Genetic Structure of Norway Spruce Ecotypes Studied by SSR Markers

Round 1

Reviewer 1 Report

The manuscript is well written, with exception of minor mistakes, and presents novel result  on genetic characterization of large  germplasm groups representing unique plant material.

Methods are suitable and widely used for this type of studies.

1. In my opinion the population structure analysis using STRUCTURE should be done with several levels of K, and the K indicated as most probable by the software should be reported.

In addition, setting used for STRUCTURE run are not described in the Methods

Figure 3 would be more informative with the individuals within each of the three ecotype-groups sorted according to their membership it the three subpopulations (for example red first,then blue,then green)

2. Authors use term nSSR in the text, without explaining the abbreviation.

Using the term nuclear SSR is more suitable when for example  chloroplast SSR are used in parallel, 

I assume that both EST SSR and genomic SSR markers used in this study are nuclear SSRs

In my opinion gSSR (genomic SSR) a term, which the Author also used in the manuscript ( l 279)should be used in the whole manuscrtipt.

3 More information about the initial screening of markers should be added (ll118-120) - brief information about the actual initial number of markers tested (at the moment Authors state that it was a "large number") and the germplasm used to screen the markers

4 Did the Authors perform AMOVA (also available in GenAlex)?

5. The section of the Discussion describing sources of SSR markers used by other Authors can be omitted (ll258-268)

6. l 300 is "Polland" , should be "Poland"

7. Please revise the sentence "In Sweden(1942) a crown architecture based on crossing....) (ll 49-51)

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Norway spruce is an important tree species in forestry in wide parts of Europe. Therefore, investigations on genetic structure are welcome.

The following corrections are needed:

Keywords:

“Ecotypes” can be deleted. The word is part of the title.

Materials and Methods, Results, Discussion:

The typing of abbreviations of the genetic traits varies from chapter to chapter and from table to table. Sometimes letters are written normally (e.g. Hobs, Na, Fis), sometimes they are subscript (H_Obs, N_a), sometimes in italics (Fis). It is necessary to standardize the spelling.

Materials and Methods:

The abbreviations LE, ME and HE should be added (and explained) in the table 1.

Results:

In Figure 1, a decimal dot should be used in the scale of the y-axis.

In line 209 the numbers needs to be checked. I could not find the range 0.128 to 0.137 in the table 4. Here the range is from 0.128 to 0.175. What is correct?

In table 4 the decimal comas needs to be changed into dots.

Discussion:

In line 242 the numbers in the bracket are not clear. It is not a mathematical formula. If you mean from … to …, than change it. On the other hand, in table 4 you give mean values why not using them hear? A discussion of the values of the different elevations is not following, just a global one.

In line 246, the number of the expected heterozygosity does not correspond with the number in table 4.

The line 254 at the beginning of the paragraph is not indented.

Is there a reason for the typing the lines 266-273 in italics?

Author Response

Please see the attachment

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript reports on analysis of putative  three Norway spruce populations from Czech Republik, representing different ecotypes, with 16 SSR markers.

The design of study is suitable, data is analyzed using widely accepted methods. The Authors obtained interesting and novel results.

The major drawbacks of the manuscript are:

1.     Poor quality of English writing. The manuscript should be thoroughly  revised  in this respect. Many sentences are difficult to understand. Examples p 11, ll 311-314; p 10 ll261-262 “ While the EST-derived SSR markers in this study where somewhat more or less variable than the genomic SSR markers” (what does it mean?); p 10  l 284 “Slightly mean higher values had in Serbian populations..”

2.     Lengthy discussion: some very general issues,  not directly related to the study,( for example: which SSR motifs are most common in plant genomes (p 10, ll245-254), what are the sources of SSR primers used in various studies on Norway spruce (p10, ll 268-278)), should be omitted,

3.     Conclusions  (pp12 ll 17) should be formulated in a more straight-forward and direct way and included in the abstract. What are the main findings of the study? What are the main conclusions with respect to plant material analyzed? Do these findings have any consequences for forest management, etc.?  Is there a genetic background to the three phenotypic forms of Norway spruce? Was it possible to verify it with SSR markers? Are 16 SSR markers a suitable tool to answer such a question?

Minor issue: various referencing styles in the manuscript (example p 12, ll 355-362)

Author Response

Dear reviewer,

we would like to thank for highly valuable and relevant comments and recommendations. We believed that we addressed all the issues and modified our manuscript into an acceptable version. Please find all our point-by-point responses in a pdf file attached.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors of “Genetic structure of Norway spruce tree forms studied by SSR markers” have produced highly valuable material that can potentially be used (among other things) to distinguished the 3 cited ecotypes of spruce occurring on Norway. The manuscript appears to be methodologically accurate although numerous omissions and unclear sentences prevented me from fully assess that. I would recommend a genetic analysis such as STRUCTURE or BAPS if the aims are to detect genetic relationship between ecotypes. The manuscript also needs to be edited substantially and some definitions are unclear. For instance (note that these are only 3 examples among many more):

Line 66-67: “we assessed the distribution of genetic variation across the three Norway spruce ecotypes in putatively autochtonous stands of the Czech Republic”. How was this done with only three sites sampled and without replicates within each type of site?

Line 75-76: “each location representing particular ecotypic form grown in relevant climatic and geographic conditions (Table 1).” I see nothing on this on table 1. What do you mean by relevant climatic and geographic conditions?

Line 85-86: “All stands of the particular tree form poses similar climatic conditions, such as mean temperature and mean annual precipitation (Table 1).” From table 1, sites varied from 4.9 to 8.2 in temperature (note that it is not clear from the table legend what you mean by temperature, here?? Average day temperature?). Anyway, this represents a high variation range so they do not have similar climatic conditions. The same goes for precipitation.

Legends of tables and figures are not clear. For instance, in table 3, ND is stated as not performed. Why?

Figure 1 legend: “Loading plot a list invisibly returned and contained the most contributing alleles. Each of the locus is represented by the observed allele reaching the argument of threshold (R package 156 “adegenet”).” What does this mean?

Figure 2 legend: “Probability of Identity (PI), Probability of Identity (PI) for each ecotype and Probability of Identity for sibling subpopulations (PIsibs) done by GenAlEx. Locus combination shows number of 168 microsatellite markers used per study data set.” I’m sorry but I don’t follow this legend.

Figure 3: where are the eigenvalues?

Figure 4 legend: “Scatter of plotting the density of individuals in single dicrimination function (R package 219 “adegenet”)”. Again, I don’t understand this legend.

Besides all these minor doubts (that I think might easily be solved by the authors), I think the data needs to be explored (and explained) in a much better way. The sampling effort is very low but it indicates a lack of genetic correlation with the ecotypes described in previous publications (it would have been good to have more details, and references about these in the manuscript). However, the authors scarcely explored this in the discussion, which is rather focused on the markers that were developed by other authors, so I don’t really see the importance of that discussion here. 

Finally, the authors conclude that SSRs are value markers for this type of studies (eg, “microsatellite markers are still applicable for population and conservation genetic studies”…” microsatellites were demonstrated to provide more precise information on population genetic structure than SNPs”…” The amplification of the SSRs Norway spruce populations revealed high level of polymorphism” but they then end conclusions stating that “Our outputs did not prove significant genetic differentiation based on microsatellite markers. Thus, consideration for future studies are non-neutral markers (SNPs) with potential to capture adaptation processes generated by any of high-throughput genotyping method such as Genotyping by Sequencing”. Besides the fact that this sentence needs to be severely edited, it is also highly misleading. Either SSRs are good to correlate genetic and morphological variation in spruce or they aren’t. But stating both things are not an option.

In sum, before this paper could be accepted it has to be re-conceived. In its current form, I fear that the paper is misleading and will generate confusion rather than insight. 

Author Response

Dear reviewer,

we would like to thank for highly valuable and relevant comments and recommendations. We believed that we addressed all the issues and modified our manuscript into an acceptable version. Please find all our point-by-point responses in a pdf file attached.

Author Response File: Author Response.pdf