Evaluation of the Composition and Accumulation Pattern of Fatty Acids in Tartary Buckwheat Seed at the Germplasm Level

Round 1

Reviewer 1 Report

In this article, the authors investigated the contents of total lipid, total flavonoid, and 10 fatty acids in the seeds of 31 different Tartary buckwheat accessions. They screened out the excellent germplasm with high contents of total flavonoid and unsaturated fatty acids. In addition, the authors also analyzed the accumulation patterns of 10 fatty acids in the developing seed and identified the biosynthesis genes of fatty acids in Tartary buckwheat. The topic is interesting.and all measurements and analyses meet the scientific standards. Some minor points need to correct before publication.

1. In line 16, add “contents of” before total lipid.

2. In lines 88-91, why were TB01 and TB04 Tartary buckwheat accessions selected to investigate the accumulation patterns of 10 fatty acids in the developing seed?

3. In Figure 4, does the FPKM value of fatty acid biosynthesis genes come from one or multiple biological replicates of transcriptome data?

4. Abbreviations should be used throughout the manuscript.

5. Grammar checks should be carried out for proper English.

 

6. Please check the structure of the format presentation for Agronomy.

Author Response

Dear Reviewer 1,

Thank you for your comments. All comments were addressed point by point as follows:

1. In line 16, add “contents of” before total lipid.

Reply: Thank you for your professional suggestion, we have corrected this error, and highlighted by RED in the revised manuscript. Please see lines 15-16 in the revised manuscript.

2. In lines 88-91, why were TB01 and TB04 Tartary buckwheat accessions selected to investigate the accumulation patterns of 10 fatty acids in the developing seed?

Reply: In our previous pre-experiment, we found they contain high contents of total lipid, oleic acid, and linoleic acid. More importantly, they have high seed setting rate (about 30-36%), while most other Tartary buckwheat accessions are 12%-20%. Therefore, we selected them to investigate the accumulation patterns of 10 fatty acids in the developing seeds, which could reduce our workload for tagging flowers.

3. In Figure 4, does the FPKM value of fatty acid biosynthesis genes come from one or multiple biological replicates of transcriptome data?

Reply: The FPKM value of each fatty acid biosynthesis gene was a mean value of three biological replicates from the same sample RNA-seq. We have stated it, and please see lines 277-278 in the revised manuscript.

4. Abbreviations should be used throughout the manuscript.

Reply: Abbreviations have been used throughout the manuscript. Please see the revised manuscript.

5. Grammar checks should be carried out for proper English.

Reply: We have performed the relative corrections as much as possible. Please see the revised manuscript.

6. Please check the structure of the format presentation for Agronomy.

Reply: We have carefully checked the structure of the format presentation for Agronomy. Please see the revised manuscript.

Reviewer 2 Report

The manuscript focuses on the analysis and comparison of 10 fatty acids content, and related gene expression, in different germplasm accessions of tartary buckwheat. The biosynthesis patterns are also explored in 2 accessions during seed development.

Overall, the work is interesting and innovative, but it needs linguistic revision and more details on material and methods especially.

Please see below my suggestions:

 

Line 40 (and title or abstract): Extended scientific name with authors should be mentioned the first time the species is mentioned.

Line 45: please name the flavonoids referred to, with their relative effect on human nutrition/health

Line 86: please explain ‘field management’ conditions.

Line 105: `song` should be majuscule; calibration curve should be shown in Supplementary materials.

Section 2.4. materials and methods should be described in more detail, e.g., fatty acid extraction methodology, type of column used for HPLC etc.

Section 2.5. It is not clear where the transcriptome analyses were taken from.

Lines 127-128: were the assumptions checked prior to ANOVA test?

Lines 128-129: please explain correlation method with assumptions.

Fig. 1: Please explain method used to perform pairwise comparisons.

Lines 211-212: why were the 2 accessions selected to perform this analysis?

Fig. 3: The description of these patterns in the text does not reflect the graphs represented in Fig. 3

 

Section 3.5: it is not clear where expression data come from (materials and methods sections), peer review cannot be performed here.

 

Author Response

Dear Reviewer 2,

Thank you for your comments. All comments were addressed point by point as follows:

1. Line 40 (and title or abstract): Extended scientific name with authors should be mentioned the first time the species is mentioned.

Reply: Thank you for your professional suggestion. We have corrected this error. Please see line 39 in the revised manuscript.

2. Line 45: please name the flavonoids referred to, with their relative effect on human nutrition/health.

Reply: We have added the related information. Please see lines 45-48 in the revised manuscript.

3. Line 86: please explain ‘field management’ conditions.

Reply: We have explained ‘field management’ conditions. Please see line 89 in the revised manuscript.

4. Line 105: `song` should be majuscule; calibration curve should be shown in Supplementary materials.

Reply: Thank you for your professional suggestion. We have corrected the error. Please see line 107 in the revised manuscript. In addition, we have provided the calibration curve of total flavonoid quantification as Supplementary Table S1. Please see line 108 in the revised manuscript.

5. Section 2.4. materials and methods should be described in more detail, e.g., fatty acid extraction methodology, type of column used for HPLC etc.

Reply: Thank you for your suggestion. We have provided detail descriptions of fatty acid fatty acid extraction methodology and column type used for HPLC. Please see lines 111-115 and 118-119 in the revised manuscript.

6. Section 2.5. It is not clear where the transcriptome analyses were taken from.

Reply: The transcriptome data from our lab, which hasn't been published yet. We have described the source of the transcriptome data. Please see line 130 in the revised manuscript.

7. Lines 127-128: were the assumptions checked prior to ANOVA test?

Reply: In our article, we directly used the one-way analysis of variance in SPASS software to analyze the significant differences and obtained relevant results.

8. Lines 128-129: please explain correlation method with assumptions.

Reply: Generally, in the time and space, there is a good correlation between the expression level of genes involved in the synthesis of a certain substance and the accumulation content of the substance. Through the correlation calculation between gene expression and substance content, many genes have been identified as candidate genes for the synthesis of some substances, and their functions have been proved to be correct. For example, (1) Boping Wu, Xiangmei Cao, Hongru Liu, et al. UDP-glucosyltransferase PpUGT85A2 controls volatileglycosylation in peach. Journal of Experimental Botany, 2019, 70(3): 925-936; (2) Yuanyuan Zhang, Xueren Yin, Yuwei Xiao, et al. An ethylene response Factor-MYB transcription complex regulates furaneol biosynthesis by activating quinone oxidoreductase expression in strawberry. Plant Physiology, 2018, 178: 189-201. Therefore, we performed the correlation analysis between fatty acids biosynthesis genes and related fatty acids accumulation in the developing seeds of Tartary buckwheat at 6 different development stages with aim to identify the key genes for fatty acid biosynthesis in Tartary buckwheat seeds.

9. Fig. 1: Please explain method used to perform pairwise comparisons.

Reply: We have stated the method used to perform pairwise comparisons in the Fig. 1 legend. Please see line 156 in the revised manuscript.

10. Lines 211-212: why were the 2 accessions selected to perform this analysis?

In our previous pre-experiment, we found they contain high contents of total lipid, oleic acid, and linoleic acid. More importantly, they have high seed setting rate (about 30-36%), while most other Tartary buckwheat accessions are 12%-20%. Therefore, we selected them to investigate the accumulation patterns of 10 fatty acids in the developing seeds, which could reduce our workload for tagging flowers.

11. Fig. 3: The description of these patterns in the text does not reflect the graphs represented in Fig. 3

Reply: We have re-described the accumulation patterns of 10 fatty acids in the developing Tartary buckwheat seeds based on Fig. 3. In addition, because the contents of the 10 fatty acids vary greatly, we put the fatty acids with similar contents in one picture instead of with the same accumulation patterns. Please see lines 222-231 in the revised manuscript.

12. Section 3.5: it is not clear where expression data come from (materials and methods sections), peer review cannot be performed here.

Reply: The transcriptome data from our lab, which hasn't been published yet. We have described the source of the transcriptome data in the materials and methods sections. Please see line 130 in the revised manuscript.

Round 2

Reviewer 2 Report

I thank the authors for addressing most of my concerns. However, I invite them to reconsider the manuscript structure.

I am not able to agree on the results linked to gene expression, as authors present correlations made with unpublished, therefore not validated, rna-seq data.

Moreover, I do not find sufficient the reply to my previous questions concerning statistical analysis:

7. Lines 127-128: were the assumptions checked prior to ANOVA test?

8. Lines 128-129: please explain correlation method with assumptions.

9. Fig. 1: Please explain method used to perform pairwise comparisons.

Given these premises, I cannot suggest acceptance in present form.

Author Response

Dear Reviewer 2,

Thank you for your professional comments. All comments were addressed point by point again as follows:

I am not able to agree on the results linked to gene expression, as authors present correlations made with unpublished, therefore not validated, rna-seq data.

Reply: Thank you for your professional comments. Our transcriptome data used in this article are true and reliable. In fact, we performed the RNA-seq of seeds of two Tartary buckwheat accessions (TB04 with larger seed and other one accession) at 8 different developing stages (4, 7, 10, 13, 16, 20, 25, and 30 DAP) with an aim to identify the genes involved in seed size (Figure 1 and Figure 2). Because the seed started grouting at 10 DAP, we only selected the seed transcriptome data of TB04 at 10, 13, 16, 20, 25, and 30 DAP to perform the expression analysis of fatty acids biosynthesis genes. At present, we have identified the key candidate genes involved in the regulation of Tartary buckwheat seed size, and are doing functional verification in progress. Since the transcriptome data have not yet been published, we have not uploaded them into NCBI. Under the premise of ensuring that the data will not be disclosed, we can send the data to you privately if you need it. Some data related to these transcriptomes, please see the file of author-coverletter-23049380.v1.docx.

7. Lines 127-128: were the assumptions checked prior to ANOVA test?

Reply: Thank you for your professional comment. Yes, we have conducted normal distribution and homogeneity of variance analysis before ANOVA test in SPSS. All data conform to normal distribution and have homogeneity of variance.

8. Lines 128-129: please explain correlation method with assumptions.

Reply: Thank you for your suggestion. The correlation method has been widely used to identify candidate genes in the molecular biological field, and the functional verification of candidate genes have also proved the reliability of the method. We have provided the description on the correlation method with assumptions. Please see lines 136-143 in the revised manuscript.

9. Fig. 1: Please explain method used to perform pairwise comparisons.

Reply: We have added the method used to perform pairwise comparisons. Please see line 164 in the revised manuscript.

Author Response File: Author Response.docx