Identification and Validation of Quantitative Trait Loci for Grain Size in Bread Wheat (Triticum aestivum L.)
Round 1
Reviewer 1 Report
Grain wt. which is one of the main component of the grain yield. The GL and GW are the major components that contribute GY. The manuscript, “Identification and validation of two major QTL regions with pleiotropic effects on grain width and length in bread wheat (Triticum aestivum L.)” add new sight to the researchers as three major qtls were identified for these traits. These three major qtls were further validated and found its contribution on increase on GW and GL.
Comments:
There is increase in GW and GL to certain limit only. By introgession of these QTLs may hampher spatial arrangement of wheat spikelet and over all spike and also lead to decrease in overall grain yield of the plant. Pl Justify.
Other comments have been mentioned on manuscript itself.
Comments for author File: 
Comments.pdf
Author Response
Point 1:
There is increase in GW and GL to certain limit only. By introgession of these QTLs may hampher spatial arrangement of wheat spikelet and over all spike and also lead to decrease in overall grain yield of the plant. Pl Justify.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
Dear review1, in the current study, QGW.yz.2D/QGL.yz.2D, QGW.yz.4B/QGL.yz.4B and QGL.yz.5A all explained major effects on corresponding traits. Among them, QGW.yz.2D/QGL.yz.2D had significantly negative effect on GNS, while QGW.yz.4B/QGL.yz.4B, and QGL-yz-5A did not have negative effect on GNS. We also find that the positive allele of QGW.yz.2D/QGL.yz.2D had been used difficulty in the Middle and Lower Yangtze Valleys Wheat Zone (MLYVWZ) and the positive alleles of QGW.yz.4B/QGL.yz.4B and QGL-yz-5A had been used widely whether in MLYVWZ and in the Yellow-Huai River Valleys Wheat Zone (YRVWZ), so further fine-mapping and map-based cloning of QGW.yz.2D/QGL.yz.2D for grain size would facilitate better use of this QTL in wheat breeding. I elaborated on this in section 4.2.
Point 2:
Why LOD threshold value was set at 2.5:
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
In section 2.4, QTL analysis was conducted using the inclusive composite interval mapping algorithm and the LOD threshold value was set at 2.5 in IciMapping V4.0 software [36–38]. We followed the method of Xu et al (2020), Jiang et al. (2020), and Zhu et al. (2020). And we also referred to “Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC. http://statgen.ncsu.edu/qtlcart/WQTLCart.htm”. A logarithm of odds (LOD) threshold of 2.5 was set for QTL mapping. We add the “with walking step = 0.001 cM and PIN = 0.0005” after the threshold to describe the process in more detail.
Point 3:
In the table 1, 3.48 is the mean value of E1 environment or else, and ** indicates on P2 not P1:
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
In section 2.4, the heritabilities (HB2) for GW and GL were 0.71 and 0.79, and we indeed identified three major QTL with larger phenotypic contribution for grain size in the current study. In the table 1, 3.48 is the mean value of E1 environment, and ** indicates different between P1 and P2 at P < 0.01.
Point 4:
In section 4.1, we corrected the wrong word in line 359. Thank you very much for your comments and kind suggestions.
Point 5:
This statement is differ from my view. The spatial arrangement of spikelet in spike is under genic control. Each Spikelet has 5-7 florets. If we increase the grain size in terms of volume, the arrangement of floret in spikelet get disturbed that might too hamper the yield. We can increase the boldness of seed to certain extent only as negative effect of other allele ll play major roles.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
In section 5, we agreed with the reviewer 1, and revised the last sentence. “In addition, three breeder-friendly markers KASP_2D, KASP_4B, and KASP_5A for QGW.yz.2D/QGL.yz.2D, QGW.yz.4B/QGL.yz.4B, and QGL-yz-5A, respectively, would be useful for marker-assisted selection in wheat breeding programs”.
Point 6:
Thank you very much for your comments and kind suggestions for some minor mistakes in the attached file. We revised the words that were not used correctly in the main text.
Reviewer 2 Report
Hu et al. conducted a good study on the identification and validation of two major QTL regions with two pleiotropic effects on grain width and length in bread wheat. They identified eleven candidate genes associated with encoding Ethylene-responsive transcription factor, Oleosin, Osmotin protein, and Thaumatin protein and reporter 3 major QTL and KASP markers that can be useful for developing new wheat cultivars with high yields. The experiment is well planned and executed and most of the necessary information is provided in the manuscript. However, I suggest the authors add a few lines about the novelty of this manuscript in the abstract and introduction. As markers have been used in this study and the authors mentioned one of their previous studies where they used SSR and SNP markers, I think that the introduction can be elaborated by adding some information on molecular markers (see the article for markers https://doi.org/10.1007/s10722-014-0114-5). Authors can also mention why KASP markers were preferred for this study. Introduction can be elaborated by adding this information. It might be better to present heading 2.3 as ‘Statistical analysis’ rather than phenotypic analysis. In 2.5, please mention the method or protocol followed for genomic DNA isolation. Please provide the list of 159 wheat cultivars/lines that were used for the validation of the major QTL either in the main text or in the supplementary material. Please elaborate on the discussion in 4.2 in the light of previous studies. The findings and their implications should be discussed in the broadest context possible.
Author Response
Point 1:
I suggest the authors add a few lines about the novelty of this manuscript in the abstract and introduction. As markers have been used in this study and the authors mentioned one of their previous studies where they used SSR and SNP markers, I think that the introduction can be elaborated by adding some information on molecular markers (see the article for markers https://doi.org/10.1007/s10722-014-0114-5). Authors can also mention why KASP markers were preferred for this study. Introduction can be elaborated by adding this information.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We add the information in the last of the introduction as follows: “SSR and SNP markers are commonly molecular markers that are based on polymerase chain reaction (PCR) [29]. Recent progress on wheat genome sequencing and availability of high-throughput chip-based markers have accelerated QTL analysis and MAS in breeding programs. Kompetitive Allele-Specific PCR (KASP) assay, which using the specific commercial master-mix without compromising data throughput, has been as an excellent breeding toolkit for high-throughput, cost-effective tracing functional genes/QTL for agronomic traits, pre-harvest sprouting resistance and biotic stress resistances in wheat [30,31].”
Point 2:
It might be better to present heading 2.3 as ‘Statistical analysis’ rather than phenotypic analysis.
Thank you very much for your comments and kind suggestions. Accepted the suggestion.
Point 3:
In 2.5, please mention the method or protocol followed for genomic DNA isolation.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
We add the sentence “Genomic DNA was extracted from fresh leaves according to Ma and Sorrells [39]” in the beginning of the 2.5.
Point 4:
Please provide the list of 159 wheat cultivars/lines that were used for the validation of the major QTL either in the main text or in the supplementary material.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
In 2.1, we talked briefly about the composition of 159 wheat cultivars/lines.
In 3.5, we mentioned “The list of 159 wheat cultivars/lines and their genotypes and phenotypes were shown in Table S3” in line 309. And we provided the name of the cultivars, the pedigree of the lines and their suitable planting areas in supplementary Table 3 (In Table S3, the red font is what I added).
Point 5:
Please elaborate on the discussion in 4.2 in the light of previous studies. The findings and their implications should be discussed in the broadest context possible.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the article as follows:
In 4.2, we referred to the previous studies, and discussed in the broadest context possible. We add a summary sentence for the current atudy : “The significant additive effect indicated that pyramiding of the major loci facilitated by using the developed KASP markers could be utilized as applicable strategy to optimize grain size in wheat breeding.” We also found that “these three positive alleles have been used more frequently in wheat breeding programs in YRVWZ, which was consistent with that the average GW and GL of the cultivars/lines from YRVWZ are 3.40 mm and 6.86 mm, which are 1.20% and 2.85% higher than those from MLYVWZ. For YZ1 allele from QGW.yz.2D/QGL.yz.2D, due to its negative effect on the GNS, it mainly exists in the cultivars/lines in YRVWZ and has been used difficulty in wheat breeding programs in MLYVWZ” (lines 416-422). If there is something wrong, please help us correct it.
Reviewer 3 Report
Hu et al. have Identified two major QTL region with pleiotropic effects on grain width and length in bread wheat.
The manuscript is written in a concise and very legible manner. In addition, the manuscript contains relevant paragraphs that have been discussed. The selection of the bibliography is appropriate to the content of the manuscript.
After analyzing the content, I included minor comments directly in the attached file.
The Abstract is a little bit lengthy; the authors should focus on the most important research results.
There is a duplication in the numeration of the references list.
Figure 2 legend is so confused, and I can’t follow the information. It should be rewritten in a simple way to understand. Figure 2 a is missed
Comments for author File: Comments.pdf
Author Response
Point 1:
The Abstract is a little bit lengthy; the authors should focus on the most important research results.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the abstract and deleted the unimportant part (lines 19-44).
Point 2:
There is a duplication in the numeration of the references list.
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We revised the references list.
Point 3:
Figure 2 legend is so confused, and I can’t follow the information. It should be rewritten in a simple way to understand. Figure 2 a is missed
Thank you very much for your comments and kind suggestions. Accepted the suggestion. We have divided Figure 2 into Figures 2 to 4, and rewritten the comments of the Figures 2 to 4. If there is something wrong, please help us correct it.
Point 4:
Thank you very much for your comments and kind suggestions for some minor mistakes in the attached file. We revised the words and the punctuation that were not used correctly in the abstract, introduction, 2.1, 2.2, 2.6, figure 1, 3.2, figure 2, 3.5, figure 4, 3.6 and 4.3. If there is something wrong, please help us correct it.
