QTL Mapping by Chromosome Segment Substitution Lines (CSSLs) Reveals Candidate Gene Controlling Leaf Sucrose Content in Soybean (Glycine max (L.) Merr.)

Round 1

Reviewer 1 Report

Dear Prof. Dr. Editor of Agronomy Journal,

 

I write you regarding Manuscript Number: agronomy- 2419199 entitled " QTL mapping reveals candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.)" which was submitted to the Agronomy journal.

In this manuscript, the author's mapped QTL reveals a candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.). The manuscript is suitable for publication in Agronomy.

I have gone through this work. My decision is accepted with minor revisions for this work. The reason for that is as follows:

The manuscript deals with the QTL mapping reveals candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.).

 

First: Title: It should change to the following:

1)  ok

 

Second Abstract, keywords and Introduction:

2) has some minor corrections as in the attached file.

 

Third: The objectives of the study

3) has some minor corrections as in the attached file.

 

Fourth: Materials and Methods

4) has some minor corrections as in the attached file.

 

Results and discussion

5)  has some minor corrections as in the attached file.

 

References

6) It has some minor corrections as in the attached file.

 

Thank you for suggesting me as a reviewer for this paper.

 

with best regards

Comments for author File: Comments.pdf

Dear Prof. Dr. Editor of Agronomy Journal,

 

I write you regarding Manuscript Number: agronomy- 2419199 entitled " QTL mapping reveals candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.)" which was submitted to the Agronomy journal.

In this manuscript, the author's mapped QTL reveals a candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.). The manuscript is suitable for publication in Agronomy.

I have gone through this work. My decision is accepted with minor revisions for this work. The reason for that is as follows:

The manuscript deals with the QTL mapping reveals candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.).

 

First: Title: It should change to the following:

1)  ok

 

Second Abstract, keywords and Introduction:

2) has some minor corrections as in the attached file.

 

Third: The objectives of the study

3) has some minor corrections as in the attached file.

 

Fourth: Materials and Methods

4) has some minor corrections as in the attached file.

 

Results and discussion

5)  has some minor corrections as in the attached file.

 

References

6) It has some minor corrections as in the attached file.

 

Thank you for suggesting me as a reviewer for this paper.

 

with best regards

Author Response

Dear review,

Many thanks for your kind suggestion and revision. It is our honor to have you help us improve our manuscript. 

We have correct the spelling and words in the manuscript. The revision has been uploaded now!

Best,

Xindong

Reviewer 2 Report

The paper deals with genetic control of the leaf sucrose content in soybeans. It is assumed that if located in the genome and properly traced (tagged) it could be used in marker assisted selection. Also, it is assumed that level of sucrose content (as function of photosynthesis) might be related and used in breeding for agronomic traits such as plant biomass and grain yield.

The QTL mapping has been performed within the population of segregating CSSL lines derived from a distant cross (G. max X G. soja) and several cycles of back-crossing aided by molecular markers. A single QTL of high LOD value for leaf sucrose content has been detected and confirmed.

Considering the material, methods and results, the manuscript has scientific merit. Based on insight in tables and figures, it provides original results that might be of interest for soybean breeding.

 

However, after careful reading, my opinion is that the presentation and discussions of results is insufficient and sometimes unclear. The level of manuscript finalization is very low. Moreover, there are some serious mistakes and missing what suggests that manuscript was nut supervised by all authors before submission. Also, my opinion is that quality of English in some chapters is not satisfactory (unclear sentences). My strong suggestion to editor is to request authors to do significant improvements in text editing according to “Instructions to authors”, as well as to pay attention to the following recommendations written bellow. I strongly advise senior authors to carefully read and approve the final version of the revised manuscript before resubmission. 

Title

The originality of the paper relies on very special mapping plant material (CSSL lines). I advise you to put this in the title. E.g. „QTL mapping by chromosome segment substitution lines (CSSLs) reveals candidate gene controlling leaf sucrose content in soybean (Glycine max (L.) Merr.)“

 

Abstract

I suggest omitting the first sentence of the Abstract because this is a very basic research. The second one is sufficient as a brief introduction to the topic.

You have to state here the exact number of CSSL lines (190) used in mapping. Also, you have to briefly explain specificity of parents (Suinong 14 is G. max variety and ZYD00006 is from G. soja species). Also, the abstract needs a sentence of explanation about used genetic markers (SNP).

The second last sentence of the abstract should be removed (Glyma.14g029100 is a member of sucrose-phosphate synthase which affects crop yield) because this is not result of this paper. This can be mentioned in the Discussion but not here.

 

1. Introduction

It is not common to put the figure from other source in the Introduction. According to me, this figure is not necessary because it explains the matter which is not subject of your research. I suggest you to keep only this sentence: „In the presence of light, sucrose is synthesized in the cytoplasm of source leaves (Stitt and Zeeman, 2012).“

In the 3rd paragraph, there is a sentence „Previous studies have already created a genome-wide chromosome segment substitution line (CSSLs) for QTL mapping, whereas is widely used for oil, protein and seed size.“ This sentence requires some citations. I suggest Balakrishnan et al, Theoretical and Applied Genetics (2019) 132:1–25; or some soybean specific papers such as Xin et al (2016) PLoS ONE 11(3):e0149380.

In the last paragraph you are citing several papers dealing with sucrose content in soybean and genomic regions (chromosomes) where QTLs have been located. E.g. „A recent paper elucidating QTL related to sucrose concentration in a 178 soybean genotype GWAS uncovered five potential QTL, located on chromosomes 4, 6, 7, 11 and 12 (Sui et al., 2020).“ You should stress here that these QTLs refer to sucrose content in the SEED.

“The objective of this study is 1) identify the candidate genes affecting the sucrose content in the leave;” > last word should be…..leaves or leaf;

 

2. Materials and Methods

2.1. Plant materials

I am very confused (and disappointed) that this sentence passed the control of all co-authors of this paper: „190 chromosome segment substitution lines (CSSLs) rice population carrying contiguous chromosomal segments of japonica Nipponbare in the genetic background of indica 9311 was developed. “

However, a similar sentence dealing with soybean should be instead of this one.

In this chapter must be clearly explained (or referred to original publication) how CSSL population has been developed. I suppose one of the authors (Dr. Qingshan Chen) should advise on this.

The last sentence in this chapter “A high-density genetic linkage map based on the CSSLs population was constructed by Zheng et,al. (Zheng et al., 2022)” is not fitting this chapter – must be erased.

 

2.2. Measurement of leaf sucrose content

“Seven samples of 190 CSSLs were collected from 4: 00 pm to 5: 00 pm…..” it is not clear why seven samples?

“The sucrose content in leaves was determined by using the sucrose content kit of Shanghai Youxuan Biological Company.” > exact commercial name of the kit, and company name must be declared. Please, read the instructions for authors on how to cite lab products.

“Under acidic conditions, sucrose was hydrolyzed to produce glucose and fructose, and fructose further reacted with m-diphenol to produce colored substances, which had a characteristic absorption peak at 480nm, and was determined by enzyme-labeled instrument.” > it is not clear at the end what is the value (unit) for “sucrose content”. You must state here what is the unit (% or mg/g?). Later in other chapters there is confusion with this.

 

2.3. Method of QTL Mapping

“The genetic map for QTL mapping was constructed by Zheng et al,. (Zheng et al., 2022) based on sequencing with an average genome depth more than 27x in the parent varieties and 5x in the CSSLs lines.” > this should be explained in more detail. I assume the genotyping has been done earlier and you did phenotyping only. Here should be stressed the number of genotypes and SNP markers used to develop genetic map.

In regard to the method of QTL mapping (use of ICIM model in ICIMMapping software) all used softwares (across entire text) have to be properly cited or version/model details have to be written (see the authors instructions).

 

3. Result (> should be Results)

3.1. Distribution of sucrose content in CSSLs population

“The correlation coefficient of sucrose content in CSSLs population from 2021 to 2022 was 0.951 (Figure 2C), which indicated that the sucrose content in CSSLs population was stable from 2021 to 2022.” > this sentence (in the middle of chapter) should be erased because it appears again at the end of chapter (second last one).

Title of Figure 2 (below the figure) is imprecise and must be clearer. E.g. instead of “Distribution histogram” better to use “Range frequencies”;  “sucrose content” must be followed by units (mg/g); “the Y axis represents the plant coefficient of the corresponding sucrose content” > Y axis represents the frequency of lines with similar sucrose content level; etc.

The titles of most tables and figures are insufficient and imprecise. It is well known that titles of tables and figures must be detailed and self-sufficient to enable readers to understand the content without reading the text. It means that e.g. title of Table 2 “QTL Mapping of Sucrose content in CSSLs population” is missing information that this is about sucrose content in the leaf; species (soybean); etc.

Figure 3. should be excluded from the manuscript. First, genetic map (figure A) was constructed in another research (Zheng et al, 2022) and is not informative at all. There is nothing useful for the reader. The same is with LOD score (figure B). This information is already present in table 2.

 

3.3. Candidate genes identification

The last sentence of this chapter “Glyma.14g029100 (Chr14:2126632..2133000) is directly or indirectly related to sucrose synthesis in soybean and plays an important role in sucrose synthesis and starch decomposition, and is considered as a hub gene affecting the leaf sucrose content.” is not the outcome of your research. In the chapter Results you are supposed to present and comment on your own results only. This sentence might be the part of Discussion.

Title of Table 4. “Annotation of candidate genes.” is too short and insufficient. A good example of how to do it is the title of Figure 5.

 

3.5. QRT-PCR analysis of candidate genes (> should be qRT-PCR)

It is not clear from the text why R149 and R194 have been selected.

Title of the Figure 6. “The relative transcription level of Glyma.14G029100 in leaves of parents and lines with high sucrose content at V4 stage. The letters at the top of the column chart indicate the results of multiple groups of comparisons, and different letters indicate significant differences.” > is not precise. What “the results of multiple groups of comparisons” means? Clarify. From the figure should be removed the name of the gene (it is already in the title) and X axis “varieties” should be replaced with “genotypes”.

Figure Sp.2 (supplement) has not been mentioned nor commented on. My opinion is that this figure should be within the manuscript.

 

3.6. Haplotype analysis of candidate genes

The names of applied softwares must be properly cited.

“The excellent haplotypes were defined as the haplotype was carried by 5% or more of the total materials.” > the term “excellent haplotype” must be better explained.

The very last sentence of this chapter “The differences of SNP--2400, SNP--1300, SNP--1254 and SNP--1060 in the excellent haplotype of Glyma.14g029100 may lead to the change of the promoter ARE element, which may affect the gene expression, thus affecting the sucrose content in leaves.” > this is not related to any presented result (table or figure)!?

The title of Figure 7 is not well understandable > should be clearer and more precise.

The title of Figure 8. “Sucrose content in leaves of GLYMA.14G029100 in different excellent haplotypes.” is not clear > should be redefined and more precise.

 

4. Discussion

Generally, large parts of the Discussion chapter are not directly related to own results but more explanation of some theoretical (general) knowledge on role of sucrose in the plant metabolism. I strongly advise that this chapter be rearranged by a senior scientist.

In the first paragraph of the discussion, as well as at several places in the text, the term “strain” (instead of line) is used. The term “strain” is usually used for microorganisms and term “line” for higher plants. I suggest using the term “line” (in context of CSSL) across entire paper.

In the first paragraph where you additionally explain usage of CSSL for QTL mapping it would be useful to explain what was the purpose of creating SN14 x ZYD00006 soybean interspecies CSSL population and to present the main achievements with this population (previously published research results obtained with this population).

In the 3^rd paragraph, the sentence “The relative transcriptional level of Glyma.14g029100 was the lowest in the female parent SN14 and higher in the male parent ZYD00006. The study on the molecular basis of heterosis (Fu et al., 2022) revealed that the expression of key regulatory genes in offspring changed significantly, leading to super-parental inheritance.”  > I do not understand the point of this comparison with Fu et al., 2022.

It should be discussed why wild parent (ZYD00006 – Glycine soja) had higher leaf sucrose content than cultivar SN14 (G. max).

The part of Discussion chapter which starts at the end of page 13 – with the sentence “Motifs, essential DNA sequences found in promoters and enhancers, play a pivotal role in the regulation of gene expression levels. In promoters, motifs facilitate the recruitment of general transcription factors (TFs) and RNA polymerase……..” > this text is mainly dealing with theoretical knowledge (without indicating the source) and is not focused on comparison with own results. Some parts of this text might be (if necessary) moved to the chapter Introduction, but these parts of the text either should be removed or discussed in the light of own results. Here are additional parts of the Discussion which are not related with the research results from this paper:

“Moreover, motifs are responsible for the targeted recruitment of transcription factors to specific genes. In our study, the promoter region of Glyma.14g029100 contained numerous cis-acting elements, including the ATBP-1 binding site, cis-acting elements of auxin, gibberellin, abscisic acid, and other plant hormones, and anaerobic-induced cis-acting regulatory elements. Cis-acting elements related to photoreaction had the largest proportion. Subsequently, the parent amino acid sequence of the candidate gene was compared, and a non-synonymous amino acid mutation was found. It was found on the website of phytozome that the amino acid site of the mutation was located in the do-main of sucrose phosphate synthase. According to the Xuemei et al,. (Dai et al., 2016) , a single amino acid mutation in the gene domain would affect its functional expression. It is speculated that the amino acid mutation site we found may affect the activity of sucrose phosphate synthase, thus affecting the accumulation of sucrose content.”

“Glyma.14g029100, also known as probable sucrose-phosphate synthase 3 (NCBI), is a member of SPS gene family. SPS is a key enzyme involved in both the instantaneous starch decomposition pathway and the sucrose anabolic pathway, serving as the rate-limiting enzyme in the latter. SPS catalyzes UDPG (glucose uridine diphosphate) and F6P (fruc-tose-6-phosphate) to produce S6P (sucrose-6-phosphate), which is then dephosphorylated by SPP (sucrose phosphatase) to generate sucrose. Its role is closely linked to the regulation of the source-sink relationship in plants, as well as the distribution of sucrose and starch content, plant growth rate, plant biological yield formation, crop yield and quality, and crop stress resistance physiology.” > this is not relevant for any of your results! This is supposed to be a research article – not textbook.

However, some parts of the text are repeating the results, without any discussion or comparison with other results:

“It was found that the leaf sucrose content of Hap_2 haplotype was higher. The Glyma.14g029100 gene of 23 strains belonging to Hap_2 comes from donor parent ZYD00006, and the Glyma.14g029100 gene of 167 strains belonging to Hap_1 comes from recurrent parent SN14. The haplotype of Glyma.14g029100 gene is the same at the 2867bp site of the CDS sequence of SNP-5293 and Glyma.14g029100 gene. The mutation of the base at this site from G to T will lead to the change of the 955th amino acid from M (methionine) to I (isoleucine), which may lead to the functional change of Glyma.14g029100 gene.”

The chapter Discussion should be seriously rearranged.

 

5. Conclusion

“A QTL related leaves sucrose was identified with CSSLs population.” > this sentence is not clear

“Nine candidate genes were screened in this area, and one hub gene, Glyma.14g029100, was identified among the candidate genes. Several deletions were found in the promoter area of hub gene in the parent (SN14 and ZYD00006) compared with the reference genome (Wil-liams82). qRT-PCR analysis shows that there was a significant difference among the offl-springs and parents and related to the leaves sucrose content closely.”

Conclusions should be simple and clear – please, redefine and check the English.

These comments were integrated with detailed comments on manuscript.

Author Response

Dear reviewer,

Many thanks for your time and careful read of our manuscript. We appreciate your hard work on the review work of our manuscript, and we learned a lot from your comments.

Now, we revised our manuscript and hope it seems better this time.

Best,

Xindong

Author Response File: Author Response.pdf