Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.)
, Barbara George-Jaeggli 1,2, Marie Bouteillé-Pallas 1, Shuaishuai Tai 3, Alan Cruickshank 2, David Jordan 1 and Emma Mace 1,2,*
Round 1
Reviewer 1 Report
This article showed meaningful results regarding the SNPs in sorghum C4 photosynthesis genes. Actually, C4 photosynthesis system has remarkable advantages compared with C3 plants, and becoming more important under global warming in this world, thus these results on diversity of C4 photosynthesis genes and SNPs information are quite important for the study of C4 photosynthesis. This article covered up to 48 genotypes of sorghum including cultivated sorghum, wild and weedy sorghum and discovered 4183 SNPs including 521 SNPs in coding sequence regions. This information would be useful for further study of sorghum C4 photosynthesis and also sorghum breeding. In these reaons, I think this article worthwhile publishing in this journal. However, there are various manuscripts and figures to be improved as follows.
L 35 “to also act as an oxygenase in the presence of oxygen (O2)” This phrase need to be modified.
L44 “ CO2 concentration in C4 bundle sheath cells are up to 10-fold higher than those found in C3 mesophyll cells” is better to write as follows “CO2 concentration in C4 bundle sheath cells is up to 10-fold higher than that found in C3 mesophyll cells”
L49 “in areas of high light intensities” is modified to “in areas with high light intensities”
L51 I think that “and are still grown in the” is better to be deleted.
L78 Comma after 2013) is not necessary
Figure 1. Letters are overlapped with borders of mesophyll and bundle sheath. These letters should be inside or outside of the borders.
Figure 1 does not represent correct photosynthesis reactions. Authors need to make better figure by citing other articles and/or books, for example https://pubmed.ncbi.nlm.nih.gov/24964784/, http://www.plantphysiol.org/content/164/4/2231
Figure S1. The number of bootstrap is missing.
L125 “moudel” is not correct. “module” is correct.
Figure 2. The size of accession numbers are too small to see. They need to be increased.
L284 Add comma after However
L380 Authors needs to follow the format to describe reference articles.
Author Response
Dear reviewer, we have revised this manuscript according to your valuable comments. Please be aware that line numbers were changed after converting to the citation format of the journal.
This article showed meaningful results regarding the SNPs in sorghum C4 photosynthesis genes. Actually, C4 photosynthesis system has remarkable advantages compared with C3 plants, and becoming more important under global warming in this world, thus these results on diversity of C4 photosynthesis genes and SNPs information are quite important for the study of C4 photosynthesis. This article covered up to 48 genotypes of sorghum including cultivated sorghum, wild and weedy sorghum and discovered 4183 SNPs including 521 SNPs in coding sequence regions. This information would be useful for further study of sorghum C4 photosynthesis and also sorghum breeding. In these reaons, I think this article worthwhile publishing in this journal. However, there are various manuscripts and figures to be improved as follows.
L 35 “to also act as an oxygenase in the presence of oxygen (O2)” This phrase need to be modified.
Response: “to also act as an oxygenase in the presence of oxygen (O2)” has been changed to “catalyse an unfavorable oxygenation reaction”, lines 40-41
L44 “ CO2 concentration in C4 bundle sheath cells are up to 10-fold higher than those found in C3 mesophyll cells” is better to write as follows “CO2 concentration in C4 bundle sheath cells is up to 10-fold higher than that found in C3 mesophyll cells”
Response: this sentence has been revised as suggested, line 49
L49 “in areas of high light intensities” is modified to “in areas with high light intensities”
Response: this sentence has been revised as suggested, line 53
L51 I think that “and are still grown in the” is better to be deleted.
Response: this sentence has been revised as suggested, line 55
L78 Comma after 2013) is not necessary
Response: the comma has been deleted as suggested, line 77
Figure 1. Letters are overlapped with borders of mesophyll and bundle sheath. These letters should be inside or outside of the borders.
Response: a new Figure 1 has been added to replace the old Figure 1. No letter is sitting on the borders now.
Figure 1 does not represent correct photosynthesis reactions. Authors need to make better figure by citing other articles and/or books, for example https://pubmed.ncbi.nlm.nih.gov/24964784/, http://www.plantphysiol.org/content/164/4/2231
Response: a new Figure 1 based on a recent study (Ermakova et al., 2020) has been added to replace the old Figure 1.
Figure S1. The number of bootstrap is missing.
Response: the number of bootstrap has been added in Figure S1 and line 158
L125 “moudel” is not correct. “module” is correct.
Response: this has been changed, line 132
Figure 2. The size of accession numbers are too small to see. They need to be increased.
Response: the size of the accession numbers has been increased in the revised Figure 2
L284 Add comma after However
Response: a comma has been added as suggested, line 291
L380 Authors needs to follow the format to describe reference articles.
Response: reference and citation style has been changed throughout this manuscript to meet the requirement of the journal
Reviewer 2 Report
„Genetic diversity of C4 photosynthesis pathway genes in Sorghum bicolor (L.)”
In this study Tao et al. investigate the genetic diversity within 48 Sorghum genotypes in regards to the core C4 genes of the NADP-ME C4 pathway. They use the availability of various cultivated and wild Sorghum varieties to determine different alleles within these genotypes. Whereas the cultivated species do not have a large variety the uncultivated species show a lot more diversity. Via the analysis of SNPs selection signals were found. The study shows that during the selection process of Sorghum the genetic diversity was reduced compared to the wild species. The bigger variation of the genetic material within the wild species opens the possibility to access new alleles that could benefit in elevating the photosynthetic efficiency.
The data presented in this study is well conducted and approaches a yet not analysed field by checking the genetic diversity of C4 genes within the cultivated and wild varieties within one species. This is an innovative idea that may help to a better understanding how to improve photosynthesis.
Only minor details need to be improve the study:
- A list of abbreviations for the analysed genes could be useful in the beginning of the text
- Figure 1 should go before Table 1, to clarify the abbreviations used in the table
Lines 79ff maybe also add the comparative studies performed in the genus Flaveria by Gowik et al. 2011, Mallmann et al. 2014 in the list
Line 125: module not moudel
Line 152: Geneious 8.1.2 needs information about the distributor, also which parameters were used?
- Figure 2: The red and green colour combination in panel A should be considered to be changed, even though the logic of the diagram makes clear which is which, it would be considerate for colour-seeing impaired people
Lines 206ff: maybe add a short explanation what “balancing and purifying selection” means exactly, to remind the reader
Figure 3: I suggest to add the gene names to the figure itself, that would be much easier than the identifiers alone.
Author Response
Dear reviewer, we have revised this manuscript according to your valuable comments. Please be aware that line numbers were changed after converting to the citation format of the journal.
In this study Tao et al. investigate the genetic diversity within 48 Sorghum genotypes in regards to the core C4 genes of the NADP-ME C4 pathway. They use the availability of various cultivated and wild Sorghum varieties to determine different alleles within these genotypes. Whereas the cultivated species do not have a large variety the uncultivated species show a lot more diversity. Via the analysis of SNPs selection signals were found. The study shows that during the selection process of Sorghum the genetic diversity was reduced compared to the wild species. The bigger variation of the genetic material within the wild species opens the possibility to access new alleles that could benefit in elevating the photosynthetic efficiency.
The data presented in this study is well conducted and approaches a yet not analysed field by checking the genetic diversity of C4 genes within the cultivated and wild varieties within one species. This is an innovative idea that may help to a better understanding how to improve photosynthesis.
Only minor details need to be improve the study:
A list of abbreviations for the analysed genes could be useful in the beginning of the text
Response: a list of abbreviations of analysed genes has been added to the manuscript, lines 32-35
Figure 1 should go before Table 1, to clarify the abbreviations used in the table
Response: the order of Figure 1 and Table 1 has been changed
Lines 79ff maybe also add the comparative studies performed in the genus Flaveria by Gowik et al. 2011, Mallmann et al. 2014 in the list.
Response: both studies have been added in the list, line 80 and bibliography.
Line 125: module not moudel
Response: this has been changed, line 132
Line 152: Geneious 8.1.2 needs information about the distributor, also which parameters were used?
Response: these information has been added, lines 158-160
Figure 2: The red and green colour combination in panel A should be considered to be changed, even though the logic of the diagram makes clear which is which, it would be considerate for colour-seeing impaired people
Response: The colours in Figure 2 have been changed.
Lines 206ff: maybe add a short explanation what “balancing and purifying selection” means exactly, to remind the reader
Response: a short explanation of balancing selection and purifying selection has been added in lines 214-216
Figure 3: I suggest to add the gene names to the figure itself, that would be much easier than the identifiers alone.
Response: the gene names have been added to Figure 3
Reviewer 3 Report
Overall, the authors have presented an excellent manuscript on the genetic diversity among Sorghum genotypes that incorporate wild and domesticated accessions. The authors have used 8 core genes and nicely showed how domestication reduced genetic diversity in the C4 core genes by 22%. Interestingly, few non-synonymous codons were identified in MDH and ME but the C4 PPDK had none. The lack of diversity is interesting given the variable genotypes analysed.
The manuscript is well written and I only have minor revisions:
Introduction:
Line 37: Zelitch et al 2009 Plant Physiology should be added as this was the first demonstration in C4 the toxicity of glycolate.
Line 77 - Check referencing here.
Figure 1 - The legend needs to add detail about the text in red and the proteins which are not defined as well - PPCK and PPDK-RP. Here you could also describe the 8 core genes. I am still unsure where you get 9 genes from?
Materials and Methods:
In the abstract the authors mention 8 core C4 genes but in the methods 9 were mentioned. Please check and adjust accordingly.
Results:
line 209 ...while no gene was identified as being under purifying selection - This sentence is confusing because as you read later in the results line 215 the authors mentioned that 8 genes were under purifying selection. Can the authors please clarify this?
How many rbcS genes are there in Sorghum? Is there differences among these? Why did you only choose 1 RbcS gene? What as the criteria for this?
Discussion:
The discussion was well written.
Author Response
Dear reviewer, we have revised this manuscript according to your valuable comments. Please be aware that line numbers were changed after converting to the citation format of the journal.
Overall, the authors have presented an excellent manuscript on the genetic diversity among Sorghum genotypes that incorporate wild and domesticated accessions. The authors have used 8 core genes and nicely showed how domestication reduced genetic diversity in the C4 core genes by 22%. Interestingly, few non-synonymous codons were identified in MDH and ME but the C4 PPDK had none. The lack of diversity is interesting given the variable genotypes analysed.
The manuscript is well written and I only have minor revisions:
Introduction:
Line 37: Zelitch et al 2009 Plant Physiology should be added as this was the first demonstration in C4 the toxicity of glycolate.
Response: the reference has been added, line 42 and bibliography.
Line 77 - Check referencing here.
Response: reference and citation style has been changed throughout this manuscript to meet the requirement of the journal
Figure 1 - The legend needs to add detail about the text in red and the proteins which are not defined as well - PPCK and PPDK-RP. Here you could also describe the 8 core genes. I am still unsure where you get 9 genes from?
Response: details explaining PPCK and PPDK-RP have been added, lines 98-106
This study focuses on 8 key proteins involved in the NADP-ME photosynthetic pathway (Figure 1). According to Williams, Aubry & Hibberd (2012) and Wang, et al. (2009), 9 genes encoding these proteins were identified in sorghum with expression and evolutionary evidence supporting their involvement in the NADP-ME photosynthetic pathway, including 2 CA (Sobic.003G234200, Sobic.003G234400), 1 PEPC (Sobic.010G160700), 1 NADP-MDH (Sobic.007G166300), 1 NADP-ME (Sobic.003G036200), 1 PPDK (Sobic.009G132900), 1 PPDK-RP (Sobic.002G324400) and 1 RbcS (Sobic.005G042000). Therefore, these genes were named C4 genes, to distinguish them from 18 homologous of these C4 genes, which were named non-C4 genes due to lack of evidence supporting their involvement in NADP-ME photosynthetic pathway.
We understand the confusion, and have revised lines 85-90 for clarity
Materials and Methods:
In the abstract the authors mention 8 core C4 genes but in the methods 9 were mentioned. Please check and adjust accordingly.
Response: as explained above,
This study focuses on 8 key proteins involved in the NADP-ME photosynthetic pathway (Figure 1). According to Williams, Aubry & Hibberd (2012) and Wang, et al. (2009), 9 genes encoding these proteins were identified in sorghum with expression and evolutionary evidence supporting their involvement in the NADP-ME photosynthetic pathway, including 2 CA (Sobic.003G234200, Sobic.003G234400), 1 PEPC (Sobic.010G160700), 1 NADP-MDH (Sobic.007G166300), 1 NADP-ME (Sobic.003G036200), 1 PPDK (Sobic.009G132900), 1 PPDK-RP (Sobic.002G324400) and 1 RbcS (Sobic.005G042000). Therefore, these genes were named C4 genes, to distinguish them from 18 homologous of these C4 genes, which were named non-C4 genes due to lack of evidence supporting their involvement in NADP-ME photosynthetic pathway.
We understand the confusion, and have revised lines 85-90 for clarity
Results:
line 209 ...while no gene was identified as being under purifying selection - This sentence is confusing because as you read later in the results line 215 the authors mentioned that 8 genes were under purifying selection. Can the authors please clarify this?
Response: This manuscript investigated selection signal at two different scales, gene-level and SNP-level. Gene level investigation analyzed genetic variation within a gene as a whole, while SNP-level investigation analyses each SNP separately, providing a higher resolution compared to gene-level investigation. A gene not under selection at the gene-level could well contain SNPs that are under selection at SNP level.
We have modified this part for clarity, lines 222-223
How many rbcS genes are there in Sorghum? Is there differences among these? Why did you only choose 1 RbcS gene? What as the criteria for this?
Response: Williams, Aubry & Hibberd (2012) reported “3 rbcS genes” in sorghum. However, only one of them is annotated as a gene in the reference genome, the other two are just DNA fragments showing high homology to rbcS. The latest annotation of the reference genome also confirmed the only rbcS gene in sorghum according to Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html#). We also blasted the sorghum rbcS gene against the reference genome to search for additional rbcS genes, but nothing came up passing our threshold (E-value<1e-10, sequence identity >60% and alignment length>80%). Therefore, only 1 rbcS gene was identified in sorghum, which was included in this study.
Discussion:
The discussion was well written.
Response: thank you.
