can serve as a reference for future screening of elite germplasm and the design of breeding parent combinations.
