Analysis of Genetic Diversity and Construction of a Core Collection of Ginkgo biloba Germplasm Using EST-SSR Markers

Round 1

Reviewer 1 Report

The manuscript of Yao et al. is well structured and most of the different sections are appropriated. However, some sections need to be improved. For instance, in Material and Methods section, some details need to be added. The results of this manuscript are interesting, however, Interpretation of some section in the results are not given, so you have to add them, to be more clear. English should be revised in the hole document with a native speaker

In the hole document: you should verify when you use the name of the tree ‘Ginkgo’ this should not be in italic. Only when you cite the specie you put it in italic.

Below are the suggested modifications to be done in the manuscript:

Title: Line 3: “a Medicinal Plant” it is bit confusing, this tree has medicinal properties and it is better to move this from the title, UP TO YOU. I suggest to modify the title as follows “Genetic diversity analysis and Core Collection construction of Ginkgo biloba germplasm using EST-SSR markers”

Abstract section

Line 26: “the retention rates of resources” is wrong it should be “the retention rates of this core collection for number of resources,  ..“

Line 28: you should add after “respectively” this sentence; “and representing the genetic diversity of the original germplasm”

Introduction section

Line 33: ‘Ginkgo’ is the common name of the tree, so don’t put it in italic !!!

Line 33: G. biloba commonly known as ginkgo and maidenhair tree.

Line 39: Add some details about this tree, its morphology description  and its diversity. If it has a particularity. Add its  genome size, and if it is sequenced. 

Line 70: "used for core collection construction" and for gentic diversity analysis in several crop species (add some new references example https://doi.org/10.3390/agronomy11061121)

Line 79: "codominance, high occurrence in genomes and high polymorphism" this is redondancy, it was mentioned the same in Lines 68-69.

Materials and Methods section

The subtitle 2.1 should be separated; (2.1 Plant material and Prospected provinces: where you talk about your accessions, you add the prospected regions, you can add a map with the 9 different locations prospected and you add GPS data of these locations).

For the DNA extraction : Lines 99-100: should be in section 2.2 and line 104-109 should be moved to the section 2.3

Section 2.2 (Lines 111-117): Is not clear what you did, a bit confusing when reading. Paragraph should be rewritten in a more comprehensive way.

For the subtitle 2.4 “Data Processing” it should be better “Molecular data analysis” up to you!

Line 130: “characterization of a matrix”: be specific and add what you did

Lines 148-154: Give more details (see my comments on the results section of core collection, to see the things that need to be added in the methodology)

Results section

Line 158: ‘15,176 SSR sequences’ wheras in the table 1 you mentioned ‘43,073’

Line 158: ‘1,150,995,496 bp’ verify this data in the Table 1 and correct the value in the table.

Verify all the data of the section 3.1 in order to be conform with data of Table 1.

In the Title of Table 1, you should add from where you determined this

In Table 2: add in the nucleotide type the repeats type

Line 169:  correcte ‘was analysis’ by ‘was determined’

Line 196: ‘were selected’ add how you selected these 20 SSRs which creteria

Table 3. Please verify the values 101.479 and 275.569 in Nm colum !!! the mean value of 2.671 in this same column is bit confusing when we see all the values you have in this column, VERIFY ALL THIS COLUMN

Lines 215-216: should be in Material and methods section.

Lines 218-2019: ‘The proportion of Na, Ne, H, I, He and Ho in the 101 accesions overall 20 loci are shown in Table 4’. YOU CAN DELETE THIS SENTENCE, as it is not necessary, just add in the sentence of line 217 (Table 4).

Table 4: after population column, add a column to show if the population is southwest or northewest China etc. and add nbre of individuos in each population. Also you have to add a column with Nm gene flow to see it for each population!!!

Lines 236-237: ‘The gene flow of Ginkgo populations is high (Nm=0.731-275.569), with an average value of 2.671 (Table 3). It indicates that the Ginkgo populations were genetically similar.” This should be moved to 3.4 section.

Line 238: delete the sentence ‘This conclusion is also supported by the analysis of molecular variance (AMOVA)’

Line 241-241: add interpretation of the results of Figure 2. The 2 structures you identified is based on what common things?

Lines 243-245: You have to add explanation of the results of PCA, UPGMA and phyllogenetic tree- and not just mentioning you have 3 genetic structure. Explain each analysis and each figure which information gives you regarding the 3 clusters, any particularity!

Figure 2: you should add (a) for the delta k and (b) for structure analysis giving a full title for each one. The membership coefficient is missing in the vertical axis, OF THE STRUCTURE analysis, you should add it.

You have to add in the title of (b) “found in the 10 provinces of collection” and add “of Ginkgo accessions in 10 different populations”. The title should be complete and self-explanatory. Add a footnote with the explanation of the abbreviation GX, GZ… etc each abbreviation to which province to be clear for reader. You should interpret this figure in the results section, you did not provide any explanation.

Figure 3: Add in the Title the explanation of each abbreviation to which province. Add in the title “studied by 20 polymorphic EST-SSR”. In the (B) you should add in the title the distance you used for this dendrogram

Figure 4: the Title is incomplete and should be self-explanatory. Such “Dendrogram of the 101 Ginkgo accessions studied by EST-SSR markers, based on- - analysis using the - - - ”

You Should add in figure 9, the (%) of each Coord. 1 and coord.2

In the results section, you should provide interpretation of these results.

Line 257: “random non-grouping method” You did not provide this detail in the Methodology section, you should add this detail. You have to provide it in the methodology section more details of each grouping principals, and each sampling strategy (P strategy, L strategy…etc

Discussion section:

Line 358: “and development methods of species” is not clear what you mean clarify!!!

Line 367: in the title of the section 4.3 you should put ‘population structure’ instead of ‘structure’

Lines 368-369: you are just reporting results Ho>He, you should discuss this and what it means

Lines 393-397: not well written, verify it

Line 405: “remote introduction” what you mean??

Line 426: “EST-SRR” instead of “SSR”, be concise

Conclusion section

Should be rewritten, you are reporting results in most of this section.

Line 460: “across China” better to add the detail “across 9 provinces of China”

Lines 462-469: not necessary in the conclusion section, delete from here. It is already in the abstract.

Lines 470 to 474: add details of the numbers. Rewrite it  

References section

Should be verified. Some reference, for instance the 1^st one, the title of this reference represent capital letter for each word, you should put them in lowercase letters

Author Response

Point to point response

Reviewer 1:

The manuscript of Yao et al. is well structured and most of the different sections are appropriated. However, some sections need to be improved. For instance, in Material and Methods section, some details need to be added. The results of this manuscript are interesting, however, Interpretation of some section in the results are not given, so you have to add them, to be more clear. English should be revised in the hole document with a native speaker

Thanks for the positive comment.

In the hole document: you should verify when you use the name of the tree ‘Ginkgo’ this should not be in italic. Only when you cite the specie you put it in italic.

Sorry for this mistake. The format has been thoroughly revised.

Below are the suggested modifications to be done in the manuscript:

Title: Line 3: “a Medicinal Plant” it is bit confusing, this tree has medicinal properties and it is better to move this from the title, UP TO YOU. I suggest to modify the title as follows “Genetic diversity analysis and Core Collection construction of Ginkgo biloba germplasm using EST-SSR markers”

Thanks for this suggestion. The title has been revised.

Abstract section

Line 26: “the retention rates of resources” is wrong it should be “the retention rates of this core collection for number of resources.

Thanks for this suggestion. This sentence has been revised.

Line 28: you should add after “respectively” this sentence; “and representing the genetic diversity of the original germplasm”

Thanks for this comment. This sentence has been rewritten.

Introduction section

Line 33: ‘Ginkgo’ is the common name of the tree, so don’t put it in italic!!!

Sorry for this mistake. The format has been thoroughly revised.

Line 33: G. biloba commonly known as ginkgo and maidenhair tree.

Sorry for this mistake. The name of Ginkgo biloba has been revised.

Line 39: Add some details about this tree, its morphology description and its diversity. If it has a particularity. Add its genome size, and if it is sequenced. 

Thanks for this suggestion. “Ginkgo is the only member of its family with no close relatives and its genome size is 10.61 Gb, therefore ginkgo germplasm resources are extremely precious”. This information has been added into the manuscript.

Line 70: "used for core collection construction" and for genetic diversity analysis in several crop species (add some new references example https://doi.org/10.3390/agronomy11061121)

Thanks for this suggestion. The content “and for genetic diversity analysis in several crop species” has been added. And we added a related reference (Chikh-Rouhou et al. Assessing the genetic diversity and population structure of a Tunisian Melon (Cucumis melo L.) collection using phenotypic traits and SSR molecular markers. Forests 2021 11, 1121.).

Line 79: "codominance, high occurrence in genomes and high polymorphism" this is redondancy, it was mentioned the same in Lines 68-69.

Thanks for pointing out this error. It has been deleted.

Materials and Methods section

The subtitle 2.1 should be separated; (2.1 Plant material and Prospected provinces: where you talk about your accessions, you add the prospected regions, you can add a map with the 9 different locations prospected and you add GPS data of these locations).

Thanks for this suggestion. We have replaced “Plant Material and DNA Extraction” by “Plant material and Prospected provinces”. A map (Figure 1) with the GPS data including the 10 different locations has been added.

For the DNA extraction: Lines 99-100: should be in section 2.2 and line 104-109 should be moved to the section 2.3

Thanks for this suggestion. These sections have been rearranged.

Section 2.2 (Lines 111-117): Is not clear what you did, a bit confusing when reading. Paragraph should be rewritten in a more comprehensive way.

Sorry for the ambiguity. This content has been revised as “The Primer 5.0 software was used to design SSR locus primers with sequence length > 20 bp, and 437,31 pairs of specific primers were successfully designed for 173,160 SSR loci.  In order to verify the effectiveness of SSR primers, 100 pairs of primers were randomly selected for synthesis (Table S2)”. This information has been added into Section 2.2.

For the subtitle 2.4 “Data Processing” it should be better “Molecular data analysis” up to you!

Thanks for this comment. We have replaced “Data Processing” by“Molecular data analysis”.

Line 130: “characterization of a matrix”: be specific and add what you did

Thanks for this comment. The reproducible and consistent PCR results of SSR primers were recorded as present (1) or absent (0) into a binary matrix separately for the 20-polymorphism locus. Then, the POPGENE 32 software was used to evaluate the parameters of ginkgo population genetic diversity. This information has been added into the manuscript.

Lines 148-154: Give more details (see my comments on the results section of core collection, to see the things that need to be added in the methodology)

Thanks for this comment. A total of 72 germplasm groups were obtained by random combination of two grouping principles (ten groups according to the geographical origins, random non-grouping method), four sampling ratios within groups (simple proportional (P strategy), logarithmic proportion (L strategy), square root proportion (S strategy) and genetic diversity proportion (G strategy)), two sampling methods (the locus-first stepwise clustering method, stepwise clustering random sampling method), and eight overall sampling ratios (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) (Table S4-1 to Table S4-5). This information has been added into the manuscript.

Results section

Line 158: ‘15,176 SSR sequences’ where’s in the table 1 you mentioned ‘43,073’

Sorry for our mistakes and this content has been revised as “There are 43,073 SSR sequences with the total length of 1,150,995,496 bp”.

Line 158: ‘1,150,995,496 bp’ verify this data in the Table 1 and correct the value in the table.

Verify all the data of the section 3.1 in order to be conform with data of Table 1.

Thanks for the comments and we have verified all the data of Table 1.

In the Title of Table 1, you should add from where you determined this

Thanks for this comment. We added “nuclear” in the title of Table 1.

In Table 2: add in the nucleotide type the repeats type

This content has been revised as “Repeat types”.

Line 169:  correcte ‘was analysis’ by ‘was determined’

Thanks for this comment. We have replaced “was analysis” by“was determined”.

Line 196: ‘were selected’ add how you selected these 20 SSRs which creteria

Thanks for this comment. In order to evaluate the amplification efficiency of the potential SSR markers, 437,31 pairs of specific SSR primers were designed. Among these, a total of 100 markers based on the SSR-containing sequences were randomly selected for validating and assessing the polymorphism in different ginkgo samples. For these selected SSR markers, PCR results showed that 50 pairs of primers generated clear and reproducible amplification products with high amplification efficiencies (Figure S1). Polymorphic primer bands of the above 50 primers were screened using 101 ginkgo germplasm genomic DNA, and 20 polymorphic primers were obtained. Moreover, their PCR products showed polymorphisms in the tested germplasms (Figure S2). Reproducible and consistent PCR results of SSR primers were recorded as present (1) or absent (0) into a binary matrix separately for the 20 ginkgo genotypes. This information has been added into the manuscript.

Table 3. Please verify the values 101.479 and 275.569 in Nm colum !!! the mean value of 2.671 in this same column is bit confusing when we see all the values you have in this column, VERIFY ALL THIS COLUMN

Thanks for this suggestion. After careful checking, we confirm that all the values were correct. Gene flow (Nm) was estimated based on the formula ?? = (1 − G_st)/4G_st. The G_st coefficients for all Ginkgo populations at loci GbSSR04447 and GbSSR27072 were 0.002 and 0.001, respectively. Therefore, the Nm values corresponding to these two sites are 101.479 and 275.569, respectively.

Lines 215-216: should be in Material and methods section.

Thanks for this comment. The content “Reproducible and consistent PCR results of SSR primers were recorded as present (1) or absent (0) into a binary matrix separately for the 20 ginkgo genotypes” has been rearranged to the Material and methods section.

Lines 218-219: ‘The proportion of Na, Ne, H, I, He and Ho in the 101 accesions overall 20 loci are shown in Table 4’. YOU CAN DELETE THIS SENTENCE, as it is not necessary, just add in the sentence of line 217 (Table 4).

Thanks for this comment and we have deleted this sentence.

Table 4: after population column, add a column to show if the population is southwest or northewest China etc. and add nbre of individuos in each population. Also you have to add a column with Nm gene flow to see it for each population!!!

Thanks for this comment. We have added population location and population size column in Table 4.

Lines 236-237: “The gene flow of Ginkgo populations is high (Nm=0.731-275.569), with an average value of 2.671 (Table 3). It indicates that the Ginkgo populations were genetically similar.” This should be moved to 3.4 section.

Thanks for this comment. This content has been moved to 3.4 section.

Line 238: delete the sentence ‘This conclusion is also supported by the analysis of molecular variance (AMOVA)’

Thanks for this comment. This sentence has been deleted.

Line 241-241: add interpretation of the results of Figure 2. The 2 structures you identified is based on what common things?

Thanks for this suggestion. An explanation of the results of Figure 2 has been added. These results indicate that there is extensive gene exchange between populations, which is consistent with the results of population genetic differentiation. We determine these two structures based on the results of ΔK.

Lines 243-245: You have to add explanation of the results of PCA, UPGMA and phyllogenetic tree- and not just mentioning you have 3 genetic structure. Explain each analysis and each figure which information gives you regarding the 3 clusters, any particularity!

Thanks for this comment. These results indicate that there is extensive gene exchange between populations, which is consistent with the results of population genetic differentiation.

Principal component analysis showed that the three principal components accounted for 87.24% of the total genetic variation. The first principal component accounted for 48.35% which could be used to divide 10 Ginkgo populations into 2 groups (first group including Guangxi, Guizhou, Shaanxi, Hubei, Hunan and Japan; second group including Zhejiang, Jiangsu, Shandong and Liaoning). The second principal component accounted for 22.99% of the variance and divided Ginkgo populations into 4 groups (the first group was Guangxi, Guizhou, Shaanxi and Hubei groups; the second group was Zhejiang, Jiangsu and Shandong groups; the third group was Liaoning group; the fourth group was Japan and Hunan groups). The third main component accounted for 15.90% and the 10 Ginkgo populations were divided into 3 groups (the first group including Guangxi, Guizhou, Shaanxi, Hubei, Zhejiang, Jiangsu, Shandong and Hunan; the second group was Liaoning; the third group was Japan).

Individual principal component analysis divided 101 ginkgo biloba resources into 3 categories. Except for Jiangsu group, the majority of members of the other groups were gathered together. Among them, Guangxi, Guizhou and Hunan are obviously clustered together, indicating that their genetic relationship is relatively close. The population in Jiangsu is relatively dispersed, and some individuals are distant from other populations, indicating that the genetic relationship between them is distant. This may be due to the large differences between individuals, the phenomenon of gene exchange or remote introduction.

The UPGMA method was used to perform cluster analysis. The cluster results showed that 10 Ginkgo populations could be divided into 2 categories and 3 subpopulations at genetic distance of 0.08, which was consistent with the results of genetic structure analysis (K=3) and principal component analysis. The first group includes Jiangsu, Shandong, Hunan, Hubei, Guangxi, Guizhou, Shaanxi and Liaoning populations. The Liaoning population is grouped as a single subpopulation, indicating that the genetic relationship between the Liaoning population and other populations is distant, while the Jiangsu and Shandong populations are grouped together first, indicating that their genetic relationship is the most recent. The second category includes only the Japan population, indicating that it is most genetically related to other populations.

The 101 germplasm resources of ginkgo were not clustered strictly according to their geographical provenances, and could be divided into 3 categories, which was basically consistent with the results of principal component analysis. The first category included 7 germplasm resources. The second category included 21 germplasm resources. Category 3 included 73 germplasm resources.

This information has been added into the manuscript.

Figure 2: you should add (a) for the delta k and (b) for structure analysis giving a full title for each one. The membership coefficient is missing in the vertical axis, OF THE STRUCTURE analysis, you should add it.

You have to add in the title of (b) “found in the 10 provinces of collection” and add “of Ginkgo accessions in 10 different populations”. The title should be complete and self-explanatory. Add a footnote with the explanation of the abbreviation GX, GZ… etc each abbreviation to which province to be clear for reader. You should interpret this figure in the results section, you did not provide any explanation.

Thanks for these comments. We added "(A) The best K was estimated from the ΔK statistics using Structure Harvester for ten ginkgo populations. Population structure bar plots from transcriptome data showed two clusters (best delta-K=2). (B) Genetic structure of ginkgo accessions in ten different populations. Each vertical bar indicates an individual, and the height of each colored bar represents the proportion of assignment to that cluster. Population abbreviations: GX, Guangxi province; GZ, Guizhou province; HB: Hubei province; JS: Jiangsu province; LN: Liaoning province; SD: Shandong province; SX: Shaanxi province; ZJ: Zhejiang province; JPN: Japan; HN: Hunan province." as a footnote to Figure 3 (original Figure 2).

Figure 3: Add in the Title the explanation of each abbreviation to which province. Add in the title “studied by 20 polymorphic EST-SSR”. In the (B) you should add in the title the distance you used for this dendrogram

Thanks for these comments. We added “(A) Principal component analysis and (B) UPGMA dendrograms show the relationships between ten ginkgo germplasm resources populations studied by 20 polymorphic EST-SSR. Population abbreviations please see Figure 3 (original Figure 2).” as a footnote to Figure 4 (original Figure 3).

Figure 4: the Title is incomplete and should be self-explanatory. Such “Dendrogram of the 101 Ginkgo accessions studied by EST-SSR markers, based on- - analysis using the - - - ” You Should add in figure 9, the (%) of each Coord. 1 and coord.2

Thanks for this comment. We added “Phylogenetic tree and principal component analysis of 101 ginkgo germplasm resources based on EST-SSR. (A) Maximum likelihood (ML) tree illustrates genetic relationships between 101 ginkgo germplasm resources. (B) Principal coordinate analysis of 101 individuals from ten ginkgo populations, assessed with 20 EST-SSR markers.” as a footnote to Figure 5 (original Figure 4).

In the results section, you should provide interpretation of these results.

Thanks for this comment. These results indicate that there is extensive gene exchange between populations, which is consistent with the results of population genetic differentiation. This interpretation has been added into the manuscript.

Line 257: “random non-grouping method” You did not provide this detail in the Methodology section, you should add this detail. You have to provide it in the methodology section more details of each grouping principals, and each sampling strategy (P strategy, L strategy…etc

Sorry for these errors. A total of 72 germplasm groups were obtained by random combination of two grouping principles (ten groups according to the geographical origins, random non-grouping method), four sampling ratios within groups (simple proportional (P strategy), logarithmic proportion (L strategy), square root proportion (S strategy) and genetic diversity proportion (G strategy)), two sampling methods (the locus-first stepwise clustering method, stepwise clustering random sampling method), and eight overall sampling ratios (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) (Table S4-1 to Table S4-5). This content has been added.

Discussion section:

Line 358: “and development methods of species” is not clear what you mean clarify!!!

Thanks for this comment. This content has been deleted.

Line 367: in the title of the section 4.3 you should put ‘population structure’ instead of ‘structure’

Thanks for this comment. We have replaced “structure” by “population structure”.

Lines 368-369: you are just reporting results Ho>He, you should discuss this and what it means

Thanks for this comment. These results showed that there was strong gene exchange in ten ginkgo populations, indicating that ginkgo germplasm resources genotype hybridization. This information has been added.

Lines 393-397: not well written, verify it

Thanks for this comment. Our results are in line with previous studies (Among pops=73.57%, Within pops=26.43%), suggesting that, most variation was maintained among ginkgo populations. This content has been rewritten.

Line 405: “remote introduction” what you mean??

Thanks. In the discussion section, “remote introduction” means it is a long distance from the planting place (such as Tianmu Mountain in Zhejiang Province) to the cultivation place (such as Hunan Province). This interpretation has been added.

Line 426: “EST-SRR” instead of “SSR”, be concise

Thanks for this suggestion. We have replaced “SSR” by “EST-SSR”.

Conclusion section

Should be rewritten, you are reporting results in most of this section.

Thanks for this suggestion. The conclusion has been thoroughly revised.

Line 460: “across China” better to add the detail “across 9 provinces of China”

Thanks for this suggestion. We added replaced “across China” by “across 9 provinces of China” .

Lines 462-469: not necessary in the conclusion section, delete from here. It is already in the abstract.

Thanks for this suggestion. This content has been deleted.

Lines 470 to 474: add details of the numbers. Rewrite it  

Thanks for this comment. The details of the numbers have been added (the mean value of I, He and H is 0.993, 0.566 and 0.563, respectively).

References section

Should be verified. Some reference, for instance the 1^st one, the title of this reference represent capital letter for each word, you should put them in lowercase letters

Thanks for this suggestion. The format of the references section has been carefully checked and revised to meet the standard of Forests.

Author Response File: Author Response.docx

Reviewer 2 Report

The paper “Genetic Diversity and Core Germplasm Resources Construction of a Medicinal Plant, Ginkgo biloba, Based on EST-SSR Markers.‘’ 

The research paper is prepared professionally. It includes a well-crafted abstract and an exhaustive introduction that justifies the research undertaken. The introduction points to the deficiencies in the literature on the subject. The aim is clearly defined. Modern analytical methods were used in the research. The discussion of the results is well prepared. The conclusions are well-defined. The illustrative material is appropriate.

In my opinion, the research paper after corrections, will be suitable for publication in journal.

Detailed comments:

Abstract: Should include some numeric data obtained from the study (if any)

Do not use abbreviations when use first time.

Introduction - The introduction is enough in my opinion. Introduction needs some minor changes.

Line 73-75 Morphological traits are easy to observe and measure. However, they are variable, subject to many limitations and particularly depend on the environment [19]. Please use more references. I suggest below ones

Akan S. 2022. Morphological characterisation and volatile analysis of Turkish garlic genotypes. Tur J Agric For. 46 (4):424-440. https://doi.org/10.55730/1300-011X.3015.,

Morelos-Flores, D.A.; Montalvo-González, E.; Chacón-López, M.A.; Santacruz-Varela, A.; Zamora-Gasga, V.M.; Torres-García, G.; de Lourdes García-Magaña, M. Comparative Study of Four Jackfruit Genotypes: Morphology, Physiology and Physicochemical Characterization. Horticulturae 2022, 8, 1010. https://doi.org/10.3390/horticulturae8111010

Line 77-79 Among molecular markers, simple sequence re- 77 peat (SSR) is widely used in ecology evaluation and population genetics with the ad- 78 vantages of codominance, high occurrence in genomes and high polymorphism [21]. Add more references. I suggested below ones.

Daler S, Cangi R (2022). Characterization of grapevine (V. Vinifera L.) varieties grown in Yozgat province (Turkey) by simple sequence repeat (SSR) markers. Turkish Journal of Agriculture and Forestry, 46 (1): 38-48.

Table 2. Please give only total percentage. Such as 50.25% delete 87017

NA

Author Response

Point to point response

Reviewer 2:

The paper “Genetic Diversity and Core Germplasm Resources Construction of a Medicinal Plant, Ginkgo biloba, Based on EST-SSR Markers.” 

The research paper is prepared professionally. It includes a well-crafted abstract and an exhaustive introduction that justifies the research undertaken. The introduction points to the deficiencies in the literature on the subject. The aim is clearly defined. Modern analytical methods were used in the research. The discussion of the results is well prepared. The conclusions are well-defined. The illustrative material is appropriate.

In my opinion, the research paper after corrections, will be suitable for publication in journal.

Thanks for your positive comments.

Detailed comments:

Abstract: Should include some numeric data obtained from the study (if any)

Thanks. The related numeric data has been added into the abstract.

Do not use abbreviations when use first time.

Sorry for this error. The manuscript has been carefully checked to revise this error.

Introduction - The introduction is enough in my opinion. Introduction needs some minor changes.

Thanks for your positive comments. The introduction section has been carefully revised.

Line 73-75 Morphological traits are easy to observe and measure. However, they are variable, subject to many limitations and particularly depend on the environment [19]. Please use more references. I suggest below ones

Akan S. 2022. Morphological characterisation and volatile analysis of Turkish garlic genotypes. Tur J Agric For. 46 (4):424-440. https://doi.org/10.55730/1300-011X.3015.,

Morelos-Flores, D.A.; Montalvo-González, E.; Chacón-López, M.A.; Santacruz-Varela, A.; Zamora-Gasga, V.M.; Torres-García, G.; de Lourdes García-Magaña, M. Comparative Study of Four Jackfruit Genotypes: Morphology, Physiology and Physicochemical Characterization. Horticulturae 2022, 8, 1010. https://doi.org/10.3390/horticulturae8111010

Thanks for these comments. These two references have been added into the list.

Line 77-79 Among molecular markers, simple sequence re- 77 peat (SSR) is widely used in ecology evaluation and population genetics with the ad- 78 vantages of codominance, high occurrence in genomes and high polymorphism [21]. Add more references. I suggested below ones.

Daler S, Cangi R (2022). Characterization of grapevine (V. Vinifera L.) varieties grown in Yozgat province (Turkey) by simple sequence repeat (SSR) markers. Turkish Journal of Agriculture and Forestry, 46 (1): 38-48.

Thanks for this comment. This reference has been added.

Table 2. Please give only total percentage. Such as 50.25% delete 87017

Thanks for this suggestion. The mentioned information has been deleted.

Author Response File: Author Response.docx

Reviewer 3 Report

Minor comments are in attached pdf it is regular experiment no much queries. 

Comments for author File: Comments.pdf

Author Response

Point to point response

Reviewer 3:

Minor comments are in attached pdf it is regular experiment no much queries. 

Lin 216: 20 SSRs not ginkgo genotypes.

Thanks for this comment. We Changed “ginkgo genotypes” into “polymorphism locus”.

Do climate can make an impact on diversity or aleleic richness L228 please just if

Thanks for this comment. This opinion is our speculation and there is no conclusive evidence to support it. Therefore, we decided to delete this sentence.

Author Response File: Author Response.docx

Reviewer 4 Report

In the presented study, the authors analyze the genetic diversity of Ginkgo biloba trees using EST-SSR markers. There are several comments to the manuscript.

How did the authors separate alleles if fragment analysis was not used? It is visually impossible to separate alleles that differ by 1-2-3 nucleotides. For example, I believe that the number of alleles in Fig. S2 is greater than 5, as indicated in Table 3.

Indicate in more detail what parameters were used to select 20 markers out of 50 (L. 195-196). 30 markers were monomorphic?

It is desirable to provide a map of the geographical distribution of Ginkgo individuals.

The report of Zhou et al. (2020) on the analysis of ginkgo populations using SSR markers (https://doi.org/10.1016/j.indcrop.2019.111942) should be mentioned in the manuscript.

The phrase “which was higher than ... (56.25%) [53]” (L. 354-356) should be removed, since it has no scientific sense. The plants listed are not related to each other and you can find dozens or hundreds of plant species in which the efficiency of SSR primers was above or below 50%.

Author Response

Point to point response

Reviewer 4:

In the presented study, the authors analyze the genetic diversity of Ginkgo biloba trees using EST-SSR markers. There are several comments to the manuscript.

How did the authors separate alleles if fragment analysis was not used? It is visually impossible to separate alleles that differ by 1-2-3 nucleotides. For example, I believe that the number of alleles in Fig. S2 is greater than 5, as indicated in Table 3.

Thanks for this comment. Microsatellite search module (MISA), a SSRs motif scanning tool written in Perl (https://pgrc.ipkgatersleben.de/misa/), was used for the identification and localization of perfect microsatellites, compound microsatellites and imperfect microsatellites which were interrupted by a certain number of bases. According to MISA, the identified motifs were one to six nucleotides in size, and the minimum repeat unit was defined as 10 for mononucleotides, 6 for dinucleotides, 5 for trinucleotides, 5 for tetranucleotides, 5 for pentanucleotides and hexanucleotides. After modifying the corresponding SSR identification related parameters in the misa.ini file, run the command: perl misa.pl genome.fasta. it is important to note that misa.ini and misa.pl need to be placed in one folder. We use POPGENE 32 software to evaluate the parameters of population genetic diversity, including number of alleles (Na), effective number of allele (Ne).

Indicate in more detail what parameters were used to select 20 markers out of 50 (L. 195-196). 30 markers were monomorphic?

Thanks for this comment. In order to evaluate the amplification efficiency of the potential SSR markers, 437,31 pairs of specific SSR primers were designed. Among these, a total of 100 markers based on the SSR-containing sequences were randomly selected for validating and assessing the polymorphism in different ginkgo samples. For these selected SSR markers, PCR results showed that 50 pairs of primers generated clear and reproducible amplification products with high amplification efficiencies (Figure S1). Polymorphic primer bands of the above 50 primers were screened using 101 ginkgo germplasm genomic DNA, and 20 polymorphic primers were obtained. Moreover, their PCR products showed polymorphisms in the tested germplasms (Figure S2). This information has been added into the manuscript.

It is desirable to provide a map of the geographical distribution of Ginkgo individuals.

Thanks for this suggestion. We added a map (Figure 1) with the 10 different locations prospected into the manuscript.

The report of Zhou et al. (2020) on the analysis of ginkgo populations using SSR markers (https://doi.org/10.1016/j.indcrop.2019.111942) should be mentioned in the manuscript.

Thanks for this suggestion. We added this study as a reference.

The phrase “which was higher than ... (56.25%) [53]” (L. 354-356) should be removed, since it has no scientific sense. The plants listed are not related to each other and you can find dozens or hundreds of plant species in which the efficiency of SSR primers was above or below 50%.

Thanks for this suggestion. This sentence has been deleted.

Author Response File: Author Response.docx

Reviewer 5 Report

The work is very interesting. A highly engaging analysis, comprehensive presentation of results, intriguing introduction, and an extensive discussion. In my opinion, it is suitable for publication in a journal after some minor revisions.

The ‘Materials and Methods’ section should include information regarding databases used by the researchers for transcriptome analysis.

Please include: Accession data for the populations of Ginkgo. Perhaps it is worth considering adding a map with marked populations subjected to analysis.

Please provide information regarding concentrations of the reaction mixture and also specify the annealing temperatures for individual primers. An explanation whether only the primer lengths were considered during primer design would be appreciated.

In the Results section:

Please explain the abbreviations used in Figure 3's legend. Additionally, the bootstrap values are missing on the dendrograms, and Figure 4a is unclear and difficult to analyze.

In the Discussion section, researchers mentioned that 101 populations from China were analyzed, but in the ‘Materials and Methods’ section, it was stated that 2 populations came from Japan - Table S1: 99 China, 2 Japan.

The conclusion section resembles more of an abstract.

Author Response

Point to point response

Reviewer 5:

The work is very interesting. A highly engaging analysis, comprehensive presentation of results, intriguing introduction, and an extensive discussion. In my opinion, it is suitable for publication in a journal after some minor revisions.

Thanks for your positive comments.

The ‘Materials and Methods’ section should include information regarding databases used by the researchers for transcriptome analysis.

Thanks. At present, we have completed the transcriptome analysis of leaves, stem and flower buds, and the transcriptome sequencing of seeds has not yet been completed. We plan to upload the transcriptome data as a whole after the completion of the transcriptome data, so we have not uploaded the existing data at present.

Please include: Accession data for the populations of Ginkgo. Perhaps it is worth considering adding a map with marked populations subjected to analysis.

Thanks for this suggestion. We added a map (Figure 1) with the 10 different locations prospected in the manuscript.

Please provide information regarding concentrations of the reaction mixture and also specify the annealing temperatures for individual primers. An explanation whether only the primer lengths were considered during primer design would be appreciated.

Thanks for this suggestion. The information regarding concentrations of the reaction mixture is revised and added into 2.3 section.

We all use 72°C as the annealing temperature. The SSR primers were designed as follows: > 20 bp in length, 40-60% GC content, 55-75 °C annealing temperature, and 100-400 bp PCR product. This information has been added into the manuscript.

In the Results section:

Please explain the abbreviations used in Figure 3's legend. Additionally, the bootstrap values are missing on the dendrograms, and Figure 4a is unclear and difficult to analyze.

Thanks for this comment. At the end of Figure 3 is added "(A) Principal component analysis and (B) UPGMA dendrograms show the relationships between ten ginkgo germplasm resources populations studied by 20 polymorphic EST-SSR. Population abbreviations are shown in Table 2.” as its footnote.

In the Discussion section, researchers mentioned that 101 populations from China were analyzed, but in the ‘Materials and Methods’ section, it was stated that 2 populations came from Japan - Table S1: 99 China, 2 Japan.

Thanks. In this study, totally 101 ginkgo individuals from nine provinces of China including Guangxi (5), Guizhou (8), Hubei (4), Jiangsu (41), Liaoning (3), Shandong (20), Shaanxi (3), Zhejiang (4), Hunan (11), and two individuals from Japan were sampled, please see 2.1 Plant Material and Prospected Provinces. The ginkgo individuals sampled from China and Japan.

The conclusion section resembles more of an abstract.

Thanks. The conclusion section has been thoroughly revised.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

All the comments were taken into account and all the requested modifications were done.

Just for the reference list you should correcte it as the journal instruction ( For documents co-authored by a large number of persons (more than 10 authors, you can either cite all authors, or cite the first ten authors, then add a semicolon and add ‘et al.’ at the end). When we look to your references we see that you use et al. even for 3 or 4 authors, so you have to correct this.

Reference #20: correct the journal it is Agronomy instead of Forests.

Author Response

Dear editor,尊敬的编辑：

It gives us pleasure to submit our revised manuscript entitled “Analysis of Genetic Diversity and Construction of a Core Collection of Ginkgo biloba Germplasm Using EST-SSR Markers” (2646401). We thank the comments from the reviewer 1 and have revised the manuscript according to the comments. The revised content is marked in green.
我们很高兴提交我们的修订稿，题为“遗传多样性分析和银杏核心种质的构建EST-SSR标记”（2646401）。我们感谢审稿人1的评论，并根据评论修改了手稿。修改后的内容用绿色标记。

Sincerely,诚挚的，

Yiqiang Wang王义强

Central South University of Forestry and Technology
中南林业科技大学

410000, Hunan, Changsha410000，湖南，长沙

Point to point response点对点响应

Reviewer 1:审核人1：

All the comments were taken into account and all the requested modifications were done.
所有的意见都得到了考虑，所有要求的修改都得到了执行。

Just for the reference list you should correct it as the journal instruction (For documents co-authored by a large number of persons (more than 10 authors, you can either cite all authors, or cite the first ten authors, then add a semicolon and add ‘et al.’ at the end). When we look to your references, we see that you use et al. even for 3 or 4 authors, so you have to correct this.
只是对于参考文献列表，你应该纠正它作为期刊说明（对于由大量的人共同撰写的文件（超过10位作者，你可以引用所有作者，或引用前十位作者，然后添加一个括号，并添加'等'。最后）。当我们看你的参考文献时，我们看到你使用了et al.即使是3或4个作者，所以你必须纠正这一点。

Thanks for this suggestion. The format of the references section has been carefully checked and revised to meet the standard of Forests.
谢谢你的建议。参考文献部分的格式经过仔细检查和修改，以符合森林标准。

Reference #20: correct the journal it is Agronomy instead of Forests.
参考文献#20：更正期刊是农学而不是森林学。

Sorry for this error. It has been revised.很抱歉这个错误。已经修订。

Author Response File: Author Response.docx