Assessment of the Geographic Origin of Romanian Common Bean (Phaseolus vulgaris L.) Landraces Using Molecular Markers and Morphological Traits

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have assessed the geographic origin of 27 common bean landraces collected from Romania using molecular markers and morphological traits. Overall, a great idea with poor presentation and interpretation. 

Introduction
- Fig. 1 shows that The Romanian yield/year is higher than the worldwide yield/year. Is that correct?
- Fig. 2: These data are results or have been published earlier. Move to results or add a reference. What are the differences to Fig. 5? 
- Lines 113-120: Why the authors chose those three genes is unclear.
- The last paragraph commonly introduces the overall aim of the article, which is missing here. 

M&M

- In Fig. 3, please explain what is meant by the abbreviations. If it is simply for states, then there is no need for the map. Better remove and keep Fig. 6
- Footnotes lines 174-176 are not clear
- Were the same seeds used for DNA extraction and morphological measurements?
- What are those traits' agronomical or evolutionary importance, H/W, L/W, etc? Especially if they don't show a significant difference

Results
- When I looked at Figure 7, I found no explanation for part b, however, after Figure 8, authors explained Figure 7. Please, correct.
- Fiugure 8: what about the two genotypes 14245 and 15078 with only 2 SNPs different from the Mesoamerican group. Any explanation?
- I believe that the three dendograms for the three genes should be compared and even maybe combined.
- Table 4, do authors mean average or mean, not range? 
- Figure 13, only one trait is significantly different between both groups and one trait is non-significantly different. Again, how important are those traits for evolutionary studies?

Discussion
- Overall, the discussion should be re-writen to be more coherent. Now it reads as separate sentences with the assumption that readers should conclude the findings. For example, In line 406, authors states that opposite to their findings, earlier reports (reference 27) found that the majority have Andean origin. However, in lines 395 and 396, they stated that their collection has not been studied until now. This needs to be better explained.  
- Lines 422 - 424, does these substitutions reveal different amino acids, it should rather be explained as synonymous and non-synonymous substitutions as explained later.
- There should be a discussion on a combined phylogenetic tree using the three genes. 

Conclusion
- Authors stats "However, the results obtained after the analysis at the molecular level are the ones that must be considered, because they show much greater credibility". Is that maybe related to the traits that might not be importance for evolution? Maybe flowering time or adaptation to stresses could show better phenotypic results. This should be considered and discussed in the experimental design.  
- Comparing the number of the populations originating from the Mesoamerican basin or Andean basin is greatly affected by the relatively small population, only 27 selected out of 3200 different populations, lines 121 and 122. This should be taken into account for driving the whole conclusion. 

 

Comments on the Quality of English Language

Some sentences are complex to read and should be restructured. For example, especially in the discussion lines 393 - 395, and lines 420-422. 

Some sentences are miswritten, for example, in line 418: "there were identified", and line 420 "it was notified an interesting point"

It should be heterozygosity not heterozygosity, lines 448 

Author Response

Reviewer 1

Thank you for review and your comments.

Comments:

Authors have assessed the geographic origin of 27 common bean landraces collected from Romania using molecular markers and morphological traits. Overall, a great idea with poor presentation and interpretation.

 

Introduction

- Fig. 1 shows that The Romanian yield/year is higher than the worldwide yield/year. Is that correct?

The data were obtained from official website of the Food and Agriculture Organisation for the worldwide yield/year, instead, for Romania we used the data provided by the National Institute of Statistics of Romania (https://insse.ro/cms/ro/content/anuarele-statistice-ale-româniei), because we could not find data on the FAO website about common bean production, yield/year and cultivated area for all considered years. At the same time, we chose to deliver data about these parameters of common bean for 21 years to highlight the fluctuations of the interest in common bean cultivation.

 

 

- Fig. 2: These data are results or have been published earlier. Move to results or add a reference. What are the differences to Fig. 5?

Figures 2 and 5 were merged as Figure 3.

The following statement was added:

Lines: 277-280

        obtained after Sanger sequencing, for the 27 common bean accessions assessed in this study, from the Romanian Gene Bank’s collection, according to the PvDREB5A gene. The genotypes  Midas, UCD 0801 (Andean genotypes and G12873, BAT 93 (Mesoamerican genototypes) were provided by Konzend et al (Konzen et al., 2019).

- Lines 113-120: Why the authors chose those three genes is unclear.

Lines 118-131

In this study, three genes (PvDREB5A, PvDREB6B and PvRPS4) were selected to analyze the geographic origin of Phaseolus vulgaris L. local populations; two nuclear markers, known to be involved in drought stress response in common bean (PvDREB5A, PvDREB6B). PvDREB5A with a sequence length of 474 bp for Andean and 483 bp for Mesoamerican genotypes, also presents a specific SNP at +33 position guanine for Andean and cytosine for Mesoamerican genotypes. Konzen et al. [25] revealed 18 SNPs among the assessed common bean genotypes, according to the PvDREB6B gene, which can prove the geographical origin of Phaseolus vulgaris L. These genes have been poorly studied in the field of phylogeography, most research being focused on gene expression assessment under different abiotic stress conditions. Another analyzed gene is PvRPS4 from the chloroplast genome, which has not been studied until now in Phaseolus vulgaris L., as a marker able to discriminate between the two gene pools. The PvRPS4 gene is involved in drought stress response, and any SNP for Andean and Mesoamerican genotypes can be associated with a superior or inferior resistance to water scarcity.

- The last paragraph commonly introduces the overall aim of the article, which is missing here.

The following statement was added:

Lines: 151-160

The aim of the article was to find out the geographic origin of Romanian common beans landraces from specific genetic pools: Andean or Mesoamerica. Phaseolus vulgaris L from these pools differ by several biological traits. Assessment of molecular and morphological characteristics of 27 Romanian landraces collected from different areas of the country and kept in Romanian Gene Bank's collection was undertaken. In this study, three genes (two from nuclear and one from chloroplast genomes) were sequenced and different levels of variability of their nucleotide’s patterns were shown. Simultaneously, 7 morphological seed parameters were measured to reveal the diversity between populations from different geographic gene pools (Andean/Mesoamerica).

M&M

- In Fig. 3, please explain what is meant by the abbreviations. If it is simply for states, then there is no need for the map. Better remove and keep Fig. 6

Figure 3 was removed and the lines 283-284 were added.

The counties’ abbreviations can be found in Table S1 in Supplementary Materials.

 

- Footnotes lines 174-176 are not clear

 

The Table 1 footnotes, lines 207-212 were replaced with:

The size of the PvDREB5A gene is different depending on the geographic origin of the common bean:

The length of the PvDREB5A gene for Mesoamerican genotypes is 483 bp;

The length of the PvDREB5A gene for Andean genotypes is 474 bp, due to an INDEL of 9 bp.

- Were the same seeds used for DNA extraction and morphological measurements?

Lines 171-176:

For each accession, twenty seeds were randomly selected and an average of these was used to measure the quantitative morphological traits per population. Eight seeds out of 20 were grown in laboratory conditions, using a growth chamber with controlled temperature and humidity (Binder, KBWF 240) with: 25°C temperature and 80% relative humidity. The seeds were maintained in these conditions fifteen days, until the first leaves were developed and finally, these served as material for DNA isolation.

 

- What are those traits' agronomical or evolutionary importance, H/W, L/W, etc? Especially if they don't show a significant difference

Lines 106 – 112

However, several parameters such as flatness index, eccentricity index, seed flatness or seed elongation can be used to assess seed shape. Cerda et al. indicate that a flatness index value of 1 shows a spherical or oval shape and a higher value is characteristic of flat seed shapes. The eccentricity index can vary from 1 (sphere and ellipsoid shapes) to values greater than 2 (for spindle seed shapes). However, seed flatness and seed elongation parameters can be used to assess common bean seed shapes (Cerdà & García-Fayos, 2002).

 

Cerdà, A., & García-Fayos, P. (2002). The influence of seed size and shape on their removal by water erosion. Catena, 48(4), 293–301. https://doi.org/10.1016/S0341-8162(02)00027-9

 

Results

- When I looked at Figure 7, I found no explanation for part b, however, after Figure 8, authors explained Figure 7. Please, correct.

Line 268: Figure 5 a).

Line 321: Figure 5 b).

 

- Fiugure 8: what about the two genotypes 14245 and 15078 with only 2 SNPs different from the Mesoamerican group. Any explanation?

The explanation was added Lines 303-309

 

However, an interesting point of view is about two populations among the 27 samples assessed, SVGB-14245 and SVGB-15078. These landraces originate from the Andean geographic gene pool, according to PvDREB5A (due to the 9 bp INDEL and the SNP at position +33), but the results after PvDREB6B sequencing show only three different SNPs from the Mesoamerican group, this fact can suggest the possible contribution of hybridization between groups of genes (Andean x Mesoamerica).

- I believe that the three dendograms for the three genes should be compared and even maybe combined.

The following statement was added:

Lines: 353-363

The cladistic arrangement of the 27 common bean landraces, inferred based on three evaluated genes (PvDRE5A, PvDREB6B and PvRPS4), shows the following differences: the nucleotide patterns for the PvDREB5A and PvRPS4 genes generated two main distinct clades, according to the geographic gene pool (Mesoamerica/Andean), (Figures 6a and 10). Differences among the mentioned dendrograms refer to the percentage of replicate trees in which associated taxa clustered together, in the bootstrap test (1000 replicates), and can be followed next to branches. The PvDREB6B gene generated a dendrogram with two main clades, for Andean and Mesoamerican basins (Figure 6b), but with a different topology which can be considered a confirmation of a hybridization process between Andean and Mesomaerica populations for the SVGB-14245, SVGB-15078 and SVGB-5749 samples.

- Table 4, do authors mean average or mean, not range?

The Average values were corrected.

- Figure 13, only one trait is significantly different between both groups and one trait is non-significantly different. Again, how important are those traits for evolutionary studies?

 Lines: 425-429:

In general, the large seeds belong to the Andean basin, while the smaller common bean seeds are specific to the Mesoamerican gene pool. The measurements of seed morphological parameters revealed some similarities between the populations of the Andean and Mesoamerican basins and even contradict those stated above.

 

Lines: 432-434

Last but not least, any conclusion about the belonging to a certain geographical basin of common bean species without molecular analysis is not conclusive.

Discussion

- Overall, the discussion should be re-writen to be more coherent. Now it reads as separate sentences with the assumption that readers should conclude the findings. For example, In line 406, authors states that opposite to their findings, earlier reports (reference 27) found that the majority have Andean origin. However, in lines 395 and 396, they stated that their collection has not been studied until now. This needs to be better explained. 

Lines 466-472:

 

The 27 populations of common bean landraces evaluated in this study, were collected from different areas of Romania, and preserved in Gene Bank collection, in optimal conditions to maintain the viability of vegetal germoplasm. These local populations have not been assessed until now considering their geographic origin or their resistance to abiotic and biotic stress factors. This research is the first which evaluated the phylogeography of Romanian common bean landraces. In general, this type of study analyses Phaseolus vulgaris L. from the European territory, less from Eastern Europe [13] .

- Lines 422 - 424, does these substitutions reveal different amino acids, it should rather be explained as synonymous and non-synonymous substitutions as explained later.

 Lines: 506-508

This point mutation represents a non-synonymous substitution, thus revealing a different amino acid in the same position of the protein sequence.

- There should be a discussion on a combined phylogenetic tree using the three genes.

 Figure 12 was added and the discussions lines 557-564

The combined phylogenetic tree according to the assessed three genes, PvDREB5A, PvDREB6B and PvRPS4, indicated similarities with the phylogenetic tree of the PvDREB6B gene, regarding cladistic arrangement. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees where associated taxa cluster together in the bootstrap test (1000 replicates) are noted next to branches. These values are different if we compare them with the phylogenetic tree of the PvDREB6B gene (Figure). At the same time, evolutionary distances were calculated using the Maximum Composite Likelihood method.

Conclusion

- Authors stats "However, the results obtained after the analysis at the molecular level are the ones that must be considered, because they show much greater credibility". Is that maybe related to the traits that might not be importance for evolution? Maybe flowering time or adaptation to stresses could show better phenotypic results. This should be considered and discussed in the experimental design. 

 

The following statement was added:

Lines:637-649

 

Measurements of 7 morphological parameters of seeds demonstrated a high diversity of common bean germplasm and some similarities between populations from different geographic genetic pools (Andean/Mesoamerica). Furthermore, results considering seed size came in contradiction with well-known fact about smaller size and Mesoamerican gene pool. It is not surprising: during domestication, similar sets of traits were selected over a wide range of plant species, including common beans (domestication syndrome), which shows numerous examples of convergent phenotypic evolution. Convergent evolution happened between the two gene pools of the common bean after the breaking of the spatial isolation of the Mesoamerican and Andean gene pools. First: this allowed spontaneous hybridization, thus increasing the possibility of novel genotypes and phenotypes. Second: the adaptation of the species to a novel agro-ecosystems which in Romania are not too extremely diverse and to human needs increased the similarity of the traits of both genepools.

 

 

- Comparing the number of the populations originating from the Mesoamerican basin or Andean basin is greatly affected by the relatively small population, only 27 selected out of 3200 different populations, lines 121 and 122. This should be taken into account for driving the whole conclusion.

The following statement was added:

Lines 652-656

The number of the evaluated common bean landraces in this research is relatively small, compared to the Genebank collection of Phaseolus vulgaris. Out of 27 common bean local populations evaluated, most of them have Mesoamerican origin, but, to certainly state the geographical origin of the Phaseolus vulgaris species from the Romanian territory, further analysis are required.

 

Comments on the Quality of English Language

Some sentences are complex to read and should be restructured. For example, especially in the discussion lines 393 - 395, and lines 420-422.

 

lines 462-464:

To find out the origin of Romanian common bean local populations from the specific geographic basins (Mesoamerica/Andean), analysis of molecular and morphological characteristics of several samples was undertaken.

 

lines 501-502

However,  an interesting fact was reported, about the type of nucleotides and amino acids within PvDREB6B gene of Andean and Mesoamerican genotypes.

Some sentences are miswritten, for example, in line 418: "there were identified", and line 420 "it was notified an interesting point"

 lines 501-502 :

However, an interesting fact was reported, about the type of nucleotides and amino acids within PvDREB6B gene of Andean and Mesoamerican genotypes.

lines 496-498:

15 SNPs within PvDRE6B gene were identified, some of them being involved in changing of the encoded amino acid.

It should be heterozygosity not heterozygosity, lines 448

We changed in line 531, as follows:

Heterozygosity

 

References

Cerdà, A., & García-Fayos, P. (2002). The influence of seed size and shape on their removal by water erosion. Catena, 48(4), 293–301. https://doi.org/10.1016/S0341-8162(02)00027-9

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of the article was to find out the origin of  Romanian common beans landraces from specific genetic pools: Andean or Mesoamerican. These pools differ by the range of biological traits. Analysis of molecular and morphological characteristics of 27 Romanian landraces from different regions was undertaken. Three genes associated with adaptation (two from nuclear and one from chloroplast genomes) were sequenced and  different level of variability of their  nucleotides was shown.

Measurements of 7 morphological  parameters of seeds revealed a high diversity of common bean germplasm and some similarities between populations from different geographic genetic pools (Andean/Mesoamerica).  Moreover results considering seed size came in contradiction with well-known fact about smaller size og Mesoamerican genepool.  It is not surprising: during domestication, similar sets of traits were selected over a wide range of plant species, including common beans (domestication syndrome), which shows numerous examples of convergent phenotypic evolution. Convergent evolution happened between the two gene pools of the common bean after the breaking of the spatial isolation of the Mesoamerican and Andean gene pools. First: this  allowed spontaneous hybridization, thus increasing of the possibility of novel genotypes and phenotypes. Second: the  the adaptation of the species to a novel agro-ecosystems which in Romania are not too extremely diverse  and to human needs increased the similarity of the traits of two genepools.

I think these reasons should be mentioned in the article to explain the controversial results.

The common conclusion of the paper:  the analysis at the molecular level must be considered more credible.

Some erratums:

Lines18-19

“...the origin  of the current species of Phaseolus genus as being in two distinct centers with different gene-pools  (Andean and Mesoamerica)” –This is not correct. Only Ph.vulgaris and Ph.lunatus have two centres of origin, all other Phaseolus species  are limited   by Mesoamerica.

Line 140

“All these selected samples are white” -  the SEEDs of all these…………

Lines 41, 72, 265, 276 and some more

Phaseolus vulgaris L.  “L.” should not be in Italics.

Author Response

Reviewer 2

Thank you for your time and suggestions to improve our manuscript.

Please find below our explanations:

Comments:

The aim of the article was to find out the origin of Romanian common beans landraces from specific genetic pools: Andean or Mesoamerican. These pools differ by the range of biological traits. Analysis of molecular and morphological characteristics of 27 Romanian landraces from different regions was undertaken. Three genes associated with adaptation (two from nuclear and one from chloroplast genomes) were sequenced and different levels of variability of their nucleotides were shown.

 

Measurements of 7 morphological parameters of seeds revealed a high diversity of common bean germplasm and some similarities between populations from different geographic genetic pools (Andean/Mesoamerica).  Moreover, results considering seed size came in contradiction with well-known fact about smaller size of Mesoamerican genepool. It is not surprising: during domestication, similar sets of traits were selected over a wide range of plant species, including common beans (domestication syndrome), which shows numerous examples of convergent phenotypic evolution. Convergent evolution happened between the two gene pools of the common bean after the breaking of the spatial isolation of the Mesoamerican and Andean gene pools. First: this allowed spontaneous hybridization, thus increasing the possibility of novel genotypes and phenotypes. Second: the adaptation of the species to a novel agro-ecosystems which in Romania are not too extremely diverse and to human needs increased the similarity of the traits of two genepools.

I think these reasons should be mentioned in the article to explain the controversial results.

We mentioned your suggestions listed above, in lines 151-160 and 637-649.

 

The common conclusion of the paper: the analysis at the molecular level must be considered more credible.

 Lines: 671-673

 

Some erratums:

 Lines18-19: “...the origin of the current species of Phaseolus genus as being in two distinct centers with different gene-pools (Andean and Mesoamerica)” –This is not correct. Only Ph. vulgaris and Ph. lunatus have two centers of origin, all other Phaseolus species are limited by Mesoamerica.

We corrected line 19, as follows: vulgaris L.

 

Line 140: “All these selected samples are white” - the SEEDs of all these…………

We corrected line 167, as follows: The seeds of all

 

Lines 41, 72, 265, 276 and some more: Phaseolus vulgaris L.  “L.” should not be in Italics.

We changed in all article L with L

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The aim of the present work “Assessment of the Geographic Origin of Romanian Common  Bean (Phaseolus vulgaris L.) Landraces Using Molecular Markers and Morphological Traits ” by  Galan et al, is to deduce the lineage of 27 landraces based on their geographical origin, as well as their morphological and molecular characteristics. Molecular analysis was performed by analyzing DNA sequences from three genes associated with abiotic stress tolerance: PvREB5A, PvDREB6B, and PvRPS4. The work is well written and worthy of publication after minor revision.

Minor issues

i)                English editing is needed

ii)              Fig 14 is not useful

 

iii)            The authors should shorten the discussion in particular the molecular part

Comments on the Quality of English Language

minor revision

Author Response

Reviewer 3

Thank you for your time and suggestions to improve our manuscript.

Please find below our explanations:

Comments:

The aim of the present work “Assessment of the Geographic Origin of Romanian Common Bean (Phaseolus vulgaris L.) Landraces Using Molecular Markers and Morphological Traits ”by  Galan et al, is to deduce the lineage of 27 landraces based on their geographical origin, as well as their morphological and molecular characteristics. Molecular analysis was performed by analyzing DNA sequences from three genes associated with abiotic stress tolerance: PvREB5A, PvDREB6B, and PvRPS4. The work is well written and worthy of publication after minor revision.

 

Minor issues

1. i) English editing is needed

We re-written and corrected several sentence lines, as follow:

We corrected lines 118-131, as follows:

In this study, three genes (PvDREB5A, PvDREB6B and PvRPS4) were selected to analyze the geographic origin of Phaseolus vulgaris L. local populations; two nuclear markers, known to be involved in drought stress response in common bean (PvDREB5A, PvDREB6B). PvDREB5A with a sequence length of 474 bp for Andean and 483 bp for Mesoamerican genotypes, also presents a specific SNP at +33 position guanine for Andean and cytosine for Mesoamerican genotypes. Konzen et al. [25] revealed 18 SNPs among the assessed common bean genotypes, according to the PvDREB6B gene, which can prove the geographical origin of Phaseolus vulgaris L. These genes have been poorly studied in the field of phylogeography, most research being focused on gene expression assessment under different abiotic stress conditions. Another analyzed gene is PvRPS4 from the chloroplast genome, which has not been studied until now in Phaseolus vulgaris L., as a marker able to discriminate between the two gene pools. The PvRPS4 gene is involved in drought stress response, and any SNP for Andean and Mesoamerican genotypes can be associated with a superior or inferior resistance to water scarcity.

 

We corrected  Line 167, as follows:

The seeds of  all these selected samples are white.

We corrected  lines 171-176, as follows:

For each accession, twenty seeds were randomly selected and an average of these was used to measure the quantitative morphological traits per population. Eight seeds out of 20 were grown in laboratory conditions, using a growth chamber with controlled temperature and humidity (Binder, KBWF 240) with: 25°C temperature and 80% relative humidity. The seeds were maintained in these conditions fifteen days, until the first leaves were developed and finally, these served as material for DNA isolation

We corrected  lines 207-212, as follows:

The size of the PvDREB5A gene is different depending on the geographic origin of the common bean:

^† The length of the PvDREB5A gene for Mesoamerican genotypes is 483 bp;

^‡ The length of the PvDREB5A gene for Andean genotypes is 474 bp, due to an INDEL of 9 bp.

We corrected  lines 460-473, as follows:

To  find out the origin of Romanian common bean local varieties from the specific geographic basins (Mesoamerica/Andean), analysis of molecular and morphological characteristics of several samples was undertaken.  The 27 populations of common bean landraces evaluated in this study, were colected from different areas of Romania and preserved in Gene Bank collection, in optimal conditions to mantain the viability of vegetal germoplasm. These local populations have not been assessed until now considering their geographic origin or their resistance to abiotic and biotic stress factors. This research is the first which evaluated the phylogeography of Romanian common bean landraces. In general, this type of study analyses Phaseolus vulgaris L. from the European territory, less from Eastern Europe [13].  This biological material  may be considered..

We corrected  lines 479-480, as follows:

The INDEL (deletion of 9 bp) codes three units of glutamine amino acids and  the..

 

1. ii) Fig 14 is not useful

We removed Figure 14.

 

iii)          The authors should shorten the discussion in particular the molecular part:

We rephrased certain sentences to shorten them in the discussion part.

Lines: 496-498

15 SNPs, within PvDRE6B gene were identified, some of them being involved in changing of encoded  amino acid.

Lines: 501-502

However, an interesting fact was reported, about the type of nucleotides and amino acids within PvDREB6B gene of Andean and Mesoamerican genotypes.

Lines:506-508

This point mutation represents a non-synonymous substitution, thus revealing a different amino acid in the same position of the protein sequence.

We removed lines 623-629

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has improved a lot and now reads much better.

Comments on the Quality of English Language

A few errors can be found, such as line 634, "there was analyzed"