Molecular Characterization of Tomato (Solanum lycopersicum L.) Accessions under Drought Stress

Round 1

Reviewer 1 Report

The manuscript by Makhadmeh et al. describes the effect of drought stress on tomato. The authors studied morphological variations and chemical alternation in tomato landraces under drought condition. Additionally, they identified SSR markers that could be useful to study genetic diversity of tomato landraces and showed the putative tissue expression pattern, subcellular localization, root cell types- and tissues-specific of selected genes by mining online databases. The manuscript is overall well presented, and the experiments well designed and support the conclusions. Therefore, I will only make some minor comments in the context of figure quality and its demonstration. It could be drawn more precisely and accurately. Additionally, the manuscript contains some typographical and grammatical errors, but could be amended.

Major Comment:

The authors predicted the expression pattern of the gene associated SSRs in tomato using online databases. I would recommend that to strengthen the findings the authors should experimentally check the expression of six genes at molecular level. It would benefit the readers to know the accurate expression of genes under drought condition.

Author Response

Comment:

The manuscript by Makhadmeh et al. describes the effect of drought stress on tomato. The authors studied morphological variations and chemical alternation in tomato landraces under drought conditions. Additionally, they identified SSR markers that could be useful to studying the genetic diversity of tomato landraces and showed the putative tissue expression pattern, subcellular localization, root cell types- and tissue-specific of selected genes by mining online databases. The manuscript is overall well presented, and the experiments are well designed and support the conclusions. Therefore, I will only make some minor comments in the context of figure quality and its demonstration.

Comment 1: It could be drawn more precisely and accurately. Additionally, the manuscript contains some typographical and grammatical errors but could be amended.

Authors’ Response: Thank you for your critical comments, the context of the figure quality and its demonstration are edited. The manuscript is edited and typographical and grammatical errors are corrected.

Comment 2: The authors predicted the expression pattern of the gene-associated SSRs in tomato using online databases. I would recommend that to strengthen the findings the authors should experimentally check the expression of six genes at a molecular level. It would benefit the readers to know the accurate expression of genes under drought conditions.

Authors’ Response: Thank you for your valuable comment. This point will be involved in our future studies. Here, this study aims to evaluate the genetic variability among 46 Jordanian tomato landraces using SSR molecular marker. Considerable genetic diversity among tomato landraces was observed at the morphological, physiological, and molecular levels, which is of importance for germplasm classification, management, and further molecular and breeding utilization. Based on our results and the available expression database, we have a list of a few contrasting landraces which will be evaluated under different stress conditions and collect samples for Expression analysis from different plant tissue. Therefore, it will be another study.

These findings provide new insights into the use of such informative molecular markers to prevent discarding alleles with low frequencies that are usually present in local/traditional varieties. Further, they are indicative of the extremely wide genetic variation present in collections from Afraa and Abeel (the southern part of Jordan).  These results will help in the establishment of core collection and better conservation of Jordanian tomato landraces. Furthermore, evaluation of these landraces under different water regimes could be useful to produce new tomato varieties coping with drought stress conditions.

Reviewer 2 Report

This article include collection of forty–six tomato landraces was evaluated based on the morpho-physiological, and molecularly characterize to detect genetic diversity. Tomato landraces seedlings were exposed to drought stress with 70% field capacity and 40% field capacity under field conditions. evaluation of these landraces under different water regimes could be useful to produce new tomato varieties coping with drought stress conditions. Before recommending this article for publication, there are some shortcomings for that should be resolve.

General comments

Check the species names in the whole MS must be italicized.     

Abstract

In abstract the authors should mention which physiological and molecular attributes were considered. Also mention the area from where the landraces were selected.  

Conclude the findings and suggest future possibilities based on the results. 

Introduction

Introduction section is well written, but information is limited.

Line 45 what does mean by this type of vegetable.

Add economic and industrial importance of tomato. The following recent articles can be cited.

https://doi.org/10.1007/s10725-021-00785-7,

Why land race study is important. How it can help to increase tomato productivity.

Add significance of landraces. What special kinds of land races are in used currently in the study area. Annual tomato productivity in Jordan.

Line 89 recent articles can be cited. https://doi.org/10.3390/ijms23084450,

What are other climatic factors influencing tomato productivity.

Which measures can be adopting to coup with drought stress in the study area.

  

Materials and Methods

Experiment is well designed, and methodology is well written.

Results

Results are well displayed

Discussion

Discussion is well justified however the authors should present and discuss some relevant studies with their results.

Conclusion

Conclusion is well presented.

Author Response

 

Check the species names in the whole MS must be italicized. 

 

 Authors’ Response: Thank you for your valuable comment, all mistakes are considered.

 

Comment 2:

In the abstract, the authors should mention which physiological and molecular attributes were considered. Also, mention the area from where the landraces were selected. 

Authors’ Response: Many thanks. Please see lines 18 and 20-23.

 

Comment 3:

Conclude the findings and suggest future possibilities based on the results.

Authors’ Response: Thank you for your critical comment. Our findings and future suggestions are added. Please see lines 34-43.

 

Comment 4:

Introduction: The section is well written, but the information is limited.

Authors’ Response: Thank you, more details are added. Please see lines 69-74.

 

Comment 5:

Line 45 what does mean by this type of vegetable.

Authors’ Response: It means tomato, we have corrected it. Please see line 47.

 

Comment 6:

Add economic and industrial importance of tomato. The following recent articles can be cited.

https://doi.org/10.1007/s10725-021-00785-7,

Authors’ Response: The reference is cited, please see line 50.

 

Comment 7:

Why landrace study is important. How it can help to increase tomato productivity.

Authors’ Response: Because landraces are adapted with large hereditary types. Therefore, they have been used for some time in breeding to make a character of adaptation to abiotic stresses.

 

Comment 8:

Add significance of landraces. What special kinds of landraces are in used currently in the study area. Annual tomato productivity in Jordan.

Authors’ Response: Thank you for your valuable comment. Please see lines 54-57. The special kinds of landraces that are used currently in our study are mentioned in Table 1.

 

Comment 9:

Line 89 recent articles can be cited. https://doi.org/10.3390/ijms23084450,

Authors’ Response: The reference is cited.

 

Comment 10:

What are other climatic factors influencing tomato productivity?

 

Authors’ Response: Many thanks. High-temperature stress causes poor pollination of tomato plants and reduces seed setting rate, plant dwarfing, aging and fruit quality decline and also corresponds to a low optimality degree and comfort ratio of the microclimate. However, temperature and humidity exist at the same time, affecting and interacting with each other. Excessive temperature and humidity are likely to cause early plant senescence, shorten the growth period, increase vulnerability to pests and diseases, and affect fruit yield and quality.

https://doi.org/10.21273/HORTSCI15145-20

 

Comment 11:

Which measures can be adopted to cope with drought stress in the study area

Authors’ Response: Several morphological and physiological attributes might cope with drought tolerance such as root architecture system, shoot-to-root ratio, and osmoregulation.

 

Comment 12:

Experiment is well designed, and methodology is well written.

Authors’ Response: Thank you so much.

 

Comment 13:

Results are well displayed

Authors’ Response: Thank you.

Comment 14:

Conclusion is well presented.

Authors’ Response: Many thanks.

Reviewer 3 Report

In this manuscript, the authors collect the local strain of tomatoes and evaluate the molecular characterization under drought stress. The aim is clear. However, I have some major concerns about the “landraces”. To my knowledge, tomatoes were domesticated in central and south America. It means the species originated in western South America and Central America but does not grow in the Middle East. The definition of “landrace” is” A landrace is a domesticated, locally adapted, traditional variety of a species of animal or plant that has developed over time, through adaptation to its natural and cultural environment of agriculture and pastoralism, and due to isolation from other populations of the species”. It means the authors use the term of “landrace” maybe not match the meaning of the word. In my suggestion, the authors may use general words like “local cultivated strains” or others, but do not directly use the “landrace”. Please use this term carefully. Second, the authors use 3% agarose to do their genotyping analysis. Recently, the SSR genotyping almost use PAGE, capillary electrophoresis, or auto-read sequencer to detect the DNA bands due to the SSR alleles with minimal differences in length. The authors use of 3% agarose gel to separate the SSR alleles may not match the standard protocol at the current time. Third, the analysis of SSR is not correct. The authors transfer their SSR data to 1/0 format. The SSR marker is a type of co-dominate marker. It can not use the 1/0 format for analysis.  Based on these major concerns, the manuscript cannot publish in the current version.    

Author Response

 

Comment 1:

However, I have some major concerns about the “landraces”. To my knowledge, tomatoes were domesticated in central and south America. It means the species originated in western South America and Central America but does not grow in the Middle East. The definition of “landrace” is” A landrace is a domesticated, locally adapted, traditional variety of a species of animal or plant that has developed over time, through adaptation to its natural and cultural environment of agriculture and pastoralism, and due to isolation from other populations of the species”. It means the authors use the term of “landrace” maybe not match the meaning of the word. In my suggestion, the authors may use general words like “local cultivated strains” or others, but do not directly use the “landrace”. Please use this term carefully.

 

Authors’ Response: Thank you for your comment. The special kinds of landraces that are used currently in our study are mentioned in Table 1 and characterized as landraces based on the information obtained from the gene bank of Jordan.

There is no doubt about the center of origin of the tomato in central and south America based on the available publications. Its also known that the domestication of the tomato was furthered in Europe during the 18th and 19th centuries, and from Europe, tomatoes were spread to the Middle East, therefore, we have landraces which are adapted to local conditions in Jordan.

 

Comment 2:

Second, the authors use 3% agarose to do their genotyping analysis. Recently, the SSR genotyping almost use PAGE, capillary electrophoresis, or auto-read sequencer to detect the DNA bands due to the SSR alleles with minimal differences in length. The authors use of 3% agarose gel to separate the SSR alleles may not match the standard protocol at the current time.

Authors’ Response:

3 % Metaphor Agarose gel (FMCBioProducts) is strongly recommended to separate small size of bands from SSRs, please have a look at the lines 249-252.

https://agris.fao.org/agris-search/search.do?recordID=PK2009000169

 

Comment 3:

Third, the analysis of SSR is not correct. The authors transfer their SSR data to 1/0 format. The SSR marker is a type of co-dominate marker. It can not use the 1/0 format for analysis.  Based on these major concerns, the manuscript cannot publish in the current version.    

Authors’ Response:

Yes we used 1) or (0) refers to the presence or absence of each recorded band allele for each genotype. Then the size of present alleles fragments that were amplified by SSR was measured and used in the next step of the analysis.

We use the size in the discussion f results, please look at the section, Genetic variation among tomato landraces revealed by SSRs, Table 4 and the supplementary figures.

Reviewer 4 Report

Dear Authors and Editors,

The research presented in the manuscript is relevant and important in examining the response of different tomato genotypes to drought stress. Of particular value is that the landraces studied, which are selected by growers over a long period of time and are likely to have valuable adaptations to local growing conditions where drought is apparently a common problem. However, the manuscript also has considerable shortcomings - first of all, it was prepared rather reluctantly and without complying with the requirements of the journal, especially when citing literature sources. A thorough English revision is needed. The manuscript authors investigates the polymorphism of tomato landraces by molecular methods. However, the relationship between the results of the SSR study and drought resistance is not discussed. Therefore, the association between SSR primer sequences and related genes is somewhat questionable. Clearly, the possible functions of these genes have been well analyzed by the authors, but the links with investigations, described in the manuscript are weak.  The conclusions are too broad and vague.

The following are specific remarks:

27 Line: number0499?

47-48 line: References should be not repeated

51 line: central bank of Jordan?

53 line: inbreeding?

54 line: instead of „Gonias, Ganopoulos, Mellidou, Bibi, 54 Kalivas, Mylona, Osanthanunkul, Tsaftaris, Madesis and Doulis [3]“  it is need to write : Gonias et al, [3]

57-58 line: not clear sentence

61 line: Cattivelli et al, [10] reported that....

77 line:  Wahb-Allah et al, [14]

89 line: 2-7 base pair

99 line: to revise the sentence is required

108 line: chambers

131 line: divided by the weight of the oven-dry soil? - should be clarified

137 line: 40% field capacity under field conditions? - should be clarified

138 line: how many seeds in one replication? 10?

147 line: why 45 days?

174 - 218 line, 237-238 line: citation not in according journal requirements. It is need to  check if all citations are included in reference list, for example: Anne et al., 2005; Cortés et al., 2012?

278-279 line: Sentence should be clarified

Table 4 It need to format the table more accurately.

301-306 line: and table 3 and 4: You mean locus or allele? SSRs are single locus markers, with several alleles.

348 line: (PIC) values were ranged from 0.00 to 0499?

Line 113, 116 (change numeration): Tam, Mhiri, Vogelaar, Kerkveld, Pearce and Grandbastien [31] citation not according requirements

Author Response

 

Comment 1:

27 Line: number0499?

Authors’ Response: Thank you, it’s a mistyping. The correct number is 0.499. Please see line 28.

 

Comment 2:

47-48 line: References should be not repeated

Authors’ Response: Thanks. The repeated references are deleted. Please see line 49.

 

Comment 3:

51 line: central bank of Jordan?

Authors’ Response: Thank you. This statement is edited, please see line 54.

 

Comment 4:

53 line: inbreeding?

Authors’ Response: This mistake is edited. Please see line 57.

 

Comment 5:

54 line: instead of „Gonias, Ganopoulos, Mellidou, Bibi, 54 Kalivas, Mylona, Osanthanunkul, Tsaftaris, Madesis and Doulis [3]“  it is needed to write : Gonias et al, [3]

 

Authors’ Response: The reference citation is edited. Please see line 57.

 

Comment 6:

57-58 line: not clear sentence

Authors’ Response: Many thanks. This statement is edited, please see lines 63-65.

 

Comment 7:

61 line: Cattivelli et al, [10] reported that....

Authors’ Response: The reference citation is edited. Please see line 65.

 

Comment 8:

77 line:  Wahb-Allah et al, [14]

Authors’ Response: The reference citation is edited.

 

Comment 9:

89 line: 2-7 base pair

Authors’ Response: Please see line 101.

 

Comment 10:

99 line: to revise the sentence is required

Authors’ Response: The sentence is edited. Please see lines 110-112.

 

Comment 11:

108 line: chambers

Authors’ Response: All mistakes are considered.

 

Comment 12:

131 line: divided by the weight of the oven-dry soil? - should be clarified

Authors’ Response: This expression is clarified. Please see line 145.

 

Comment 13:

137 line: 40% field capacity under field conditions? - should be clarified

Authors’ Response: 40% field capacity under field conditions was clarified. Please see lines 142-144, and 152-154.

 

Comment 14:

138 line: how many seeds are in one replication? 10?

Authors’ Response: Ten healthy seeds for each replicate (Three replications) were used for this investigation. Please see line 155.

 

Comment 15:

147 line: why 45 days?

Authors’ Response: This indicates the vegetative stage.

 

Comment 16:

174 - 218 line, 237-238 line: citation not in according journal requirements. It needs to check if all citations are included in the reference list, for example, Anne et al., 2005; Cortés et al., 2012?

Authors’ Response: Thank you. All mistakes are considered.

 

Comment 17:

278-279 line: Sentence should be clarified

Authors’ Response: Thank you.

 

Comment 18:

Table 4 It needs to format the table more accurately.

Authors’ Response: The format of the table is adjusted.

 

Comment 19:

301-306 line: and tables 3 and 4: You mean locus or allele? SSRs are single-locus markers, with several alleles.

Authors’ Response: We mean locus with several alleles.

 

Comment 20:

348 line: (PIC) values ranged from 0.00 to 0499?

Authors’ Response: Thank you, it’s a mistyping. The correct number is 0.499.

Comment 21:

Line 113, 116 (change numeration): Tam, Mhiri, Vogelaar, Kerkveld, Pearce and Grandbastien [31] citation not according requirements

Authors’ Response: All mistakes in the reference citations are considered.

Round 2

Reviewer 1 Report

The manuscript entitled ‘Molecular characterization of tomato (Solanum lycopersicum L.) landraces under drought stress’ by Ibrahim Makhadmeh and co-authors, have satisfactorily revised and improved the manuscript. Thank you for their efforts and quick response. My only suggestion is replacement of figure 1 by a new figure with more clarity. The authors should remove the tomato pictures from the dendrogram and can mention the accession numbers used in this study.   

Author Response

Dear Reviewer,

Thanks for your comments, the pictures have been removed from figure 1.

A new figure had been added to the manuscript.

Reviewer 3 Report

In the prior comments, the authors do not focus on the major concerns and do not revise the major problems. The “landraces” is not a good term that use in this manuscript. As the reference that I put below. Bai and Lindhout (2007) already figure out the point “ Tomato (Solanum lycopersicum) originated from the Andean region now encompassed by part of Chile, Bolivia, Ecuador, Colombia, and Peru. The time and place of domestication of the tomato are not known with certainty. The tomato had reached a fairly advanced stage of domestication before being taken to Europe in the 15th century and further domestication on a much more intense level occurred throughout Europe in the 18th and 19th centuries.” It means the tomato was already domesticated before the European bring back to their countries. They should be the cultivated strain. I know several genebanks still use the term of “landrace”, but it is not used precisely. Second, the old system to separate and identify the alleles by agarose cannot apply to current studies. The authors responded to the prior comment based on the reference published in 2008. Recently, the SSR genotyping almost use PAGE, capillary electrophoresis, or auto-read sequencer to detect the DNA bands due to the SSR alleles with minimal differences in length. Third, the analysis of codominant markers cannot use the 1/0 format.  

Bai Y, Lindhout P. Domestication and breeding of tomatoes: what have we gained and what can we gain in the future?. Ann Bot. 2007;100(5):1085-1094. doi:10.1093/aob/mcm150

Author Response

Dear Reviewer,

Thanks for your critical comments.

We understood your concern about the landraces term and agreed to change it.

Therefore, now we use the "accession" term instead of "landrace" to avoid any conflict with the other terms.

Regarding agarose, this study had been done years ago when the only option in the lab was agarose.

We also removed the 0/1 table from the manuscript.

 

 

Reviewer 4 Report

Dear Authors and Editors,

The errors in the manuscript have been corrected, but the text requires more detailed corrections by an English language specialist. The conclusions remain very broad and vague.

Author Response

Dear Reviewer,

 

thanks for the comments.

 

The manuscript had been critically revised by an English specialist and the conclusion is re-written and structured. 

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 paper is of interest but the number of studied loci is low. It would be of interest to analyse HRM patterns using qPCR

Reviewer 2 Report

This paper used tomato landraces in Jordan to investigate the various phenotypic traits grown under drought stress conditions, and to investigate SSR diversity. In addition, putative associated genes were selected from the SSR information, and the functions of these genes were searched using public databases.

 

The paper has major problems in completeness as an academic paper due to the technical problems with SSR analysis and the fact that the information on landrace morphology, SSR polymorphisms, and genes are independent and not closely related to each other.

 

One of the major concerns is the SSR analysis: it is considered technically difficult to correctly identify the difference of a few bp listed in Table 2 with 3% Metaphore Agarose. The band in Figure S2 is described as a single band, but it appears to be polymorphic. Also, the scores in Table 5 do not necessarily match the information in Figures S2-S7. Furthermore, it is also an obvious mistake that Figure S7 has data (polymorphism) of 1 and 0, but Table 3 and Table 4 have a diversity index score of 0 (H, PIC and I, for example). The part "primers LEct004 and LEat018 were most significant as they can recognize all 46 landraces." in the abstract does not match results in Table 5 because there were multiple landraces of the same genotype.

 

Another concern is that the process by which candidate genes related to SSR markers were selected is unclear, which gives the impression that the discussion is a leap forward, with little relevance to phenotypic or SSR analysis. As mentioned in the cited reference, many SSR markers have already been developed, but the selection criteria for the 14 SSR markers is also unclear. The process that led to the selection of specific candidate genes needs to be described since many candidate genes are assumed to be in the vicinity of SSR loci. Furthermore, it is unclear what criteria were used to select the materials. Many of the sites listed in Table 1 are quite biased geographically within Jordan.

 

The entire paper is redundant, and the information should be consolidated in a more compact manner. For example, Figures 2, 3, and 4 are the browser search results themselves, and I do not believe that they should be included as is without taking the trouble to devote more paper space to them. Tables 3, 4, and 5 also contain raw data and can be combined into a single table.

Reviewer 3 Report

This article examined a collection of forty–six tomato landraces was evaluated based on the morpho-physiological, and molecularly characterize to detect genetic diversity. It will help the genetic breeders to study cultivars and also help the conservation strategies of different plants specifically tomato crops. Before recommending this article for publication, there are some shortcomings for that should be resolve.

General comments

I would suggest revising the MS with an expert English writer it will help to convey clear information to readers of this journal.

Abstract

In abstract section some sentences are not clear and must be revised. Such as “ as they can recognize all 46 landraces” should be revise like recognize in all 46 land races.

Methods must be clearly summarized in the abstract section.

Add quantitative results.

The authors must mention future recommendation based on results of this study in conclusion of the abstract.

I strongly recommend writing small and clear sentences.

Introduction

Introduction section is well written, sentences and paragraphs coherence are balanced. But English of the MS is very poor which must be revised.

Line 43-44 add reference or link.

Line 53-57 provide reference, which several studies. https://doi.org/10.1007/s10725-021-00785-7,  

Line 67-68 add reference after phylogenetic https://doi.org/10.3390/ijms22179175, https://doi.org/10.1016/j.plaphy.2021.01.042,  

Include mechanism of how drought effect tomato physiological factors. Also add literature review which land races are suitable for Jordan specifically in drought regions

Materials and Methods

 

Experiment is well designed, and methodology is well written. English must be improved to clarify the sentences.

Results

Line 268 provide the for tomato analyzer.

Discussion is well written.

Conclusion is well justified. The authors should discuss some points for the future studies molecular level studies are required to know about the involved mechanism and improvement of productivity.