Exploring Genetic Variability among and within Hail Tomato Landraces Based on Sequence-Related Amplified Polymorphism Markers

Round 1

Reviewer 1 Report

The study "Exploring Genetic Variability among and within Hail Tomato Landraces Based on Sequence-Related Amplified Polymorphism Markers" showed a method to assess the genetic diversity in Hail tomato populations. I want to say that in this work resulted confusing for the reader and some part of the experimental design is not clear. I am worried that in the present form is not acceptable. I reports my doubt in the case of major revision.

Firstly in the introduction, you cited the SRAP (without the acronym that is fundamental). The reader expects an explaination of this type of marker that lacks in the text (you reported only some citation. Please add clearly the information in the Introduction.

Consulting the Supplementary materials I note some difficulties to understand them in a single file. Please divide each supplementary in a single file.

Material and methods. I did not understand the plant material. A brief explaination on how you selected the different populations.

Material and methods. You reported only the tools you used for the statistical analysis but you do not reported what kink of analyses you have done. In the text, for example I found the "Shannon index", that is not specified what is, the PcoA is not mentioned, as for the Cluster analysis, ANOVA and so on.

Results. You reported the PCoA analysis and you see that the first two components explain the 26 %. I think that is a too low value to say that your populations are well-differentiated. I think that you have to consider at least the third component, since you distinguished only the 747 members from each other.

The references are old. Only seven out of 35 are at least of 5 years ago. You have to underline the novelty of your research that lacks in this study

English editing are required

Author Response

Dear Prof.

Thank you very much for your efforts added to improve our work.

We considered every point you suggested and raised. The corrections are inserted in a different color.

Best regards

Author Response File: Author Response.docx

Reviewer 2 Report

In the “Abstract” there is no clear information about the biological material. How many landraces (accessions) were studied, and how many accessions were used for each of the three Hail (548, 747, 1072) landraces?

The “Introduction” can be improved, considering that includes only nine references (three from last decade and one self-citation). Reformulate the aim of the study from lines 64-67 (the two sentences can be combined into one).

In the M&M section there is absolutely no information about the plant material (landraces/accessions). Fill with the name and geographical origin of the landraces/accessions, accessions number for each of the three Hail (548, 747, and 1072) landraces and type of tomato (cultivars, tomatillo, cherry). Give information about the plant samples used for DNA analysis. Leaves were randomly collected from …. plants / seedlings (…days old) for each landraces/accessions. DNA was extracted using …..protocol of ….(Reference). It is also appropriate to insert in this section the ssupplementary Table 1 containing SRAP primers sequences. There is no information about the statistical method (software) used for analysis of molecular variance (AMOVA).

Pay attention to the English language and style used in the description (for some) of the experimental results! E.g. the expression “Primers showed an average of 0.68 (PIC) values ranging from 0.17 for primer SRAP3 to 0.82 for primer SRAP2” can be reformulated as “The average PIC of the primers was 0.68, with values ranging from 0.17 for SRAP3 to 0.82 for SRAP2”. The title of Table 1 can be (more clearly) reformulated as follows: Features (Summary) of SRAP amplified products used for analysis of genetic diversity in Hail tomato accessions. Also, the title of Figure 1 can be (more clearly) reformulated as follows: UPGMA dendrogram generated by SRAP markers, showing the relationships among tomato Hail accessions from Saudi Arabia, based on Jaccard's coefficient. For a better visibility of the dendrogram rotate it with 180 degrees, so that the accessions names will be at the bottom of the Figure 1. Reformulate the title of Table 4 as follows: Features (Summary) of SRAP amplified products used for analysis of genetic diversity in Hail tomato landraces.

Pay attention to the English language and style used in the Discussions and Conclusions sections! E.g. the expression “Polymorphism%, (PIC) and (DP) were over many research results using SRAP Marker “can be reformulated as ‘The polymorphism (%), PIC and DP, were over the results of many SRAP marker research”. There are many other expressions that need to be checked!

Specify the references related to the sentence from lines 187-188. Specify the references related to the discussions from lines 203-213. The information from line 294 to 297 is also found in the Results section. Specify the accession carrying rare alleles (lines 301-303).

Author Response

Dear Prof.

Thank you very much for your efforts added to improve our work.

We considered every point you suggested and raised. The corrections are inserted in a different color.

Best regards

Author Response File: Author Response.docx

Reviewer 3 Report

The work is very interesting and can provide valuable information on the accessions studied

Tthe authors should provide more information on the accesions studied and number of plants per accesion, in Materials and Methods. In line 96 of Results section apperas that “96 accessions of three Hail tomato landraces”. The authors should clarify the cultivar types and the accesion number per cultivar type. Authors can also add information about accesions, such as the origin, the germplasm bank, etc.

The authors can add as supplementary figures pictures of the fruits and an electrophoresis gel

The authors should indicate the meaning of the numbers near the nodes in the dendrogram (Figure 1).

The authors can add the Bootstrap values in the dendrogram nodes, in Figure 4. With these values we can estimate the robustness of the dendrogram.

Author Response

Dear Prof.

Thank you very much for your efforts added to improve our work.

We considered every point you suggested and raised. The corrections are inserted in a different color.

Best regards

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript "Exploring Genetic Variability among and within Hail Tomato Landraces Based on Sequence-Related Amplified Polymorphism Markers" was revisioned following the reviewers' suggestion. So, I approve in the present form.

Author Response

Dear respective Profesor

Thank you very much for your efforts and help to improve our work.

Best regards

Reviewer 2 Report

In the revised form (v2) the value of the paper is considerably higher than v1. However, I consider that some changes are still needed.

Reformulate the text  “We assess inter and intra-genetic variability among 96 accessions represented three Hail tomato landrace using DNA-based marker sequence-related amplified polymorphism (SRAP).” from lines 14-16, as follow: “We assess the inter- and intra-genetic variability among 96 accessions representing three Hail tomato landraces using sequence-related amplified polymorphism (SRAP) markers.”

Reformulate the text “Seven SRAP primer combinations generated 17 alleles with a polymorphism of 100%, with an average of 7.86 polymorphic alleles per pair of primers.” From lines16-17, as follow: “Seven SRAP primer combinations generated 17 alleles with a polymorphism of 100%, and an average of 7.86 polymorphic alleles per pair of primers.”

The description of SRAP marker technique is too detailed for the “Introduction” section. This information can be grouped into two parts: one for Introduction “Sequence-related amplified polymorphism (SRAP) is an efficient and straightforward marker technique system for genetic diversity characterization because it possesses high reproducibility and discriminatory power, it discloses many codominant markers and it targets open reading frames.”, and the second part for “Material and Methods” “It is based on two-primer amplification. The primers are comprising the core sequences, which are 13 to 14 bases long, where the first 10 or 11 bases starting at the 5’ end, are sequences of no specific constitution (“filler” sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. Three selective nucleotides follow the core at the 3’ end. The filler sequences of the forward and reverse primers must differ from each other. For the first five cycles, the annealing temperature is set at 35°C. The following 35 cycles are run at 50°C”

Reformulate the text “Out of over 100 accessions for each landrace, we randomly selected 32 accessions from each landrace and were evaluated at the molecular level using SRAP markers. The accessions were labeled and numbered from 1 to 32” from lines 97-99 as follow: “Out of over 100 accessions for each landrace, we randomly selected 32 (labeled and numbered from 1 to 32) which were subsequently evaluated at the molecular level using seven SRAP markers.”

Reformulate the text “An example electrophoretic pattern…” from line 121, as follow: “An example of electrophoretic pattern….”

Replace the text “pair-wise comparisons…” from line 121, with “Pair-wise comparisons…”

Reformulate the text”…using PAST 3.11...” from line 125, as follow: “ …using PAST software version 3.11. …” or “…using PAST (v. 3.11) software. …”. Also in line 127 replace “…using the PAST (v.3.15) program” with “…using the PAST (v.3.15) software” or “…using the PAST software version 3.15”

Reformulate the text “Principal coordinate analysis (PCoA) was used the matrix of genetic distances to produce a…” from lines 127-128, as follow: “Principal coordinate analysis (PCoA) based on the genetic distances matrix was used to produce a … “

Reformulate the text “…clear visualization of the accessions' genetic diversity level…” from line 130 as follow: “…clear visualization of the genetic diversity level of accessions”

The formula for PIC from line 133 must be resized (decreased), because it is too large compared to the size of the text.

Reformulate the text “Analysis of molecular variance (AMOVA) was performed using [21] with 999 permutations.” from lines 143-144, as follow: “Analysis of molecular variance (AMOVA) was performed using GenAlEx 6.503 complement for MSExcel [21] based on 999 permutations.”

Reformulate the text “The primers' average PIC value was 0.68, with values ranging from 0.17 for SRAP3 to 0.82 for SRAP2.”  from lines  158-159, as follow: “The primers' average PIC value was 0.68, with values ranging from 0.17 for SRAP3 to 0.82 for SRAP2.”

Reformulate the text from lines 138-143 “The total number of different alleles (Na), the total number of effective alleles (Ne) = 1 / (p^2 + q^2), Shannon’s information index (I) is -1* (p * Ln (p) + q * Ln (q)), expected heterozygosity (He) = (2 * p * q), where p is the allele present and q is the absent of the allele. Percentage of polymorphic loci (% P) and private alleles per population were calculated using this approach.” as follows:   “Percentage of polymorphic loci (% P) and private alleles per population were calculated using different statistical parameters such as: the total number of different alleles (Na); the total number of effective alleles (Ne) = 1 / (p^2 + q^2); Shannon’s information index (I) is -1* (p * Ln (p) + q * Ln (q)); expected heterozygosity (He) = (2 * p * q); where p is the allele present and q is the absent of the allele.”

Reformulate the text “The narrow genetic differences between accessions used could be behind the lower values of polymorphism and PIC values, and the moderate values could be because of the narrow genetic base of the tomato cultivars and highly informative markers used in these studies [30].” from lines 264-267, as follow: “The narrow genetic differences between used accessions could be behind the lower values of polymorphism and PIC values, and the moderate values could be due to the narrow genetic base of the tomato cultivars and to highly informative markers used in this study [30].

Reformulate the text “The high percentage of polymorphism (100%), combined with a high number of polymorphic alleles generated per pair of primers (7.86), could explain by both wide range of genetic diversity, and geographical collection area and the efficiency of SRAP markers …” from lines 267-271, as follow: “The high percentage of polymorphism (100%), combined with a high number of polymorphic alleles generated per pair of primers (7.86), could be explain by both wide range of genetic diversity and geographical collection area, and by the efficiency of SRAP markers….”

Reformulate the text “Although the small number of accessions screened (96 accessions) representing three landraces, we reported an average of 7.86 polymorphic alleles, which exceeded that reported in other studies.” from lines 272-274, as follow: ”Although the number of screened accessions (96) representing three landraces was small, we found an average of 7.86 polymorphic alleles, which exceeded that reported in other studies (specify the references)”

Reformulate the text “Genetic diversity parameters and AMOVA showed that most of the genetic variation was because of differences within populations (87%), while the variability among populations contributed 13%.” from lines 355-357, as follow: “Genetic diversity parameters and AMOVA showed that most of the genetic variation was due to the differences within populations (87%), while the variability among populations has a significantly lower contribution (13%).”

Reformulate the text “The difference between the number of alleles in each locus and the number of effective loci shows rare alleles presented in Hail 1072 accessions.”  from lines 358-360, as follow: “The difference between the number of alleles in each locus and the number of effective loci shows the presence of two rare alleles in Hail 1072 accessions.”

Author Response

Dear Professor

Thank you for the valuable comments you suggested to improve our work. All corrections are inserted in the text in different colors.

Best regards

Author Response File: Author Response.docx